Professional Documents
Culture Documents
Antifungal Compounds VI
provide the tools for disease control expands. In order to
Spectrum Phytomedizin
Probedruck
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ISBN: 978-3-941261-10-5
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Contents
Page
Contents III
List of Participants X
Preface XVII
OVERVIEW ARTICLES
The Impact of the New European Regulations on the Management of 1
Crop Diseases
A. Leadbeater
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FUNGICIDE RESISTANCE -
MOLECULAR ASPECTS AND GENETICS
Molecular Approaches to Elucidate Pathways and Sites of Fungicide 91
Resistance in Oomycetes
S.C. Whisson, R. Fonné-Pfister, M. Csukai
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COMPLEX II INHIBITORS –
MODE OF ACTION AND RESISTANCE
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VI
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VII
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VIII
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Index 434
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Participants
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XI
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XII
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XIII
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XIV
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XV
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16. International
Reinhardsbrunn Symposium
April 25-29, 2010
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Preface
It has become increasingly difficult for growers to control crop diseases. Genetic
resistance of crop plants towards diseases has been in many cases short-lived and GMOs
have only limited success for disease control and acceptability. With more intensive
cropping, new diseases, new races and more aggressive pathotypes of diseases may arise.
All these changes require chemical control measures to prevent economic disaster, since
reliance on genetic resistance, biological control and cultural techniques have been
insufficient. The control of crop diseases with chemical fungicides has a successful
history for more than a century. The use of fungicides is an integral part of crop
production in many countries of the world, resulting in increased yields and farmer
income. Economic benefit studies have shown conclusively that without fungicides for
control of plant pathogens, production of some crops would be impossible in parts of the
world. Intensive use of chemical control measures has in turn led to its own challenges,
including resistance to pesticides. The sustainable use of pesticides to prolong their
effectiveness and usefulness to growers is key, and the implementation of resistance
management strategies an essential part of this.
The discovery and development of new fungicides and the technical support of
market products require a broad range of scientific investigations including studies on the
biological and biochemical mode of action, the genetic and molecular mechanism of
resistance, aspects of population genetics in controlled and natural field conditions. The
tri-annual series of the International Reinhardsbrunn Symposia on “Modern Fungicides
and Antifungal Compounds” is one of the few scientific platforms for a thorough
discussion of fungicide science. As in previous years, the 16th Symposium was held over
4 days (April 25 to 29, 2010) in Friedrichroda, Germany, and was visited by 114
delegates from 21 (mostly European) countries. The organizing committee with Prof.
H.W. Dehne (Bonn, Germany), Prof. H.B. Deising (Halle, Germany), Prof. U. Gisi
(Syngenta Crop Protection, Switzerland), Dr. K.-H. Kuck (Germany) and Prof. P.E.
Russell (UK), who acted also as reviewers of the submitted manuscripts of the
proceedings, put together a scientifically broad programme containing 48 talks and 37
poster presentations. The opening session covered general aspects such as the impact of
new EU regulations, crop protection markets, plant breeding and climate change on
disease control strategies. A session on new fungicides and new modes of action was
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XVIII
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1
The Impact of the New European Regulations
on the Management of Crop Diseases
A. LEADBEATER
Syngenta Crop Protection, Research and Development, Schwarzwaldallee 215, CH-4058 Basel,
Switzerland
Abstract
In November 2009, Regulation (EC) No 1107/2009, concerning the placing of plant protection
products on the market, was published, replacing Directives 91/414/EEC and 79/117/EEC. This
Regulation could, if implemented in an over-precautionary way, have a significant impact on the
future of pesticides in the European Union. The new Regulation represents a significant change
to the way pesticides are registered in the EU and is likely to result in a further decline in the
number of effective plant protection products, including fungicides, available to the farmer. It
will therefore result in a further reduction in the diversity of solutions to manage diseases in crops.
In addition, the new provisions for comparative assessment means that there is likely to be a still
further decline in the availability of fungicides in the future with no or low associated benefits in
terms of additional protection to humans or the environment.
Preliminary impact assessments of the new Regulation and of comparative assessment
indicates that several key fungicides which are cornerstones of disease management programmes,
for example members of the triazole group, are likely to be lost to the market. As a consequence,
the products available for farmers to control crop diseases will be reduced. In addition to this,
there is likely to be an impact on the resistance management of the remaining products on the
market. Maintaining sufficient diversity in mode of action is one of the most important
considerations for effective resistance management. The reduction in the number and diversity of
fungicide products maintained in the European market in future years, combined with the
increased burden on industry to bring new solutions to market is expected to have overall a
significant, negative impact on the management of crop diseases in the future and may affect the
sustainability of growing some crops in Europe.
Introduction
Crop production in Europe is efficient and sustainable, and a key factor is the effective
management of a broad range of diseases, weeds and insects. European agriculture and
horticulture are thus dependent on a variety of inputs including a diverse range of readily
available, safe and highly efficacious pesticidal crop protection products. Their
responsible and effective use has led to Europe becoming one of the most efficient areas
of crop production in the world. In July 1991, Council Directive 91/414 concerning the
placing of plant protection products on the market was introduced. In 1993, as required
by the Directive, the European Commission launched the work programme on the
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A. Leadbeater
Community-wide review for all active substances used in plant protection products
within the European Union. In this review process, each substance had to be evaluated as
to whether it could be used safely with respect to human health (consumers, farmers,
local residents and passers-by) and the environment, in particular considering
groundwater and non-target organisms, such as birds, mammals, earthworms, bees. There
were about 1,000 active substances (and tens of thousands of products containing them)
on the market at the time the Directive was adopted.
Since 1993, the European Commission has created an EU list of approved active
substances and Member States may authorise only plant protection products, containing
such substances, which are included in this list. A consequence of the review is that there
has been a dramatic decrease in the number of active substances for crop protection
authorised in the European Union. Over the last decade a large number of plant
protection products have been eliminated from the market and there are now only around
350 of the original 900 - 1000 active substances remaining on the market. This has
already had an impact on the ability of growers to efficiently manage their crops against
devastating pests, weeds and diseases, in particular there are now very few products (if
any) available to the growers of many "minor crops".
In July 2006, the European Commission issued a proposal which initiated a process to
revise and replace Directive 91/414 with a new regulation in the EU. The stated purpose
of this revision included reinforcement of the high level of protection of health and the
environment, to safeguard the competitiveness of European agriculture and the chemical
industry, to give better harmonisation and availability of Plant Protection Products (PPP)
and to update and simplify approval processes. This proposal was then the subject of
intensive review and debate, in particular because it introduced a fundamental departure
from the well-established and scientifically supported principles of risk-based assessment
to a new hazard-based regulatory assessment with defined cut-off criteria. This debate
included scientists, farmers, advisers and the chemical industry who made clear their
concerns that the result was likely to be a reduction in the number of PPPs available in
the market to allow sustainable crop production in the EU.
In November 2009, Regulation (EC) No 1107/2009, concerning the placing of plant
protection products on the market, was published, replacing Directives 91/414/EEC and
79/117/EEC. The Regulation had an entry into force date of January 2010 and is
applicable from the 3rd Quarter of 2011. This Regulation is likely to have a significant
impact on the future of PPPs in the European Union. Although there are some provisions
in the Regulation to reduce the burden of registering products (mutual recognition, zonal
registration), as mentioned above, it represents a fundamental change from science-based
risk assessment, to hazard-based regulatory cut-off criteria and will certainly result in a
further decline in the number of effective plant protection products, including fungicides,
available to the farmer and therefore a further reduction in the diversity of solutions to
manage diseases in crops.
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Although during the review period before the new Regulation was finalised there were
various calls for a EU-wide assessment of the likely impact of the new Regulation on
European agriculture, this was not approved or carried out. Following agreement with
Parliament on the new Regulation, there has been much communication about the active
substances that may be affected by the cut-off criteria that are included in the Regulation.
It should be noted that these lists are speculative and the authorities who have carried out
the evaluations have clearly stated that this is the case. As an example, the Chemicals
Regulation Directorate (CRD, formerly PSD) in the United Kingdom published two
impact assessments in early and late 2008 which give an indication of what the
consequences of the new Regulation might be for the UK (depending on the final
definitions of some criteria, notably endocrine disruptors) (PSD, 2008). Assessments
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A. Leadbeater
Table 1: Summary of the findings of the analysis of the impact of the new Regulation on fungicide
availability, by BMELV (Germany), KEMI (Sweden) and CRD (UK).
Number of N/A 94 82 82 82
fungicides
examined
*= Additional fungicides that may be eliminated depending on definition of cut-off criteria for endocrine
disruption
The three assessments are largely in agreement concerning the fungicides which are
most likely to be eliminated due to the hazard criteria, as far as they are understood today.
The consensus is that somewhere around 12-16% of fungicide active ingredients are
likely to be lost to agriculture based solely on the cut-off criteria. The CRD evaluation
goes into more depth and considers further fungicides that might be at risk depending on
the definition of endocrine disruption, which would lead to a total of 32% of fungicides
at risk of being eliminated. Furthermore, considering the number of active substances
that may be at risk of being identified as candidates for substitution the number rises still
higher, although there is some double-counting in the data (Table 2). Just considering the
candidates for substitution equates to 22% of the EU market by value (ECPA data).
When considering the impact of Comparative Assessment, it should be remembered
that many fungicide products in each country contain mixtures of active substances (for
example for broad-spectrum disease control, or for resistance management) of which one
or more may be candidates for substitution. Therefore, on a product level the likely
adverse effect in the markets will be much greater than would be expected by simply
looking at the numbers of active substances affected (Table 2).
It can be seen from the information in Table 2 that some major fungicides of great
importance to European agriculture today appear to be at risk of being eliminated, and
thus a large number of products would not be available to farmers to enable them to
avoid crop losses. Of particular concern regarding the sustainability of disease control
would be the loss of a large number of triazoles, and other fungicides such as mancozeb,
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Table 2: The potential impact of the new regulation on fungicides as assessed by the UK CRD.
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A. Leadbeater
alone, even with fungicide protection, losses in wheat production due to M. graminicola
amount to £49 million (HGCA, 2010).
The elimination of multi-site fungicides such as mancozeb or chlorothalonil would
also have significant economic effects on agriculture in Europe (and indeed worldwide)
since they are very cost-effective solutions in themselves, and in addition the majority of
fungicides used in crops such as grapes and potatoes are used in mixtures with such
fungicides for security in disease control and resistance management.
Although resistance to several important fungicides occurred only a few years after
market introduction, most of these are still successfully used and contribute significantly
to effective and sustainable disease management around the world. Notable are the
benzimidazoles, dicarboxamides and phenylamides, all of which are still widely used
more than 30 years after resistance problems were identified. More recent examples are
the strobilurins which, 10 years after significant resistance issues were first identified, are
still widely used and rank in the top two economically most important fungicide groups
used worldwide. In cases such as these (and there are many more examples, also
insecticides and herbicides) resistance was not prevented but rather, successfully
managed. This successful management has been largely due to proactive measures taken
by the Fungicide Resistance Action Committee (FRAC) and other groups, which include
the limitation of spray numbers, use of fungicides only where they are required, and
alternation or mixtures with other fungicides having a different mode of action on the
target disease(s) (FRAC 2007a, FRAC 2007b).
Such disease management (and resistance management) strategies have only
succeeded because of the availability to date of a quite wide diversity of fungicides with
different modes of action, some of which have an inherently low risk of resistance
developing (for example the multisite fungicides chlorothalonil, mancozeb and copper
salts). Effective resistance management requires the portfolio to contain a range of
available modes of action such that each has a different target site within the target
organism, and the grower does not have to rely on a single mode of action. It has been
well demonstrated in practice that the repeated use of crop protection products with just
one mode of action can readily lead to resistance to those products in the target
pathogens. The potential reduction in the numbers of available fungicide active
substances as a result of the new Regulation would seriously affect the capability for
resistance management, a concern that was raised by scientific experts in the field as well
as industry during the revision process of the Directive (Bielza et al., 2008). Without a
sufficient number of unique fungicides in terms of modes of action, the effective life of
many remaining fungicides will be shortened due to increasing resistance problems and
therefore the management of serious diseases in many crops will be very difficult if not
impossible (with concomitant impacts on the economic production of crops in Europe).
On top of this, without sufficient diversity in fungicides available on the market, should
any new invasive species occur in Europe (e.g. black stem rust of wheat) similar to when
the entire Brazilian soybean harvest was threatened in 2003 by Asian rust caused by
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Phakopsora pachyrhizi then the tools available might be insufficient to secure key food
crops.
The 2010 edition of the FRAC Code List shows there to be a portfolio of more than
180 fungicides, grouped in 54 mode of action groups (www.FRAC.info). This seems to
be a fortunate situation in terms of the required diversity to manage resistance problems.
It must however be remembered that not all products are registered or available in all
countries of the world and not all are effective against the same crop/pathogen
combinations. Therefore the options to the farmer for their particular situation are often
much more limited. An analysis of the FRAC Code list shows that around 28% of
fungicides are currently classified as being of high or high to medium risk of resistance
problems arising (Table 3). A further 26% are classified as medium risk, with the
remaining 46% classified as either low, low to medium or unknown resistance risk. This
seems to be a reasonably comfortable situation at first consideration and the assumption
could be made that there is already today a favourable situation regarding diversity and
resistance risk. However, when global sales are used as an indicator of the actual use in
agriculture of these fungicides (popularity due to effectiveness, benefits, ease of use etc.)
a quite different picture can be seen. According to industry sales figures for 2008, sales
of high to medium risk fungicides represents around 69% of the market, with low
resistance risk fungicides only representing 21% of sales by value. This is due to the
immense success and popularity of the strobilurin (QoI) and triazole (DMI) fungicides,
which are high and medium resistance risk fungicides, respectively.
Table 3: Resistance risk classification of fungicides according to the FRAC Code List 2010.
This analysis shows clearly that we cannot afford to dismiss important fungicides,
without fully considering their benefits, not only as stand - alone products, but also with
their role in supporting the continued effectiveness of other fungicides and the
sustainable growing of European food crops. The potential impact of the EU Regulation
has been considered in depth by the agrochemical industry including FRAC (Nauen et al.,
2008) and concern expressed over the likely impact of active substance reductions on
resistance management and therefore on sustainable control of crop pests and diseases.
The Resistance Action Committees have given clear recommendations that no fewer than
3, and in the case of multi-spray crops (e.g. potatoes, bananas), preferably 5, different
modes of action are required per crop/target pest to ensure effective resistance
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management and sustainable pest, disease and weed management for the future. To
complement this recommendation, work is being done by the European Plant Protection
Organisation (EPPO) to give technical guidance on chemical diversity and resistance
management.
One solution to fill the gaps created by the loss of active substances is for industry to
invent and develop new fungicides which would meet all the requirements of the new
regulatory criteria. However, the rate at which industry can research and introduce new
fungicides is realistically much slower than the rate of removal of valuable fungicides
from the market due to the new legislation. Although historically industry has been very
successful with a steady stream of new innovative fungicides brought to the market
(Leadbeater and Gisi, 2010), the increasing costs of Research and Development of new
products and the long timelines required to carry out the studies required to show the
efficacy and safety of new active substances all act against the ability of industry to
innovate quickly.
A recent study into the costs of new agrochemical product discovery, development
and registration (Philips McDougall, 2010) concludes the Agrochemical industry
invested $2328 million in Research and Development during 2007 and that this is
expected to rise by at least 26% to $2943 million in 2012. The 2007 figure represents
6.7% of sales of the R&D companies, underlining that this industry continues to be a
heavy investor into R&D. The study also concludes that costs have risen by 39% during
the years 2000 - 2008 to $256 million. The study showed that much of this increase can
be attributed to increasing costs of toxicology, environmental chemistry and efficacy
studies required for regulatory authorities. The study also shows that the number of new
molecules required to be made and screened in order to bring a single successful product
to the market has almost tripled, from 52,500 to 140,000 and the number of years
required to bring a new active substance to the market has increased from 8.3 to 9.8 years.
These high costs and longer timelines are working against the quest for successful
innovation. It is rather likely that due to the new constraints imposed by the new
Regulation the rate of new product introduction will in fact slow down, rather than
become faster (as is one stated intention of the new Regulation, see earlier in this paper).
It will become increasingly challenging for new molecules to be discovered that are both
highly effective as fungicides and at the same time satisfy the raised hurdles required to
satisfy the new legislation. Since the investments required to deliver a new product to the
market are very large and are increasing, it becomes even more important to ensure that
these new inventions have a long and successful life and are available to agriculture for a
lasting period of time.
An additional concern is that because of such constraints to innovation, many new
active substances will be closely related to each other and themselves suffer from a lack
of diversity in mode of action, requiring effective management strategies to ensure their
long-term and sustainable use. An example of this can be seen in the pipelines of the
major agrochemical companies today. There has been much success recently in the
discovery and development of new fungicides of the carboxamide class. These new
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As has been indicated in this paper, the future for the management of crop diseases in
Europe under the new Regulation is far from clear. There are many uncertainties
concerning the final impact of the Regulation on the availability of active substances in
the EU and therefore it is difficult to draw firm conclusions.
One driving force will be to reduce the dependency on fungicides in crop production
in Europe through the use of more resistant plant cultivars and different agronomic
techniques to reduce disease pressure. These approaches are likely to meet with some
success although durability of disease resistance in most crop plants has so far been
difficult to achieve. Probably with fewer chemical options for disease management crop
yields are likely to fall (Offermann and Nieburg, 2000). Another requirement may be that
farmers and growers will have to settle for lower levels of disease control (and therefore
yields) than have been the norm over the past few decades. It is possible that some of the
most efficaceous fungicides will be lost to European farmers, which would mean that the
available portfolio of products may well be of a lower level of efficacy than today's
products. And the increasing threats of resistance mean that disease control may slip still
further.
There could be many effects seen on the product development strategies of the
agrochemical R&D companies. We are already seeing some major manufacturers
investing in products with hazard profiles more compatible with the new cut-off criteria
(for example biologicals). Such products will not increase safety for users, consumers or
the environment, but they are more in line with the societal preferences in the EU.
Discovery strategies for Europe will have the new cut-off criteria and comparative
assessment criteria built into the early selection processes; this will lead to a greater
attrition rate for new molecules (and therefore probably reduced success rates). However,
if achieved, the resulting products are likely to be quickly successful in the market,
providing the biological effectiveness is sufficient. It is interesting to speculate whether
companies will continue to see Europe as a key driving market for new product discovery
or if the focus will instead be more on growth regions such as Asia and Latin America.
Of course, the world is watching what happens in Europe and it is likely that ultimately
there will be harmonisation globally in regulatory requirements.
What is clear is that without a sufficient diversity of fungicide products being
available to farmers in the future to use in integrated production programmes, the ability
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of the grower to continue in business and to produce sufficient food would be endangered.
With this in mind it is important that all efforts are made to preserve the currently
available portfolio of fungicides, and that industry continues to be supported to enable it
to continue to invent and deliver a wide range of innovative new fungicides and indeed
other tools, including non-chemical approaches, to the market to safeguard against crop
losses due to diseases in the future.
References
Bielza, P., Denholm, I., Ioannidis, P., Sterk, G., Leadbeater, A., Leonard, P., Nistrup Jorgensen,
L. (2008): Declaration of Ljubljana. The Impact of a Declining European Pesticide
Portfolio on Resistance Management. Outlooks on Pest Management, 19 (6), 246-248.
Cook, R.J., Jenkins, J.E.E. (1988): The contribution and value of pesticides to disease control in
cereals. pp. 1-12 in B. C. Clifford and E. Lester, eds., Control of Plant Diseases: Costs and
Benefits. Blackwell Scientific Publications, Oxford, UK.
Clarke, J., Wynn, S., Twining, S., Berry, P., Cook, S., Ellis, S., Gladders, P. (2008): Pesticide
availability for cereals and oilseeds following revision of Directive 91/414/EEC; effects of
losses and new research priorities. HGCA research review No. 70, London.
FRAC (2007a): Fungicide Resistance in Crop Pathogens: How can it be managed? FRAC
Monograph No.1, second revised edition. Available at: www.FRAC.info
FRAC (2007b): Fungicide Resistance: The Assessment of Risk. FRAC Monograph No. 2, second
revised edition. Available at: www.FRAC.info
HGCA (2010): The Wheat Disease Management Guide 2010. Home Grown Cereals Authority
Guide 38: www.hgca.com
Leadbeater, A., Gisi, U. (2010): The challenges of chemical control of plant diseases. Chapter 1
(pp. 3-17) in: Gisi. U., Chet, I., Gullino, M.L. eds., Recent Developments in Management
of Plant Diseases, Plant Pathology in the 21th Century, Vol. 1. Springer Science +
Business Media, Dordrecht, Netherlands.
Nauen, R., Leadbeater, A., Thompson, A. (2008): Proposal on the Revision of EU Directive
91/41. The impact on Resistance Management and Sustainable Crop Production in Europe,
Outlooks on Pest Management, 19 (4), 150-151.
Offermann, F., Nieberg, H. (2000): Economic performance of organic farms in Europe. Organic
farming in Europe: Economics and Policy. Volume 5. Universität Hohenheim, Germany.
PSD (2008): Revised Assessment of the impact on crop protection in the UK of the ‘cut-off
criteria’ and substitution provisions in the proposed Regulation of the European Parliament
and of the Council concerning the placing of plant protection products in the market.
Pesticides Safety Directorate, 37 pp.: www.pesticides.gov.uk.
Ruske, R.E., Gooding, M.J., Jones, S.A. (2003): The effects of adding picoxystrobin,
azoxystrobin and nitrogen to a triazole programme on disease control, flag leaf senescence,
yield and grain quality of winter wheat. Crop Protection, 22 (7), 975-987.
Self, M. (2006): Disease control programmes using triazole and strobilurin fungicides on winter
wheat. HGCA Project Report No. 385, London.
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2
Crop Protection Markets in a Changing World
Abstract
Today’s challenge for global agricultural is to provide safe, affordable food for a growing world
population, at a time when natural resources such as water and land are becoming increasingly
scarce. Indeed, we can only increase the availability of farming land by sacrificing hitherto
unused natural surfaces which contain reservoirs of biodiversity. The best option is therefore to
grow more from less, which means a sustainable intensification of agricultural productivity on
existing farmland. The productivity of the land must be improved and our use of limited natural
resources made more efficient through intelligent use and application of all available agricultural
technologies and farming practices. By doing so, agriculture will rise to the production challenge
whilst also protecting our environment and biodiversity, and contribute to the stabilization of
yield, prices and farmer income.
Introduction
According to FAO (Food and Agriculture Organization), the global population will reach
9 billion people by 2050. At the same time, driven by increased urbanization and
affluence, nutritional habits in developing countries will shift towards a more protein rich
diet. Based on these expectations FAO has concluded that by 2050 the Global food
requirement will be 70% higher than today. Quite a challenge when one considers the
fact that almost 40% of global agricultural production is lost in the field or during storage.
Meeting this challenge will not be easy since the availability of agricultural land is
limited and the additional demand for food puts more pressure on already scarce natural
resources.
The response to this increased demand needs to be carefully managed. If it is not,
then this could lead to the depletion of natural resources (water and soil) and an increase
in pollution and emissions of greenhouse gases, which are widely acknowledged as
contributors to global warming. The use of natural resources, if not responsibly managed,
can permanently damage the ecosystems on which we rely for our food production. A
delicate balance must be found between the need to achieve food security, on the one
hand, and the use of natural resources and the environment, on the other. Two choices are
often cited in this respect: get more out of existing farmland or bring more land under
cultivation. The latter option cannot be easily achieved for several reasons: the best land
is already being cultivated; bringing more ‘marginal’ land under cultivation would
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further reduce the space for biodiversity and undermine many other environmental
protection goals.
In this context, the objective for European agriculture is clear: produce more, pollute
less, and enhance the quantity and quality of goods and services provided to society.
Europe’s Responsibility
Figure 1: EU land grabbing (adapted from von Witzke and Noleppa, 2010).
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Industry Position
The crop protection industry is one of the largest private investors in agricultural
research in Europe, with around 10% of sales going into the research and development of
new innovative products which have unprecedented safety profiles both for human health
and the environment.
This level of R&D investment exceeds that found in many other industries (where
the industrial average is 1-6%); puts it on a par with IT (10%) and only slightly behind
pharmaceuticals at 12%. The crop protection industry should therefore be considered as a
pillar of the EU’s ‘Innovation Union’, particularly when one considers that public
funding for agricultural research, education and extension services has constantly
decreased over the last 30 years.
Industry’s investment does not only aim at the discovery and development of new
and safer solutions with increased toxicological and environmental safety testing as
provided for in the new European Regulation 1107/091. A large portion of the investment
goes also into the development of integrated programs (Figure 2). These aim at
optimizing the use of individual technologies and their combinations, to improve
operator and environmental safety; minimize the risk of resistance development; promote
1
Regulation (EC) No 1107/2009 of the European Parliament and of the Council, concerning the placing of
plant protection products on the market and replacing Council Directives 79/117/EEC and 91/414/EEC
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safe use principles; or minimize residues in produce. Not surprisingly, the cost of
bringing a new crop protection product to the market has doubled in the last ten years to
around € 200 m.
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Numbers approximately
Sustainability in Practice
Simple adjustment in farmer behavior and farm management can considerably increase
the sustainability of farm operations, improve risk management and promote biodiversity.
The EU Sustainable Use Directive, presently in the implementation phase in Member
States, is a critical document in this process, providing the legal framework for the
promotion of safe use behavior beyond common practice. Below are a few examples
which illustrate sustainability in practice:
Operation Pollinator from Syngenta: A simple system of field margins planted with
flower rich seed mixtures provides additional nutrition for pollinator insects, so important
to agricultural productivity, at a time when fields are relatively poor in flowering plants.
Scientific research over ten years has shown these strips to be highly effective in
boosting not only key pollinators such as bees, but also bumble bees, butterflies and other
useful insects. At the same time the strips create safe havens for many small mammals
and birds. Presently, about 2000 ha of pollinator margins have been established across
Europe.
TOPPS, PROWADIS and SUI projects from ECPA: The European Crop Protection
Association (ECPA) has a number of multiyear projects running to research causes of
contamination from pesticides (TOPPS and PROWADIS). The learning is then
transferred into guidance principles for farmers to promote safe use of pesticides (SUI)
and is promoted through brochures, farmer training sessions and intensive collaboration
with local authorities. Market research has shown considerable progress in the
application of safe use principles following such training and many of the principles now
represent a fertile discussion basis for the preparation of the National Action Plans under
the Sustainable Use Directive.
Multifunctional Field Margins: OPERA (OPERA Research Center, a think tank of the
University of Piacenza focusing on the promotion of sustainable intensive agriculture)
and ELO (European Landowner Organization) have provided a guidance brochure on the
use of multifunctional field margins to manage risks such as water run-off and
contamination, erosion or spray drift, while at the same time providing refuge and space
for biodiversity on farm. Educating farmers in the proper management of their field
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margins and providing rewards for the delivery of public goods on their farms are critical
elements for success that need to be addressed in the running CAP reform process.
The concept of sustainability is multifaceted and must deliver economic,
environmental and social benefits in a balanced way. The cornerstones of tomorrow’s
CAP must be to promote the competitiveness of European agriculture, to contribute to
Global market stability, and to advance environmental sustainability of farming through
the recognition and reward of public goods and services delivered by farmers.
Innovation strongly contributes to the enhancement of sustainability, efficiency, and
production capacity. This may range from investments into technology research;
knowledge sharing of best management practices; or incentivizing organizational and
process innovation to the benefit of the market and society. Good risk management based
on farmers’ pro-active behavior, production diversification and investment in mitigating
mechanisms will underpin income stability.
The CAP framework should provide the basis for a closer link between the value
generated by farmers for society’s benefit and the support they receive.
Conclusion
European farmers have been incredibly successful in responding and adapting to the
changing needs and demands of society in the past. Today, with the planned evolution of
the CAP and the productive potential of sustainable intensive agriculture, Syngenta
believes they are better placed than ever to meet the new demands for food and
environmental security, whilst at the same time helping to drive a thriving rural economy
in Europe.
Farmers must, however, be motivated to maintain and improve upon the service they
provide to society and – in the future – this is going to depend on finding the optimal
balance between economic, social and environmental considerations. New management
practices and technologies must be available that make work on farms easier, more
productive, and stable. Technology alone, however, will not be sufficient to meet the new
challenges. The integration of all available technologies with best-in-class farm
management practices is imperative, supported by a network of excellence and
collaboration including all actors, from farmers to academia, politicians, industry,
authorities and NGO’s and a joined up approach to rural development and land
management.
In summary, the success of tomorrow’s agricultural policy in Europe will depend on
promoting farmer competitiveness and environmental stability. This should be done
through a fair compensation of farmers for their delivery of public good and services;
providing training and advice in the face of increasing regulation and growing
expectations from society; regulations based on scientific evidence and sound risk
management principles; and engagement of all stakeholders in an open dialogue on the
values and benefits of agriculture for the European economy and land management.
References
von Witzke H., Noleppa S. (2010): EU agricultural production and trade: Can more efficiency
prevent increasing “land-grabbing” outside of Europe? Agripol network for policy drive.
Humboldt University, Berlin.
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3
Climate Change and Arable Crop Disease
Control: Mitigation and Adaptation
Abstract
Global food security is threatened by crop diseases that account for average yield losses of 16%.
Climate change is exacerbating the threats to food security in many areas of the world,
emphasising the need to improve crop yields to increase food production in northern European
countries such as the UK. However, to mitigate climate change, the crops must be grown in such
a way as to minimise greenhouse gas emissions (GHG) and optimise inputs associated with their
production. As examples, it is estimated that UK production of winter oilseed rape and winter
barley is associated, respectively, with GHG of 3300 and 2617 kg CO2 eq. ha-1 of crop,
with >70% of the GHG associated with the use of nitrogen fertiliser. Furthermore, it is estimated
that control of diseases by use of fungicides in UK oilseed rape and barley is associated,
respectively, with decreases in GHG of 100 and 50 kg CO2 eq. t-1 of seed. These results
demonstrate how disease control in arable crops can make a contribution to both climate change
mitigation and sustainable arable crop production. Climate change will affect both growth of
agricultural crops and diseases that attack them but there has been little work to study its
combined effects on crop-disease interactions to guide strategies for adaptation to climate
change. For example, it may take 10-15 years to develop a new fungicide and it is important to
identify future target diseases now. As examples, the impact of climate change on UK epidemics
of phoma stem canker and light leaf spot on winter oilseed rape and fusarium ear blight on winter
wheat is investigated by combining weather-based disease models, crop growth models and
simulated weather for different climate change scenarios. It is predicted that climate change will
increase the risk of oilseed rape phoma stem canker and wheat fusarium ear blight epidemics but
decrease the risk of light leaf spot epidemics by the 2050s. Such predictions illustrate unexpected,
contrasting impacts of climate change on complex plant-disease interactions in agricultural and
natural ecosystems. They can provide guidance for government and industry planning for
adaptation to effects of climate change on crops to ensure future food security.
Introduction
Crop diseases directly threaten global food security because diseases cause crop losses,
estimated at 16% globally despite efforts to control the diseases (Oerke, 2006), in a world
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
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where more than 1 billion people do not have enough food (Anonymous, 2009). Thus,
food production must be increased by controlling crop diseases more effectively. Food
security problems are most serious in developing countries, where diseases can destroy
crops and cause famine for subsistence farming families (Strange and Scott, 2005). Food
security problems associated with crop diseases are exacerbated by climate change
(Garrett et al., 2006; Gregory et al., 2009; Stern, 2007). There is a need to evaluate
impacts of climate change on disease-induced losses in crop yields to guide government
and industry policy and planning for adaptation to climate change.
Since the threats of climate change to food security are particularly severe in
marginal areas (Schmidhuber and Tubiello, 2007), there is pressure on farmers in fertile
areas that may benefit from climate change, such as northern Europe (Butterworth et al.,
2010), to produce more food to ensure global food security (Stern, 2007). Thus, it is
essential to include methods to control disease problems in strategies for adaptation to
impacts of climate change (Evans et al., 2008; Gregory et al., 2009). However, it is also
necessary to grow crops in countries such as the UK in a manner that decreases
emissions of greenhouse gases (GHG) to contribute to climate change mitigation from
agriculture (Jackson et al., 2007). To decrease the contribution of agriculture to global
warming, possible options include decreasing the use of fossil fuels and nitrogen
fertilisers, decreasing methane emissions from livestock and increasing the sequestering
of carbon from the atmosphere (Glendining et al., 2009). This paper reports work to
study the contribution to climate change mitigation from disease control in arable crops
through fungicide treatment, using UK oilseed rape and barley crops as examples, and
estimates the impact of climate change on oilseed rape and losses from phoma stem
canker and wheat anthesis date and fusarium ear blight across the UK.
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Climate Change
Figure 1: Differences in greenhouse gas (GHG) emissions per tonne of yield between winter oilseed rape
crops (means of 24-39 cultivars at 4-7 different sites) treated with fungicides to control phoma stem canker
and light leaf spot diseases (■) and untreated crops (■) in the HGCA trials), at sites differing in epidemic
severity. The numbers of sites where the data were available for both treated and untreated crops were 5
(2004), 7 (2005), 6 (2006) and 4 (2007). The numbers of cultivars used in different years were 26 (2004),
39 (2005), 24 (2006) and 29 (2007) (adapted from Mahmuti et al., 2009).
1200
1000
kg CO2 eq. t-1
800
600
2005 2006 2007 2005 2006 2007
Rothamsted ADAS
Figure 2: Differences in greenhouse gas (GHG) emissions per tonne of yield between winter oilseed rape
crops treated with fungicides to control phoma stem canker and light leaf spot diseases (■) and untreated
crops (■). Results are for field experiments done at Rothamsted (2005-2007) and by ADAS at Teversham
(2005) and Boxworth (2006-2007), at sites differing in epidemic severity. Rothamsted experiments tested
19 different cultivars in 2005 and 20 cultivars in 2006 and 2007, in all cases with three replicates of each
untreated and treated plot (six plots per cultivar). ADAS experiments tested 20 cultivars with three
replicates of each treated and untreated cultivar (6 plots) for 2005 and four replicates (8 plots) for 2006-
2007 (adapted from Mahmuti et al., 2009).
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Effects of fungicide treatment on GHG emissions t-1 winter or spring barley grain
were also calculated in kg CO2 eq. t-1 using data from HGCA trials, experiments in
England and Scotland as part of a BBSRC LINK project and some ADAS trials done for
Bayer CropScience. In the HGCA dataset, fungicide treatment increased the 8-year mean
yield for winter barley by 1.38 t ha-1 (19%) and for spring barley by 0.91 t ha-1 (14%).
Yield always responded to fungicide treatment, with increases of 0.98 - 2.04 t ha-1 for
winter barley and 0.60 - 1.14 t ha-1 for spring barley. In the LINK dataset, fungicide
treatment increased the 3-year mean yield for winter barley by 1.03 t ha-1 (14%) and the
4-year mean for spring barley by 0.57 t ha-1 (9%). In the ADAS dataset, fungicide
treatment increased the 7-year mean yield for all winter barley experiments by 1.41 t ha-1
(19%).
Average yield across all 2,400 plots (treated and untreated) in the HGCA data for
winter barley was 7.8 t ha-1 and for spring barley was 7.0 t ha-1. In terms of the grain
yield, the total GHG emissions were 355 kg CO2 eq. t-1 for winter barley and 318 kg CO2
eq. t-1 for spring barley. Fungicide treatment reduced average GHG emissions of
producing 1 t of winter or spring barley for each UK data set (HGCA, LINK, ADAS).
For winter barley, fungicide treatment reduced GHG emissions by 42 - 60 kg CO2 eq. t-1
(11-16%) and for spring barley, fungicide treatment reduced GHG emissions by 29 - 39
kg CO2 eq. t-1 (8-11%). Disease control in winter oilseed rape decreased GHG emissions
more than disease control in winter or spring barley or winter wheat (60kg CO2 eq.t-1,
Berry et al., 2008). However, these calculations underestimate the climate change
mitigation benefits of disease control since the fungicide treatments did not completely
control diseases and disease epidemics can be much more severe than those in these
experiments.
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Climate Change
Table 1: Effects of climate change on the yield of treated oilseed rape (OSR) (Tr) and untreated oilseed
rape (Unt) after phoma stem canker losses, calculated by region. The untreated oilseed rape was calculated
as the mean of susceptible and resistant cultivars. The area grown per region (2006) and the predicted
average regional yield are given for the baseline (1960-1990) scenario. The predicted regional yield as a
percentage of the baseline scenario is given for the 2020LO (low CO2 emission), 2020HI (high CO2
emission), 2050LO and 2050HI climate scenarios. The figures were calculated after interpolating the
results from the treated oilseed rape yield predictions and the stem canker yield loss predictions according
to UK government regionc.
England total 462764 3.09 2.52 99.3 94.1 99.5 93.4 102.6 92.9 104.8 88.9
Scotland 35780 3.15 3.06 104.8 103.2 107.1 105.0 109.7 96.9 111.5 103.6
UK total 498544 3.12 2.77 101.8 98.7 103.0 99.3 105.9 94.9 107.9 96.4
a
Government regions can be found at http://www.statistics.gov.uk/geography/downloads/uk_gor_cty.pdf
b
Area of winter oilseed rape grown in each region in harvest year 2006 (www.defra.gov.uk)
c
Based on Butterworth et al. (2010), with corrected data for Scotland and UK total
The predicted effects of climate change in the 2020LO scenario are to decrease the
untreated yields in all regions of England by 5% (South West) to 10% (North East);
conversely, the effect of climate change in Scotland will be to increase the yield by 3%
(Evans et al., 2010). Under the 2020HI scenario, it is predicted that the untreated yield
will decrease by more than in the 2020LO scenario in some English regions (e.g. 16%,
North West) but by less in other regions (e.g. 2%, North East), so that the overall
decrease is similar for both scenarios. By contrast, in Scotland there will be a further
predicted increase in yield (5%). In the 2050LO scenario, it is predicted that there will be
an increase in the treated yield but a decrease in the untreated yield for both England and
Scotland. In the 2050HI scenario, there is a predicted increase in yield for treated yield
for both England (5%) and Scotland (12%) but a predicted decrease in untreated yield for
England (11%) by contrast with a predicted increase for Scotland (4%). These
predictions suggest that climate change will increase total production of fungicide-treated
crops from the baseline of 2.69 Mt to 2.90 Mt in the 2050HI scenario, with the amount
produced in Scotland increasing. However, they suggest that total production of
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untreated winter oilseed rape in England will decrease from 1.17 Mt (baseline) to 1.04
Mt (2050HI). Such predictions illustrate unexpected, contrasting impacts of climate
change on complex plant-disease interactions in agricultural and natural ecosystems.
The baseline yield indicates that the annual value of the total oilseed rape output for
the UK, calculated at a price of £195.60 t-1, was over £302M (Table 2), if phoma stem
canker and light leaf spot were controlled with fungicides. This value is predicted to
increase under all climate change scenarios, with highest increases under high CO2
emissions and in Scotland rather than England, so that under the 2050HI emissions
scenario, the value of the crop will be £13M more than the baseline scenario in England
and £2.5M more in Scotland. Average annual losses caused by phoma stem canker and
light leaf spot were estimated at £74M under the baseline scenario (Table 3) and climate
change is predicted to increase these losses, with further losses of £6-8M in England and
£0.6-0.9M in Scotland by the 2020s. By the 2050s, losses in England are predicted to
increase by £16M in the low emissions scenario and by £28M in the high emissions
scenario. This is in contrast to Scotland, for which losses are predicted to increase by
£2.2M for the 2050HI scenario and by £3.1M for the 2050LO emissions scenario. The
UK total losses are predicted to increase by £30M in the 2050s. The price in autumn
2010 is now nearer £300 t-1, so these values are underestimates.
Table 2: Effects of climate change on the output of winter oilseed rape (treated with fungicide), calculated
by region. The area grown per region (2006) and the predicted regional output are given for the baseline
(1960-1990), 2020LO (low CO2 emissions), 2020HI (high emissions), 2050LO and 2050HI climate
scenarios and presented in thousands of pounds (£000s). The yield figures were calculated after inter-
polating the results from the oilseed rape yield predictions according to UK government region and then
multiplied by an average price of £195.60 t-1.
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Climate Change
Table 3: Effects of climate change on losses from phoma stem canker and light leaf spot (for cultivars
with average resistance) in winter oilseed rape crops not treated with fungicide. Values are given for the
baseline (1960-1990), 2020LO (low CO2 emissions), 2020HI (high emissions), 2050LO and 2050HI
climate scenarios and presented in thousands of pounds (£000s). Figures were calculated after interpolating
results from stem canker and light leaf spot yield loss predictions according to UK government region and
then multiplied by an average price of £195.60 t-1.
Value of losses caused by phoma stem canker and light leaf spot (£000s)b
Region a Baseline 2020LO 2020HI 2050LO 2050HI
North East 3,431 3,526 3,934 4,208 4,630
North West 520 533 501 602 676
Yorks & Humberside 7,804 8,118 9,074 9,661 10,874
East Midlands 15,116 16,869 17,567 18,871 21,748
West Midlands 5,038 5,539 4,716 6,244 7,308
Eastern 14,481 16,179 16,582 18,454 21,359
London & South East 12,388 13,540 13,874 15,381 17,882
South West 7,910 8,198 8,337 8,996 10,191
England total 66,690 72,502 74,584 82,417 94,668
Scotland 7,109 7,663 7,901 10,240 9,067
UK total 73,890 80,165 82,485 92,657 103,735
a
Government regions can be found at http://www.statistics.gov.uk/geography/downloads/uk_gor_cty.pdf
b
The stem canker and light leaf spot loss predictions depend on the crop yield predictions in Table 2 of
Evans et al. (2010). This table is based on a table in Evans et al. (2010), with corrected data for Scotland
and UK total
Further work has investigated how impacts of climate change on wheat anthesis date
will influence fusarium ear blight in the UK. The timing of wheat anthesis affects
severity of wheat fusarium ear blight (head blight, scab) because the wheat is susceptible
to infection only at anthesis, when there is rainfall (Xu et al,. 2007). In the UK, the
disease is caused by several pathogens, including Fusarium graminearum and F.
culmorum of which some chemotypes produce mycotoxins (www.hgca.com; Madden
and Paul, 2009; Xu and Nicholson, 2009). A wheat growth model was used for
predictions of anthesis dates, and a weather-based model was developed for use in
predictions of incidence of fusarium ear blight in the UK. Daily weather data, generated
for 14 sites in arable areas of the UK for the baseline scenario and for high and low CO2
emissions in the 2020s and 2050s, were used to predict wheat anthesis dates and
fusarium ear blight incidence for each site for each scenario. It was predicted that, with
climate change, wheat anthesis dates will be earlier and fusarium ear blight epidemics
will be more severe, especially in southern England, by the 2050s. These predictions
suggest that industry and government strategies for adaptation to climate change should
prioritize improved control of fusarium ear blight to ensure future food security.
Discussion
These results show that disease control in arable crops can contribute to both climate
change mitigation and global food security. They suggest that disease control should be
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included in policy options for decreasing GHG emissions from agriculture (Smith et al.,
2008). Thus, controlling diseases in UK winter oilseed rape and barley gives benefits in
terms of decreased GHG per tonne of crop produced and increased yield to contribute to
food production in northern Europe in response to climate change threats to global food
security (Stern, 2007). These decreases in GHG are especially associated with more
efficient use of nitrogen fertiliser applied to the crop (Glendining et al., 2009).
Furthermore, the climate change mitigation benefits associated with disease control in
UK winter oilseed rape are considerably greater than those associated with disease
control in winter wheat (Berry et al., 2008) or winter or spring barley. It is also likely that
there will be climate change mitigation benefits from disease control in other arable crops
in different regions of the world.
These results with diseases of UK oilseed rape demonstrate how climate change can
increase losses from crop diseases. For UK winter oilseed rape, the increase in losses is
associated with the increase in range and severity of phoma stem canker with global
warming (Butterworth et al., 2010; Evans et al., 2008). Predicted losses from canker are
substantial even though they may be offset by decreasing losses from light leaf spot. This
work illustrates how, worldwide, increased disease losses may be associated with
increases in severity of existing diseases or spread of diseases to new areas to threaten
crop production (Garrett et al., 2006; Gregory et al., 2009). Thus, there is a risk that the
16% of crop production lost to diseases (Oerke, 2006) may increase, with serious
consequences for the 1 billion people who do not have enough to eat (Anon., 2009;
Strange and Scott, 2005), unless appropriate strategies for adaptation to this effect of
climate change are put in place. To guide government and industry strategies for
adaptation to climate change, there is an urgent need for reliable predictions of impacts of
climate change on different diseases, obtained by combining impacts on crop growth and
on disease epidemics with predicted future weather patterns (Barnes et al., 2010). Since it
may take 10-15 years to develop a new fungicide or incorporate resistance to a crop
pathogen from a novel source of resistance, it is important to identify future target
diseases now.
In a world where climate change is exacerbating the food security problems for
communities farming in marginal environments (Schmidhuber and Tubiello, 2007), it is
essential to develop better strategies for controlling crop diseases as a contribution to
global food security. There is an urgent need to decrease current global average crop
losses to diseases from 16% (Oerke, 2006), especially since disease losses are often
much greater in crops grown by subsistence farmers in marginal areas. It is
environmentally preferable to increase food production by decreasing losses to diseases
rather than by expanding the area cultivated with crops, which will lead to destruction of
rainforests and other natural ecosystems and increases in HGH emissions. Disease
resistance breeding, fungicides and cultural methods can all contribute to strategies to
decrease disease losses but they need to be carefully integrated into disease management
strategies appropriate for the relevant farming system. There is a need to optimise disease
control to maximise crop production in northern Europe both as a contribution to global
food security in the face of climate change (Stern, 2007) and to maintain the yields and
profitability of European farms and thus provide food security for their farming families.
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Climate Change
Acknowledgements
The authors thank the UK Biotechnology and Biological Sciences Research Council (BBSRC,
Bioenergy and Climate Change ISPG) and Department for Environment, Food and Rural Affairs
(Defra, OREGIN, IF0144), HGCA and the Sustainable Arable LINK programme (PASSWORD,
LK0944; CORDISOR, LK0956; CLIMDIS, LK09111) for funding this research, and the
British Society for Plant Pathology for supplementary funding. We thank Michael
Butterworth, Martin Mahmuti, Mikhail Semenov, Rodger White (Rothamsted), Andreas
Baierl (University of Vienna), Andrew Barnes, Dominic Moran (SAC) and Jack Watts
(HGCA) for their contributions to this work. We are grateful to many colleagues for
supplying data or advice for this work.
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Xu, X.-M., Monger, W., Ritieni, A., Nicholson, P. (2007): Effect of temperature and duration of
wetness during initial infection periods on disease development, fungal biomass and
mycotoxin concentrations on wheat inoculated with single, or combinations of Fusarium
species. Plant Pathology, 56, 943-956.
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4
Breeding for Resistance as a Component of
Integrated Crop Disease Control and Sustainable
Food Production
J.K.M. BROWN
Department of Disease and Stress Biology, John Innes Centre, Norwich, NR4 7UH, England
Abstract
Global climate change is expected to alter the severity of established diseases and cause the
emergence of new pathogens. New legislation in Europe, however, will make it harder to cope
with unpredictable disease threats by restricting the use of pesticides in agriculture. This and
other changes in the political and economic context of agriculture will make it increasingly
challenging to maintain food security in the next few decades. Disease resistant crop varieties
play an important role in crop protection. Breeders aim to reduce farmers’ costs by selecting
varieties with adequate resistance, or at least acceptable susceptibility, to diverse diseases and
stresses. There is an increasing need, however, for methods of rapidly selecting new varieties
with reduced susceptibility to new diseases. Association genetics is a powerful new tool for crop
improvement, which can be used to increase the genetic diversity of disease resistance in
breeding programmes and detect useful variation in current germplasm. Resistant varieties are
most effective in providing robust disease control when they are used as a component of
integrated crop management. Their use avoids excessive reliance on pesticides, reducing
selection pressure for pathogen insensitivity to these chemicals. Conversely, pesticides
supplement varietal resistance, buffer against environmental variation and, increasingly, allow
time for breeders to develop varieties resistant to new pests and diseases.
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
J.K.M. Brown
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resistance which is effective over a long time in an area prone to the disease, and is not
readily overcome by pests or pathogens, is one of its most significant achievements. It is
sometimes forgotten that the Green Revolution began not with the release of semi-dwarf
wheat varieties, as is often supposed, but with breeding for durable resistance to stem
(black) rust. This was achieved by Norman Borlaug and his colleagues working at
CIMMYT, the International Maize and Wheat Improvement Centre in Mexico, in the
1940s and 1950s, who bred the genes Sr2 and Sr26 into a wide range of spring wheat
varieties (Hoisington et al., 1999). Yet there is no single model for durable resistance
(Johnson, 1984). Durable resistance to powdery mildew has been a major achievement in
wheat breeding in northern Europe, but in contrast to the case of stem rust, little is known
about either its genetics or physiology.
A desire for excellent resistance has been used to justify countless grant applications
for research particular diseases but what is actually required in breeding for resistance?
For the plant breeding company, a desirable variety is one which achieves high sales.
This means it must meet the needs of customers – farmers – better than competing
varieties do. Resistance to disease as a whole, not merely one disease, must be
subordinate to yield and quality as an element in farmers’ choice of varieties, and even to
traits which affect agronomy, such as plant height and standing power. Priorities vary:
for fruit and vegetables sold raw, visual appearance is crucial to the value of the produce
so resistance to diseases which cause surface blemishes is crucial as an element of quality.
Fusarium toxins in wheat or barley destroy the value of shipments of grain so again,
resistance to this fungus can be considered as a quality factor in environments where the
disease may be severe. The general point is that disease is significant if it threatens the
profitability of a crop.
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J.K.M. Brown
desirable.
Probably the most challenging aspect of resistance breeding is the need to have
adequate resistance, or at least acceptable susceptibility, to the full range of important
diseases. The first aspect of the challenge is that, with few important exceptions,
resistances to different diseases are controlled by different genes, each of which must be
handled separately in a breeding programme. To make the challenge harder, there is
emerging evidence that improving resistance to one disease often comes at the expense of
increasing susceptibility to others. This has been studied in depth for mlo resistance to
powdery mildew (Blumeria graminis f.sp. hordei) in spring barley, which has provided
durable control of mildew for over 30 years but also enhances susceptibility to
Magnaporthe, Cochliobolus and Fusarium (Jarosch, 1999; Kumar et al., 2001; Jansen et
al., 2005). And as if that were not hard enough, the direction of the interactions between
resistances to different diseases can vary in different environments. mlo mildew
resistance alleles were associated with increased resistance to Ramularia leaf spot in a
field trial (Makepeace et al., 2007) but with increased susceptibility under artificial
lighting in a growth cabinet experiment (Brown and Makepeace, 2009).
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for this was that many of the well-known sources of Septoria resistance actually had the
same gene, named Stb6 (Chartrain et al., 2005). Secondly, selection processes must be
effective, so that a line’s performance in a breeder’s nursery reflects its likely
performance in farmers’ fields. Thirdly, a faster generation time not only speeds up the
commercial release of varieties but accelerates the progress of variety improvements over
several generations in a long-term breeding programme.
Selection in breeders’ nurseries is therefore more effective when variation is
relevant to farming, selection is predictable and the breeding process is quick. The last of
these is generically relevant to improving all traits. Plant pathologists have an important
role to play in improving the effectiveness of selection but, like breeders, can only work
with diseases which are already known to be relevant to farming. An area where
significant progress appears likely is in diversifying sources of resistance in breeding.
Association genetics is a powerful, relatively new tool for crop improvement, based
largely on advances in human genetics (Rafalski, 2010). It combines information about
phenotypes, markers and relatedness of many varieties to conduct simultaneous analysis
of the genetics of several traits. One of its most useful applications is to identify lineages
of a crop species which are not only sources of resistance but carry different resistance
genes. Varieties from such lineages can be crossed to select progeny with resistance
superior to that of either parent. The association genetics approach can also be used to
identify unusual varieties with genes which can be introgressed into the mainstream of a
plant breeding programme and to rediscover sources of resistance which have fallen into
disuse. If the databases and seed stocks required for association genetics are kept up-to-
date, they will enable breeders to respond rapidly to new pathogens and other threats to
food production.
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J.K.M. Brown
necessarily have yield, quality or resistance to other diseases equal to that of the best
current varieties. As the process of resistance breeding advances, fungicides are
necessary to protect yield on varieties which have moderate resistance as well as elite
standards of yield or quality. This is the current situation with Septoria tritici blotch of
wheat in the UK, after around 25 years of breeding for resistance (Angus and Fenwick,
2008).
Thirdly, not all resistance is durable. Many well-known resistance genes follow the
gene-for-gene relationship but this class of resistance has been well studied because it is
amenable to scientific research, with single genes conferring strong resistance
phenotypes, not because it is especially useful. While occasional gene-for-gene
resistances have lasted longer, the great majority have only remained effective for a few
years before the target pathogens have mutated to virulence. However, new gene-for-
gene resistances are superficially attractive because they provide almost complete control
of a disease, albeit for only a short time. In the case of yellow (stripe) rust of wheat
(Puccinia striiformis f.sp. tritici), breeding for durable resistance has had some success
(Johnson, 1984; Park, 2008) but breeders have repeatedly introduced new gene-for-gene
resistances, although not always intentionally. The main long-term effect of this has been
to mask the effects of durable resistance, making it harder to select in breeders’ nurseries.
This has resulted in occasional severe outbreaks of yellow rust, as virulence to a hitherto
effective resistance has evolved in the fungal population and varieties which have that
resistance therefore become highly susceptible. The more severe epidemics have
occurred when leading varieties have had few ‘background’ genes for durable resistance,
such as, for example, varieties Slejpner, with the gene Yr9 in the late 1980s or Brigadier
(Yr17) in the late 1990s (Hovmøller et al., 2002). Such so-called breakdowns of host
resistance – more accurately, evolution of virulence in pathogens – is also well-known in
cereal powdery mildew (Blumeria graminis), late blight of potato (Phytophthora
infestans) and many other diseases (Hovmøller and Brown, 2002). Fungicides are
essential to maintain food production when pathogens overcome resistance genes in this
way.
Fourthly, even when breeding for durable resistance has made very good progress, it
may not provide sufficient control in all areas and varietal resistance may need to be
supplemented by fungicides. Almost all UK wheat varieties released in the last 25 years
have had at least moderately high partial resistance to powdery mildew, which has
provided good control of the disease in the dryer eastern half of the country. In the wetter
west of England, however, mildew can still be a significant problem even on modern
wheat varieties and be a target for fungicide applications. Fungicide applications help to
buffer yields against the challenges posed by pathogens and thus help to make crop
production more sustainable.
Lastly, changes in climate and agronomy are likely to cause changes in the incidence
and severity of diseases. When a new disease appears, control by application of broad-
spectrum chemicals is essential for at least as long as it takes breeders to produce elite
varieties with acceptable resistance to it, say 20 years in the case of cereals. As climate
change accelerates and local seasonal weather becomes less predictable, changes in the
prevalence and severity of crop diseases are likely to become more frequent. Crop
management will become more challenging as supplies of phosphate become less
accessible and inorganic fertilisers more expensive to produce. Plant variety
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improvement will be more important than ever in protecting food production against
attack by the known threats, where genetic variation is plentiful and selection for
resistance is effective. Pesticides, however, will have an even more vital role than they do
now in defending crops against new threats. It is deeply unfortunate that the introduction
of highly restrictive regulations on the use of pesticides, for very little if any benefit to
the environment or human health, will severely jeopardise the security of future food
supplies, especially for the world’s poorest people, who spend a high proportion of their
income on food.
Perspective
In this article, I have taken a short-term view of the challenges facing disease control in
agriculture. Farmers are currently able to protect an elite variety against the threats
presented by an unpredictable environment by using controlled applications of fertilisers
and pesticides. This desirable situation may last for perhaps a further 30 to 50 years.
(Some authors are more pessimistic; the UK Government Chief Scientist has warned of a
“perfect storm” of global events striking at our capacity to sustain supplies of fresh water,
energy and food by 2030 [Beddington, 2009]. Still, even 30-50 years is scarcely a blink
in the lifespan of the human species.) More radical approaches to crop protection within
a robust, long-term system of food production will surely be needed. One thing that can
be asserted with confidence is that control of diseases, principally through growing
resistant – or less-susceptible – varieties, will continue to ensure that crops continue to
feed people, not pests, weeds and pathogens.
Acknowledgements
The author’s research is and has been supported by BBSRC, DEFRA, HGCA, the EU
Framework 6 Programme and plant breeding and agrochemical companies.
References
Angus, W.J., Fenwick, P.M. (2008): Using genetic resistance to combat pest and disease threats.
In: Arable Cropping in a Changing Climate. Proceedings, HGCA R & D Conference, 21-
27. HGCA, London, England.
Arraiano, L.S., Balaam, N., Fenwick, P.M., Chapman, C., Feuerhelm, D., Howell, P., Smith, S.J.,
Widdowson, J.P., Brown, J.K.M. (2009): Contributions of disease resistance and escape to
the control of Septoria tritici blotch of wheat. Plant Pathology, 58, 910-922.
Beddington, J. (2009): Food, energy, water and the climate: A perfect storm of global events?
Speech at: Sustainable Development UK Conference. [http://www.
govnet.co.uk/news/govnet/professor-sir-john-beddingtons-speech-at-sduk-09].
Brown, J.K.M. (1996): Fungicide resistance in barley powdery mildew: from genetics to crop
protection. In: Proceedings, 9th European and Mediterranean Cereal Rusts and Powdery
Mildews Conference, 259-267. Eds. Kema, G.H.J., Niks, R.E. and Daamen, R.A.,
European and Mediterranean Cereal Rust Foundation, Wageningen, The Netherlands.
Brown, J.K.M., Hovmøller, M.S. (2002): Aerial dispersal of pathogens on the global and
continental scales and its impact on plant disease. Science, 297, 537-541.
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J.K.M. Brown
Brown, J.K.M., Makepeace J.C. (2009): The effect of genetic variation in barley on responses to
Ramularia collo-cygni. Aspects of Applied Biology, 92, 43-47.
Chartrain, L., Brading, P.A., Brown, J.K.M. (2005): Presence of the Stb6 gene for resistance to
Septoria tritici blotch (Mycosphaerella graminicola) in cultivars used in wheat-breeding
programmes worldwide. Plant Pathology, 54, 134–143.
Friesen, T.L., Stukenbrock, E.H., Liu, Z.H., Meinhardt, S., Ling, H., Faris, J.D., Rasmussen, J.B.,
Solomon, P.S., McDonald, B.A., Oliver, R.P. (2006): Emergence of a new disease as a
result of interspecific virulence gene transfer. Nature Genetics, 38, 953-956.
Hoisington, D., Khairallah, M., Reeves, T., Ribaut, J.-M., Skovmand, B., Taba, S., Warburton, M.
(1999): Plant genetic resources: what can they contribute toward increased crop
productivity? Proceedings of the National Academy of Sciences of the USA, 96, 5937-5943.
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Management Science, 65, 1156-1163.
Hovmøller, M.S., Justesen, A.F., Brown, J.K.M. (2002): Clonality and long-distance migration of
Puccinia striiformis f.sp. tritici in north-west Europe. Plant Pathology, 51, 24-32.
Jansen, C., von Wettstein, D., Schafer, W., Kogel, K.H., Felk, A., Maier, F.J. (2005): Infection
patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase
gene disrupted Fusarium graminearum. Proceedings of the National Academy of Sciences
of the USA, 102, 16892-16897.
Jarosch, B., Kogel, K.H., Schaffrath, U. (1999): The ambivalence of the barley Mlo locus:
mutations conferring resistance against powdery mildew (Blumeria graminis f.sp. hordei)
enhance susceptibility to the rice blast fungus Magnaporthe grisea. Molecular Plant-
Microbe Interactions, 12, 508-514.
Johnson, R. (1984): A critical analysis of durable resistance. Annual Review of Phytopathology,
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Kumar, J., Hückelhoven, R., Beckhove, U., Nagarajan, S., Kogel, K.H. (2001): A compromised
Mlo pathway affects the response of barley to the necrotrophic fungus Bipolaris
sorokiniana (teleomorph: Cochliobolus sativus) and its toxins. Phytopathology, 91, 127-
133.
Makepeace, J.C., Oxley, S.J.P., Havis, N.D., Hackett, R., Burke, J.I., Brown, J.K.M. (2007):
Associations between fungal and abiotic leaf spotting and the presence of mlo alleles in
barley. Plant Pathology, 56, 934-942.
Oxley, S., Havis, N., Hunter T., Hackett, R. (2007): Impact of fungicides and varietal resistance
on Ramularia collo-cygni in spring barley. In: Ramularia collo-cygni, a new Disease and
Challenge in Barley Production. Proceedings, 1st European Ramularia Workshop, 103-
112. Eds. Koopmann, B., Oxley, S., Schützendübel, A., von Tiedemann, A., Georg-
August-Universität, Göttingen, Germany.
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5
Fungicide Resistance in Rice
H. ISHII
National Institute for Agro-Environmental Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki
305-8604, Japan
Abstract
Four decades have passed since fungicide resistance first occurred on rice in Japan; that was the
resistance of blast fungus Magnaporthe oryzae to kasugamycin. Thereafter, fungicide resistance
was not always very serious on rice although some other cases were reported as reviewed by
Uesugi (1982). However, the new problem of resistance to MBI-D fungicides caused a decrease
in control efficacy and there is now a concern whether QoI resistance may occur in the field
populations of rice blast fungus in near future. In this paper, the history of fungicide resistance in
rice and current topics related are reviewed briefly.
Resistance to Kasugamycin
Blast, caused by the fungus Magnaporthe oryzae, is the most important disease on rice,
the traditionally major crop in Japan. In early 1970s, the antibiotic kasugamycin was the
most common fungicide for rice blast control, sprayed four to five times per growing
season, and approximately 90% of the applications for blast were occupied by this
antibiotic in Shonai district, Yamagata Prefecture (Miura et al., 1975).
The field occurrence of fungicide resistance was found in Japan when control
efficacy of kasugamycin against this disease was lost causing a serious problem in
Shonai in 1971 (Miura, 1984). Resistant isolates of M. oryzae exhibited reduced
sensitivity to kasugamycin on rice-straw decoction agar medium. Control efficacy of
kasugamycin was extremely low in inoculation tests when resistant isolates were used as
an inoculum source.
Importantly, the problem of resistance to another antibiotic, polyoxin, also occurred
in 1971. Resistant isolates of Alternaria alternata Japanese pear pathotype, the cause of
black spot disease, appeared and field performance of this fungicide decreased drastically
in Yonago City, Tottori Prefecture (Nishimura et al, 1973).
However, a decline of kasugamycin-resistant strains was observed in Yamagata
(Miura, 1984) and Akita prefectures (Fukaya and Kobayashi, 1982) after withdrawal of
this fungicide and blast control efficacy of kasugamycin was recovered gradually.
H. Ishii
efficacy decreased (Katagiri et al., 1978; Yaoita et al., 1978). Two levels of IBP
resistance were reported but most of the resistant isolates showed a moderate level of
resistance. IBP-resistant isolates exhibited cross resistance to another organophosphorus
fungicide EDDP and an organosulfur fungicide isoprothiolane (Katagiri and Uesugi,
1977). The frequency of IBP-resistant strains also decreased when the selection pressure
with the fungicide was removed (Iijima and Terasawa, 1987).
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oryzae. The third resistance inducer isotianil was released to the blasticide market in
2010 (Figure 1).
Interestingly, results from monitoring tests suggested that MBI-D resistant strains seem
to be less fit to the environment as their populations decreased after withdrawal of the
fungicides (Yasunaga, 2007; Table 1). Possibility of the reuse of MBI-D fungicides is
under consideration at present.
Tiadinil Isotianil
Table 1: Fluctuations of MBI-D-resistant isolates of rice blast fungus in paddy fields after the use of
MBI-D fungicides was stopped.
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H. Ishii
blast as well as sheath blight disease caused by Rhizoctonia solani was marketed on rice,
the Japan Fungicide Resistance Action Committee (J FRAC) made a guideline indicating
how to use orysastrobin and other QoI fungicides which had already been in the market.
Shortly after that, the Research Committee on Fungicide Resistance under the
Phytopathological Society of Japan also proposed a guideline on this subject (So and
Yamaguchi, 2008). It was proposed to use QoIs only once per year on rice if necessary. It
was also recommended that QoIs to be used in alternation with other unrelated fungicides
such as MBI-R fungicides or resistance inducers every 2 to 3 years when QoIs were
employed in seedling box treatment. As rice blast fungus is disseminated not only by
wind but also by seeds, it was not recommendable to use QoIs in a paddy field where
commercial seeds were produced. The same strategies were also proposed for MBI-D
fungicides, if they were still effective. How widely this guideline will be distributed will
be a future discussion subject.
Monitoring of QoI resistance in M. oryzae has been done carefully but truly resistant
isolates have not been found so far. Only two isolates which caused slight decrease of
azoxystrobin efficacy were found in artificial inoculation tests performed on potted rice
plants (ZEN-NOH R&D Center, unpublished).
In QoI-resistant isolates of P. grisea found on turf, two types of point mutation,
G143A and F129L, were detected in fungicide-targeted cytochrome b gene (Kim et al.,
2003). As reviewed earlier (Gisi et al., 2002), the mutation of G143A and F129L was
closely associated with high and moderate level of resistance, respectively, also in P.
grisea. Based on these mutations, PCR-RFLP (restriction fragment-length polymorphism)
methods were developed.
As QoI-resistant isolates of M. oryzae were not detected from rice fields, site-
directed mutagenesis was employed and the G143A point mutation was introduced into a
plasmid containing the cytochrome b gene sequence of rice blast fungus (Wei et al.,
2009). Subsequently, molecular diagnostic methods such as PCR-RFLP and PCR-
Luminex were developed for identifying QoI resistance in rice blast fungus which may
appear in future. In addition, PCR products of cytochrome b gene of blast fungus could
be amplified from DNA directly extracted from infected leaves and seeds of rice.
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Biotechnol. Biochem., 68, 615–621.
Yamaguchi, J. Kuchiki, F., Hirayae, K., So, K. (2002): Decreased effect of carpropamid for rice
blast control in the west north area of Saga Prefecture in 2001. Jpn. J. Phytopathol., 68,
261.
Yaoita, T., Goh, N., Aoyagi, K., Sakurai, H. (1978): Frequency distribution of sensitivity in rice
blast fungus to an organophosphorus fungicide in Niigata Prefecture. Ann. Phytopath. Soc.
Jpn., 44, 401-402.
Yasunaga, T. (2007): MBI-D-resistant isolates of rice blast fungus: their fluctuations and reuse of
fungicides. Plant Protect., 61(4), 88-92.
Yoshino, R. (1988): Current status of Bakanae disease occurrence and its control. Plant Protect.,
42, 321-325.
Zhang, C.Q., Zhu, G.N., Ma, Z.H., Zhou, M.G. (2006): Isolation, characterization and
preliminary genetic analysis of laboratory tricyclazole-resistant mutants of the rice blast
fungus, Magnaporthe grisea. J. Phytopathol., 154, 392-397.
Zhang, C.Q., Xin, H. Wang, J.-X., Zhou M.-G. (2009): Resistance development in rice blast
disease caused by Magnaporthe grisea to tricyclazole. Pestic. Biochem. Physiol., 94, 43-47.
40
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6
Disruption of Botrytis cinerea Genome
Highlights the Possibility that Non-protein
Coding Regions may be Involved in Controlling
Infection and Sporulation
Abstract
Attempts to “knockout” an external alternative NADH dehydrogenase (NDE) gene in Botrytis
cinerea generated transformants with an interesting “loss of function” phenotype, which
sporulated poorly and failed to infect unwounded tomatoes. The resident wild-type NDE gene
was unaltered, and instead ectopic insertion was into a 5Kb intergenic region of the Botrytis
genome. Although insertion was into an open reading frame coding for 104 amino acids, there
was no evidence that this coded for a known, or putative, protein. RT-PCR showed that the
region was transcribed into an RNA of between 606 to 804 nucleotides during both conidial
germination and early conidial formation. Normal development of a plant pathogenic fungus may
depend on transcription of a non-coding RNA.
Introduction
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
In this paper we identify where the NDE disruption cassette inserted into the
Botrytis genome, show that RNA is transcribed in this region, and deduce that it probably
does not have a coding function.
The fungus
The haploid Botrytis cinerea strain B05.10 was maintained on V-8 juice agar at 25ºC in
the dark. For liquid cultures conidia were washed from 7 day-old cultures with water
containing Tween 20 (0.05% v/v), filtered through muslin, collected by centrifugation,
and rinsed twice with water before inoculating Malt Extract Broth (MEB; 50 ml; Oxoid,
Basingstoke, UK) to give a final concentration of 5 x 104 conidia ml-1. Cultures were
incubated on a rotary shaker (150 rpm) at 25ºC.
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frozen mycelium to a powder. RNA was extracted using a Qiagen RNeasy Plant Mini-kit
(Qiagen, Hilden, Germany) according to the manufacturer’s instructions. On-column
DNase digestion using the RNase-free DNase Set (Qiagen) failed to remove all DNA, so
eluted RNA was also treated in solution with this DNase Set according to the “RNA
clean-up” protocol. RNA was dissolved in RNase-free water and stored at -80ºC.
Results
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evidence that the missing part of the disruption cassette inserted elsewhere in the Botrytis
genome. PCR confirmed that ectopic insertion in the other four stable transformants was
at a similar point in the Botrytis genome.
A B
TTCGGGAAGAAGCAATTGAGTCAACGAATCCTACAACATCAGATGTGTCCCTGGGACCAATCTCC
――――――――――――26――――――――――――――› ――――――――27
CCGTCCATCTCTCCGCGCTGCGCTGCCCTGCGCTGCCCTGCCTTGCCGATACTTGCTCATACTG
――――――――› ――――――――――――――34
ctatctacaTGTGATCTGCTCTCATCCCACCGGCAACGACTCAATGCGATCGCGATGTCGACCG
――――――――――――› ‹
ACGATGAATCATTAATCGTATTTCAGATACACTACTATGCTACGGGCGATACTCGATTGACCAT
――29―――――――――――
CTTGGTATTAATCCGTTCCTCAAATGATGTGCGTCCCTGCATGGATCAGGAAGGCTAA
‹――――――――――――28――――――――――――
Figure 2: Botrytis cinerea genomic sequence surrounding the point of ectopic insertion of the NDE
disruption cassette. The 9bp sequence deleted from the sequence at the point of insertion is shown in lower
case. The sequence shown covers an ORF of 104 amino acids. Primer sequences used in RT-PCR are
shown in bold.
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Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
Figure 3: RT-PCR using RNA from B. cinerea germlings 24 hours after inoculation.
Discussion
The attempted disruption of the NDE gene impacted on conidial germination, infection
and sporulation, but insertion was ectopic, and not within the resident NDE gene. PCR
analysis confirmed that insertion was into a 5 Kb intergenic genomic region, and
although this included a putative ORF of 104 amino acids, motifs normally found in
upstream promoter or downstream terminator regions were not evident in the flanking
sequences. Furthermore, a BLAST search failed to identify homology with known
proteins. RT-PCR confirmed that insertion was into a region of the genome which is
transcribed into RNA of between 606 and 842 nucleotides during both conidial
germination, and the early stages of conidial formation. RNA may be cleaved into shorter
micro(mi) RNA fragments (Kim 2005) but, as yet, these have not been identified in fungi.
The transformation rate using circular DNA was low compared with other
filamentous fungi, but in line with other reports using B. cinerea (Patel et al., 2008).
Attempts to increase the frequency of hygromycin stable transformants using linearised
DNA were unsuccessful. Whole genome sequencing of filamentous fungi has shown
that only a small fraction codes for proteins, whilst about half is transcribed into ncRNAs
(Mattick, 2007), which vary from small 20-25nt to 500nt or larger fragments. Functions
of nc RNAs include regulation of developmental pathways through a variety of methods
affecting messenger RNA processing. A few classes of ncRNAs with a known function
can be identified by common sequence motifs, but many ncRNAs are too diverse to be
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assigned, as yet, to a particular class. A recent bioinformatic study of a part of the RNA
transcriptome of Aspergillus fumigatus identified 45 ncRNAs, fifteen of which were
novel (Jöchl et al., 2008). Although related to brown algae and not fungi, the
Phytophthora infestans genome contains over 400 closely related ncRNA sequences,
which may be involved in the co-ordinated transcription of infection specific genes
(Avrova et al., 2007).
“Knockout” experiments such as described here are useful to assess the function of
ncRNAs. It is tempting to suggest, since both infection and sporulation are disrupted, that
the candidate ncRNA in some way regulates the expression of these two key
developmental processes. Revealing the function of this candidate ncRNA may identify
novel ways to interfere with infection and sporulation, either directly by chemical
treatment, or more subtly perhaps by expression of an appropriate anti-sense RNA in
genetically modified crops. A greater diversity in modes of action is urgently needed to
combat the loss of fungicides through resistance and regulatory changes.
Acknowledgements
References
Avrova A.O., Whisson S.C., Pritchard L., Ventner E., De Luca S., Hein H., Birch P.R.J. (2007):
A novel non-protein-coding infection-specific gene family is clustered throughout the
genome of Phytophthora infestans. Microbiology, 153, 747-759.
Jöchl C., Fischer S.E.J., Hertel J., Stadler P.F., Hofacker I.L., Schrettl M., Haas H. (2008): Small
ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for
regulation of protein synthesis. Nucleic Acids Research, 36, 2677-2689.
Kim V.N. (2005): MicroRNA biogenesis: co-ordinated cropping and dicing. Nature Review
Molecular Cell Biology, 6, 376-385.
Lee S.B., Milgroom M.G., Taylor JW. (1988): A rapid, high yield mini-prep method for isolation
of total genomic DNA from fungi. Fungal Genetics Newsletter, 35, 23-24.
Mattick J.S. (2007): A new paradigm for developmental biology. Journal Experimental Biology,
210, 1526-1547.
Melo A.M.P., Duarte M., Videira. A. (1999): Primary structure and characterisation of a 64KDa
NADH dehydrogenase from the inner membrane of Neurospora crassa mitochondria.
Biochimica Biophysica Acta, 1412, 282-287.
Patel R.M., Heneghan M.W., Van Kan J.A.L., Bailey A.M., Foster G.D. (2008): The pOT vector
system: improving ease of transgene expression in Botrytis cinerea. Journal General
Applied Microbiology, 54, 367-376.
Van Kan J.A.L., Van’t Klooster J.W., Wagemakers C.Q.M., Dees D.C.T., Van der Vlugt-
Bergmans C.J.B. (1997): Cutinase A of Botrytis cinerea is expressed, but not essential,
during penetration of gerbera and tomato. Molecular Plant Microbe Interactions, 10, 30-38.
von Tiedemann A. (1997): Evidence for a primary role of active oxygen species in induction of
host cell death during infection of bean leaves with Botrytis cinerea. Physiological
Molecular Plant Pathology, 50, 151-166.
46
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7
New Fungicides and New Modes of Action
H. WALTER
Syngenta Crop Protection Muenchwilen AG, Research Chemistry, Schaffhauserstrasse, 4332
Stein, Switzerland
Abstract
The introduction of new fungicides is an essential element to sustain control of major pathogens
in agriculture. New fungicides can be discovered either within established mode of action (MoA)
groups, ideally with low resistant risk (robust MoAs), or in areas with a novel MoA. Compounds
having a novel MoA are of special interest, as they play a key role in resistance management
strategies. A review will be presented on the market share of major players in the Crop Protection
business, current and future market needs and new fungicidal compounds in late development or
recently introduced to the market. These compounds are divided into four groups: QoIs, SDHIs,
compounds against Oomycetes and ‘other’ fungicides. Some key features of the new compounds
will be discussed including biological target segments (as known so far), business potential and
expected launch data. New MoAs are rare in the cereals segment (major introductions are all
SDHIs), but seem to be more frequently discovered for the control of Oomycetes. New
compounds with blockbuster potential (peak sales > 250 mio US$) are only seen in the SDHI
area.
Introduction
Currently, the six major players in the Crop Protection (CP) area are Monsanto, Syngenta,
Bayer CropScience, DuPont, BASF and Dow. Based on Phillips McDougall data
published in 2009, Monsanto is overall the biggest company with total sales in 2008 of
11965 mio US$, closely followed by Syngenta (sales 2008: 11673 mio US$). Bayer
CropScience is on the third rank (sales 2008: 9344 mio US$), followed by DuPont (sales
2008: 6632 mio US$), BASF (sales 2008: 4991 mio US$) and Dow (sales 2008: mio
4535 US$). Monsanto is clearly on top in the seeds/traits area (sales 2008: 6632 mio
US$), followed by DuPont (sales 2008: 3992 mio US$). Syngenta is ranked third (sales
2008: 2442 mio US$). In the area of agrochemicals, Syngenta is on top (sales 2008: 9231
mio US$), followed by Bayer (sales 2008: 8682 mio US$). In the fungicide area, there
are currently three major players: Syngenta (sales 2008: 3142 mio US$), Bayer
CropScience (sales 2008: 2501 mio US$) and BASF (sales 2008: 2297 mio US$). The
sales figures include lawn and garden as well as the seed care area. The biggest generics
company in the fungicide area is MAI (sales 2008: 415 mio US$). As there are a lot of
compounds running out of patent protection, MAI will have further opportunities to grow.
Five major current and future market needs may be defined as important in the fungicide
area: 1) triazole and strobilurin follow-up compounds with curative Septoria control in
wheat (potentially with weak cross resistance to existing compounds), 2) triazole and
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
H. Walter
strobilurin follow-up compounds for rust control in soybean, 3) novel contact products
for fruit and vegetable market, 4) novel mode of action for leaf spot control in a broad
range of crops to follow strobilurins and 5) novel fungicides that meet future EU hazard
driven registration criteria. An example for a successful introduction of a new fungicide
within an “old” MoA group is the DMI prothioconazole. The major driving elements for
Bayer’s success with prothioconazole were the excellent control of Fusarium spp. and
Septoria leaf blotch in cereals (sales 2008: 360 mio US$). Further growth for
prothioconazole is expected, especially due to the strong activity against Fusarium, for
which no adequate competitor is currently on the market.
New Fungicides
Respiration inhibitors
Respiration inhibitors (Figure 1) represent clearly the most important class of fungicides
in the last 20 years, and major commercial breakthroughs have been seen in the CP area.
Complex III inhibitors (QoIs) – the strobilurin family (FRAC Code 11)
The strobilurins (QoIs) represent the most successful class of respiration inhibitors; all
major crop protection companies have a strobilurin in their portfolio. According to
Phillips McDougall data published in 2009, the biggest QoIs worldwide are azoxystrobin
(sales 2008: 895 mio US$), pyraclostrobin (sales 2008: 670 mio US$) and trifloxystrobin
(sales 2008: 474 mio US$). New developments in the QoI area such as pyribencarb,
pyrametostrobin and pyraoxystrobin planned to be launched by Kumiai and Shenyang,
respectively, are not expected to be big products worldwide as they seem to be cross
resistant to the existing market strobilurins and thus, might be only of local importance.
Due to the high resistance risk with strobilurins for major plant pathogens, the big CP
companies will probably not invest further into this area.
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O F O F O
H H
N F N F N
H H H
N Cl N N
N Cl N F
Me Me F
Cl F
Boscalid Cl Bixafen Fluxapyroxad
(BASF) (Bayer CS) (BASF)
F O S F O O
F H
F N F N N
H H H
N Me N N
N Me N N F
Me
Me Me Me
Penthiopyrad Sedaxane Penflufen
(Mitsui/DuPont) (Syngenta) (Bayer CS)
Other carboxamides:
F
F
Cl F
F O F O
F F N
H
F N N
H H
N
N Isopyrazam Fluopyram
Me (Syngenta) (Bayer CS)
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H. Walter
The two major subclasses of SDHIs are the bisphenyltype carboxamides and the o-
alkyl/cycloalkylsubstituted carboxamides. Isopyrazam may be classified as a
benzonorbornene carboxamide and Fluopyram as a pyridinylethylamide. Bixafen and
fluxapyroxad are follow-ups of boscalid. Substitution of the pyridine by a pyrazole
moiety in the molecule resulted in activity against all major cereal diseases including leaf
spots and rusts in wheat and barley. Bixafen is a broad spectrum compound with major
strength in the cereals area and will be used mainly in mixture with prothioconazole. The
mixture is claimed to show positive effects on plant physiology resulting in higher yields
and survival rates of cereal plants after drought stress (Suty-Heinze et al., 2011). The
Bayer CS business forecast for bixafen is up to 300 mio € (Bayer CS, 2009).
Fluxapyroxad (Xemium) presented by BASF (Semar et al., 2011) shows broad spectrum
of activity and is announced to be a mixing partner for epoxiconazole and pyraclostrobin
to be used in many crops including cereals. The compound provides preventive, curative
and long lasting efficacy and is expected to have significant sales potential and the
market introduction in 2012. As a common feature, bixafen and fluxapyroxad contain the
same difluoromethylsubstituted pyrazole acid building block, and the bisphenyl parts
look similar. However, bixafen contains a fluorine atom in the phenyl ring bearing the
aminogroup (Figure 2).
The o-alkyl/cycloalkylsubstituted carboxamides contain either a 1.3-dimethylbutyl
or a biscyclopropyl group (Figure 2). Penthiopyrad is a broad spectrum compound with
potential use in many segments (Yanase et al., 2007). It is the only compound having a
thiophene ring instead of a phenyl ring. The compound is described to have preventive
and curative properties with major uses in fruits and vegetables but probably also in
cereals. The EU registration (cereals) is expected for 2011 (DuPont, 2010). Penflufen
(Bayer CS) and sedaxane (Syngenta) are both broad spectrum seed treatment compounds
with potential uses in many crops. Penflufen, recently presented at the IUPAC
conference in Melbourne (Riek, H., 2010) has several strengths, e.g. the excellent
Rhizoctonia control; the business forecast is more than 100 mio € (Bayer CS, 2009). The
seed treatment activity for sedaxane (Syngenta) is associated with the difluoromethyl
acid part of the molecule (corresponding to a modified pyrazole acid part in penflufen,
Figure 2), and a novel aniline part, the 2-biscyclopropylaniline, was introduced to get a
patentable new compound. Sedaxane will be sold as a mixture of trans and cis isomers.
Both seed treatment compounds are foreseen to enter the market 2011-2012.
Isopyrazam (Syngenta) is a carboxamide with a unique, innovative structural
element, the benzonorbornene part, not known in any other fungicide structure.
Isopyrazam will be sold as a mixture of syn and anti isomers, it shows a broad spectrum
of activity with major strengths in the cereal segment (leaf spots and rusts). The first
commercial product based on isopyrazam was recently introduced in the UK as Bontima,
a mixture of isopyrazam and cyprodinil. Further introductions in other segments will
follow in 2010/11. The compound is expected to become a major pillar in Syngenta’s
fungicide portfolio (Syngenta, 2010). Fluopyram (Bayer CS) is a pyridinylethylamide
and the only new SDHI compound, which is not derived from an aromatic amine (e.g
aniline). All the other compounds discussed before have a pyrazole acid part in common,
with the difluoromethylpyrazole acid part dominating (bixafen, fluxapyroxad,
isopyrazam and sedaxane). Fluopyram is the only compound of the SDHI series derived
from a benzoic acid. It shows a broad spectrum of activity including activity against
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Botrytis, Sclerotinia and powdery mildews (Labourdette et al., 2011). Foliar as well as
seed treatment uses are foreseen by Bayer CS. The major use is expected to be in the
fruits, vegetables and field crop segments; Luna is the brandname for all mixtures based
on fluopyram. Mixtures of fluopyram with trifloxystrobin and tebuconazole have been
announced and sales of up to 200 mio € are expected for this compound (Bayer CS, 2009)
with first sales in 2011 (NAFTA and EAME).
In summary, boscalid is so far the best selling SDHI (sales 2008: 215 m US$).
Bixafen, fluopyram and isopyrazam have significant sales potential, and with sedaxane
and penflufen two broad spectrum seed treatment compounds with high potential are
close to market introduction.
NH2 F O
n-octyl H
N Cl Cl N
N F
H F
N N O
N N Cl O
Cl O O
Ametoctradin Fluopicolide Mandipropamid
(BASF) (Bayer CS) (Syngenta)
O Cl
S N
N N
O
N
O O O
S H
O O N
F N N O
H
O
Amisulbrom Valiphenalate
Br (Nissan) (Isagro)
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H. Walter
The structure of fluopicolide (FRAC Code 43, Bayer CS) is closely related to
fluopyram (an SDHI) containing a benzoic acid and a pyridine methylamine part
(pyridinylmethylbenzamide). The MoA of this compound is new and not related to
SDHIs; it affects spectrin-like proteins believed to play a role in maintaining the
membrane stability in oomycetes. (Tafforeau et al., 2010). The compound is claimed to
have long lasting and systemic activity but only limited curative potential. It is used in
mixtures with either propamocarb hydrochloride (against P. infestans in potatoes) or with
fosetyl-Al (against downy mildew in grapevine). The compound was introduced in 2006,
the sales in 2008 were < 30 mio US$ (Phillips McDougall, 2009) which are expected to
grow further in the next years. Mandipropamid (CAA fungicide, FRAC Code 40) was
announced in 2005 (Syngenta, 2005). It shows strong, long lasting and protective control
of late blight in tomatoes and potatoes as well as downy mildews in vines and a number
of vegetable crops. The compound is a mandelamide (Lamberth et al., 2008), the MoA
was elucidated recently as inhibiting cellulose biosynthesis by affecting the CesA3
protein (Blum et al., 2010). Mandipropamid was launched in 2007 and is currently sold
under the brandnames Revus (solo formulation) and Pergado (e.g mixture with folpet),
the sales in 2008 were 40 mio US$, they are expected to grow significantly in the next
years (sales potential of up to 200 mio US$ for mandipropamid based products).
Amisulbrom (FRAC Code 21) was introduced in 2008 by Nissan, it is a QiI (quinone
inside inhibitor in complex III), which combines a sulfamoyltriazole with an indole
moiety. The compound shows good activity against grapevine downy mildew and potato
late blight (Hugues et al., 2006). Amisulbrom has preventive activity, inhibits the
infection process at different levels and offers long lasting protection and anti-sporulation
effects. Potential mixing partners are mancozeb or folpet (Hugues et al., 2006); the sales
in 2008 were < 10 mio US$ (Phillips McDougall, 2009).
Ametoctradin (Initium) is a new BASF compound active against oomycetes (Gold et
al., 2011), which is planned to be introduced in 2010/11. The MoA is not yet fully
elucidated, but is claimed to be within complex III (but outside the Qo site) of the
respiration chain. Ametoctradin is a triazolopyrimidine and the structure is similar to
inhibitors of tubulin polymerization which were synthetized by BASF. Ametoctradin is
claimed to have strong protective activity (Gold et al., 2011) and will be sold only in
ready formulations together with other fungicides such as dimethomorph, mancozeb or
metiram. Ametoctradin controls late blight in tomatoes and potatoes and downy mildews
in grapes and vegetables (e.g. curcurbits, brassicas, onions and lettuce), but there is no
control of Pythium spp.. Valifenalate (valiphenal) is a valinamide carbamate, which was
launched by Isagro in Italy and France in 2009. It is a CAA fungicide with preventive,
curative and eradicative activities against late blight in potatoes and downy mildews in
grapes and vegetables.
In summary, several new MoAs against oomycetes have been introduced recently
with good activity against P. infestans, P. viticola and other downy mildews, but without
control of Pythium species.
‘Other’ fungicides
Three compounds of ‘other’ fungicide groups are listed in this chapter (Figure 4):
isotianil, an isothiazolecarboxamide from Bayer CS; flutianil, a methylidenethiazolidine
from Otsuka; and tebufloquin, a quinoline derivative from Meiji Seika. Isotianil is
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claimed to be a host plant defense inducer (FRAC Code P) used against rice diseases
(Pyricularia oryzae, Helminthosporium miyabeanus and Xanthomonas campestris).
Flutianil is specifically active against powdery mildews, and tebufloquin against rice
diseases such as P. oryzae. The MoAs for flutianil and tebufloquin are unknown. All
compounds have a narrow spectrum of activity and therefore, the sales potential may be
limited. Bayer CS gave a sales forecast for isotianil of < 50 mio US$ (Bayer CS, 2009).
N
F
Cl Cl
H
N O S
N
S N F
O F N
F
S N F
Isotianil Flutianil Tebufloquin
(Bayer CS) (Otsuka) (Meiji Seika)
Conclusions
All new broad spectrum compounds are respiration inhibitors (SDHIs) offering
significant sales potential. Major innovations of the last years include new seed treatment
compounds, which are all SDHIs, and several compounds with different MoAs
specifically active against oomycetes except the genus Pythium. Outside the oomycetes
area, new MoAs seem to be pretty hard to find.
References
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Conference, Monheim, Germany.
Blum M., Boehler M., Randall E., Young V., Csukai M., Kraus S., Moulin S., Scalliet G., Avrova
A.O., Whisson S.C., Fonné-Pfister R. (2010): Mandipropamid targets the cellulose
synthase like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen,
Phytophthora infestans. Molecular Plant Pathology, 11, 227-243.
DuPont (2010): Registration Applications Submitted for Two New Fungicides. Press Release,
Wilmington, US.
Gold R.E., Schiffer, H., Speakman J., Stammler, G., Klappach, K., Brix, H.D., Schlehuber S.,
(2011): Initium: A new innovative fungicide for the control of Oomycetes in speciality
crops. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H. Kuck, P.E. Russell, H. Lyr (Eds).
Modern Fungicides and Antifungal Compounds VI, DPG-Verlag, Braunschweig, Germany,
55-61.
Hugues R., Hasunuma N., (2006): Amisulbrom: nouveau fongicide contre les oomycetes. pp.
818-825 in Proceedinggs 8ème Conference International sur les maladies des plantes Tours,
France.
Isagro (2006): Isagro: a small global player founded on R&D. ‘Star’ Event, Milan Italy.
Labourdette G., Lachaise H., Rieck H., Steiger D. (2011): Fluopyram: Efficacy and beyond on
problematic diseases. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H. Kuck, P.E. Russell, H.
Lyr (Eds.). Modern Fungicides and Antifungal Compounds VI, DPG-Verlag,
Braunschweig, Germany, 75-80.
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H. Walter
Lamberth C., Jeanguenat A., Cederbaum F., De Mesmaeker A., Zeller M., Kempf H.-J., Zeun R.
(2008): Multicomponent reactions in fungicide research: The discovery of mandipropamid.
Bioorganic and Medicinal Chemistry, 16, 1531-1545.
Phillips McDougall Agriservice (2009): Product Section.
Riek. H. (2010): New chemistries leading to SDHI fungicides with different spectra. 12th IUPAC
International Congress of Pesticide Chemistry, Melbourne, Australia.
Semar M., Strobel D., Strathmann S., Groeger U., (2011): Xemium: The BASF fungicide
innovation. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H. Kuck, P.E. Russell, H. Lyr (Eds).
Modern Fungicides and Antifungal Compounds VI, DPG-Verlag, Braunschweig, Germany,
63-68.
Suty-Heinze A., Dunkel R., Krieg U. Rieck H. (2011): Bixafen – The new cereal fungicide with
yield boosting effects. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H. Kuck, P.E. Russell, H.
Lyr (Eds). Modern Fungicides and Antifungal Compounds VI, DPG-Verlag, Braunschweig,
Germany, 69-73.
Syngenta International AG (2005): Syngenta presents new fungicide at British Crop Production
Council. Press Release Basel, Switzerland.
Syngenta International AG (2010): Syngenta launches isopyrazam with first registration in the
UK. Press Release, Basel, Switzerland.
Tafforeau S., Wegmann T., Latorse M.P., Gouot J.M., Duvert M., Bardsley E., (2005):
Fluopicolide, a novel fungicide with a unique mode of action, setting a new standard for
Oomycete control. BCPC International Congress – Crop Science and Technology, pp. 79-
86. Glasgow, UK.
Yanase Y., Yoshikawa Y., Kishi J., Katsuta, H. (2007): The history of complex II inhibitors and
the discovery of penthiopyrad. pp. 295-303, in Ohkawa, H., Miyagawa, H., Lee P.W., eds.,
Pesticide Chemistry, Crop Protection, Public Helath, Environmental Safety.WILEY-VCH,
Weinheim, Germany.
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8
Initium® – A New Innovative Fungicide for the
Control of Oomycetes in Speciality Crops
Abstract
Initium (common name = ametoctradin) is an innovative new fungicide recently introduced by
BASF; it belongs to a new class of chemistry, the triazolo-pyrimidylamines. The biochemical
mode of action of Initium is the inhibition of mitochondrial respiration in Oomycetes. Initium is a
highly effective inhibitor of zoospore formation, release, motility and germination. In addition,
Initium inhibits the direct germination of zoosporangia. Initium adsorbs rapidly to the
epicuticular wax layer of plant surfaces and exhibits very good rainfastness. Numerous field trials
have proven this new fungicide to be highly effective against late blight and downy mildews in
preventive spray applications. In addition, these studies have shown Initium to be highly selective
in a wide range of speciality crops. Initium has an excellent toxicological and ecotoxicological
profile and is highly suitable for use in integrated crop management programmes.
Introduction
Late blight and downy mildews are plant diseases due to the Oomycetes that cause
devastating diseases of speciality crops worldwide and play an important economic role
in commercial food production (Strange and Scott, 2002). Initium is an innovative new
fungicide recently introduced by BASF; it belongs to a new class of chemistry, the
triazolo-pyrimidylamines. Initium exhibits a high intrinsic activity against Oomycetes
(Gold et al., 2009). Since its discovery in 2004, Initium has undergone detailed
evaluations in laboratory tests and global field programmes. The aim of this paper is to
give an overview of Initium’s mode of action, biological profile and field performance,
and to review resistance management strategies.
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Table 1: Products and active ingredients used in laboratory studies and field trials.
Microscopical studies
Grape leaves (cv. Riesling) infected with Plasmopara vitcola were washed with water
and filtered (50 µm) to separate sporangia from leaf pieces and other debris. The
zoospore suspension was filtered again (10 µm) to separate the released zoospores from
the remaining sporangia. Initium was added to the zoospore suspension and observed
under the light microscope in comparison to the untreated spore suspension.
Rainfastness study
The rainfastness trials were conducted on leaves of 3 month old glasshouse grown grape
plants (cv. Cabernet Sauvignon) with 10 to 12 expanded leaves. 6 replicates were used
for these studies. Applications with Initium SC were made in a spray cabinet equipped
with 5 nozzles to ensure good coverage on upper and lower leaf surfaces. Simulated rain
of 40 mm over 2 hrs was applied with overhead nozzles at 1, 6 or 24 hrs after application
of Initium SC. The leaves were inoculated with a zoospore suspension of P. viticola 18
hours after rain simulation and evaluated for disease severity 8-10 days thereafter.
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Mode of action
Laboratory studies have shown that Initium interferes with mitochondrial respiration in
complex III of sensitive Oomycetes as demonstrated by biochemical studies with
mitochondrial particles isolated from Pythium ultimum (Gold et al., 2009). This
inhibition leads to a strong reduction in the synthesis of ATP needed by the cell. The
exact binding site of Initium in complex III has not been fully elucidated, but it does not
appear to be the Qo-site, known for other complex III inhibitors (Sauter, 2007). This
conclusion is based on studies which show that Initium inhibits P. viticola isolates
carrying the G143A target site mutation (Stammler, unpublished data). Initium is not
cross-resistant to Oomycete fungicides with confirmed field resistance based on target-
site mutations (e.g. phenylamides, Qo inhibitors or carboxylic acid amides). Cross-
resistance to cyazofamid or amisulbrom, that are known to bind to the Qi site in complex
III (Mitani et al., 2001), was not tested since strains with target-site mutations for these
AIs were not available.
Microscopical studies
Initium is a highly effective inhibitor of zoospore formation, release, motility and
germination. Even at very low concentrations (0.05 mg/l), Initium rapidly led to bursting
of zoospores (Figure 1).
Figure 1: Light microscope images of Plasmopara viticola zoospores. Top: Untreated zoospore remained
intact and formed a germ tube within 2 hours. Bottom: At time 0 seconds, Initium (0.05 ppm) was added to
the zoospore suspension and observed in comparison to the untreated spore suspension. The Initium-treated
zoospore ruptured within 4 seconds and cytoplasm leaked from the spore.
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100%
Leaf surface,
water wash
80% Adsorbed on/in
epicuticular wax
60% Leaf uptake
40%
20%
0% Time
1 hr 3 hr 1d 3d 7d
Figure 2: Uptake of 14C-Initium in grape leaves. The distribution of radio-labelled Initium was determined
following application of droplets to the upper leaf surface of grape leaves. Measurements were made at 1
hr, 3 hr, 1 d, 3 d and 7 d after application.
Based on 2 experiments, 6.0% and 5.1% of the applied compound was detected in
water wash and leaf uptake fractions, respectively (Figure 2). The majority of the applied
Initium (86.9%) was adsorbed on/in the epicuticular wax layer of the grape leaves. No
statistical differences were observed between any samples taken between 1 hour and 7
days after application (Figure 2). These characteristics indicate that Initium is a non-
systemic fungicide. The high affinity of Initium to the epicuticular wax is linked to its
high log Pow value of 4.4. This characteristic allows Initium to form a stable protective
film on plant surfaces and results in an effective barrier against pathogen attack, as
previously described (Gold et al., 2009).
Rainfastness
Disease severity was low on plants treated with Initium SC and efficacies ranged from 87
to 91% (Figure 3). The efficacy remained high when an artificial rain of 40 mm was
simulated at 1, 6 or 24 hrs after application. These results show that Initium SC exhibits
very good rainfastness. The high affinity of Initium to the waxy surfaces of plants
described above is important for the favourable rainfastness characteristics of Initium.
Field trials
Initium SC is an excellent tool to control grape downy mildew on both leaves and
clusters (Table 2). Strong protection of grape clusters was observed in trials performed
throughout Europe, which confirmed the results reported earlier by Aumont et al. (2009)
from trials in France.
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100
90
80
% Efficacy 70
60
50 No water
40
40 mm
30
(over 2 hr)
20
10
0
Rain simulation Rain simulation Rain simulation
after 1 hr after 6 hr after 24 hr
Figure 3: Rainfastness test with Initium SC (200 g/l SC) on grape leaves following 40 mm rain simulated
at 1, 6 or 24 hr after application. Disease severity in the untreated plants was 71.3%, 80.0% and 84.0% for
1, 6 and 24 hr, respectively. High efficacies were observed in all treatments.
Table 2: Control of Plasmopara viticola in grapes in France, Italy, Hungary, Germany, Greece,
Spain and Portugal in 2006 – 2008.
Number of trials 28 28
1
5-9 applications at BBCH 69-85 using 10-15 day spray intervals. Orthogonal ave. of 28 trials.
2
Statistical analysis: SNK-Test
In the European trials reported here, Initium SC was significantly more effective
than cyazofamid on clusters (Table 2). The high efficacy of Initium SC on grape clusters
appears to be linked to several factors. These include the high affinity of the AI to the
epicuticular wax layer, the relatively short period of berry susceptibility and the fine
structure of the epicuticular wax crystalloids on the berries to which Initium binds.
Studies on the last point are currently ongoing.
Initium SC also displayed very good potato late blight control in trials performed in
several countries (Table 3). These trials demonstrate the superior activity of Initium SC
compared to the reference product.
Depending on target disease, Initium will be used at 200 to 300 g active
ingredient/ha. Initium is characterised by excellent crop safety. At the recommended
rates, no crop injury has been observed in numerous trials performed over several years
in a broad variety of crops.
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Table 3: Control of Phytophthora infestans in potatoes in Spain, Germany, Brazil and Taiwan in
2004 – 2005.
Number of trials 8
1
3-7 applications at BBCH 19-85 following a 5-11 day spray interval. Ave. of 8 trials.
2
Statistical analysis: SNK-Test
Resistance management
To ensure the long-term efficacy of Initium, it will only be available in ready
formulations combined with other fungicidal active ingredients of a different mode of
action group. Registrations in Europe will allow a maximum of 3-4 applications of
Initium-containing products per crop and year. These should be made in alternation with
non-cross-resistant fungicides. Initium shows the best performance when it is applied as
a preventive spray before disease is established in the crop. Selection pressure for
resistance is lower through preventive applications, compared to a curative or eradicant
approach, because the pathogen population is smaller at disease onset than when it is
already established in the field. Therefore, Initium should be applied in a preventive
manner following the recommendations on product labels.
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Conclusions
Initium is a highly active preventive fungicide from a new chemical class for use against
Oomycete diseases in different crops. The favourable toxicological and ecotoxicological
properties of Initium described previously (Gold et al., 2009) makes it ideal for use in
integrated crop management programmes. BASF plans worldwide registrations for
Initium. Initium will be marketed in ready formulations with other Oomycete active
compounds, like dimethomorph, metiram and mancozeb, to improve disease control,
complement the spectrum of activity and to provide resistance management.
References
Anon. (2001): Growth stages of mono-and dicotyledonous plants BBCH Monograph, 2nd edition,
ed. U. Meier, published at www.jki.bund.de/fileadmin/dam_uploads/_veroeff
/bbch/BBCH-Skala_englisch.pdf.
Anon. (2010): FRAC Code List©: Fungicides sorted by mode of action, published at
www.frac.info/frac/publication/anhang/FRAC_Code_List_2010.pdf.
Aumont, C., Gauthier, C., Cousin, A., Fritz-Piou, S., Lardier, P.A., Morvan, Y. (2009) : BAS
650F: Caracterisation d’une molecule issue d’une nouvelle familie chimique pour la lutte
contre les Oomycetes. AFPP – 9th Conférence Internationale sur les maladies des plantes.
Tours, France, 8-9 December.
Baker, E.A., Huntz, G.M., Stevens, P.J.G. (1983): Studies of plant cuticle and spray droplet
interactions: A fresh approach. Pesticide Science, 14, 645-658.
Gold, R., Scherer, M., Rether, J., Nave, B., Levy, T., Storer, R., Marris, D. (2009): Initium: An
innovative fungicide of a new chemical class for the control of Oomycetes. BCPC
Congress. Glasgow, UK 9-11 November.
Mitani, S., Araki, S., Takii, Y., Oshima, T., Matsuo, N., Miyoshi, H. (2001): The biochemical
mode of action of the novel selective fungicide cyazofamid: Specific inhibition of
mitochondrial complex III in Pythium spinosum. Pestic. Biochem. Phys., 71, 107-115.
Sauter, H. (2007): Strobilurins and other complex III inhibitors. In Modern Crop Protection
Compounds, Vol. 2, eds. W. Krämer, U. Schirmer, Wiley-VCH Verlag, Weinheim,
Germany, 457-495.
Strange, R.N., Scott, P.R. (2005): Plant Disease: A Threat to Global Food Security. Annual
Review of Phytopathology, 43, 83-116.
Wood, P.M., Hollomon, D.W. (2003): A critical evaluation of the role of alternative oxidase in
the performance of strobilurin and related fungicides acting at the Qo site of complex III.
Pesticide Management Science, 59,499-511.
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9
Xemium® – the BASF Fungicide Innovation
Abstract
Xemium (common name Fluxapyroxad) is a new broad-spectrum carboxamide fungicide
belonging to the group of succinate dehydrogenase inhibitors (SDHIs; complex II inhibitor of the
mitochondrial respiration chain). It economically controls important diseases of the classes
Basidiomycetes, Ascomycetes and Deuteromycetes. After being applied to the crop, the molecule
is systemically (acropetally) distributed in the plant. In addition to its preventative and long
lasting activities, Xemium also provides high curative activity. Besides important cereal
pathogens including Mycosphaerella graminicola (Septoria tritici), the most important pathogen
of wheat in northern Europe, Xemium also controls a broad range of fungi in various arable and
specialty crops. The Xemium project is global, with a family of optimized formulations including
mixtures with epoxiconazole and F 500®.
Introduction
Modern agriculture depends on efficient tools for controlling fungal diseases that can
have a strong impact on yield and quality. Better, innovative fungicides are key for
sustainable management of such diseases. BASF continues to develop its portfolio of
highly active fungicides for use in crop protection globally. With Xemium (common
name Fluxapyroxad), we present the latest fungicide further extending the spectrum of
active compounds available for agricultural usage. Xemium is the result of BASFs
ongoing research on succinate dehydrogenase inhibitors (SDHIs) having started with
benodanil, a carboxamide fungicide introduced in the early 70s of the last century, which
mainly provides control of rusts (Puccinia spp.) (Pommer et al., 1973).
This chemical group was further developed with boscalid, another SDH inhibitor
being introduced in a wide range of crops including fruits, vegetables, oilseed rape and
cereals in the mid 2000s (Ammermann et al., 2002; Stammler et al., 2008). With
Xemium, researchers at BASF were able to further improve the biological performance
of SDHIs on a wide range of pathogens.
In this paper, information on physical and chemical properties, systemicity, a
summary on toxicological and ecotoxicological properties and results on field
performance in cereals will be given.
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Systemicity
Systemicity in wheat was measured by applying radioactive (14C) spiked product close to
the leaf axil and by visualizing its distribution from 2 hours to 9 days after application
with exposing the plants on Fuji Imaging Plates (BAS-MS 2040). After 24h, the plates
were read on a Fujifilm radioimager FLA-7000. For comparison, standard strips carrying
known amounts of radioactivity were exposed together with the samples.
Mode of action
Xemium is an inhibitor of the mitochondrial succinate-dehydrogenase (SDH), also
known as complex II of the mitochondrial respiration chain. The enzyme links the
carboxcylic acid cycle and the cellular respiration by catalysing the oxidation of
succinate to fumarate and inserting the electrons via ubiquinone into the respiration chain.
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The competitive inhibition prevents the reduction of ubiquinone and interrupts the
mitochondrial respiration and finally the energy production within fungal cells (Keon et
al., 1991; Matsson and Hederstedt, 2001; Glaettli et al., 2009).
Figure 1: Systemic distribution of Xemium. Wheat leaves treated with formulated active ingredient.
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M. Semar et al.
Performance
SDHI fungicides of the first generation were mainly used as seed treatment products and
controlled only a very narrow range of pathogens, mainly from the group of the
Basidiomycetes. In contrast to second generation SDHIs like boscalid, Xemium shows an
extraordinary high activity at low dose rates against a broad spectrum of diseases.
Figure 2: Curative efficacy of Xemium on Septoria tritici in wheat (UK, 2008, 5% initial attack, 1
application at GS 59, full dose rate).
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Figure 3: Long lasting efficacy of Xemium on Septoria tritici in wheat (Germany, 2009, 4% initial attack,
1 application at GS 33, half dose rates; CTL: Chlorothalonil).
Figure 4: Consistent control of Septoria tritici and yield protection across varieties with Xemium and
epoxiconazole (EPX).
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M. Semar et al.
mainly known for their preventative properties, Xemium shows in addition excellent
curative and long lasting efficacy. These effects are clearly observed when Xemium is
applied against the most important pathogen in European wheat production, S. tritici
(Figures 2-4).
Acknowledgements
The authors would like to thank Steve Waterhouse, BASF plc, for organizing, supporting
and evaluating the variety trials with NIAB and his valuable contributions for drafting
this publication. We also would like to thank Helmut Schiffer, BASF SE, for his
intensive studies leading to a better understanding of the systemic behaviour of Xemium.
References
Ammermann, E., Stierl, R., Hanke, W., Scherer, M., Ypema, H., Bardinelli, T. (2002):
Nicobifen* – The foundation of a new fungicide family. Phytopathology, 92, S4, P-2002-
0023-AMA. *=boscalid.
Glättli, A., Stammler, G., Schlehuber, S. (2009): Mutations in the target proteins of succinate-
dehydrogenase inhibitors (SDHI) and 14α-demethylase inhibitors (DMI) conferring
changes in the sensitivity – structural insights from molecular modelling. 9th International
Conference on Plant Diseases, Tours, France, 670-681.
Keon, J.P.R., White, G.A., Hargreaves, J.A. (1991): Isolation, characterization and sequence of a
gene conferring resistance to the systemic fungicide carboxin from the maize smut
pathogen, Ustilago maydis. Current Genetics, 19, 475-481.
Matsson, M., Hederstedt, L. (2001): The carboxin-binding site on Paracoccus denitrificans
succinate:quinone reductase identified by mutations. Journal of Bioenergetics and
Biomembranes, 33, 99-105.
Pommer, E. H., Jung, K., Hampel, M., Löcher, F. (1973): BAS 3170 F, (2-Jodbenzoesäureanilid),
ein neues Fungizid zur Bekämpfung von Rostpilzen in Getreide. Mitt. Biol. Bundesanst.
Land- und Forstwirtsch., 151, 204.
Stammler, G., Brix, H.D., Nave, B., Gold, R., Schoefl, U. (2008): Studies on the biological
performance of boscalid and its mode of action. Modern fungicides and Antifungal
Compounds V. Eds: Dehne, H.W., Deising H.B., Gisi, U. Kuck, K.H. Russell, P. Lyr,
H., DPG, Braunschweig, Germany. 45-51.
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10
Bixafen – The New Cereal Fungicide with Yield
Boosting Effects
Abstract
Bixafen is a novel systemic fungicide of the chemical class of the pyrazole-carboxamides within
FRAC resistance Group 7. It has a broad spectrum of activity against the most economically
important diseases of cereal crops caused by fungi from the classes of Ascomycetes,
Basidiomycetes and Deuteromycetes. Bixafen has excellent crop safety in all formulations and on
all cereal crops. It demonstrates a high level of activity as well as an outstanding long-lasting
control against Septoria tritici, Puccinia triticina, Puccinia striiformis, Oculimacula spp. and
Pyrenophora tritici-repentis in wheat and against the main diseases in barley including
Pyrenophora teres, Ramularia collo-cygni, Rhynchosporium secalis and Puccinia hordei. In
addition, bixafen shows very favourable toxicological and ecotoxicological profiles. Bixafen has
full systemic activity, being absorbed by the cuticle and translocated in the plant via the xylem.
Translocation speed is moderate which is optimal to provide an even distribution of the active
ingredient in the whole leaf. The biochemical mode of action is based on the inhibition of
succinate dehydrogenase, an enzyme of complex II within the fungal mitochondrial respiration
chain. For resistance management purposes and to minimize the risk of selection of resistant
fungal strains, bixafen is combined with highly active fungicides with different biochemical
modes of action such as sterol biosynthesis inhibitors (DMIs or Amines). Based on physico-
chemical complementarities of bixafen and the DMI prothioconazole, the mixture of the two
compounds (the basis of the Xpro technology) sets new standards in disease control and provides
high yields.. Studies under both controlled conditions and in the field demonstrated the benefits
of Xpro technology in terms of extended green leaf period, delay of senescence and a high
survival rate of the crop plants after drought stress. Significant yield increase was observed in the
field which was higher than with similar products of the same biochemical modes of action group.
Introduction
Fungicide research in Bayer CropScience has always put special emphasis on the
development of innovative active ingredients for the use in cereal crops. In the 1980’s
and 1990’s, efforts were focused on fungicide classes such as SBI’s (especially triazoles)
and strobilurins. These research activities led to the launch of leading compounds such as
fluoxastrobin and prothioconazole. The intensive and repeated use of SBI’s and
strobilurins for the control of cereal pathogens led to a reduction and, in some cases, a
total loss of efficacy based on reduced sensitivity or resistance of several key pathogens
in cereals (e.g. powdery mildew, Septoria, DTR). For this reason and because of the lack
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of suitable chemical classes, new solutions showing distinct modes of action from SBI’s
and strobilurins were required in order to avoid a further erosion of their activity.
In this context, Bayer CropScience started to work intensively on the chemical
family of carboxamides. The goal was to find a compound with broad spectrum of
activity, high level of efficacy, long lasting control and good plant compatibility. The
bixafen molecule, a representative of the pyrazole-carboxamide family, was discovered
in 2001 and further characterized in a series of lab, greenhouse and field trials. Bixafen
gave particularly outstanding results against diseases in monocotyledons and, for this
reason, was specifically developed as a foliar fungicide for cereal crops. Its market
introduction is planned from 2010 onwards. The purpose of this paper is to present a
summary of available information on the specific characteristics of the active ingredient,
the mode of action, and its conditions of application.
Spectrum of Activity
The efficacy against many different cereal pathogens was evaluated worldwide in
numerous field trials. Bixafen demonstrated excellent levels of activity under protective
and curative conditions against economically most important diseases of cereal crops
caused by fungi from the classes of Ascomycetes, Basidiomycetes and Deuteromycetes.
Bixafen provided excellent crop safety in all formulations and on all cereal crops and
good to excellent levels of activity against Septoria tritici, Puccinia triticina, Puccinia
striiformis, Oculimacula spp. and Pyrenophora tritici-repentis in wheat and against
Pyrenophora teres, Ramularia collo-cygni, Rhynchosporium secalis and Puccinia hordei
in barley. Under practical conditions, bixafen will be used in mixture with other active
ingredients, such as the DMI prothioconazole, providing powerful solutions for an
effective resistance management. Moreover, the combination of the two active
ingredients and the use of a unique formulation technology results in a significant
advance in fungicide performance, called “Xpro technology”.
Mode of Action
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the host tissue. When applied in curative conditions, bixafen is able to inhibit further
fungal growth and drastically reduce sporulation.
appressoria
Systemic Properties
Systemicity was studied with radiolabeled bixafen. Bixafen is rapidly taken up by the
cuticle and is steadily translocated throughout plant tissues via the xylem system.
Translocation speed is moderate, similar to that of prothioconazole, which is optimal for
providing an even distribution of the active ingredient in the whole leaf. The continuous
redistribution from the cuticle to the transpiration stream combined with the moderate
translocation pattern of both active ingredients is responsible for the outstanding and
long-lasting activity of the compounds in cereal crops.
Sensitivity monitoring with bixafen was performed in the laboratory for the main
pathogens of wheat and barley. The concentration providing 50% of growth inhibition
(IC50) was determined in a microtest or a detached leaf test. The results conducted with
hundreds of isolates each year showed that no shift of sensitivity was observed for the
major cereal pathogens. However, cases of resistance to succinate dehydrogenase
inhibitors (SDHI’s) have been reported for plant pathogenic fungi in dicotyledonous
crops. Consequently, particular attention will be paid to sound anti-resistance strategies
for bixafen which will be similar to those of other SDHIs. The recommendations
provided by the SDHI FRAC working group will be applied, because bixafen shows an
overall positive cross-resistance to all other molecules of this chemical group.
In practice, bixafen will be used in cereal crops only in combination with molecules
of other chemical classes with a different biochemical mode of action. In this respect,
prothioconazole, the leading molecule of the family of sterol biosynthesis inhibitors
(SBI’s) will play a key-role. By combining the two active ingredients at effective dose
rates, a high level of activity against major cereal pathogens is observed. A strong anti-
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The beneficial physiological effects of both bixafen and prothioconazole are visible
also in the field. The duration of green leaf period is extended contributing to prolonged
photosynthetic activity and thus to a significant yield increase in barley and wheat
(Figure 3).
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100
Wheat 85
Barley
+ 3.5 dt/ha + 2.4 dt/ha
95
80
90
Yield (dt/ha)
Yield (dt/ha)
18.3 10.8
85 75
14.8 8.4
80
70
75
UTC = 76.5 dt/ha UTC = 69.9 dt/ha
70 65
REF* BIX&PTZ REF* BIX&PTZ
113 trials (2005-2008) 2 applications 74 trials (2006-2008) 1-2 application
Figure 3: Yields provided by Xpro, the mixture of bixafen and prothioconazole, in wheat and barley
(UTC = untreated control).
Conclusion
Bixafen is a highly active, protective and curative fungicide with excellent and long
lasting efficacy, developed especially for the use in cereal crops. Bixafen is a new
generation SDHI belonging to the chemical class of pyrazole-carboxamides. In numerous
field trials implemented around the world, bixafen has shown broad spectrum of activity
against all key pathogens in wheat and barley including strobilurin resistant populations.
Bixafen will be combined with other highly active fungicides with different biochemical
mode of action in order to develop a powerful resistance management strategy. The
combination of excellent intrinsic properties of bixafen and the DMI prothioconazole
builds the basis for the “Xpro technology” concept. Xpro delivers exceptional disease
control combined with physiological benefits expressed as delayed senescence, positive
effects on morphogenesis and extended greening periods of crop plants leading to higher
yields and improved quality of cereal production.
Acknowledgments
The authors would like to thank all colleagues world-wide who contributed to the
development of bixafen.
References
Berdugo, C.A., Steiner, U., Oerke, E.-C., Dehne, H-W. (2011): Effects of the new SDHI-
fungicide bixafen on the physiology and yield of wheat plants. In: Modern Fungicides and
Antifungal Compounds, Vol. VI, Eds: Dehne, H.W., Deising, H.B., Gisi, U., Kuck, K.H.,
Russell, P.E., Lyr, H., DPG, Braunschweig, Germany. 81-84.
Dutzmann, S., Suty-Heinze, A. (2004): Prothioconazole: a broad-spectrum demethylation-
inhibitor (DMI) for arable crops – Pflanzenschutz Nachrichten Bayer, 57 (2), 249-264.
FRAC code list© 2010: Fungicides sorted by mode of action (including FRAC code numbering),
www.frac.info
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11
Fluopyram: Efficacy and Beyond on
Problematic Diseases
Abstract
Fluopyram, a fungicide from the new chemical class of pyridinyl ethyl benzamide has been
specifically developed for the control of problematic diseases in a broad range of crops.
Fluopyram affects the fungi at all growth stages from germination to sporulation. Its biochemical
mode of action has been shown to rely on the inhibition of succinate dehydrogenase (complex II)
within the fungal mitochondrial respiratory chain. Thus, fluopyram is a succinate dehydrogenase
inhibitor (SDHI) with a unique chemical structure and an attractive spectrum of activity against
problematic pathogens such as Sclerotinia spp., Botrytis spp. and Monilia spp. but also against
powdery mildews and some leaf spot diseases in many crops. It exhibits an excellent level of
efficacy at low dose rates. When applied to the plant, fluopyram is taken up and transported
translaminarly and acropetally thus protecting the entire plant throughout all growth stages, even
during fast growth periods.
For the “at risk” target pathogens, sensitivity studies have been carried out to evaluate the risk
of resistance development and to establish an anti-resistance strategy. Currently, in the majority
of the target pathogens, no shift in sensitivity has been detected. In addition, recent sensitivity
studies from independent laboratories on Alternaria alternata in pistachio and in Podosphaera
xanthii (cucurbit powdery mildew) have shown compound specific resistance patterns and
resistance factors.
Fluopyram has also been developed in combination with other fungicides to create a family of
products which offer a broad spectrum of activity and robust resistance management. Thanks to
the excellent field efficacy, fluopyram and its family of products provide outstanding in-season
disease control resulting in improved quantity and quality of yield coupled with better storage
stability and increased shelf life of the harvested produce.
Introduction
Bayer CropScience has always been very active in the development of innovative
products for crop protection. The new active substance fluopyram, a molecule discovered
in 2001, is the latest innovation from Bayer CropScience fungicide research. It has been
specifically developed for the control of problematic diseases in a broad range of crops
and will be globally available from 2011 onwards in more than 70 horticultural and
industrial crops.
This paper presents a synthesis of the available information regarding the
characteristics of the active substance, its mode of action as well as the use conditions.
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
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G. Labourdette et al.
Physico-Chemical Properties
N N
Structural formula
Mode of Action
Fluopyram is the only representative of a new and unique class of fungicides, the
pyridinyl ethyl benzamides. Its mode of action has been shown to rely on the inhibition
of the enzyme succinate dehydrogenase (complex II) within the fungal mitochondrial
respiratory chain. This enzyme is involved in the Krebs cycle (also called the citric acid
cycle), which contributes to fungal cell energy production by the process of ATP
synthesis. In addition, the Krebs cycle is an important source of various metabolic
precursors used in different biosynthetic pathways such as amino acids biosynthesis. The
succinate deshydrogenase complex is bound in the inner mitochondrial membrane
catalyzing the oxidation of succinate to fumarate coupled with the reduction of
ubiquinone to ubiquinol. The inhibition of succinate dehydrogenase by fluopyram
disrupts the respiratory chain thus blocking the energy production as well as the synthesis
of biosynthetic precursors used for the synthesis of key cellular components such as
amino acids. As a result, fluopyram affects the fungi at all growth stages from
germination (Figure 1) to sporulation.
Figure 1: Germination of Erysiphe necator spores without (left) or with fluopyram (right) treatment.
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Efficacy of Fluopyram
Bioavailability on Crops
Fluopyram behaviour on plants has been studied using the foliar formulation prepared
with 14C radio-labelled fluopyram. The application of the product was done by depositing
10 µl droplets on the leaf surface or on the stem. The mobility of the product from the
deposit points into plant tissues was investigated by using high resolution imaging
techniques designed to rapidly image levels of 14C active substances.When applied on the
plant surface, the major part of the active substance remained on the surface followed by
a multidimensional distribution. A continuous penetration into the leaf and diffusion in
the tissue were observed (Figure 2) resulting in the protection of the non treated parts.
14 14
C fluopyram 1 day C fluopyram 2 days
after application after application
When applied to the stem, an acropetal distribution of fluopyram to leaves and buds
was observed (Figure 3) providing protection of new emerging leaves.
14 14
C fluopyram 1 day C fluopyram 2 days
after application after application
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G. Labourdette et al.
Resistance Management
1.6 2009
2008
Base line
1.2
Density
0.8
0.4
log IC50
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Efficacy of Fluopyram
Activity Spectrum
Fluopyram is a fungicide with a broad spectrum of activity (Figure 5). Based on a high
number of field trials it has been shown that against problematic diseases such as
Sclerotinia spp., Botrytis spp. and Monilia spp., fluopyram at 250 g/ha exhibits a high
level of efficacy. Fluopyram at 100 g/ha provides also very good levels of control of
powdery mildews, e.g. Erysiphe necator, and some leaf spots such as Sigatoka species.
Gradient of
Leaf spot Powdery mildew
fruit trees
efficacy
Scab Brown rot
In order to create a family of products which offer a broad spectrum of activity and a
robust resistance management, fluopyram has also been developed in combination with
other fungicides. Fluopyram mixtures with for example trifloxystrobin or tebuconazole
enlarge the spectrum of activity to additional disease such as rusts, scab (early stages)
and leaf spots (Figure 6).
Powdery
Powderymildew
mildewgrape
grape
Powdery mildew grape
Rusts
Rusts Powdery
Powderymildew
mildew
Rusts vegetables
Powdery vegetables
mildew
vegetables
Leaf
Leafspot
spot Powdery
Powderymildew
mildew
Leaf spot fruit
Powdery trees
fruit trees
mildew
fruit trees
Scab
Scab Brown
Brownrot
rot
Scab Brown rot
Early
Earlyblight
blight Grey
Greymould
mould
Early blight Grey mould
Sigatoka
Sigatokasp.
sp. White
Whitemould
mould
Sigatoka sp. White mould
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G. Labourdette et al.
Conclusion
Fluopyram is the only representative of a new and unique class of fungicides, the
pyridinyl ethyl benzamides which has been developed specifically for the control of
problematic diseases in a broad range of crops. It exhibits at low application rates an
outstanding efficacy against Botrytis, Sclerotinia, powdery mildews and other diseases
responsible for quality losses. In addition, it presents benefits for the food chain industry
through a better storability and a longer shelf-life of the harvested produces. Co-
formulations provide farmers with innovative and complete solutions including built-in
resistance management. The fluopyram based family of products will be available
globally in 2011 for the use in foliar application and seed treatment on more than 70
horticultural and industrial crops.
References
Grouet, D., Montfort, F., Leroux, P., (1981): Resistance to carboxin and fenfuram in Ustilago
nuda (Jens.) Rostr., the causal agent of loose smut. Phytiatrie - Phytopharmacie. 30, 3-12.
Leroux, P., Berthier, G. (1988): Mise en evidence, en France, d’une souche de Puccinia horiana
résistante à l’oxycarboxine. Crop Protection, 7, 16-19.
Avenot, H.F., Michailides, T.J., (2007): Resistance to Boscalid Fungicide in Alternaria alternata
isolates from Pistachio in California. Plant Disease, 91, 1345-1350.
Avenot, H.F., Michailides, T.J., (2009): Monitoring the sensitivity to boscalid of Alternaria
alternata populations from California pistachio orchards. Phytopathology, 99, 6.
Stammler, G., Brix, H.-D., Glaettli, A., Semar, M., Schoefl, U., (2007): Biological properties of
the carboxamide boscalid including recent studies on its mode of action. Proceedings, 16th
International Congress of Plant Protection, Glasgow, Session 2A, 16-21.
Ishii, H., Miyamoto, T., Ushio, S., Kakishima, M., (2009): Sensitivity of boscalid-resistant and
sensitive isolates of Corynespora cassiicola and Podosphaera xanthii to a novel succinate
dehydrogenase inhibitor fluopyram. Abstracts from the 2009 KSPP fall meeting and the
first Japan – Korea joint symposium, Jeju, 28-31 October 2009, D-19, 135.
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12
Effects of the New SDHI-Fungicide Bixafen on
the Physiology and Yield of Wheat Plants
Abstract
Bixafen, a pyrazole carboxamide inhibiting fungal succinate dehydrogenase in the respiratory
chain, is a new broad-spectrum fungicide from Bayer CropScience developed for the control of
pathogens in cereals. In experiments under disease-free growth conditions the compound proved
to have positive effects on the morphogenesis and yield formation of wheat plants. When applied
twice (growth stages 39 and 59), bixafen increased the size of the upper wheat leaf layers,
delayed plant senescence and promoted grain filling of ears.
Introduction
Damage caused by fungal diseases on wheat (Triticum aestivum L.) may produce an
important drop in grain yield and quality, resulting in a considerable reduction of the
income. Foliar diseases due to fungal pathogens like Puccinia triticina, Septoria tritici
and Blumeria graminis f. sp. tritici decrease the photosynthetic active leaf area and
affects plant growth. In order to achieve an optimum crop yield, it is important to have an
effective control of foliar diseases during the period between flag leaf emergency and
milky ripeness, due to the fact that flag leaf photosynthesis is essential for an optimum
grain filling (Gooding et al., 2000). In addition to fungicidal effects, some fungicide
classes like Qo-inhibitors have been reported to induce physiological modifications in
crops, like increased tolerance against abiotic stress, darker green appearance of leaves,
delayed senescence of photosynthetic leaf area and modifications in the balance of plant
growth regulators (Pepler et al., 2005). These effects were frequently associated with a
positive influence on yield (Beck et al., 2002). Bixafen (Bayer CropScience), a pyrazole
carboxamide inhibiting succinate dehydrogenase in the fungal respiratory chain, is a new
broad-spectrum fungicide developed for the control of cereal pathogens. Bixafen was
tested for positive effects on yield formation of wheat in disease-free conditions.
The effects of bixafen on the physiology and yield of wheat plants were studied and
compared to those caused by triazoles and strobilurins in a disease-free environment
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Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
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under greenhouse conditions. Twenty kernels of spring wheat (Triticum aestivum L.),
cultivar Passat, were sown 2 cm deep per pot (20x20x30 cm). Ten pots per treatment
were used, and were randomized in the greenhouse. Fungicides were applied at two
growth stages (GS), first when the flag leaf ligule was visible (GS 39) and again at the
end of heading, when the inflorescence was fully emerged (GS 59). After the second
fungicide application, green leaf area duration (GLAD) was assessed weekly as
percentage of green area of the blades of the top leaves. Digital IR-thermal images were
taken at four growth stages: GS 75, GS 80, GS 85 and GS 90. The images were obtained
by a Stirling-cooled infrared scanning camera (VARIOSCAN 3201 ST, Jenoptic Laser,
Jena, Germany). The measurements were conducted between 5:00 pm and 7:00 pm in
order to avoid physiological and environmental changes among measurements. The
following parameters were analysed as well: senescence of leaves and maturation of ears,
photosynthetic activity and yield parameters such as grain yield, thousand kernels mass
and numbers of kernels per ear.
Effects of bixafen on morphogenesis of wheat was more pronounced than those produced
by the other fungicides, it increased the length and width of flag leaf (Figure 1).
Length Width
30 b
a a a 2,0
a c
b
1,5 a ab
20 a
cm
1,0
10
0,5
0 0,0
Untr. Bixafen Fluox. Proth. Spirox.
Figure 1: Effect of fungicide treatments on the flag leaf width and length at GS 75. Different letters
indicate significant differences according to Tukey's test (p ≤ 0.05). Error bars represent standard error of
the mean. Unt. Untreated; Fluox. Fluoxastrobin; Proth. Prothioconazole; Spirox. Spiroxamine.
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Figure 2: Effect of fungicide treatments on the temperature of wheat ears and leaves (temperature
difference to untreated). Error bars represent standard error of the mean.
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(Figure 3). The combination of the positive effects produced by bixafen application on
morphogenesis and physiology of wheat resulted in a clear yield benefit.
Figure 3: Effect of fungicide treatments on the yield of wheat. Different letters indicate significant
differences according to Tukey's test (p ≤ 0.05). Error bars represent standard error of the mean.
References
Beck, C., Oerke, E.-C., Dehne, H.-W. (2002): Impact of strobilurins on physiology and yield
formation of wheat. Med. Fac. Landbouww. Univ. Gent, 67/2, 181-187.
Gooding, M.J., Dimmock, J., France, J., Jones S.A. (2000): Green leaf area decline of wheat flag
leaves: the influence of fungicides and relationships with mean grain weight and grain
yield. Ann. Appl. Biology, 136, 77-84.
Pepler, S., Gooding, M.J., Ford, K.E., Ellis, R.H., Jones S.A. (2005): A temporal limit to the
association between flag leaf life extension by fungicides and wheat yields. Eur. J. Agron.,
22, 405-416.
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13
Studies on the Heterologous Expression of
Dihydroorotate Dehydrogenase from
Stagonospora nodorum
G. GUSTAFSON
Dow AgroSciences LLC, Discovery Research, 9330 Zionsville Rd., Indianapolis, IN 46268
Abstract
Studies were conducted on the heterologous expression of native or N-truncated dihydroorotate
dehydrogenase (DHODH) from the phytopathogenic fungus Stagonospora nodorum. The studies
encompassed several protein expression systems, numerous incubation conditions, N-terminal
fusions and chimeric DHODH proteins. Although significant quantities of insoluble and inactive
Sn DHODH were often produced, no more than minute amounts of soluble native or N-truncated
Sn DHODH were ever detected. In contrast, soluble native DHODH from Escherichia coli and
soluble N-truncated DHODH from Drosophila melanogaster could be produced and isolated in
quantities sufficient for activity assays and structural studies.
Introduction
G. Gustafson
inhibitors have little activity against fungal plant pathogens. Although LY214352 does
not have commercial levels of activity, a clear understanding of the interaction between
LY214352 and DHODH might promote the design and commercialization of DHODH-
inhibiting agricultural fungicides. The initial step in achieving this goal is the
heterologous expression and crystallization of DHODH from an LY214352-sensitive
fungal plant pathogen. N-terminally truncated DHODH from the plant pathogen Ustilago
maydis (Um), which is not sensitive to LY214352, has already been expressed as a
soluble and active GST-fusion protein in E. coli (Zameitat et al., 2007). The results of
studies on the heterologous expression of DHODH from an LY214352-sensitive
organism, Stagonospora nodorum (Sn), are presented in this contribution.
Dm ----------------------------------MDQDHLKNAKNATRRVGRLRSLGIVTVGGAALVAGITAYKNQDQLFRTFVMPAVRLLPAEASHQLA
Ec ------------------------------------------------------------------------------MYYPFVRKALFQLDPERAHEFT
Sn MLSAANMRALRVRPSMLRVGASRSLTRHASTDAVIAPQTASNVRSAGTRSKNLIFGTVLTLG--LSFGYLYVTDTRASIHEWLLVPALRQIYPDGEDAHH
Dm VLACKYRLCPVSQYHDDQNLHTS--------FFGRMLSNPIGIAAGFDKNAEAVDGLQDLGFGFIEVGTVTPAAQEGNPKPRVFRLTEDKAIINRYGFNS
Ec FQQLRRITGTPFEALVRQKVPAKPV-----NCMGLTFKNPLGLAAGLDKDGECIDALGAMGFGSIEIGTVTPRPQPGNDKPRLFRLVDAEGLINRMGFNN
Sn AGTAMLKALHSFGMNPRERGPGDSAGDLAVEVFGHTLANPIGTSAGIDKNAEVPTPLFELGPAIVEVGAVTLLPQEGNPKPRVFRIPSQNALINRYGFNS
Dm DGHQAVLQRLRL---------------LRKKENFNGVVGVNLG-------------RNKTTMSP-----IADYVQGVRVFGPVADYLVINVSSPNTKGLR
Ec LGVDNLVENV-------------------KKAHYDGVLGINIG-------------KNKDTPVEQG---KDDYLICMEKIYAYAGYIAINISSPNTPGLR
Sn EGAELVATRLRQRVREFAYHSGLGLDEEAERKVLDGEAGVPPGSLKQGRLMAVQVAKNKTTSENDIDAVVRDYATSVSHVGKYADILVVNVSSPNTPGLR
Dm DMQSKEKLRELLEQVNDTKSSLDKN--KNVPILLKLSPDLSLDDMKDIVWVIKRKKSRVDGLIVSNTTVSR---------ENIEKNKLAEETGGLSGPPL
Ec TLQYGEALDDLLTAIKNKQNDLQAMHHKYVPIAVKIAPDLSEEELIQVADSLVRHN--IDGVIATNTTLDR---------SLVQGMKNCDQTGGLSGRPL
Sn TLQNVEPLTRLLTGVVQAVNKIDRK--TKPAIMVKVSPDEDSEEQVSGICEAVWDSG—VDGVIVGNTTKKRPDPLPKGYLLPASEAQVLLEQGGYSGPQL
Dm KARSTEMIAQMYQLTDG----------------------------------------------------------------------------KIPIIGV
Ec QLKSTEIIRRLSLELNG----------------------------------------------------------------------------RLPIIGV
Sn FERTLALVSRYRKALDEGPKSVPSQQAESEGPNTSIIEKLQPSKQTGSSKTETTVEVGSASPALSSNVDDLQESAPTLTLHSKPVTAPAPKNPQKVIFAT
Dm GGVASGYDAYEKIEAGASYVQIYTALVYEGPALVEDIKAELSALITRLGHTNVADVVGTNSKFYLPK
Ec GGIDSVIAAREKIAAGASLVQIYSGFIFKGPPLIKEIVTHI
Sn GGITNGRQAREILDAGASVAQVYTALIYGGAGTISRIKQEMRDDAKKQTTATKR
Figure 1: Homology between the amino acid sequences of DHODH from Drosophila melanogaster (Dm),
Escherichia coli (Ec) and Stagonospora nodorum (Sn).
DHODH genes from E. coli (Ec), D. melanogaster (Dm) and S. nodorum (Sn) were
cloned by PCR and sequenced. A truncated version of the Dm gene, reported to express a
soluble and active DHODH, and two truncated versions of the Sn gene were prepared
(Figure 2). All full-length and truncated versions of the various DHODH genes were
ligated into a pET E. coli expression vector that adds six histidine residues to the C-
terminus of the expressed protein. Proteins were expressed in E. coli BL21(DE3) under a
variety of conditions. Most typically, transformed cells were incubated at 37˚ C until the
OD600 was about 0.5. IPTG was added to a final concentration of 1-20 µM and cells
were incubated for an additional 16 hr at 25-28˚ C and 125 rpm. Cells were harvested by
centrifugation and lysed by sonication. The lysate was centrifuged to produce pellet
(insoluble) and supernatant (soluble) fractions. In many cases his-tagged proteins in the
supernatant fraction were isolated by affinity chromotography. Protein samples were
analyzed by SDS/PAGE. In some cases, Western blots were used for detection of
expressed DHODH.
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Ec DHODH MYYP…
-36
M
Dm DHODH …SLGIVTVGGAALVAGITAYKNQDQLFRT…
-64 -73
M M
Sn DHODH …LIFGTVLTLGLSFGYLYVTDTRASIHEW…
-93
M
Um DHODH …LIYLVFAIAAGTLGIAYYADSRSAIHRW…
Figure 2: Constructs were prepared for expression of full-length Ec and Sn DHODH and truncated
versions of Dm DHODH (-36 = N-terminal 36 amino acids eliminated) and Sn DHODH (-64 and -73
versions). The previously published construct used for expression of Um DHODH is also shown. The
underlined amino acids form a predicted N-terminal membrane-spanning region that is not present in the
soluble Ec DHODH.
Results
66.3 kD
55.4 kD 66.3 kD
55.4 kD
36.5 kD
Figure 3 (left): SDS/PAGE analysis of proteins isolated from E. coli cells transformed with vectors for
expressing full-length (FL) or truncated (-73) Sn DHODH. Transformed cells were induced with 20 µM
IPTG and incubated for 16 hr at 25˚ C. The expected size for (-73) Sn DHODH with his-tag is 51.9 kD.
The expected size for full-length Sn DHODH with his-tag is 59.7 kD. Pel = insoluble proteins, sup =
soluble proteins, elu = soluble proteins eluted from a his-tag affinity column and MK = marker proteins.
Figure 4 (right): SDS/PAGE analysis of proteins isolated from E.coli cells transformed with vectors
expressing no protein (pET), truncated Dm DHODH (Dm), or one of two truncated forms of Sn DHODH (-
63 or -74). Transformed cells were induced with 1 µM IPTG and incubated for 16 hr at 25˚ C. The
expected sizes for (-73) Sn DHODH with his-tag, (-64) Sn DHODH with his-tag and (-36) Dm DHODH
with his-tag are 51.9 kD, 52.9 kD and 41.8 kD, respectively. Pel = insoluble proteins, sup = soluble
proteins, elu = soluble proteins eluted from a his-tag affinity column and MK = marker proteins.
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G. Gustafson
truncated Sn DHODH was insoluble and inactive (Figures 3 and 4). Native Ec DHODH
and truncated Dm DHODH were produced as soluble proteins in E. coli. (Figures 4 and
5). The activity of soluble Ec DHODH was suppressed by a known thiadiazolidinedione
inhibitor (Marcinkeviciene et al., 2000).
pET Ec Ec
MK pel sup pel MK MK pel elu
55.4 kD
36.5 kD 36.5 kD
Figure 5 (left): SDS/PAGE analysis of proteins isolated from E. coli cells transformed with vectors
expressing either no protein (pET) or full-length E. coli (Ec) DHODH. Transformed cells were induced
with 20 µM IPTG and incubated for 16 hr at 25˚ C. The expected size for Ec DHODH is 36.9 kD. Pel =
insoluble proteins, sup = soluble proteins and MK = marker proteins.
Figure 6 (right): SDS/PAGE analysis of proteins isolated from E. coli cells transformed with a vector
expressing an N-terminal truncated (-73) Sn DHODH with an additional internal deletion of a 76 amino
acid region that is absent from Ec and Dm DHODH (Figure 1). Transformed cells were induced with 1 µM
IPTG and incubated for 16 hr at 25˚ C. The expected size for (-73) Sn DHODH with the internal deletion
of 76 amino acids and a C-terminal his-tag is 42.9 kD. Pel = insoluble proteins, elu = soluble proteins
eluted from a his-tag affinity column and MK = marker proteins.
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MK DS SD DS SD MK
- pel pel elu elu
Truncated Sn MASIHEWLLVPALRQIYPDGEDAHHAGTAMLKALHSFGMN
Truncated Dm MAYKNQDQLFRTFVMPAVRLLPAEAS------HQLAVLACKYRLC
Truncated Sn PRERGPGDSAGDLAVEVFGHTLANPIGTSAGIDKNAEVPTPLFEL
Truncated Dm PVSQYHDDQN-LLHTSFFGRMLSNPIGIAAGFDKNAEAVDGLQDL
66.3 kD
36.5 kD
IP
Truncated Dm GFGFIEVGTVTPAAQEGNPKPRVFRLTEDKAIINRY……
Figure 7 (left): Alignment of the N-terminal regions of N-truncated Sn DHODH (-73) and Dm DHODH
(-36) depicting proteins encoded by chimeric DHODH genes containing the N-terminal region of Dm
DHODH and the C-terminal region of Sn DHODH (clone DS) or the N-terminal region of Sn DHODH
with the C-terminal region of Dm DHODH (clone SD).
Figure 8 (right): SDS/PAGE analysis of proteins isolated from E. coli cells transformed with vectors for
expressing chimeric Sn/Dm DHODH genes. Transformed cells were induced with 20 µM IPTG and
incubated for 16 hr at 25˚ C. The expected size for DS chimeric DHODH with his-tag is 52.2 kD. The
expected size for SD chimeric DHODH with his-tag is 41.6 kD (see Figure 7 for definition of DS and SD).
Pel = insoluble proteins, elu = soluble proteins eluted from a his-tag affinity column and MK = marker
proteins.
Conclusions
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G. Gustafson
References
Davies, M., Heikkila, T., McConkey, G. A., Fishwick, C. W., Parsons, M. R., Johnson, A. P.
(2009): Structure-based design, synthesis and characterization of inhibitors of human and
Plasmodium falciparum dihydroorotate dehydrogenases. J. Med. Chem., 52, 2683-2693.
Deng, X., Gujjar, R., El Mazouni, F., Kaminsky, W., Malmquist, M. A., Goldsmith E. J., Rathod,
P. K., Phillips, M. A. (2009): Structural plasticity of malaria dihydroorotate dehydrogenase
allows selective binding of diverse chemical scaffolds. J. Biol. Chem., 284, 26999-27009.
Gustafson, G., Davis, G., Waldron, C, Smith, A., Henry. M. (1996): Identification of a new
antifungal target site through a dual biochemical and molecular genetics approach. Current
Genetics, 30, 159-165.
Heikkila, T., Ramsey, C., Davies, M., Galtier, C., Stead, A., Johson, A. P., Fishwick, C., Boa, A.
N., McConkey, G. A. (2007): Design and synthesis of potent inhibitors of the malaria
parasite dihydrorortate dehydrogenase. J. Med. Chem., 50, 186-191.
Marcinkeviciene, J., Rogers, M. J., Kopcho, L., Jiang, W., Wang, K., Murphy, D. J., Lippy, J.,
Link, S., Chung, T., Hobbs, F., Haque, T., Trainor, G. L., Slee, A., Stern A. M., Copeland,
R. A. (2000): Selective inhibition of bacterial dihydroorotate dehydrogenases by
thiadiazolidinediones. Biochem. Pharmacol, 60, 339-342.
Rawls, J., Knecht, W., Diekert, K., Lill, R, Loffler, M. (2000): Requirements for the
mitochondrial import and localization of dihydrorotate dehydrogenase. Eur. J. Biochem.,
267, 2079-2087.
Ullrich, A., Knecht, W., Fries, M., Loffler, M. (2001): Recombinant expression of N-terminal
truncated mutants of the membrane bound mouse, rat and human flavoenzyme
dihydroorotate dehydrogenase. Eur. J. Biochem., 268, 1861-1868.
Zameitat, E., Freymark, G., Dietz, C. D., Loffler, M., Bolker, M. (2007): Functional expression
of human dihyroorotate dehydrogenase (DHODH) in pyr4 mutants of Ustilago maydis
allows target validation of DHODH inhibitors in vivo. Appl. Environ. Microbiol., 73, 3371-
3379.
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14
Molecular Approaches to Elucidate Pathways
and Sites of ‘Fungicide’ Resistance in
Oomycetes
Abstract
Oomycetes cause some of the most damaging diseases of crop plants worldwide. Although they
are superficially similar to true fungi, they are evolutionarily distinct and are more closely related
to the brown algae and diatoms. Oomycete diseases may be controlled by resistance in plants or
through applications of chemical control agents. Numerous compounds may be used to control
oomycete diseases, but the mode of action for very few has been conclusively demonstrated. The
understanding of oomycete biology has undergone a renaissance in recent years, with the
sequencing of several genomes and the development of molecular biology tools to test
hypotheses regarding the roles of specific genes. To aid in the determination of the mode of
action for existing and new oomycete-active compounds, these recently developed tools and
resources are being exploited. This chapter summarises the molecular biology resources and tools
available for mode of action determination, with case studies of how molecular genetic strategies
have been used to determine mode of action for oomycete-active compounds in the model
oomycete, Phytophthora infestans.
Introduction
Plant pathogenic oomycetes are responsible for some of the most devastating diseases of
crops worldwide. Phytophthora infestans alone, causing late blight disease on potato and
tomato, is responsible for losses and control costs of over $5 bn per year worldwide
(Haas et al., 2009; Haverkort et al., 2008). Additional examples of oomycete diseases of
economic importance are cocoa black pod (P. palmivora; Guest, 2007), soybean root rot
(P. sojae; Schmitthenner 1985), grape downy mildew (Plasmopara viticola; Emmet et al.,
1992), and tobacco blue mould (Peronospora hyoscyami f.sp. tabacina; Borras-Hidalgo
et al., 2010). Oomycetes such as the tree pathogens P. cinnamomi and P. ramorum
(Hardham, 2005; Rizzo et al., 2005) can also cause significant damage in natural
ecosystems. The genera encompassing Phytophthora, downy mildews (eg Bremia,
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Plasmopara, Peronospora), and white blister rusts (Albugo) are exclusively plant
pathogens, while the genera Pythium and Aphanomyces comprise plant and animal
pathogenic species, including the human pathogen P. insidiosum (Phillips et al., 2007).
Saprolegnia and Lagenidium species are not plant pathogens, but are known to infect fish
and insects, respectively (Phillips et al., 2007).
Oomycetes infecting crops are superficially similar to fungi in that they have a
hyphal growth habit and produce spores (called sporangia) or conidia. Elements of their
infection biology also resemble fungi in the production of an appressorium-like structure
to initiate infection (Hardham, 2006; Grenville-Briggs et al., 2008), and the production of
biotrophic intracellular haustoria formed inside infected plant cells (Hardham, 2006;
Avrova et al., 2008). However, other morphological features, biochemistry, and
molecular phylogenetic analyses place the oomycetes within the stramenopiles. This
phylogenetic group also includes the golden brown algae and diatoms, and is distantly
rooted in the same lineage as the apicomplexan parasites of animals (includes malaria
parasites) (Burki et al., 2007). Some characteristics of oomycetes are a cell wall
composed predominantly of cellulose with only a minor chitin component, biflagellate
swimming zoospores, coenocytic hyphae (no or few septa), sterol and thiamine
auxotrophy (Phytophthora), and diploidy in asexual stages (reviewed in Erwin and
Ribeiro, 1996).
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studies has been the identification of mutant variants of the target proteins that are shown
to confer insensitivity. Only for one compound, mandipropamid, has this been
determined (Blum et al., 2010a). One contributing factor in the paucity of information
about mode of action for oomycete active compounds may be a perceived lack of
resources or defined protocols, or complicated nature of conducting this work in
oomycetes.
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transformation remains the most widely used. Genetic transformation is a crucial tool in
demonstrating the role of genes and their encoded proteins, either through
complementation of a recessive phenotype (e.g. chemical sensitivity) with a dominant
allele (e.g. chemical insensitivity), or removal of gene activity through gene silencing.
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enantiomer found in mefenoxam) is active against oomycetes. It has also been relatively
straightforward to produce laboratory mutants insensitive to metalaxyl using a variety of
mutagenic agents (Young et al., 2001 for example). Genetic studies have shown that
insensitivity to metalaxyl is not always simply inherited, may be semi-dominant, and that
the action of minor genes may act to condition variable levels of insensitivity (Fabritius
et al., 1997; Lee et al., 1999; Knapova et al., 2002). However, Judelson and Senthil
(2006) investigated the expression of ABC transporters in P. infestans, which may act to
actively expel toxic compounds from the cell, and found no correlation between their
mRNA levels and insensitivity to metalaxyl. Using a genetics approach and bulked
segregant analysis, Fabritius et al. (1997) also identified DNA markers linked to the
locus conditioning metalaxyl insensitivity in some, but not all genetic crosses examined
in that study. These genetic markers can be transferred to a physical region of DNA,
either by screening of a large DNA insert library, sequencing of the marker and
identification of a genomic region spanning the metalaxyl insensitivity locus, or a
combination of both strategies. Identifying a candidate gene for metalaxyl insensitivity
from a defined region of the P. infestans genome sequence will rely on knowledge of the
biochemical and phenotypic symptoms induced after metalaxyl treatment. The strongest
indication to date for the target of metalaxyl has been from biochemical evidence that
treatment leads to a marked decrease in the levels of ribosomal RNA (rRNA) (Davidse et
al., 1983; Wollgiehn et al., 1984). In eukaryotes, rRNA is transcribed by the RNA
polymerase I complex, which comprises at least 14 subunits, and may share up to 5
subunits with RNA polymerase II (Kuhn et al., 2007). Thus, in searching for a candidate
gene conditioning mefenoxam (active R enantiomer) insensitivity, the most robust
candidate genes would likely be those subunits specific to the RNA polymerase I
complex. Recent results from a genetic approach similar to that of Fabritius et al. (1997),
using the steps outlined above and combined with genomics and genome resources, have
led to the identification of a region of the P. infestans genome conditioning mefenoxam
insensitivity (Whisson, Randall, Csukai, Fonne-Pfister, unpublished data). Discovery of
this region was accelerated through use of a P. infestans BAC library (Whisson et al.,
2001) pooled in multiple dimensions to enable rapid screening for DNA markers. This
genomic region encodes an RNA polymerase I subunit that, when transferred by
transformation into a sensitive isolate of P. infestans, leads to a shift towards mefenoxam
insensitivity.
New Tools for Mode of Action Determination in Oomycetes, and Targets for
Development
As shown in the determination of mandipropamid mode of action (Blum et al., 2010a),
accurate descriptions of the cellular effects of oomycete active compounds can prove
critical in the development of hypotheses for mode of action. However, if no obvious
symptoms are visible by light microscopy, additional avenues need to be explored for
insights into the mode of action. One such avenue may be to use transformation of
Phytophthora to tag specific cellular compartments and processes using fluorescent
protein fusions to Phytophthora proteins. This, coupled with confocal laser scanning
microscopy, can provide insights into subcellular organisation upon a variety of pathogen
lifecycle transitions, pathogenesis, host plant resistance, and chemical insult. To date,
only secreted avirulence and pathogenicity proteins have been localised by this method
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(Whisson et al., 2007; Avrova et al., 2008), but such a strategy, targeted to many
subcellular compartments and processes, could prove to be a valuable resource to
augment mode of action studies in oomycetes. Phytophthora proteins, such as an ATPase,
tubulin, and zoospore vesicle protein have been localised by using antibody detection
(Young and Slawecki 2001; Robold and Hardham, 2004; Shan et al., 2006). In the case
of tubulin, localisation by antibody has been used to help determine the mode of action,
or mode of action hypothesis, for zoxamide (Young and Slawecki, 2001). The advantage
of fluorescent protein tagging over antibody detection is largely that detection and
visualisation of fluorescent proteins is non-destructive and can be carried out in real time
on live specimens rather than the fixed samples used for antibody detection.
Since its description in the nematode Caenorhabditis elegans more than a decade
ago (Fire et al., 1998), RNA interference (RNAi), also known as gene silencing, has
proved a valuable tool in determining the roles of many eukaryotic genes. This strategy
for determining the role of a given gene and its encoded protein relies on the triggering of
the endogenous silencing pathway by double stranded RNA (dsRNA) arising from
aberrant RNA molecules, inverted repeats, transcription from both DNA strands, or
genome invaders such as viruses and transposons (reviewed in Whisson et al., 2009a).
The degraded dsRNA fragments then target homologous RNA molecules for degradation,
in a sequence specific manner. Thus, by introducing a dsRNA molecule homologous to a
gene of interest, the action of the encoded protein may be removed by destruction of its
mRNA. In oomycetes, gene silencing has been best characterised in P. infestans
(reviewed in Whisson et al., 2009a) and has been used to identify the role of several
genes in spore development and pathogenicity (Ah Fong and Judelson, 2003; Blanco and
Judelson 2005; Avrova et al., 2008). In addition to this, the application of gene silencing
holds promise in aiding mode of action determination in oomycetes. This could function
through linkage of observed chemical treatment symptoms with phenotypes observed
when specific Phytophthora genes are silenced. Such an approach could be useful, as
exemplified by the cellulose synthase gene targeted by mandipropamid. Grenville-Briggs
et al. (2008) used transient gene silencing to knock down the expression of these genes,
yielding partially silenced phenotypes that were similar in appearance to the symptoms of
mandipropamid treatment.
Presently, it is possible to silence Phytophthora genes in a stable or transient manner.
Stable silencing operates at the transcriptional level in Phytophthora and may be initiated
by transformation with sense, antisense, or inverted repeat gene constructs (Ah Fong et
al., 2008). Silenced transformants are effectively null mutants, as no transcription of the
targeted gene occurs. Partially silenced transformants are also occasionally identified (Ah
Fong et al., 2008). Partial silencing allows phenotypic characterisation of genes that are
essential for cellular survival, such as chemical control targets, the complete silencing of
which would otherwise be lethal.
An assay for transient silencing of target genes has also been developed for use in
Ph. infestans. Transient silencing is achieved through treatment of protoplasts with in
vitro synthesised dsRNA. The silencing in this assay is partial, and persists for up to 15
days, allowing time for phenotypic effects to be assessed (Whisson et al., 2009a). Several
genes may be assayed in parallel, and so it is particularly well suited to higher throughput
screening. Such an approach could be used to identify a range of phenotypes that may be
associated with symptoms from chemical treatment. Further, a silencing screen of P.
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infestans genes, predicted from the genome sequence and expression profiled by
microarray, could also yield potential targets for future control chemicals. To identify
pathogenicity genes from P. infestans by transient gene silencing, Whisson et al. (2009b)
selected over 50 genes for screening, identified from a microarray as upregulated during
infection. Not only did this study identify numerous P. infestans-specific secreted
effector proteins as required for pathogenicity, but several other genes encoding highly
conserved proteins were also identified.
In 1983, the oomycetes were described as a fungal geneticist’s nightmare (Shaw, 1983).
Since then, many advances have been made in developing the resources and tools to gain
a deeper understanding of these fungus-like organisms. This has culminated in recent
years with the sequencing of genomes and development of molecular biology tools to
determine the involvement of specific genes in various stages of oomycete biology. This
has been best developed for the hemibiotrophic Phytophthora sp., for which the
molecular events underlying sporulation and pathogenicity are intensively studied. These
molecular biology tools have now also been applied to the determination of mode of
action for oomycete-active compounds such as mandipropamid and mefenoxam. There
remains scope for further use of molecular genetic strategies in this field. For example,
effects of the applied chemicals may be better described if transgenic Phytophthora
strains were available with specific cellular components and processes labelled with
fluorescent proteins to determine the primary disruption to the Phytophthora cell upon
chemical exposure. Further, gene silencing represents a presently underutilised tool in
aiding in determination of chemical mode of action; knockdown of gene expression for
specific genes may mimic the symptoms observed for chemical exposure, allowing
hypotheses for mode of action to be refined. Further, gene silencing experiments
examining aspects of oomycete biology may define targets for development of future
control chemicals. Further development of molecular genetic tools such as targeted gene
disruption, currently not possible in oomycetes, will continue to accelerate the
understanding of oomycete biology, contribute towards determining mode of action
studies for existing control chemicals, and yield targets for future chemical control.
Acknowledgements
I thank my lab colleagues at SCRI, and collaborators at Syngenta for discussion and
advice. SCW is funded by the Scottish Government Rural and Environment Research
and Analysis Directorate (RERAD) and the Biotechnology and Biological Sciences
Research Council (BBSRC), UK.
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15
Sensitivity to CAA Fungicides and Frequency of
Mutations in Cellulose Synthase (CesA3) Gene
of Oomycete Pathogen Populations
Abstract
Recently the molecular mechanism of resistance to the carboxylic acid amide (CAA) fungicide
mandipropamid (MPD) has been described to be due to one recessive mutation in the cellulose
synthase gene, CesA3, causing inheritable resistance in Plasmopara viticola. Mode of action
studies in Phytophthora infestans confirmed that mandipropamid inhibits cellulose biosynthesis.
Sequencing the CesA3 genes of CAA sensitive and resistant isolates of P. viticola and
Pseudoperonospora cubensis revealed three different amino acid exchanges at the same position:
In P. viticola, mainly G1105S but also G1105V were detected, whereas in P. cubensis, both
G1105V and G1105W were found depending on the origin of isolates. The sensitivity towards
different CAA fungicides of these genotypes is presented. Based on the molecular knowledge,
quantitative methods were developed to test the frequency of the mutations in populations of P.
viticola. The quantitative measurement of the mutations in populations is assessed by
pyrosequencing and quantitative PCR. The correlation between the mutation frequency and the
CAA sensitivity phenotypes is discussed.
Introduction
The carboxylic acid amide (CAA) fungicide dimethomorph, has been used to control
downy mildew in grapes and cucurbits since the late 1980’s (Albert et al., 1991). It was
subsequently followed by other compounds of the CAA class, such as mandipropamid,
iprovalicarb and benthiavalicarb (Gisi et al., 2007). Resistance to dimethomorph in
Plasmopara viticola was first discovered in 1994 in France (Chabane et al., 1996), but
created no problem for growers (relatively low area coverage and use in mixtures) until
selection pressure increased due to the introduction of new fungicides containing CAA’s.
Resistance has been described as being disruptive, monogenic, and recessive based on a
nuclear gene (Gisi et al., 2007). Early reports suggested that the mode of action is
associated with cell wall deposition and/ or biosynthesis (Jende et al., 2002). Recent
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H. Sierotzki et al.
Pathogens
P. viticola isolates were retrieved from leaf samples collected in different European
countries in 2008 and 2009. The method is described in: www.frac.info.
Allele quantification
Total genomic DNA extraction from the samples or isolates was performed according to
standard CTAB protocol (Zolan and Pukkila 1986) or using the MagAtract 96 DNA
Plant Core Kit (Qiagen). The CesA3 G1105S/V/W allele quantifications (G: glycine, S:
serine, V: valine and W: tryptophane) were performed with Q-PCR and pyrosequencing.
Quantitative PCR for CesA3 G1105S/V in P. viticola: Based on the wild type sequence,
three allele specific MAMA-forward primers (Cha et al., 1992) and a common reverse
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primer have been designed (wild type forward: 5’-CCT TTA CGG CAA ATG TGT TAG
G-3’, Serine allele forward: 5’-ACC TTT ACG GCA AAT GTG TTG A-3’, Valine allele
forward: 5’-ACC TTT ACG GCA AAT GTG TTC TT-3’ and common reverse: 5’-CCA
ACA AGT TGC CCT CGT AAT-3’; mismatch base underlined). Each allele has been
determined in separate reactions on a ABI 7900HT real-time PCR system using the
Power SYBR Green PCR Master Mix from ABI at 12 µl reaction volume and primer
concentration of 0.5 µM (PCR conditions: 10 min at 95°C, 40 cycles 15 sec at 95°C, 30
sec at 60°C, 30 sec at 72°C).
Pyrosequencing was performed on a PyroMark ID system from Qiagen according to
the manufacturer’s recommendations. The PCR fragment was amplified using the
primers: 5’-TCG TCA TGA GCC AGT TTT ACC-3’ and 5’-biotin-GCC ACA GCT
GCA CAA ACA-3’. The product was transferred after single strand preparation (as
described by the manufacturer), to the annealing buffer containing the sequencing primer
5’-ACC ACA CGG CTG CTA-3’ at a final concentration of 0.4 µM. Allele
quantification was performed by calculating the ratios of the peak heights of the 2nd
nucleotide (G for serine and T for valine) over the 3rd base (C) in the codon.
Plasmopara viticola
Fungicide sensitivity analysis
Sensitivity monitoring of P. viticola isolates for mandipropamid, which was
commercially introduced in 2005, started on large scale in 2003. Each year 100 to 200
samples of infected grape leaves from different European countries were tested. The
distribution of EC50 values (concentrations that reduce the downy mildew attack by 50%)
shows that resistance to CAA fungicides is disruptive. There was a sensitive part in the
population with EC50 values between 0.01 and 10 mg/L and a resistant part with EC50
values of higher than 100 mg/L. The highest concentration used in the tests was 100
mg/L. Interestingly, since 2006 downy mildew samples with EC50 values between 10
and 100 mg/L were detected. Although these samples were interpreted as mixtures of
sensitive and resistant isolates, they were classified as resistant in this study. The overall
frequency of resistance in P. viticola populations collected in European countries varied
from 15 to 25%, with no tendency to increase over the years. There were significant
differences in frequency between regions, e.g. high frequency in Armagnac and Mosel
compared to low frequency in Spain and some parts in Italy. Significant differences were
observed within regions. In some regions (Champagne, Cognac, and Lombardy) the
frequency of resistance has increased.
Gene sequencing
The discovery of the molecular mechanism responsible for resistance in P. viticola
towards CAA fungicides (Blum et al., 2009), was used to develop molecular assays to
measure and quantify the frequency of resistance alleles in field populations of downy
mildew. As mentioned above, inheritance of CAA resistance in crossing studies was
recessive (Gisi et al., 2007). Most molecular methods can determine the zygocity of
diploid individuals in single spore isolates (by allele discrimination tests), but not in
mixtures of putatively homozygous and heterozygous individuals in which only the allele
frequency can be measured.
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H. Sierotzki et al.
The detection of the amino acid change at position 1105 (glycine to serine), based
on a single nucleotide polymorphism in the CesA3 gene enabled the development of
molecular assays to quantify the allele frequency in bulk samples. Frequency
measurements in single individuals should follow the rules of homozygous or
heterozygous traits in diploid organisms. However, in field samples where mixtures of
different individuals often occur, the relation between quantitative allele measurements
and phenotype is rather complex. Q-PCR analysis of the frequency of the allele
responsible for CAA resistance in P. viticola should theoretically produce overlapping,
non-discriminating ranges of sensitive and resistant phenotypes (Table 1), due to the
recessive inheritance of resistance. Additionally, the phenotype measurement by bioassay
is biased by the fact that only about 10% to 20% of resistant spores in a bulk isolate may
cause a completely resistant result. Therefore, only samples with 10% or less resistance
allele will result in a sensitive response to CAA’s. Samples with a frequency of 10% to
70% might be either sensitive or resistant, whereas samples with 10% to 100% can be
resistant (Table 1).
In a first molecular test the frequency of the G1105S allele was measured. In 2008,
resistance was detected predominantly in samples with a resistance allele frequency of
70% or higher, with exception of one sample. The samples with more than 70% of
resistance allele were interpreted as predominantly homozygous for serine. On the other
hand, resistance to CAA’s was also found in samples with as low as 45% resistance allele
frequency, indicating the presence of heterozygous and homozygous sensitive or resistant
individuals. Samples with less than 40% resistance allele frequency were predominantly
sensitive, being homozygous or heterozygous at position 1105. Two resistant samples
(EC50 >100) contained a low amount of serine allele; these samples were proved in
subsequent studies to contain the G1105V allele (Figure 1).
The results also showed that samples with up to 70% of resistance allele (either
serine and/or valine) can produce a sensitive phenotype, albeit being heterozygous.
Additionally, these findings show that different mutations in CesA3 gene of P. viticola
can cause resistance to CAA fungicides (G1105S and G1105V). Both mutations produce
a similarly strong resistant phenotype.
Based on these results, the molecular test was broadened to measure both alleles in a
quantitative manner in bulk samples. The two technologies used were Q-PCR and
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pyrosequencing. Each technology has its own advantages: Q-PCR is highly sensitive,
whereas pyrosequencing detects simultaneously different SNP’s. On the other hand, the
disadvantage of Q-PCR is the need for separate PCR for each SNP and the reduced
sensitivity of pyrosequencing at low allele frequency. Comparisons showed that
pyrosequencing for the G1105S allele correlates well with the Q-PCR assay although
significant deviations occur at frequencies below 10%. Additionally, it was not possible
to design a pyrosequencing that measured appropriately a high frequency of G1105V.
Consequently, we continued analyzing the relation between resistant phenotype and
allele frequency for both alleles together with the Q-PCR assay.
100 100
L/ 90
Log EC50 value MPD in mg/L
g
m 80
in 70
D10
P 60
M
e 50
u
la 40
v 1
0
5 30
C
E 20
g
o
l 10
0.1 0
0 10 20 30 40
log EC50 value MPD in mg/L
Number of sample
G1105S frequency in each sample
Figure 1: Relation between G1105S allele frequency (dark grey, diamonds) and CAA resistance (grey,
square) in samples of Plasmopara viticola from 2008. Two resistant samples with low frequency of
G1105S frequency (highlighted by circle) showed G1105V substitution. Allele frequency was measured by
Q-PCR.
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H. Sierotzki et al.
the valine allele was more frequent than the serine allele in the Spanish samples
compared to samples from other countries. In order to resolve the issue of homozygous
vs. heterozygous samples, a new method has to be developed to distinguish homo- from
heterozygous bulk isolates.
L/
g
m 10
in Predominantly
0 homozygous
5
C
E resistant
g 1
o
l
Predominantly heterozygous sensitive
0.1
Figure 2: Frequency of mutations leading to serine and valine in relation to EC50 values towards
mandipropamid in bulk isolates of Plasmopara viticola collected in2009 in Europe, measured by Q-PCR.
Pseudoperonospora cubensis
Resistance towards CAA fungicides in P. cubensis has been reported from trial
experiments in the United States and in samples collected in Israel (Table 2).
The P. cubensis isolates were obtained from different locations and hosts, such as
cucumber, zucchini, watermelon or cantaloupe. The bioassay showed clear differences in
EC50 values and RF values higher than 100 (data not shown) between sensitive and
resistant isolates. Interestingly, when sequencing the CesA3 gene, two different
resistance alleles were found. In US isolates the G1105W (Trp, tryptophane) mutation
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was detected, whereas in the isolates from Israel the G1105V (Val, valine) mutation was
present. Both mutations lead to resistance to all tested CAA fungicides (Figure 3).
Table 2: Sensitivity to mandipropamid (MPD) and amino acid configuration at position 1105 in the
CesA3 gene of Pseudoperonospora cubensis isolates collected in the United States and Israel.
100
MPD Gly
Sensitivity log EC50 in mg/L
MPD Val
MPD Gly and Val
L/ 10 CAA resistant isolates from US and Israel
g MPD Trp
m
n
i DMM Gly
0
5
C
E DMM Val
g
o
l 1 DMM Gly and Val
yt
iv DMM Trp
it
is
n Bent Gly
e
S CAA sensitive isolates from Switzerland, US and Israel
Bent Val
0.1
Bent Gly and Val
0 5 10 15 20 25
Bent Trp
Number of isolate
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Despite the long history of CAA fungicide usage for the control of P. cubensis, the
evolution of resistance towards CAA’s is difficult to explain. It might be based on the
very rare occurrence of oospore formation in this oomycete (Bedlan 1989, Cohen,
unpublished data). However, a recent evaluation of population structures of P. cubensis
in North America revealed a rather high diversity (Mitchell et al., 2009). It is not known
whether the genetic difference in the CesA3 mutations between isolates from US and
Israel are related to different population dynamics in the two countries.
In conclusion, we show that several mutations at position 1105 in the CesA3 gene
may occur within one oomycete pathogen species leading to recessive resistance to CAA
fungicides. These mutations can be either in geographically separated populations or in
the same population. It is not known, how these mutations mechanistically influence the
inhibitory effect of CAA fungicides, but the phenotypic effect is similar for all mutations
towards all CAA fungicides.
Acknowledgments
Alyssa Mulcahy is acknowledged for screening the P. cubensis isolates in the United
States.
References
Albert, G., Thomas. A., Gühne, M., 1991: Fungicidal activity of dimethomorph on different
stages in the life cycle of Phytophthora infestans and Plasmopara viticola. In: The 3.
International Conference on Plant Diseases, Bordeaux. Bordeaux, France: ANPP, 887–94.
Bedlan, G., (1989): First detection of oospores of Pseudoperonospora cubensis (Berket Curt.)
Rost. on glasshouse cucumbers in Austria. Pflanzenschutzberichte, 50,119–120 (German,
with English summary).
Blum, M., Waldner, M., Gisi, U., (2010): A single point mutation in the novel PvCesA3 gene
confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara
viticola. Fungal Genetics and Biology, 47, 499-510.
Blum, M., Boehler, M., Randall, E., Young, V., Csukai, M., Kraus, S., Moulin, F., Scalliet, G.,
Avrova, A., Whisson, S., Fonne-Pfister, R., (2010): Mandipropamid targets the cellulose
synthase like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen,
Phytophthora infestans. Molecular Plant Pathology, 11, 227- 243.
Cha, R.S., Zarbl, H., Keohavong, P., Thilly, W.G., 1992: Mismatch amplification mutation assay
(MAMA): Application to the c-H-ras gene. Genome Research, 2, 14-20.
Mitchell; M.N., Ocamb, C., Gent, D., 2009: Addressing the relationship between
Pseudoperonospora cubensis and P. humuli by multigenic characterization and host
specificity. Phytopathology, 99, S87.
Gisi, U., Waldner, M., Kraus, N., Dubuis, P. H., Sierotzki, H., 2007: Inheritance of resistance to
carboxylic acid amide (CAA) fungicides in Plasmopara viticola. Plant Pathology, 56, 199-
208.
Jende, G., Steiner, U., Dehne, H.-W., 2002: Microscopical characterization of fungicidal effects
on infection structures and cell wall formation of Phytophthora infestans. In: H.W. Dehne,
U. Gisi, K.H. Kuck, P.E. Russell, H. Lyr (Eds.), Modern Fungicides and Antifungal
Compounds III (pp. 83–90). AgroConcept, Bonn, Germany.
Zolan, M.E., Pukkila, P.J., 1986: Inheritance of DNA methylation in Coprinus cinereus.
Molecular Cell Biology, 6,195-200.
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16
Fenhexamid Resistance in Botrytis
pseudocinerea: Target Modifications and
Fungicide Detoxification
Abstract
The hydroxyanilide fenhexamid is a sterol biosynthesis inhibitor blocking the 3-keto reductase
(Erg27) involved in the C4 demethylation process. Among field isolates causing grey mould and
exhibiting reduced sensitivity to fenhexamid some of them were detected before the introduction
of this botryticide. In fact they were recently identified as belonging to a new species in the
Botrytis spp. complex, named Botrytis pseudocinerea. This species is naturally resistant to
fenhexamid mainly at the mycelial stage. Twelve simultaneous amino acid substitutions in Erg27
from B. pseudocinerea compared to that of Botrytis cinerea seem to have a weak effect on
fenhexamid susceptibility. A second mechanism related to detoxification of this hydroxyanilide
seems to be more significant. A strong synergism was found between the fenhexamid and sterol
14α-demethylation inhibitors (DMIs), inhibiting the Cyp51 protein. Sixty Cyp51 orthologues
were identified from the B. cinerea genome. The gene with the highest similarity to Cyp51,
named Cyp67, was deleted in B. pseudocinerea. Cyp67 knock out mutants exhibit an increase in
fenhexamid sensitivity, showing that Cyp67 encoding a cytochrome P450 is responsible, at least
partially for the B. pseudocinerea’s natural resistance to fenhexamid. Differences in Cyp67
protein composition or its regulation between B. cinerea and B. pseudocinerea may account for
difference in the fenhexamid detoxification and their respective fenhexamid susceptibilities.
Introduction
Fenhexamid is a sterol biosynthesis inhibitor (SBI) used against grey mould. This disease
was recently found to be caused by a complex of two related fungal species living in
sympatry: Botrytis group II (= Botrytis cinerea sensus stricto) and Botrytis group I (=
Botrytis pseudocinerea; Fournier et al., 2005). The target of fenhexamid is the sterol 3-
keto reductase (encoded by the erg27 gene) involved in the C4 demethylation process in
ergosterol biosynthesis (Debieu et al., 2001). Field isolates exhibiting in vitro reduced
susceptibility to fenhexamid can be classified into two main categories. In those
belonging to B. cinerea, acquired resistance with high to moderate levels is determined
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Botrytis transformation
Transformations of B05.10 and B900 strains were carried out according to Levis et al.
(1997) with 5 - 10 μg of DNA. Transformed protoplasts were plated on selective medium
with 50 μg ml-1 of hygromycin B (Sigma-Aldrich) and cultivated at 20°C under
continuous white light until conidiation.
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Results
Table 1: Sensitivity (EC50) to fenhexamid and edifenphos of B. cinerea sensitive (B05.10) and B.
pseudocinerea wild type strains compared to B. cinerea erg27 replacement and B. pseudocinerea Cyp67
deletion mutants.
EC50a
Fungicides
B. cinerea B05.10:Erg27B.pseudocinerea B. pseudocinerea ∆cyp67
Fenhexamid
0.05 0.1 0.1 -b
(Germ Tube)
Fenhexamid
(mycelium) 0.015 0.1 > 10 0.25
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Table 2: Joint action of five different compounds towards fenhexamid in wild type strains of Botrytis
cinerea and Botrytis pseudocinerea (A: Antagonistic; AD: Additive; I: Independent; S: Synergistic effect).
Botrytis
Synergistsa Target activity Botrytis cinerea
pseudocinerea
Glutathione S-
DEM A A
transferases
DEF Esterases A A
PBO Cytochrome P450 A A
Prochloraz Cyp51 AD/I S
Tebuconazole Cyp51 AD/I S
a
DEM (Diethyle maleate); DEF (S,S,S-tributyl phosphorotrithioate); PBO (Piperonyl butoxyde)
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Figure 1: Expression pattern of cyp67 after 1 hour of a 10 mg l-1 fenhexamid treatment in Botrytis cinerea
and Botrytis pseudocinerea. The tubA gene serves as reference.
Figure 2: Joint action between fenhexamid (vertical) and prochloraz or tebuconazole (horizontal strip) on
a Botrytis pseudocinerea strain and a ∆Cyp67 mutant.
Recent studies indicated that grey mould is caused by two sympatric species: B. cinerea
sensus stricto (syn Botrytis group II) and B. pseudocinerea (syn Botrytis group I or
HydR1) (Fournier et al., 2005). In all the B. pseudocinerea strains showing a reduced
susceptibility to fenhexamid, altered target site and increased detoxification can be
observed (Leroux et al., 2002; Albertini and Leroux, 2004).
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References
Albertini, A., Leroux, P. (2004): A Botrytis cinerea putative 3-keto reductase gene (ERG27) that
is homologous to the mammalian 17 beta-hydroxysteroid dehydrogenase type 7 gene.
European Journal of Plant Pathology, 110, 723-733.
Debieu, D., Bach J., Hugon, M., Malosse, C., Leroux. P. (2001): The hydroxyanilide fenhexamid,
a new sterol biosynthesis inhibitor fungicide efficient against the plant pathogenic fungus
Botryotinia fuckeliana (Botrytis cinerea). Pest Management Science, 57, 1060-1067.
Dellaporta, S. L., Wood, J., Hicks, J. B. (1983): A plant DNA minipreparation: version2. Plant
Molecular Biology Rep, 1, 19-21.
Fillinger, S., Leroux, P., Auclair, C., Barreau, C., Al Hajj, C., Debieu, D. (2008): Genetic
analysis of fenhexamid-resistant field isolates of the phytopathogenic fungus Botrytis
cinerea. Antimicrobial Agents and Chemotherapy, 52(11), 3933-40.
Fournier, E., Giraud, T., Albertini, C., Brygoo. Y. (2005): Partition of the Botrytis cinerea
complex in France using multiple gene genealogies. Mycologia, 97, 1251-1267.
Katagiri, M., Uesugi, Y. (1997): Similarities between the fungicidal action of isoprothiolane and
organophosphorous thiolate fungicides. Phytopathology, 67, 1415-1417.
Leroux, P., Debieu, D., Albertini, C., Arnold, A., Bach, J., Chapeland, F., Fournier, E., Fritz, R.,
Gredt, M., Giraud, T., Hugon, M., Lanen, C., Malosse, C., Thebaud. G. (2002): The
Hydroxyanilide Botryticide Fenhexamid/ Mode of Action and Mechanism of Resistance.
In: H.W. Dehne, U. Gisi, K.H.Kuck, P.E. Russell and H. Lyr (Eds.) Modern Fungicides
and Antifungal Compounds III, AgroConcept GmbH, Bonn, Germany 29-40.
Levis, C., Fortini, D., Brygoo, Y. (1997): Transformation of Botrytis cinerea with the nitrate
reductase gene (niaD) shows a high frequency of homologous recombination, Current
Genetics, 32, 157-162.
Suty, A., Pontzen R., Stenzel, K. (1999): Fenhexamid - sensitivity of Botrytis cinerea:
determination of baseline sensitivity and assessment of the resistance risk. Pflanzenschutz-
Nachrichten Bayer, 52, 149-161.
Werck-Reichhart, D., Feyereisen, R. (2000): Cytochromes P450: a success story. Genome
Biology, 1, 3003.1-3003.9.
Yu, J.H., Hamari, Z., Han K.H., Seo J.A., Reyes-Dominguez, Y., Scazzocchio, C. (2004):
Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous
fungi. Fungal Genetics and Biology, 41, 973-981.
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17
Fitness Measurements of Fenhexamid Resistant
Strains in Botrytis cinerea
Abstract
Fenhexamid (hydroxyanilide) is a sterol biosynthesis inhibitor blocking the sterol 3-keto
reductase (Erg27) involved in the C4 demethylation process. In Botrytis cinerea several
fenhexamid resistant phenotypes have been characterized. Field isolates exhibiting the highest
resistance levels (HydR3+) show target changes (F412 S, I or V). We have generated artificial
fenhexamid resistant mutants by site-directed mutagenesis, via homologous recombination.
These isogenic strains were used to quantify under controlled conditions the impact of the allelic
mutations on the fitness of the fungus. Classical parameters (sporulation capacity, radial growth,
sclerotia production, freezing resistance and pathogenic aggressiveness) were quantified in
laboratory conditions. Significant differences were observed on some characters between mutant
and parental strains. In particular, reduced growth, variations in sclerotia production according to
the temperature tested in isogenic mutants and a susceptibility to freezing, underline a potential
impact of F412 mutations in reducing the survival ability of B. cinerea under field conditions.
Introduction
Fenhexamid target is the sterol 3-keto reductase, an enzyme (encoded by the Erg27 gene)
involved in the biosynthesis of ergosterol (Debieu et al., 2001). This fungicide is mainly
used in grapevine to control grey mould caused by Botrytis cinerea. The monitoring
conducted in French vineyards allowed the identification of strains highly resistant in
vitro to fenhexamid (HydR3+). This was due to replacement of a phenylalanine by
isoleucine, valine or serine at position 412 in the Erg27 protein (Fillinger et al., 2008).
The first HydR3+ strains were detected in 2003, their average frequency reached approx.
10 % in 2009 and they are overall distributed in French vineyards. In some regions,
notably in the Loire valley (France), where fenhexamid is annually used, a wide inter-
annual variation of HydR3+ frequencies has been observed (Lachaise et al. unpublished
data). This fact and the relatively slow evolution of HydR3+ strains in French vineyards
suggest that the F412 mutations are causing a reduced fitness especially during winter
time. In this paper we describe comparisons of in vitro fitness parameters of isogenic
HydR3+ strains obtained through reverse genetics in comparison to the parental sensitive
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A. Billard et al.
strain. Particular attention has been directed to temperature and nutritional factors when
measuring fitness parameters under laboratory conditions.
Freezing assays
50 mycelial plugs from each condition were frozen at -20°C during 15, 30 or 40 days in
2.5 % of glycerol solution. After thawing, each plug was inoculated on MY medium.
Survival and mycelial growth were quantified after two days.
Pathogenicity assays
The infection of bean (Phaseolus vulgaris) by B. cinerea was made by inoculating
detached leaves with non sporulating mycelial plugs as described previously by Pinedo et
al., (2008).
Results
This study aimed to analyze the impact of the point mutations characteristic for the
HydR3+ fenhexamid resistance phenotype on the fitness of B. cinerea. Artificial HydR3+
resistant mutants (Erg27F412 mutants) were generated in order to avoid the effects of
different genetic backgrounds encountered in field isolates.
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Figure 1: Mycelial growth of erg27F412 mutants and the wild type strain B05.10ku70 at 11°C, 20°C
(minimal medium) and 26°C (rich medium).
Figure 2: Sclerotia production of Erg27F412 mutants compared to wild type strain B05.10ku70 on
minimal medium at three different temperatures.
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Figure 3: Radial growth at 2 days after thawing for Erg27F412 mutants compared to the wild type strain
B05.10ku70 on MY media compared to non-frozen mycelial plugs (control).
Fitness studies are usually carried out by comparing natural isolates and chemically
mutated strains. To reduce the impact of individual genetic backgrounds, large sample
sizes and a sophisticated statistical analysis are needed in this case.
The generation of artificial resistant mutants by reverse genetics allows to quantify
more exactly the real impact of causal mutations on fitness. Erg27F412 isogenic mutants
showed similar in vitro sporulation and similar pathogenic aggressiveness on detached
bean leaves. On the other hand, they showed reduced growth and sclerotia production on
limited nutrient resources and at low temperatures (Figures 1, 2, 3). These results indicate
that the Erg27F412 mutations generate some fitness costs. A fitness cost had been found
on chemical mutants of B. cinerea resistant to fenhexamid by Ziogas et al. (2003). The
disadvantage of the Erg27F412 mutants mainly observed under restricted conditions (i.e.
poor medium, low temperature) suggest that with HydR3+ isolates winter survival
decreases. These findings provide insight into the present evolution of HydR3+ resistant
strains and suggest a moderate impact on the fenhexamid efficacy in field practice to
control grey mould disease.
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References
Choquer, M., Robin, G., Le Pêcheur, P., Giraud, C., Levis, C., Viaud, M. (2008): Ku70 or Ku80
deficiencies in the fungus Botrytis cinerea facilitate targeting of genes that are hard to
knock out in a wild-type context. FEMS Microbiology Letters, 289 (2), 225-32.
Billard, A., Fillinger, S., Leroux, P., Solignac, P., Lachaise, H., Beffa, R., Debieu D. (2011) :
Fenhexamid Resistance in Botrytis pseudocinerea : Target Modifications and Fungicide
Detoxification. In: Modern Fungicides and Antifungal Compounds, Vol. VI, Eds: Dehne,
H.W., Deising, H.B., Gisi, U., Kuck, K.H., Russell, P.E., Lyr, H., DPG, Braunschweig,
Germany, 110-116.
Debieu, D., Bach J., Hugon, M., Malosse, C., Leroux. P. (2001): The hydroxyanilide fenhexamid,
a new sterol biosynthesis inhibitor fungicide efficient against the plant pathogenic fungus
Botryotinia fuckeliana (Botrytis cinerea). Pest Management Science, 57, 1060-1067.
Fillinger, S., Leroux, P., Auclair, C., Barreau, C., Al Hajj, C., Debieu, D. (2008): Genetic
analysis of fenhexamid-resistant field isolates of the phytopathogenic fungus Botrytis
cinerea. Antimicrobial Agents and Chemotherapy, 52 (11), 3933-40.
Pinedo, C., Wang, C.M., Pradier, J.M., Dalmais, B., Choquer, M., Le Pêcheur, P., Morgant, G.,
Collado, I.G., Cane, D.E., Viaud, M. (2008): Sesquiterpene synthase from the botrydial
biosynthetic gene cluster of the phytopathogen Botrytis cinerea. ACS Chemical Biology, 19
(12), 791-801.
Ziogas, B.N., Markoglou A.N., Malandrakis A.A. (2003): Studies on the inherent resistance risk
to fenhexamid in Botrytis cinerea. European Journal of Plant Pathology, 109, 311–317.
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18
Functional Characterisation of Mycosphaerella
graminicola Sterol 14α-Demethylase Variants
Resistant to Azole Fungicides
H.J. COOLS1, J.E. PARKER2, D.E. KELLY2, J.A. LUCAS1, S.L. KELLY2 and
B.A. FRAAIJE1
1
Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden,
Hertfordshire, AL5 2JQ, United Kingdom.
2
Institute of Life Science and School of Medicine, Swansea University, Swansea, Wales SA2 8PP,
United Kingdom
Abstract
In Western Europe control of Mycosphaerella graminicola, the ascomycete fungus causing
Septoria Leaf Blotch of winter wheat is currently dependent on the programmed application of
azole fungicides. The reliance on azoles, and the consequent selection pressures imposed by their
widespread use, has led to the emergence of resistance to some azoles and a shift in sensitivity to
others. The mechanism predominantly associated with this change in sensitivity is mutation of
the gene (MgCYP51), resulting in amino acid alterations in the target enzyme, sterol 14α-
demethylase. Analogous to the development of azole resistance in other fungi, for example the
opportunistic human pathogen Candida albicans, MgCYP51 alterations in M. graminicola are
most often found in combination, with isolates most resistant to azoles carrying multiple amino
acid substitutions compared to the wild type. To study the impact of both individual and
combinations of MgCYP51 alterations on azole sensitivity, we have introduced mutations by site
directed mutagenesis and expressed mutated MgCYP51 proteins in a Saccharomyces cerevisiae
strain carrying a regulatable promoter controlling native CYP51 expression. We have shown the
wild type MgCYP51 gene complements the function of the orthologous gene in S. cerevisiae and
that introduction of some mutations, for example those encoding amino acid alterations between
Y459-Y461, substantially reduce azole fungicide sensitivity. Some substitutions, including I381V,
destroy MgCYP51 function in the S. cerevisiae mutant when introduced alone. However, this can
be partially rescued by combining I381V with alterations between Y459-Y461. Therefore, these
studies provide functional evidence underlying the sequence in which MgCYP51 alterations in
the Western European M. graminicola population emerged.
Introduction
Site-directed mutagenesis
Expression of “wild type” MgCYP51 in S. cerevisiae strain YUG37:erg11 in yeast
expression vector pYES2/CT (pYES2-Mg51wt), complements the function of the S.
cerevisiae CYP51 (Cools et al., 2010). Individual and combinations of mutations
identified in M. graminicola isolates were introduced into pYES2-Mg51wt using the
QuikChange II Site-Directed Mutagenesis Kit (Stratagene) according to the
manufacturer’s instructions using 100 ng of target (pYES-Mg51wt) plasmid and 5 ng of
each primer.
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MgCYP51 Variants
GAL + RAFF agar plates with or without 3 µg ml-1 doxycycline which suppresses native
CYP51 expression. Plates were photographed after 96 hr incubation at 30 ºC
Results
Figure 1: Complementation of S. cerevisiae strain YUG37:erg11 with wild-type (Mg51wt) and mutated
variants of MgCYP51. Growth in the absence (-DOX) and presence (+ DOX) of doxycycline which
suppresses native CYP51 expression shown.
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MgCYP51 Variants
does not support this suggestion. The prevalence of these alterations in modern M.
graminicola populations may simply be a consequence of contingent evolution, as amino
acid substitutions conferring decreased azole sensitivity occurred, and were selected, in
combination with L50S, S188N and/or N513K.
Figure 2: Sequence alignment of members of the CYP51 family from different phyla. Abbreviations are
Mycosphaerella graminicola CYP51 (MgrCYP51F1), Candida albicans CYP51 (CaCYP51F1), Homo
sapiens CYP51 (HsCYP51A1), Sorghum bicolor CYP51 (SbCYP51G1), Mycobacterium tuberculosis
CYP51 (MtCYP51B1) and Trypanosoma brucei (TbCYP51E1). Filled triangles indicate alterations that
have no impact on MgCYP51 function on azole sensitivity when expressed in S. cerevisiae. Empty
triangles indicate alterations that decrease azole sensitivity without affecting MgCYP51 function in yeast.
Grey filled triangles indicate alterations that destroy MgCYP51 function in yeast. Filled diamonds indicate
uncharacterised alterations.
Discussion
Until recently, evidence implicating MgCYP51 mutation in the recent decline in the
effectiveness of azole fungicides in controlling M. graminicola has been correlative
(Fraaije et al., 2007; Leroux et al., 2007). Here we describe the functional
characterisation of both individual and combinations of MgCYP51 changes by
heterologous expression in S. cerevisiae. Using this experimental system we have shown
the substantial reduction in azole sensitivity conferred by changes between Y459-Y461,
thereby providing evidence for the rapid selection of these changes in M. graminicola
populations in the late 1990s. We have shown that some MgCYP51 substitutions, such as
V136A, Y137F, and I381V, prevalent in recent M. graminicola populations, prevent
protein function in yeast, and that the lethality of these alterations can be partially
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Acknowledgements
This work was supported by the Biotechnology and Biological Sciences Research
Council (BBSRC) of the UK, project numbers BB/E02257X1 and BB/E0218321.
Rothamsted Research receives grant-aided support from the BBSRC.
References
Clark, W.S. (2006): Septoria tritici and azole performance. In: Fungicide resistance: are we
winning the battle but losing the war? Aspects Appl Biol., 78, 127-132.
Cools, H.J., Fraaije, B.A. (2008): Spotlight: Are azole fungicides losing ground against Septoria
wheat disease? Resistance mechanisms in Mycosphaerella graminicola. Pest Manag Sci.,
64, 681-684.
Cools, H.J., Parker, J.E., Kelly, D.E., Lucas, J.A., Fraaije, B.A., S.L. Kelly: (2010), Heterologous
expression of mutated eburicol 14α-demethylase (CYP51) proteins of Mycosphaerella
graminicola to assess effects on azole fungicide sensitivity and intrinsic protein function.
Appl. Environ. Microbiol., 76, 2866-2872.
Fraaije, B.A., Cools, H.J., Fountaine, J., Lovell, D.J., Motteram, J., West, J.S., Lucas, J. A.
(2005): The role of ascospores in further spread of QoI-resistant cytochrome b alleles
(G143A) in field populations of Mycosphaerella graminicola. Phytopathol., 95, 933-941.
Fraaije, B.A., Cools, H.J., Kim, S-H., Motteram, J., Clark, W.S., Lucas, J. A. (2007): A novel
substitution I381V in the sterol 14-demethylase (CYP51) of Mycosphaerella graminicola
is differentially selected by azole fungicides. Mol Plant Pathol., 8, 245-254.
Lepesheva, G.I., Waterman M.R. (2007): Sterol 14α-demethylase cytochrome P450 (CYP51), a
P450 in all biological kingdoms, Biochim. et Biophys. Acta, 1770, 467-477.
Leroux, P., Albertini, C., Gautier, A., Gredt, M., Walker, A.S. (2007): Mutations in the cyp51
gene correlated with changes in sensitivity to sterol 14α-demethylation inhibitors in field
isolates of Mycosphaerella graminicola. Pest Manag Sci., 63, 688-699.
Revankar, S.G., Fu, J., Rinaldi, M.G., Kelly, S.L., Kelly, D.E., Lamb, D.C., Keller, S.M., Wickes,
B.L. (2004): Cloning and characterization of the lanosterol 14α- demethylase (ERG11)
gene in Cryptococcus neoformans. Biochem Biophys Res Commun., 324, 719-728.
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19
Molecular Mechanisms of Altered Triazole
Sensitivity in Rhynchosporium secalis
Abstract
Barley leaf blotch or scald, caused by the fungus Rhynchosporium secalis, is a highly
economically damaging foliar disease of barley, causing annual yield losses estimated at £4.8
million in 2005 in the UK. Fungicides are a major component of control programmes for this
disease, with triazoles plus a mixing partner such as a QoI fungicide widely recommended.
However, a reduction in sensitivity to some triazole fungicides has been found in the field. This
study aims to identify genetic changes responsible for reduced fungicide sensitivity in R. secalis,
and to investigate their occurrence and spread in populations.
Triazole sensitivity tests have revealed a 100-fold reduction in in vitro sensitivity to some
triazoles over the last 10-15 years, but the resistance mechanism is not yet known. R. secalis has
two copies of the gene, CYP51, encoding the triazole target site. These genes have been cloned
and sequenced from a range of isolates with different triazole sensitivities. Some isolates carried
either a T67S substitution in CYP51B or an A111V or P170S substitution in CYP51A. However,
none of these mutations was correlated with triazole sensitivity differences, suggesting that other
mechanisms are responsible.
Introduction
Isolates used
Isolates studied are listed in Table 1. Isolates were stored as spores in silica gel at -80°C.
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measured (averaged over 12 points per well) and EC50 values calculated from a 4-
parameter fit dose-response curve.
K1124 1993 UK
FI12-63 1996 Finland
788 1997 France
SAC 1-4-8 2000 UK (Scotland) Scottish Agricultural College
QUB 12-3 2001 UK (Northern Ireland) ARINI
QUB 30-10 2001 UK (Northern Ireland) ARINI
R 9528.4 2001 UK (Northern Ireland) ARINI
R 9522.3 2001 UK (Northern Ireland) ARINI
GKII 18-2-3 2002 UK (England) Rothamsted Research
GKII 18-3-2 2002 UK (England) Rothamsted Research
SAC 09/943/14 2007 UK (Scotland) Scottish Agricultural College
RS 219 2004 UK Syngenta
RS 783 2004 UK Syngenta
Propiconazole 0 0.00508 0.0152 0.0457 0.137 0.412 1.235 3.70 11.1 33.3 100 300
Tebuconazole 0 0.00508 0.0152 0.0457 0.137 0.412 1.235 3.70 11.1 33.3 100 300
Epoxiconazole 0 0.00524 0.0131 0.0327 0.0819 0.205 0.512 1.28 3.2 8 20 50
Prothioconazole 0 0.00169 0.00508 0.0152 0.0457 0.137 0.412 1.235 3.70 11.1 33.3 100
DNA analysis
Isolates were grown in Sabouraud liquid medium at 18°C for ten days, filtered and
freeze-dried. DNA extractions were carried out as described in Fraaije et al., (1999), with
the following modifications: the DNA extraction buffer was amended with 0.1 M 1,10-
phenanthroline monohydrate and 2% polyvinylpyrrolidone (molecular weight 40000);
and samples were homogenised at room temperature, with the extraction buffer added,
with a ball bearing using a FastPrep instrument.
PCR reactions were carried out using Phusion High-Fidelity DNA Polymerase
(Finnzymes Oy, Finland) according to manufacturer’s instructions, in 30µl reactions with
HF buffer, 0.5mM primers and 1.67 µg ml-1 template. The PCR programme was as
follows: 2 min at 95°C; followed by 40 cycles of 10 s at 95°C, 20 seconds at 60°C and 50
s at 72°C; followed by 4 min 10 s at 72°C. CYP51B was amplified with primer pair
Cyp51B 1, CYP51A was amplified with primer pair Cyp51A 1, and each gene was
sequenced with the corresponding nested and internal primers (Table 3).
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Table 3: Primers used for amplification and sequencing of R. secalis CYP51 genes.
Results
EC50 values for the four triazole fungicides tested, and mutations in the two CYP51 genes,
are shown in Table 4. For isolates RS 219, K1124 and FI12-63, the CYP51A gene could
not be amplified.
Table 4: Triazole EC50 values and CYP51 alterations for the R. secalis isolates studied.
R 9528.4 0.037 (0.01) -0.361 (0.01) -1.433 (0.08) -0.164 (0.01) WT T67S
QUB 30-10 0.057 (0.01) -0.556 (0.09) -0.893 (0.02) 0.033 (0.01) WT T67S
R 9522.3 0.117 (0.00) 0.013 (0.06) -1.287 (0.05) -0.268 (0.01) P170S WT
GKII 18-3-2 0.629 (0.01) 1.143 (0.03) -0.747 (0.00) -1.280 (0.03) WT WT
GKII 18-2-3 0.706 (0.01) 1.137 (0.05) -0.580 (0.02) -0.347 (0.05) WT WT
SAC 1-4-8 0.712 (0.02) 1.111 (0.10) -0.380 (0.04) 0.111 (0.06) WT WT
788 0.725 (0.02) 0.979 (0.06) -0.284 (0.04) 0.104 (0.06) WT WT
SAC 1.228 (0.00) 1.705 (0.13) -0.243 (0.13) 0.041 (0.08) A111V WT
09/943/14
QUB 12-3 1.462 (0.06) 0.866 (0.05) 0.198 (0.15) 0.874 (0.03) A111V WT
RS 783 >2.0 >1.70 1.305 (0.42) 0.801 (0.30) WT WT
a
Numbers in brackets indicate standard error of Log10 EC50
b
WT indicates Wild-Type, whereas None indicates that the gene could not be amplified from that isolate
Discussion
Triazole sensitivity profiles of the R. secalis isolates against the four fungicides tested
fall into three main groups. The first group, comprising isolates K1124, FI12-63 and RS
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219, have the lowest EC50 values for all four triazoles tested, and can be considered as
sensitive. The second group, containing isolates QUB 30-10, R 9528.4 and R 9522.3,
show some shifts in sensitivity levels, with an approximate tenfold increase in EC50
values to propiconazole and tebuconazole, but smaller shifts in epoxiconazole and
prothioconazole sensitivity. Most isolates in this group were collected in Northern
Ireland in 2001. Kendall et al. (1993) reported an eightfold shift in mean propiconazole
sensitivity in field trials between 1988 and 1990, with some cross-resistance, but greater
sensitivity, to tebuconazole. A similar shift in propiconazole sensitivity is apparent in
these isolates, but with a greater shift in sensitivity to tebuconazole compared to the
Kendall study.
The remaining isolates are further reduced in triazole sensitivity. Propiconazole and
tebuconazole EC50 values are around 100-fold higher than those of the sensitive reference
isolates. Prothioconazole and epoxiconazole EC50 values are also increased relative to the
sensitive reference isolates, but this is generally a less than tenfold increase. This shift in
epoxiconazole sensitivity is consistent with shifts observed by Cooke et al. (2004),
although actual sensitivity values are not comparable due to their use of MIC rather than
EC50 values. The smaller sensitivity shifts for epoxiconazole and prothioconazole are
also consistent with HGCA monitoring showing that these compounds remain effective
in the field (Oxley and Burnett, 2010).
Sequencing of CYP51B revealed one mutation, encoding the substitution T67S, in
sensitive isolate FI12-63 and two of three isolates with intermediate sensitivities: QUB
30-10 and R 9528.4. Alterations at this residue, corresponding to amino acid position 63
in C. albicans and 68 in M. graminicola, have not been previously reported, and the
presence of this substitution does not correlate with differences in sensitivity, suggesting
that it does not affect fungicide binding.
Sequencing of CYP51A revealed two point mutations, encoding the substitution
A111V in isolates SAC 09/943/14 and QUB 12-3, and the substitution P170S in isolate
R9522.3. Residue 111 corresponds to amino acid position 117 in C. albicans and 122 in
M. graminicola, at which no alterations have been previously reported. Residue 170 lies
in a 16 amino acid region only found in R. secalis CYP51A, so has no equivalent in other
species. Isolates SAC 09/943/14 and QUB 12-3 did not show clear sensitivity differences
compared to other less sensitive isolates, nor isolate R 9522.3 from other intermediate
isolates, suggesting that these alterations do not affect fungicide sensitivity, and the
observed sensitivity differences in R. secalis are not due to point mutations in CYP51A.
However, in the three sensitive isolates, a functional CYP51A gene could not be
amplified. The role of the presence of a functional CYP51A and expression levels of
CYP51A and CYP51B genes in triazole sensitivity are currently being investigated further.
Acknowledgements
Work carried out by N.J.H. was part of a CASE postgraduate studentship funded by the
Biotechnology and Biological Sciences Research Council of the UK and Syngenta Crop
Protection.
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References
Blake, J., Paveley, N., Fitt, B., Oxley, S., Bingham, I., Cockerell, V. (2010): The barley disease
management guide 2010. Eds. Edwards, C. and Dodgson, G. Home Grown Cereals
Authority, London, UK.
Caldwell, R. (1937): Rhynchosporium scald of Barley, Rye and Other Grasses. Journal of
Agricultural Research, 55 (3), 175-198.
Cooke, L.R., Locke, T, Lockley, K.D., Phillips, A.N., Sadiq, M.D.S., Coll, R., Black, L., Taggart,
P., Mercer, P.C. (2004): The effect of fungicide programmes based on epoxiconazole on
the control and DMI sensitivity of Rhynchosporium secalis in winter barley. Crop
Protection, 23 (5), 393-406.
Cools, H.J., Fraaije, B.A. (2008): Are azole fungicides losing ground against Septoria wheat
disease? Resistance mecchansims in Mycosphaerella graminicola. Pest Management
Science, 64, 681-684.
Edlind, T.D. (2008): Emergence and Evolution of Antifungal Resistance. Evolutionary Biology of
Bacterial and Fungal Pathogens, 297-306. Eds. Baquero, F., Nombela, C., Cassell, G. and
Gutièrrez-Fuentes, J.A. ASM Press, Washington DC, USA.
Fraaije, B.A., Lovell, D, Rohel, E., Hollomon, D.W. (1999): Rapid detection and diagnosis of
Septoria tritici epidemics in wheat using a polymerase chain reaction/PicoGreen assay.
Journal of Applied Microbiology, 86, 701-708.
Fraaije, B.A., Cools, H.J., Kim, S.H., Motteram, J., Clark, W.S., Lucas, J.A. (2007): A novel
substitution I381V in the sterol 14 alpha-demethylase (CYP51) of Mycosphaerella
graminicola is differentially selected by azole fungicides. Molecular Plant Pathology, 8
(3), 245-254.
Hollomon, D.W. (1984): A laboratory assay to determine the sensitivity of Rhynchosporium
secalis to the fungicide triadimenol. Plant Pathology, 33, 65-70.
Jones, D.R. (1990): Sensitivity of Rhynchosporium secalis to DMI fungicides. Brighton Crop
Protection Conference - Pests and Diseases, 1990: Proceedings, Volume 3, 1135-1140.
British Crop Protection Council, Farnham, U.K.
Kendall, S.J., Hollomon, D.W. (1990): DMI resistance and sterol 14-alpha-demethylation in
Rhynchosporium secalis. Brighton Crop Protection Conference - Pests and Diseases,
1990 : Proceedings, Volume 3, 1129-1134. British Crop Protection Council, Farnham, U.K.
Kendall, S. J., Hollomon, D.W., Cooke, L.R., Jones, D.R. (1993): Changes in sensitivity to DMI
fungicides in Rhynchosporium secalis. Crop Protection, 12 (5), 357-362.
Miyazaki, T., Miyazaki, Y., Izumikawa, K., Kakeya, H., Miyakoshi, S., Bennett, J. E., Kohno, S.
(2006): Fluconazole treatment is effective against a Candida albicans erg3/erg3 mutant in
vivo despite in vitro resistance. Antimicrobial Agents and Chemotherapy, 50 (2), 580-586.
Oxley, S.P., Burnett, F.J. (2010): Barley Disease Control. SAC Technical Notes TN627. The
Scottish Agricultural College, Edinburgh, U.K.
Robbertse, B., van der Rijst, M., van Aarde, I.M.R., Lennox, C., Crous, P. (2001): DMI
sensitivity and cross-resistance patterns of Rhynchosporium secalis isolates from South
Africa. Crop Protection, 20, 97-102.
Sanglard, D., Kuchler, K., Ischer, F., Pagani, J.L., Monod, M., Bille, J. (1995): Mechanisms of
resistance to azole antifungal agents in Candida albicans isolates from aids patients
involve specific multidrug transporters. Antimicrobial Agents and Chemotherapy, 39 (11),
2378-2386.
Schnabel, G., Jones, A.L. (2001): The 14 alpha-demethylase (CYP51A1) gene is overexpressed in
Venturia inaequalis strains resistant to myclobutanil. Phytopathology, 91 (1), 102-110.
Wyand, R.A., Brown, J.K.M. (2005): Sequence variation in the CYP51 gene of Blumeria
graminis associated with resistance to sterol demethylase inhibiting fungicides. Fungal
Genetics and Biology, 42 (8), 726-735.
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20
Exploring the Molecular Basis of Azole
Resistance in Powdery Mildew Fungi
Abstract
Powdery mildew is a serious disease responsible for significant crop losses in both cereals and
cucurbits throughout major areas of cultivation. Sterol demethylation inhibitors (DMIs) are
widely-used fungicides for broad-spectrum control of diseases caused by fungal pathogens of
agricultural and clinical importance, targeting the sterol C14α-demethylase (CYP51) enzyme
which catalyses demethylation of eburicol. Several mechanisms of DMI resistance operate in
plant pathogens, including mutation of the CYP51 protein and overexpression of the CYP51 gene.
In Erysiphe necator (grapevine powdery mildew) and Blumeria graminis f.spp. hordei and tritici
(barley and wheat powdery mildew), mutations in the open reading frame of the CYP51 gene
have been associated with decreased sensitivity to triazoles. Homology modelling of the B.
graminis CYP51 protein showed that, nonetheless, all the mutations were clustered in the channel
that gives access to the catalytic site of the enzyme, indicating a potential role in DMI resistance.
To assess the role of CYP51 gene expression in DMI resistance, 26 isolates of Podosphaera
fusca, differing in their responses to DMI fungicides fenarimol, myclobutanil and triadimenol,
were analysed by a quantitative PCR assay. CYP51 expression in DMI-resistant isolates was
found not to be significantly different from sensitive ones in the absence of a DMI fungicide.
Introduction
most common and widespread diseases affecting barley and cucurbits crops and
responsible for significant losses each year worldwide (Backes et al., 2003; Pérez-García
et al., 2009). None of the current commercially available cultivars is fully resistant to the
disease so the programmed application of fungicides is an essential component of
integrated disease management. However, both pathogens have quickly adapted to
fungicides and resistance to several mode-of-action classes, including QoI and DMI, is
now a major threat to control of both Bgh and P. fusca (Fernández-Ortuño et al., 2006;
López-Ruiz et al., 2010).
A wide variety of mechanisms confer resistance to DMIs, including decreased
affinity of CYP51 for the fungicide substrate (Van den Bossche and Koymans, 1997),
defects in sterol 14-demethylation (Sanglard, 2002), and increased level of CYP51 gene
expression (Stergiopoulos et al., 2003; Luo and Schnabel, 2008). Point mutation in the
CYP51 gene is probably the most common mechanism of resistance to DMIs. A number
of mutations have been linked to this phenomenon in human pathogens and
phytopathogenic fungi. In the powdery mildew fungi of grapevine, barley and wheat, the
mutation Y136F has been associated with isolates resistant to DMI fungicides (Délye et
al., 1997; 1998; Wyand and Brown, 2005).
The recent spread of resistance to strobilurins (QoIs) in many crop pathogenic fungi
means that broad-spectrum disease control is once again substantially dependent upon
DMI fungicides. The study reported here aimed to gain an insight into resistance
mechanisms to DMI fungicides in powdery mildew fungi of cereals and cucurbits at the
molecular level. Knowledge of these mechanisms may be useful for identifying
molecular markers for DMI fungicide resistance and may help in designing anti-
resistance strategies.
Homology modelling
The 3D model of B. graminis CYP51 protein was based on a structure template of the
protein from Mycobacterium tuberculosis (Mt CYP51, 1e9x.pdb), using the FUGUE
module in the Swiss-PdbViewer 4.0.1 (Guex and Peitsch, 1997) and PyMOL 0.99rc6
(DeLano Scientific LLC, San Francisco, California, USA) programmes.
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β-tubulin gene from P. fusca were identified and partially sequenced (data not shown).
RNA from P. fusca isolates was subjected to DNase treatment according to the
manufacturer’s protocol (DNase I FPLCpureTM, Amersham, Piscataway, USA). Reverse
transcription reaction was performed in 20 µl volume using SuperScriptTM III Reverse
Transcriptase (Invitrogen, California, USA) and Anchored oligo(dT)20 primer (Invitrogen,
Carlsbad, USA) following the manufacturer’s recommendations. P. fusca cDNA was
subjected to qPCR in a DNA Engine Opticon 2 Continuous Fluorescence Detector
System (MJ Research, San Francisco, USA) by using the DNA stain SYBR Green
JumpStartTM Taq ReadyMixTM (Invitrogen, St. Louis, USA). Amplifications were
conducted in 20 μl volumes containing 10 μl SYBR Green, 1 μl reverse transcription
product, and 1 μl each of the forward and reverse primers at 10 pmol each. For the P.
fusca CYP51 gene the primers 147FqPCR (5’-CATGAGCCGCCTGTC GTGTT-3’) and
356RqPCR (5’-CTGAAGGATGTCAATGCCGA-3’) were used, giving an amplified
fragment of 209 bp. A 118 bp-fragment of the P. fusca β-tubulin gene amplified by
specific primers tub15F (5’- TTCCCTGATCGAATGATGGCAACC-3’) and tub14R (5’-
CGTCGGAGTTTTCGACCAACTGATG -3’) was included in each experiment as a
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reference to normalize the quantification of P. fusca CYP51 mRNA expression. The PCR
cycling conditions were as follows: 2 min of initial denaturation step at 94°C followed by
40 cycles each consisting of 94°C for 15 s, 67°C for 45 s, 72°C for 1 min, and a final
extension step at 72°C for 10 min. Data were analyzed by using the Opticon Monitor
analysis software version 2.02.24 (MJ Research, San Francisco, USA).
Amplification efficiencies of both cDNAs diluted over a 100-fold range were shown
to be equivalent (slope= 0.045), allowing use of the comparative CT method 2 CT (Livak
and Schmittgen, 2001). For each isolate, the CT value was determined by subtracting the
average β-tubulin CT from the average CYP51 CT value. Expression levels of both genes
in different cDNA samples was calibrated by subtracting the CT from the CT from SF8,
the isolate with the highest CT value and thus the lowest amount of CYP51 mRNA.
There were three replicates for each sample, and the experiment was performed three
times. Assays were repeated on independent occasions and with independently isolated
RNA.
Results
Homology modelling
Based on the structure template of 1e9x.pdb from M. tuberculosis CYP51, a three-
dimensional model of Bgh CYP51 was established. The overall conformation of the Bgh
CYP51 model was very similar to the template. A well-defined access channel enables
entry and exit of the substrate as well as the antifungal triazoles (Figure 1). To find out
whether or not these changes might affect affinity for triazole fungicides through
alteration of interaction points or repositioning of the tertiary structure, mutations were
placed on the Bgh CYP51 model. Thus, substitutions Y136F and K147Q were found to
be part of this access channel which could interfere with the entry or positioning of
triazole molecules.
Figure 1: Backbone representation of the three-dimensional model of Bgh CYP51 structure after merging
the heme group (shown as black sticks) to the active site. Mutations Y136F and K147Q are shown as solid
black spheres. Predicted access channel is depicted as semi-transparent dark grey spheres.
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2,5
1,5
0,5
0
Sm3
22717
SF48
SF45
SF26
SF222
2208
3161
72168
3168
SF56
98Sm32
SF213
31430
SF29
71178
31426
22812
SF8
SF60
22318
22317
21394
21385
21817
2086F
Figure 2: Relative expression of the P. fusca CYP51 gene. Strains of P. fusca sensitive to DMI (white),
resistant to fenarimol (grey) and resistant to both fenarimol and triadimenol (black) obtained from different
locations and years were used. Values shown are the means of three independent experiments, and the bars
show the standard deviation from the mean.
Discussion
Homology modelling
In the absence of crystal structures, homology modelling is a valuable tool for gaining
insight into the interaction between substrates and P450 enzymes (Zhao et al., 2007). The
postulation of a well-defined channel in B. graminis CYP51 suggests that both the
substrate and the azole molecule should enter through this channel and position below
the I helix and proximal to the haem site, competing for the substrate binding pocket.
Thus, changes in this access channel (i.e. changes in the amino acids in direct contact
with some part of the azole molecule or changes in the three-dimensional arrangement of
structures) would result not only in a reduction in affinity but also less efficient binding
of the azole to the modified protein (Van den Bossche and Koymans, 1997). Mutations
Y136F and K147Q are probably linked to a structural rearrangement due to their
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predicted positions at the opening of the access channel that could affect fungicide
molecule entry (Figure 1). The same was hypothesized for the Y132H substitution in
Candida albicans corresponding to Y136F in the phytopathogenic fungi Penicillium
digitatum, E. necator and Bgh. In C. albicans this mutation is situated in the B-B9 helix
cluster, a region that configures the gate of the access channel and is believed to play a
role in the entry of the substrate in the binding pocket (Sanglard et al., 1998).
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Livak, K.J., Schmittgen, T.D. (2001): Analysis of relative gene expression data using real-time
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López-Ruiz, F.J., Pérez-García, A., Fernández-Ortuñno, D., Romero, D., García, E., de Vicente,
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Luo, C.X., Schnabel, G. (2008): The cytochrome P450 lanosterol 14-demethylase gene is a
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Ma, Z., Proffer, T.J., Jacobs, J.L., Sundin, G.W. (2006): Overexpression of the 14-demethylase
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Pérez-García, A., Romero, A., Fernández-Ortuño, D., López-Ruiz, F.J., de Vicente, A., Torés,
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Sanglard, D., Ischer, F., Koymans, L., Bille, J. (1998): Amino acid substitutions in the
cytochrome P-450 lanosterol 14-demethylase (CYP51 A1) from azole-resistant Candida
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Schnabel, G., Jones, A.L. (2001): The 14-demethylase (CYP51A1) gene is overexpressed in
Venturia inaequalis strains resistant to myclobutanil. Phytopathology, 91, 102-110.
Stergiopoulos, I., Van Nistelrooy, J.G.M., Kema, G.H.J., de Waard, M.A. (2003): Multiple
mechanisms account for variation in base-line sensitivity to azole fungicides in field
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Van den Bossche, H., Koymans, L. (1997):, Cytochromes P450 in fungi. Mycoses, 41, 32-38.
Wyand, R.A., Brown, J.K.M. (2005): Sequence variation in the CYP51 gene of Blumeria
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Genetics and Biology, 42, 726-735.
Zhao, L., Liu, D., Zhang, Q., Zhan, S., Wan, J., Xiao, W. (2007): Expression and homology
modeling of sterol 14-demethylase from Penicillium digitatum. FEMS Microbiology
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21
A Group I Intron Located Downstream of the
G143 Position of the Cyt b Gene in Monilinia
fructicola is Present in Genetically Diverse
Populations from China
M.J. HU1, L.F. YIN1,2, Y. CHEN1, S.N. CHEN1, X.L. LIU3, F.G. CHEN3 and
C.X. LUO1,2*
1
Department of Plant Pathology, College of Plant Science and Technology, Huazhong
Agricultural University, Wuhan 430070, China
2
The Key Lab of Crop Disease Monitoring & Safety Control in Hubei Province, Huazhong
Agricultural University, Wuhan 430070, China
3
Department of Plant Pathology, College of Agriculture and Biotechnology, China Agricultural
University, Beijing 100193, China
*Corresponding author
Abstract
RAPD technique was applied to analyze the genetic diversity of Monilinia fructicola isolates
from Beijing (BJ), Shandong (SD), Hubei (HB), Zhejiang (ZJ), Fujian (FJ) and Yunnan (YN)
provinces in China. Diversity was low among isolates of geographical populations, but high
between populations. SD isolates were genetically closest to HB isolates; FJ isolates were
genetically most distant. PCR and sequence analysis revealed all isolates contained a 1166 bp in
length intron located just downstream of the G143 position of the cytochrome b (Cyt b) gene.
These results indicate that the 1166-bp intron is present in genetically diverse M. fructicola
populations from China and confirm our hypothesis that the G143A mutation conferring high
levels of QoI resistance may not develop in M. fructicola.
Introduction
M.J. HU et al.
al. 2002; Sierotzki et al. 2002). Other point mutations leading to amino acid changes
F129L and G137R conferred lower levels of resistance compared to G143A (Gisi et al.
2002; Kim et al. 2003; Sierotzki et al. 2007). The analysis of the Cyt b gene is critically
important to evaluate the inherent resistance risk and to demonstrate the resistance
mechanisms to QoI fungicides.
The Cyt b gene of M. fructicola was isolated previously and a 1166 bp group I intron
(1166-bp intron) was detected immediately downstream the G143 position in 6 isolates
from three states of the United States (Luo et al. 2010). It was hypothesized that the
intron cannot be properly spliced if position 143 changes from A to G, making the
G143A mutation unlikely to occur in M. fructicola from the US. It is not known if this
intron is a firm part of different M. fructicola genotypes outside the United States as well.
In the present study, 47 single spore M. fructicola isolates from different provinces in
China were genetically characterized using random amplified polymorphic DNA (RAPD)
and the presence of the 1166-bp intron was determined.
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phylogenetic tree was established by using the Tree plot program of the software package
NTSYS-pc 2.1.
Results
M 1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 20 21 22
23
1.6 kb
1.2 kb
0.4 kb
Figure 1: Example of electrophoresis patterns of RAPD profiles from 23 Chinese isolates with primer
S385. M = size marker, the largest to smallest bands are 5.0, 3.0, 2.0, 1.0, 0.75, 0.5, 0.35 and 0.1 kb in
length. Lanes 1-23 represent isolates YM09-1a, YM09-1c, SD-5a, SD-5b, MPA13, MSA9, MSB10,
MBJA8, MTA4, 0907-a, 0907-b, 0907-d, 0908-a, 0908-b, PeachMF-1, PeachMF-2, BM09-1c, BM09-3c,
BM09-6a, ZM09-1a, ZM09-2a, ZM09-3a, ZM09-4a, respectively.
amplified 75 bands, yielded in 25.33 % percent of polymorphic loci for all isolates
combined. Within populations, the percentage of polymorphic loci was lower and ranged
from 1.5 to 24.0%. The BJ population showed the highest (24.0%), whereas the SD and
YN populations showed the lowest (1.5 and 1.6%) polymorphism. The Nei’s gene
diversity (H) and Shannon's Information index (I) were 0.3709 and 0.5487 for all isolates
combined, ranging from 0.0263 to 0.2724 and 0.0365 to 0.4218, respectively, for
geographical populations. The Nei’s gene diversity and Shannon's Information indexes
revealed similar tendencies in that the BJ population had highest genetic diversity and
SD and YN populations had the lowest.
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M.J. HU et al.
Table 2: Unbiased measures of genetic identity (below diagonal) and genetic distance (above diagonal)
among different geographic groups in China.
Population YN SD BJ FJ HB ZJ
YN 0.2007 0.2808 0.2699 0.2161 0.3306
SD 0.8182 0.3408 0.4583 0.0280 0.2236
BJ 0.7551 0.7112 0.5428 0.2789 0.3205
FJ 0.7634 0.6324 0.5811 0.5364 0.5046
HB 0.8057 0.9724 0.7566 0.5849 0.2308
ZJ 0.7185 0.7997 0.7258 0.6037 0.7939
YN
SD
HB
ZJ
BJ
FJ
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Discussion
Azoxystrobin was the first QoI fungicide introduced into the Chinese fungicide market
for control of Colletotrichum capsici, Pseudoperonospora cubensis and Phytophthora
infestans. Since then, more QoI fungicides including several Chinese independent brands
such as enostrobilurin, SYP-1620, SYP-3375 and ZJ0721 etc. were registered for control
of vegetable, fruit, and row crop diseases. Some producers started to use QoI fungicides
in peach orchards in north-eastern and eastern areas of China. In our preliminary
investigation, all of the 23 tested isolates were still sensitive to the QoI fungicide
azoxystrobin with EC50 values below 1.0 µg/ml (Luo et al. 2010). Whether these isolates
had been exposed previously to QoI fungicides is unknown.
The RAPD technique has been one of the most commonly used molecular
techniques to analyze the genetic diversity and to establish phylogenetic trees in bacteria,
protozoa, fungi, plants and animals (Chalmers et al. 1992; Kambhampati et al. 1992;
Megnegneau et al. 1993; Tibayrenc et al. 1993; Borowsky et al. 1995; Martinez et al.
2003). RAPD analysis is relatively cheap and can efficiently yield a large number of
polymorphic markers in a short time. The technique does not require knowledge of
genomic DNA sequences, thus is valuable for fungi like M. fructicola the genome of
which has still not been sequenced. However, the method has been discussed
controversially due to reproducibility issues of RAPD profiles, especially for bands with
low intensity. In this study, only the RAPD bands with high intensity were used and all
others were ignored. Based on the Nei’s locus diversity and Shannon’s index data, the BJ
population had the higher genetic diversity, while the SD, YN and HB isolates had the
lowest genetic diversity. Altogether 26 BJ isolates from different hosts (peach and
nectarine) and locations (3 locations), but only 2 peach isolates from SD-, YN- and HB
provinces were used (we only have 2 isolates from each of these provinces). A higher
genetic diversity might have been observed if more isolates from different hosts and
locations had been studied. Although individual geographic populations did not show
high genetic diversity, high diversity was observed between all isolates studied with
Nei’s locus diversity and Shannon’s index as 0.3709 and 0.5487, respectively.
The SD population showed the closest relationship with HB and YN isolates. In
China, the SD province produces most of the fresh market peaches, and many are
shipped to other provinces, including YN and HB province. HB and YN isolates were
isolated from fruits obtained from local markets and it is possible that they originated
from SD province.
This study shows that the 1166 bp intron is present in genetically diverse M.
fructicola populations suggesting that an intron-less version of the fungus may not exist.
These results provide further evidence that the powerful G143A mutation may not
develop in M. fructicola. It is still unknown if the intron exists in its close relatives,
Monilinia fructigena and Monilinia laxa which also cause brown rot of stone fruits in
China.
Acknowledgements
We thank Dr. Guido Schnabel, Department of Entomology, Soils and Plant Sciences,
Clemson University, USA for critical comments and review of the manuscript. This
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M.J. HU et al.
material is based upon work supported by Program for New Century Excellent Talents in
University (NCET-08-0783) and partially sponsored by the Scientific Research
Foundation for the Returned Overseas Chinese Scholars, Huazhong Agricultural
University.
References
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Borowsky, R.L., Mcclelland, M., Cheng, R., Welsh, J. (1995): Arbitrarily primed DNA
fingerprinting for phylogenetic reconstruction in vertebrates: the Xiphophorus model.
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Chalmers, K.J., Waugh, R., Sprent, J.I., Simons, A.J., Powell, W. (1992): Detection of genetic
variation between and within populations of Gliricidia sepium and G. maculata using
RAPD markers. Heredity, 69, 465-472.
Gisi, U., Sierotzki, H., Cook, A., Mccaffery, A. (2002): Mechanisms influencing the evolution of
resistance to QoI inhibitor fungicides. Pest Management Science, 58, 859-867.
Heaney, S.P., Hall, A.A., Davies, S.A., Olaya, G. (2000): Resistance to fungicides in the QoI-
STAR cross-resistance group: current perspectives. Brighton Crop Protection Conference:
Pests and Diseases, Brighton, UK.
Kambhampati, S., Blavk, W.C., Rai, K.S. (1992): Random Amplified Polymorphic DNA of
mosquito species and populations (Diptera: Culicidae): techniques, statistical analysis, and
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Kim, Y.S., Dixon, E.W., Vincessi, P., Farman, M.L. (2003): Field resistance to strobilurin (QoI)
fungicides in Pyricularia griesea caused by mutations in the mitochondiral cytochrome b
gene. Phytopathology, 93, 891-900.
Kim, Y.S., E. W. Dixon, E.A. (2003): Field resistance to strobilurin (QoI) fungicides in
Pyricularia griesea caused by mutations in the mitochondiral cytochrome b gene.
Phytopathology, 93, 891-900.
Luo, C.-X., Hu, M.-J., Jin, X., Yin, L.-F., Bryson, P.K., Schnabel, G. (2010): An intron in the
cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to
QoI fungicides. Pest Management Science, 66 (12), 1308-1315.
Martinez, L.M., Rørvik, V., Brox, J., Lassen, M., Seppola, L., B., G.A. (2003): Fonnesbech-
Vogel, Genetic variability among isolates of Listeria monocytogenes from food products,
clinical samples and processing environments, estimated by RAPD typing. International
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Megnegneau, B., Debets, F., Hoekstra, R.F. (1993): Genetic variability and relatedness in the
complex group of black Aspergilli based on random amplified polymorphic DNA. Current
Genetics, 23, 323-329.
Sierotzki, H., Frey, R., Wullschleger, J., Palermo, S., Karlin, S., Godwin, J., Gisi, U. (2007):
Cytochrome b gene sequence and structure of Pyrenophora teres and P-tritici-repentis and
implications for QoI resistance. Pest Management Science, 63, 225-233.
Sierotzki, H., Parisi, S., Steinfeld, U., Tenzer, I., Poirey, S., Gisi, U. (2000): Mode of resistance
to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella
fijiensis field isolates. Pest Management Science, 56, 833-841.
Sierotzki, H., Schlenzig, A., Wullschleger, J., Windass, J., Stanger, C., Burbidge, J., Al., E.
(2002): Cytochrome b gene in fungi: Phylogenetic relationships and a mutation for QoI
resistance. In: H.W. Dehne, U. Gisi, K.H.Kuck, P.E. Russell and H. Lyr (Eds.) Modern
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Sierotzki, H., Wullschleger, J., Gisi, U. (2000): Point mutation in cytochrome b gene conferring
resistance to strobilurin fungicides in Erysiphe graminis f. sp. tritici field isolates. Pestic.
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Steinfeld, U., Sierotzki, H., Parisi, S., Gisi, U. (2002): Comparison of resistance mechanisms to
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22
Comparison of Cellulose Synthase 3 (CesA3)
Gene Structure in Different Oomycetes
Abstract
Cellulose is the most abundant polymer on earth, occurring in plants, bacteria, tunicates, slime
molds as well as oomycetes. Cellulose biosynthesis was recently shown to be the target for
mandipropamid, a member of the CAA (carboxylic acid amide) fungicides, which strongly
interfere with the function of the CesA3 protein. A specific SNP (single nucleotide
polymorphism) in the cellulose synthase 3 (CesA3) gene was detected in the grapevine downy
mildew Plasmopara viticola causing an amino acid change from glycine to serine at position
1105 (G1105S), leading to CAA resistance. Additional mutations (G1105V, G1105W) in the
CesA3 gene were detected in P. viticola and P. cubensis that correlated with resistance to CAAs.
The present study describes the CesA3 gene structure of several plant pathogenic oomycetes. The
full-length nucleotide sequences of the CesA3 gene was identified in Bremia lactucae,
Phytophthora capsici, Pseudoperonospora cubensis, and Pythium ultimum and compared to those
of other oomycetes. The sequences of all species were interrupted by one 76-136bp intron located
at the 5’-end. All four predicted CesA3 sequences contained several transmembrane domains and
a conserved set of motifs (D, D, D, QXXRW) known to be essential for processive
glycosyltransferases. The previously described Pleckstrin domain located in CesA1, CesA2 and
CesA4, was absent in the CesA3 sequences of the four pathogen species. Phylogenetic
comparison with the other members of the CesA family revealed that CesA3 genes of oomycetes
form a distinct clade. ClustalW sequence alignment showed a highly conserved amino acid motif
around position 1105 in all species included in this study, except for P. ultimum, being generally
insensitive to CAAs. Compared to other members of the Peronosporales, five out of the 23
amino acids, forming the last predicted transmembrane domain, were dissimilar in P. ultimum.
Based on our data we conclude that the CesA3 sequences in oomycetes are highly conserved and
that amino acid changes at position 1105 in the CesA3 protein affect sensitivity to CAA
fungicides.
Introduction
and host infection (Grenville-Briggs et al., 2008). The existence of four CesA genes
encoding putative cellulose synthases was reported in P. infestans, Phytophthora sojae,
Phytophthora ramorum Plasmopara viticola and Saprolegnia monoica (Blum et al.,
2010a, Grenville- Briggs et al., 2008, Fugelstad et al., 2009). Recently, it was shown that
the CAA (carboxylic acid amide) fungicide, mandipropamid (MPD) interferes with
cellulose synthesis by targeting the CesA3 protein in Peronosporales (Blum et al.,
2010b), but not in Pythiales. Studies with P. viticola reported that a recessive mutation in
the CesA3 gene causes resistance to this fungicide class (Blum et al., 2010a).
The aim of this study was to identify and characterize, in different oomycetes, the
CesA3 gene, the translated protein of which was described as target of MPD. For this
task, we used the CODEHOP PCR approach to identify the full length nucleotide
sequence of this gene in important plant pathogens like Bremia lactucae, Phytophthora
capsici, Pseudoperonospora cubensis and Pythium ultimum.
For DNA extraction, four isolates (B. lactucae HS, P. capsici 188, P. ultimum 71 and P.
cubensis 365) from the Syngenta culture collection were used. DNA was extracted
according to a standard CTAB protocol. CesA3 gene orthologs were identified using the
CODEHOP PCR approach described by Rose et al. (1998). Full length nucleotide
sequences were obtained by genome walking using DNA Walking SpeedUp Kit
(Seegene). Obtained PCR fragments were cloned in pCR4-TOPO vectors, sequenced and
analyzed on a 3130 Genetic Analyser (Applied Biosystems) according to manufacturer
instructions. Putative CesA3 sequences were blasted in the NCBI database and complete
ORFs were predicted using the NCBI ORF finder program. For analysis of the predicted
amino acid sequences the NCBI Conserved Domain Database was used. Prediction of
putative transmembrane domains was done using SOSUI software.
The cellulose synthase sequences used for phylogenetic analysis were obtained from
the NCBI protein database. Alignments were made by ClustalW and dendrograms
constructed using MEGA 4 software, with the minimum evolution algorithm using 1000
bootstrap replications.
In oomycetes, cellulose synthases are encoded by up to four CesA genes with specific
features. In this study, we identified the complete CesA3 gene sequence in four different
oomycetes. All four CesA3 gene sequences were about 3.5 Kb in size and were
interrupted by one conserved 76-136bp intron (length dependant on species) located at
the 5’-end (Figure 1A). The D,D,D,QXXRW motif found to be conserved in processive
β-glycosyltransferases was also present in these four sequences. Proteins belonging to the
cellulose synthase family are integral membrane proteins, characterized by one or more
transmembrane domains at the N-terminus and several transmembrane segments at the
C-terminus. Structural prediction with SOSUI software confirmed the presence of
transmembrane domains in these proteins (Figure 1A). Conserved domain searches with
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the CesA3 gene products revealed, at the C-terminal end, similarity to cellulose synthase
domains found in plants and bacteria (Figure 1A). The previously described oomycete
specific Pleckstrin domain located in CesA1, CesA2 and CesA4 of P. infestans and P.
viticola (Grenville-Briggs et al., 2008, Blum et al., 2010a), was absent in the CesA3
sequences of the four species.
Phylogenetic analysis of the CesA3 proteins with cellulose synthases from other
kingdoms showed that oomycete CesAs form a distinct clade, split into subclades for
CesA1, CesA2, CesA3 and CesA4 (Figure 1B). Based on the constructed phylogenetic
tree, the oomycete CesA gene products show the closest relationship to a cellulose
synthase from the Rhodophyta Porphyra yezoensis.
Figure 1: (A) Intron-/exon-structure of CesA3 gene from B. lactucae (Bl), P. capsici (Pc), P. cubensis
(Pcub) and P. ultimum (Pu). Total sequence length in base pairs (bp) including introns, is given in
parentheses. Introns are marked by a triangle. Putative transmembrane domains (vertical lines), QXXRW
motif (dotted line) and cellulose synthase domain (grey box) are indicated.
(B) Minimum evolution phylogram of cellulose synthases derived from Oomycetes, Plants, Rhodophyta,
Amoebozoa and Prokaryotes. The analysis is based on the complete amino acid sequence of the cellulose
synthases. CesA3 sequences identified in this study are marked by an asterisk. Bootstrap values are
indicated if >60.
For further analysis of the mutation at position 1105 in CesA3, ClustalW sequence
alignment was performed. Results showed that the mutation site 1105 is located in a
predicted transmembrane domain (Figure 2). The amino acid motif within this domain is
highly conserved in Peronosporales, but differs at 7 positions in Saprolegniales and 5
positions in Pythiales (Figure 2), the latter order being generally insensitive to CAAs. In
addition to the G1105S mutation identified in P. viticola, two other mutations in this
domain (G1105V, G1105W) were found in P. cubensis that conferred resistance to
CAAs.
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Figure 2: Multiple sequence alignment of oomycete CesA3 segments flanking position 1105.
CesA3 sequences of P. infestans (Pi), P. ramorum (Pr), P. sojae (Ps), P. viticola (Pv), B. lactucae (Bl),
P. capsici (Pc), P. cubensis (Pcub), S. monoica (Sm) and P. ultimum (Pu) were aligned using ClustalW.
Identical residues are colored in grey, differences are marked by triangles. The amino acid glycine at
position 1105 is marked by an asterisk. The last predicted transmembrane domain in CesA3 is marked by a
black bar above the alignment.
References
Blum M., Waldner M., Gisi U. (2010a): A single point mutation in the novel PvCesA3 gene
confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara
viticola. Fungal Genetics and Biology, 47, 499-510.
Blum, M., Boehler, M., Randall, E., Young, V., Csukai, M., Kraus, S., Moulin, F., Scalliet, G.,
Avrova, A., Whisson, S., Fonne-Pfister, R. (2010b): Mandipropamid targets the cellulose
synthase like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen,
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Grenville-Briggs, L.J., Anderson, V.L., Fugelstad, J., Avrova, A.O., Bouzenzana, J., Williams, A.,
Wawra, S., Whisson, S.C., Birch, P.R., Bulone, V., van West, P. (2008): Cellulose
synthesis in Phytophthora infestans is required for normal appressorium formation and
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Bulone, V. (2009): Identification of the cellulose synthase genes from the Oomycete
Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and
enzyme activity. Fungal Genetics and Biology, 46, 759-767.
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23
Assessment of G143A Mutation and Type I Cytb
Intron Frequencies in Botrytis cinerea Isolates
from Strawberry in Greece
Abstract
The study was conducted to investigate the frequency of the G143A mutation and the presence of
the type I cytb intron within Botrytis cinerea isolates (n=82) collected from strawberry fields
during 2008 and 2009 in Greece. Determination of resistance frequency to pyraclostrobin using a
discriminatory concentration revealed the wide spread presence of QoI-resistant isolates during
both years. Measurements of EC50 values to pyraclostrobin showed a bimodal sensitivity
distribution. All the isolates that were phenotypically resistant to QoIs carried the G143A
mutation. Some isolates without the G143A mutation carryied the type I cytb intron while others,
at frequencies of 20 to 24%, did not carry neither the mutation nor the intron. The results of the
study suggest that a high risk for selection of QoI-resistant strains exists in strawberry fields
when extensively treated with QoIs.
Introduction
Chemical control is the main strategy to control gray mould caused by Botrytis cinerea;
however, development of fungicide resistance is an important shortcoming (Leroux,
2007). Pyraclostrobin, a QoI fungicide, has been recently registered for the use against B.
cinerea in several crops. Resistance of B. cinerea to QoIs has been associated to the
presence of the G143A mutation in the cytb gene. Fungal populations were divided into
two groups according to the presence or absence of a type I intron in the cytb gene
(Banno et al., 2009; Jiang et al., 2009; Ishii et al., 2009). The current study was
conducted to investigate the frequency of resistance and sensitivity to pyraclostrobin in B.
cinerea isolates collected from strawberry. Furthermore, the frequency of the G143A
mutation and the presence of the type I cytb intron within fungal genome was
investigated.
Figure 1: Detection of type I cytb intron (A) and G143A mutation (B) in Botrytis cinerea isolates.
Results
Fungal sensitivity
Using the discriminatory concentration of pyraclostrobin, QoI-resistant isolates were
detected at frequencies higher than 40% during both years of the study. Measurements of
sensitivity to pyraclostrobin, expressed as EC50 values showed that there was a bimodal
sensitivity distribution, i.e. a sensitive and a resistant part (Figure 2). During both years
of sampling EC50 values ranged from 0.002 to >50 μg ml-1, however during 2009 there
were clearly more isolates with lower sensitivity (Figure 2).
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16
2008
14 2009
Number of isolates 12
10
0
<0.005 0.005-0.1 0.1-0.5 0.5-1 1-10.0 >10
-1
EC50 (μg ml ) pyraclostrobin
Figure 2: Distribution of EC50 values to pyraclostrobin in Botrytis cinerea isolates collected from
strawberry during 2008 and 2009.
60
G143A+ Intron-
G143A- Intron+
50 G143A- Intron-
40
Frequency %
30
20
10
0
2008 2009
Figure 3: Frequency of G143A mutation, associated with QoI resistance and presence or absence of type I
cytb intron in Botrytis cinerea isolates from strawberry.
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Conclusions
The results of the study suggest that in strawberry fields there is a wide spread presence
of QoI-resistant strains. The presence of fungal strains carrying the type I cytb intron
does apparently not prevent resistance since under fungicide selection they can be
eliminated, in favor of strains carrying the G143A mutation. Molecular detection of the
G143A mutation should be accompanied by sensitivity measurements since detection of
the mutation does not always correlate with high levels of resistance. This may be due to
the heteroplasmic status of cytb in B. cinerea, an observation which requires further
investigations.
References
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Fujimura, M. (2009): Characterization of QoI resistance in Botrytis cinerea and
identification of two types of mitochondrial cytochrome b gene. Plant Pathol., 58, 120–
129.
Ishii, H., Fountaine, J., Chung, W-H., Kansako, M., Nishimura, K., Takahashi, K., Oshima M.
(2009): Characterization of QoI-resistant field isolates of Botrytis cinerea from citrus and
strawberry. Pest Man. Sci., 65, 916-922
Jiang, J., Ding, L., Michailides, T.J., Li, H., Ma, Z. (2009): Molecular characterization of field
azoxystrobin-resistant isolates of Botrytis cinerea, Pest. Biochem. and Physiol. 93, 72-76.
Leroux P. (2007): Chemical control of Botrytis and its resistance to chemical fungicides. In:
Botrytis: Biology, Pathology and Control, ed. by Elad Y, Williamson B, Tudzynski P and
Delen N, Springer, Dordrecht, The Netherlands, pp.195 -222.
Myresiotis, C.K., Bardas, G.A., Karaoglanidis, G.S. (2008): Baseline sensitivity of Botrytis
cinerea to pyraclostrobin and boscalid and control of anilinopyrimidine- and
benzimidazole-resistant strains by these fungicides, Plant Dis., 92, 1427-1431.
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24
SDH-Inhibitors: History, Biological
Performance and Molecular Mode of Action
Abstract
This paper gives an overview of the succinate dehydrogenase inhibitors (SDHIs) class of
fungicides. The history of SDHIs, their biological performance and mode of action are reviewed.
Emphasis is put on the structural analysis of SDHIs by applying standard molecular modelling
techniques: Their common structural features and binding mode to the target, complex II of the
mitochondrial respiratory chain, are discussed. The results of this modelling study show that all
commercial SDHIs developed through conventional research share common chemical features
which are essential for fungicidal activity, and they hence bind to their target in the same manner.
These results also imply that some of the observed target alterations conferring resistance to
SDHIs have a direct impact on the binding behavior of SDHIs, whereas other mutations influence
SDHI binding by long-range structural rearrangement in the transmembrane part of complex II.
Introduction
In 1966, von Schmeling and Kulka described the systemic activity on basidiomycete
fungi of carboxin and oxycarboxin, which were subsequently launched as seed
disinfection and foliar spray agents by Uniroyal in 1969 and 1975 respectively (von
Schmeling and Kulka, 1966). Soon after this discovery a number of structural analogues
were introduced such as benodanil by BASF and fenfuram by Shell, both in 1974,
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
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Figure 1: Market entry of selected fungicidal SDHIs and their main biological targets.
Over the years, the structural complexity of SDHIs increased in parallel to the
broadening of their disease control spectrum. Still, as a comparison of their chemical
structures shows (Figure 2), they share a number of common features essential for
fungicidal activity: The central amide moiety which is essential for hydrogen-bond
interactions in the ubiquinone binding-site of SDH; the aromatic ring in the aniline part
ensures optimal hydrophobic contacts or - interactions to the binding site.
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SDH Inhibitors
Figure 2: Chemical structures of different SDHI fungicides (A) and their structural alignment (B)
illustrating common chemical features essential for fungicidal activity. The alignment suggests an identical
binding mode at complex II.
Mode of Action
Early mode of action studies indicated that carboxin has profound effects on fungal
respiration (Mathre, 1970; Ragsdale and Sisler, 1970). Further investigations pointed at
complex II of the mitochondrial respiration chain as a possible target of the
carboxamides (Mathre, 1971; Ulrich and Mathre, 1972), which has been confirmed by
the detection of mutations in the gene encoding SDH in carboxin-resistant strains of U.
maydis (Georgopoulos et al., 1972).
Complex II is a membrane-anchored protein and represents the link between the
mitochondrial respiration and the Krebs cycle (Cecchini, 2003). It consists of four
subunits: the flavoprotein subunit (SDH A) catalyzing the oxidation of succinate to
fumarate, the iron-sulfur protein (SDH B) containing the three iron-sulfur clusters
responsible for the electron transfer from succinate to ubiquinone and the two membrane
anchor subunits (SDH C and SDH D) with the heme b located between two antiparallel
helices of SDH C and SDH D (Figure 3).
The questions of the carboxamides mechanism of inhibition and site of action have
been addressed by various studies (Kuhn, 1984): Electron paramagnetic resonance (EPR)
spectroscopy studies proposed that carboxin interrupts the electron transfer between the
[3Fe-4S] cluster and the ubiquinone (Ackrell et al., 1977). Photoaffinity labeling studies
revealed binding of azidocarboxin to the subunit C and D (Ramsay et al., 1981). Both
findings point at the ubiquinone binding pocket (Q-site) as a possible site of action which
was later confirmed by X-ray studies on complex II (Yankovskaya et al., 2003, Huang et
al., 2006).
Figure 3: Homology model of SDH from B. cinerea with subunits A (flavoprotein, blue), B (iron-sulfur
protein, red), C and D (membrane anchors, yellow and green) (A). The arrow points at the Q-site. SDH
catalyzes the oxidation of succinate to fumarate, transports the released electrons from the flavin via the
three iron-sulfur clusters to the Q-site where the reduction of ubiquinone to ubiquinol is taking place (B).
Technical details of the homology model construction are described elsewhere (Glättli et al., 2009).
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Figure 4: Ubiquinone binding-site with carboxin (green), boscalid (orange), bixafen (cyan), isopyrazam
(yellow) and xemium (magenta) bound as suggested by computational docking experiments (A).
Comparison of the binding modes of ubiquinone (grey) and SDHIs represented by boscalid (orange), side-
view and top-view, showing that SDHIs bind deeper into the Q-site than ubiquinone itself (B).
Computational details on the docking experiments are reported elsewhere (Glättli et al., 2009).
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Carboxin-resistant strains have been reported and characterized soon after the
introduction of carboxin (Bochow et al., 1971; Georgopoulos et al., 1972, 1975; Matsson
et al., 1998; Matsson and Hederstedt, 2001) and recently reviewed (Avenot and
Michailides, 2010). Similarly, field and laboratory mutants resistant to boscalid have
been reported (Stammler et al., 2007; Avenot et al., 2008; Ishii, 2008; Miyamoto et al.,
2009; Stammler et al., 2011). Table 1 summarizes the target alterations in field isolates
conferring reduced SDHI sensitivity of different pathogens relevant for specialty crops.
Locating some of these mutations in the three-dimensional homology model of SDH
(Figure 5) shows that all amino acid exchanges found in B. cinerea isolates are situated
in direct proximity of the ubiquinone binding-site (Q-site) of SDH B. Proline at position
225 is an integral part of the Q-site contributing to carboxamide binding through
hydrophobic contacts. The exchange of proline by an amino acid with a bulkier side-
chain such as phenylalanine and leucine or into a slightly more polar residue such as
threonine could result in a decreased binding affinity for carboxamides. The observed
exchange of histidine at position 272 (homologous to H272 in B. elliptica, to H277 in A.
alternata and to H278 in C. cassiicola), with its side-chain located at the furthest point
from the opening of the Q-site, will also have a direct impact on the carboxamide binding
affinity, as SDHIs bind deeper into the Q-site than ubiquinone and are in direct contact to
H272, possibly via hydrogen-bonding (Horsefield et al., 2006, Huang et al., 2006,
Ruprecht et al., 2009, see also Figure 4). The observed difference in binding mode
between ubiquinone and carboxamides could explain why, in almost all fungi which
developed resistant strains, amino acid exchanges are found at this particular histidine.
Furthermore it is noteworthy that both mutations, H272Y/R and P225L/F/T, are in close
vicinity to the [3Fe-4S]-cluster. This could alter the reduction potential of the [3Fe-4S]-
cluster and consequently affect the electron transfer from succinate to ubiquinone.
In addition to the mutations in the SDH B subunit, pathogens such as A. alternata
and C. cassiicola show also mutations in the membrane anchor of SDH (subunit C & D).
The histidine at position 134 in subunit C of A. alternata is a highly conserved residue
located about 12-13 Å from the Q-site. It is involved in the iron coordination of heme B.
A mutation at this position may result in some structural rearrangement indirectly
affecting the topology of the Q-site and hence the binding affinity of carboxamides. A
comparable structural effect could be expected from the H133R mutation in subunit D of
A. alternata and the homologous D-H132R of S. sclerotiorum, which correspond to the
second axial histidine coordinating the iron of heme B.
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Figure 5: Location of selected resistance mutations listed in Table 1 in the structural model of SDH from
B. cinerea (subunit SDH A not shown) (A). B-H272Y/R (H277Y in A. alternata, B-H278Y/R in C.
cassiicola) and B-P225L/F/T (B), C-H134R and D-H133R in A. alternata (D-H132R in S. sclerotiorum)
(C), C-S73P in C. cassiicola (D) and D-S89P in C. cassiicola (E).
To date, the exact role of heme B in the electron transfer is still unclear and matter
of scientific debate (Horsefield et al., 2004; Oyedotun et al., 2007; Maklashina et al.,
2010). Three possible functions for heme b have been proposed: (a) as an essential
participant in the electron transfer from succinate to ubiquinone, (b) as an electron-sink
to protect against reactive oxygen species (ROS) formation (Yankovskaya et al., 2003) or
(c) for proper assembly and structural stabilization of the protein complex (Maklashina et
al., 2001).
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Chemistry, 280, 33331-33337.
Avenot H.F., Sellam, A., Karaoglanidis, G., Michailides T.J. (2008): Characterization of
mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with boscalid
resistance in Alternaria alternata from California pistachio. Phytopathology, 98, 736-742.
Avenot, H.F., Michailides, T.J. (2010): Progress in understanding molecular mechanisms and
evolution of resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in
phytopathogenic fungi. Crop Protection, 29, 643-651.
Bochow, H., Luc, L.H., Sung, P.Q. (1971): Development of tolerance by phytopathogenic fungi
to systemic fungicides. American College of Physicians, Bulletin 6, 399-414.
Cecchini, G. (2003): Function and structure of complex II of the respiratory chain. Annual
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type, coordination number and the amino-acid residues involved in the coordination. Acta
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Georgopoulos, S.G., Alexandri, E., Chrysayi, M. (1972): Genetic evidence for the action of
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Glättli, A., Stammler, G., Schlehuber, S. (2009): Mutations in the target proteins of succinate-
dehydrogenase inhibitors (SDHI) and 14α-demethylase inhibitors (DMI) conferring
changes in the sensitivity – structural insights from molecular modelling. 9th International
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von Heijne, G. (1991): Proline kinks in transmembrane -helices. Journal of Molecular Biology,
218, 499-503.
Horsefield R., Iwata S., Byrne B. (2004): Complex II from a structural perspective. Current
Protein and Peptide Science, 5, 107–118.
Horsefield R., Yankovskaya V., Sexton G., Whittingham W., Shiomi K., Omura S., Byrne B.,
Checchini G., Iwata S. (2006): Structural and computational analysis of the quinone-
binding site of complex II (succinate-ubiquinone oxidoreductase). The Journal of
Biological Chemistry, 281, 7309-7316.
Huang L., Sun G., Cobessi D., Wang A.C., Shen J.T., Tung E.Y., Anderson V.E., Berry E. A.,
(2006): 3-Nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon
oxidation by complex II, forms a covalent adduct with a catalytic base arginine in the
active site of the enzyme. The Journal of Biological Chemistry, 281, 5965–5972.
Ishii H. (2008): Fungicide research in Japan – an overview. In: H.W. Dehne, H.B. Deising, U.
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Maklashina, E., Rothery, R.A., Weiner, J.H., Checchini, G. (2001): Retention of heme in axial
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T., Inokuchi, H., Kojima, S. (1996):Two hydrophobic subunits are essential for the heme b
ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase)
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Oyedotun, K.S., Sit, C.S., Lemire, B.D. (2007): The Saccharomyces cervisiae succinate
dehydrogenase does not require heme for ubiquinone reduction. Biochimica et Biophysica
Acta, 1767, 1436-1445.
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Ragsdale, N.N., Sisler, H.D. (1970): Metabolic effects related to fungitoxicity of carboxin.
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Reaction site of carboxanilides and of thenoyltrifluoroacetone in complex II. Proceedings
of the National Academy of Sciences, USA, 78, 825-828.
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Escherichia coli succinate:quinone oxidoreductase with an occupied and empty quinone-
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carboxamide boscalid including recent studies on its mode of action. In: Proc. 16th Int.
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conferring resistance to SDHI fungicides. In H.W. Dehne, H.B. Deising, U. Gisi, K.H.
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Checcini G., Iwata S. (2003): Architecture of succinate dehydrogenase and reactive
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25
SDHIs and the Fungal Succinate Dehydrogenase
Abstract
Carboxamides (SDHI fungicides) inhibit succinate dehydrogenase (SDH) by binding to the
ubiquinone binding site (Qp site) of the SDH enzyme. In this contribution a range of biochemical
and molecular strategies are presented to study the mode of action and differential binding
properties of this class of inhibitors to the SDH enzyme in fungal pathogens. To gain deeper
insight into the binding properties of carboxamides to the Mycosphaerella graminicola SDH, we
aimed to purify the fungal enzyme with the objective of obtaining SDH-carboxamide co-crystals.
Attempts to purify the enzyme directly from the pathogen or by adopting bacterial expression
systems were mostly unsuccessful. A second strategy was the generation of target site mutants
using random mutagenesis followed by selection on agar amended with carboxamides of various
structures. This strategy delivered key information concerning differential binding properties
across the class of carboxamides allowing first predictions for resistance development to this
class of inhibitors.
Introduction
G. Scalliet et al.
SDHA is a flavoprotein carrying the succinate binding and oxidation site (Huang et al.,
2006), SDHB is an iron sulphur cluster protein which is involved in the two-step electron
transfer from the reduced FAD to the ubiquinone substrate (Cheng et al., 2006), and
SDHC and SDHD carry a prosthetic b-type heme which might also have a role in the
electron transfer to ubiquinone as a cofactor stabilizing the ubiquinone semi-radical
formed during the course of the reaction (Anderson et al., 2005). Ubiquinone reduction is
a complex process not yet fully understood, occurring at the ubiquinone binding site (Qp
site) which is structurally defined by the interface between the SDHB, SDHC and SDHC
subunits (Yankovskaya et al., 2003; Sun et al., 2005; Horsefield et al., 2006; Huang et al.,
2006). Carboxamides belong to the succinate dehydrogenase inhibitors (SDHIs) class of
fungicides; they specifically inhibit SDH by binding to the enzyme at the Qp site in place
of the ubiquinone substrate (Huang et al., 2006; Horsefield et al., 2006). Huge variation
of the biological profile can be observed across carboxamides which suggest that the
structure of the Qp site might account for these differences. Interestingly, SDHC and
SDHD protein sequences, except for a few residues of the Qp site, display very weak
degree of conservation across species which contrasts with the other subunits SDHA and
SDHB (Yankovskaya et al., 2003).
A range of biochemical techniques were adopted to better understand carboxamide
binding and mechanism of toxicity in fungi. Mycosphaerella graminicola was used both
as an important plant pathogen and as model organism to study SDH function and to
purify the functional enzyme for co-crystallisation experiments with the final aim of
performing drug design.
In this study, various attempts to purify M. graminicola SDH are described using
bacterial and fungal expression systems as a source for protein production. The nature of
the functional complex was confirmed and various promising approaches were developed.
Finally, random mutagenesis followed by carboxamide selection was used to possibly get
more insight into differential binding across carboxamides. The outcome of these
experiments allows making first predictions on resistance development towards the SDHI
class of fungicides.
Figure 1: General structure and enzymatic activity of the mitochondrial SDH enzyme.
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S O
F
F
O
no inh 0.75 µM 10.9 µM 17 nM 423 nM 43 nM 3 nM
OD (595nm)
F N
N
at 100µM
N
Cl
F
F
O no inh
F N
at 100µM 68.7 µM 1.8 µM 27 nM 31 nM 17 nM 5.9 nM
N
Time
Figure 2: (A) Carboxamide driven Qp site inhibition as shown by the absence of inhibition of the
succinate dependent PMS-mediated reduction of MTT (upper panel) and inhibition of the ubiquinone-
mediated reduction of DCPIP (lower panel).
Figure 2: (B) I50 values (μM) for different carboxamides as determined with sub-mitochondrial particles
and the DCPIP reduction test for Botrytis cinerea (B. cinerea), Ustilago nuda (U. nuda), Mycosphaerella
graminicola, (M. gram.) and Alternaria solani (A. solani), E. coli (purified SDH) and mitochondrial preps
from yeast and chicken.
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G. Scalliet et al.
A B
Figure 3: Blue native (A) and SDS-page (B) analysis of M. graminicola mitochondrial complexes. Panel
A: Blue native gel lane run with digitonin solubilised mitochondrial protein extract. The first dimension gel
was a 5-17% acrylamide gradient, functional SDH was histochemically stained as reported by Bullis and
Lemire (1994). B: Second dimension (12% acrylamide) SDS-gel stained with colloidal coomassie blue.
Rows of spots correspond to the individual polypeptides of respiratory chain complexes (and
supercomplexes) and are annotated according to MSMS analysis results.
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therefore the mitochondrial SDH enzyme) are not very abundant in fungal cells.
Secondly, M. graminicola SDH is rapidly disassembled during solubilisation (>80% loss)
and subsequent purification steps, even in the presence of mild non-ionic detergents like
lauryl, decyl, octyl-sarcosyl or polyoxyethylene. This is in contrast to the very good
stability we observed with E.coli SDH using similar detergents.
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G. Scalliet et al.
Figure 4: Affinity column (nickel) purified fraction from a M. graminicola SDHB-His tag expressing
strain. Affinity purification was performed with digitonin extracted mitochondrial proteins.
Discussion
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References
Anderson, R.F., R. Hille, S.S. Shinde, G. Cecchini. (2005): Electron transfer within complex II.
Succinate: ubiquinone oxidoreductase of Escherichia coli. Journal of Biological Chemistry,
280, 33331-33337.
Avenot H., Michailides T.J., (2010): Progress in understanding molecular mechanisms and
evolution of resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in
phytopathogenic fungi. Crop Protection, 39, 643-641.
177
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G. Scalliet et al.
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binding site in Escherichia coli succinate dehydrogenase is required for electron transfer to
the heme b. Journal of Biological Chemistry, 281, 32310-32317.
Weiner, J.H. (1974): The localization of glycerol-3-phosphate dehydrogenase in Escherichia coli.
J. Membr. Biol., 15, 1-14.
White, G.A., G.D. Thorn, S.G. Georgopoulos. (1978): Oxathiin derivatives highly active against
carboxin-resistant Succinic Dehydrogenase complexes from Carboxin-selected mutants of
Ustilago maydis and Aspergillus nidulans. Pesticide Biochemistry and Physiology, 9, 165-
182.
Yankovskaya, V., R. Horsefield, S. Tornroth, C. Luna-Chavez, H. Miyoshi, C. Leger, B. Byrne,
G. Cecchini, S. Iwata. (2003): Architecture of succinate dehydrogenase and reactive
oxygen species generation. Science, 299, 700-704.
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26
Sensitivity of Fungal Pathogens to SDHI
Fungicides
Abstract
Baseline sensitivity to isopyrazam, a new SDHI fungicide, has been determined for
Mycosphaerella graminicola, Pyrenophora teres, Rhynchosporium secalis and Puccinia
recondita. Isopyrazam has a strong binding affinity towards a broad range of fungal succinate
dehydrogenase enzymes, which inhibit electron transfer from succinate to ubiquinone;
consequently, the mitochondrial TCA cycle is arrested. In vivo, strongest inhibition effects are
observed at the spore germination or germ-tube elongation stage. The sensitivity distributions
were determined over several years in different countries and in relation to different treatment
strategies. In M. graminicola populations, the baseline sensitivity variation was up to 1000 fold.
Among more than 1200 tested field isolates, no resistance or reduced sensitivity was detected
when compared to the sensitivity of artificial mutants as reference. Also for P. teres, R. secalis
and P. recondita, baseline sensitivities were determined. No resistant isolates were detected
among 1000, 500 and 200 tested isolates for the three pathogens, respectively. Preliminary
sensitivity studies with several SDHI fungicides in Alternaria alternata, for which SDHI resistant
isolates were available, showed cross resistance among all tested SDHI fungicides. Finally, a
resistance risk assessment is presented for the entire SDHI group.
Introduction
H. Sierotzki et al.
maydis (sdh b, H257L) (Keon et al., 1991; Broomfield and Hargreaves, 1992) and in lab
mutants of Mycosphaerella graminicola (sdh b, H267Y) (Skinner et al., 1998), Coprinus
cinereus (sdh c, N80K) and Aspergillus nidulans (Ito et al., 2004). Recently, mutations in
subunit sdh b were detected in boscalid resistant field isolates of three additional
pathogens: P225L (or 225F or T) and H272Y (or 272R) in Botrytis cinerea (detected
three years after boscalid use in a vineyard in Germany; Stammler et al., 2007); several
different mutations in Alternaria alternata (detected in pistachio in California; Avenot
and Michailides, 2007, and 2010; Avenot et al., 2009) and a yet unknown mutation in
Corynespora cassiicola (Miyamato et al., 2009).
In this paper, the results on the sensitivity towards SDHI’s in several cereal
pathogens are reported. In addition, a first attempt is made to investigate possible
selection schemes for different SDH-inhibitors.
Fungal strains
The sampling and sensitivity testing of Pyrenophora teres and Puccinia recondita was
performed by Epilogic (Weihenstephan, Germany). The detailed methods are available
from the FRAC webpage (www.frac.info). The isolates were collected in the air with a
spore trap mounted on a car.
Isolates of Rhynchosporium secalis and Mycosphaerella graminicola were generated
from leaf samples. The method is also available from the FRAC webpage.
Ramularia collo-cygni isolation was performed according to Frei et al., (2000). The
sensitivity testing protocol was as follows: Leaf samples with symptoms were wrapped in
a paper towel and sent immediately to the lab. Conidia were transferred to PDA plates
containing tetracycline (25 mg/L). After addition of 2 drops of sterile H2O (bidest), the
conidia were spread out with a Drigalski spatula; then, the plates were closed with
parafilm and incubated at 20°C under a light/darkness cycle of 12h/ 12h. After 1-2 days,
the spores germinated, and the isolates could be transferred to a fresh PDA plate. The
plates were closed with parafilm and incubated for 10-14 days under the same conditions
as above. The sensitivity test was performed on AE medium (see FRAC methods) in 24
well plates. Conidia from one agar plate, grown under near UV light for 21 days at 20°C,
were suspended in 5ml of water. 100µl spore suspension was sprayed in each well. The
plates were incubated for 5 days at 20°C in the dark.
Alternaria isolates were collected from pistachio in a field trial in California, USA.
Samples were taken from plots treated in different ways: Before treatment, untreated
check, treatments with boscalid, isopyrazam, boscalid + pyraclostrobin, or
difenoconazole + cyprodinil. Two treatments were made at recommended rates and
samples taken 48 days after the second treatment. In total, 476 isolates were generated
from all plots. The isolates were tested for sensitivity to isopyrazam and boscalid in a
bioassay. The presence of mutations in sdh b, sdh c and sdh d was determined in all
isolates by pyrosequencing. On pistachio, different Alternaria species can occur. In 70%
of the isolates, the expected sdh gene sequence for A. alternata was found, whereas in
30%, the sdh c or the sdh d sequence was different, suggesting the presence of another
species (e.g. A. tenuissima and/or A. arborescens).
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Pyrenophora teres
Air borne spores of P. teres were collected throughout Europe. In 2009, 213 isolates have
been tested for sensitivity towards isopyrazam. Only 31 isolates grew more than 10%
compared to the control on 3 mg/L, and only two slightly more than 10% on 10 mg/L
isopyrazam. The calculated EC50 value was below 3 mg/L for all isolates. Therefore, all
isolates from all regions tested in 2009 can be considered as sensitive. Sensitivity
monitoring carried out within Syngenta (based on 50 to 100 leaf samples per year with
three isolates per sample) revealed no significant variation for the median EC50 values of
the populations from 2006 to 2009 (median EC50 0.8 to 2.3 mg/l). Furthermore, no
isolates with reduced sensitivity to SDHI’s have been detected in that period. In contrast,
a significant part of these isolates contained the F129L mutation in the cyt b gene leading
to (moderate) resistance towards QoI’s (data not shown).
Rynchosporium secalis
The isolates of R. secalis tested were all generated from leaf samples. Isopyrazam
baseline monitoring started in 2007 with 91 isolates, was continued in 2008 with about
100 isolates, and procured in 2010 with more than 300 isolates (Figure 1). The median
sensitivity in all three years was between 0.03 and 0.05 mg/l, and no isolate could be
detected with an EC50 value higher than 1 mg/l. Therefore, all the isolates can be
considered as sensitive to SDHI’s; in addition, they were also sensitive towards QoI
fungicides.
Figure 1: Sensitivity of Rhynchosporium secalis isolates to isopyrazam collected in 2009 from CZ, D, E,
F, IR, PL and UK.
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H. Sierotzki et al.
Ramularia collo-cygni
In 2008, about 13 isolates of R. collo-cygni were tested for sensitivity towards
isopyrazam and azoxystrobin. The sensitivity range was very narrow for isopyrazam,
from 0.1 to 1 mg/L; however, about half of the isolates were QoI resistant with a
resistance factor of 100 compared to the sensitive isolates (EC50 values of sensitive
isolates to azoxystrobin were between 0.05 and 0.1 mg/L).
Mycosphaerella graminicola
Since 2004, more than 1000 isolates of M. graminicola have been tested for sensitivity to
isopyrazam (IZM). The EC50 values of isolates tested so far varied from 0.001 to about 1
mg/L (baseline sensitivity). There were no differences in sensitivity between the years of
sampling (Figure 2).
1e+1
1e+0
log EC50 value IZM mg/L
1e-1
1e-2
1e-3
1e-4
1e-5
2004 2005 2006 2007 2008 2009
Years
Puccinia recondita
Although P. recondita is classified by FRAC as pathogen with a low risk to develop
resistance to fungicides, a baseline monitoring has been initiated to determine the
sensitivity of the rust population in Europe to isopyrazam. More than 200 isolates have
been tested, originating from different regions throughout Europe (GB, F, D). Initial
testing of reference isolates showed that the dose-response curve is very steep between
0.01 and 0.3 mg/L. All isolates were in a narrow sensitivity range and almost no growth
on concentration higher than 1 mg/L was observed. The discriminary dose for reduced
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sensitivity to IZM was defined as pathogen growth > 50% at 0.30 mg/L relative to the
untreated control (Epilogic data). No isolates with reduced sensitivity to isopyrazam have
been detected, they can be regarded as sensitive (data not shown).
Figure 3: Sensitivity of isolates of Alternaria alternata possessing different mutations in the sdh genes
towards isopyrazam (left) and boscalid (right).
Among all the tested isolates, 8 different amino acid changes were detected: In the
sdh b: H277Y, H277L; in the sdh c: H134R, S135R; in the sdh d: D123E, H133R/P/T. In
a few isolates, a combination of sdh b H277Y and sdh c H134R and a combination of sdh
b H277Y and sdh d H133R was found at low frequency. The wild type isolates (277H in
sdh b) were spread over the entire sensitivity distribution; however, only very few
isolates displayed a high EC50 value (higher than 100). The reason for this observation is
not clear. All mutations caused an increase in the EC50 value against boscalid, but within
these genotypes a few isolates were still sensitive. The mutation sdh d D123E (123E)
mediated only weak resistance to boscalid (Figure 3). In tendency, the relationship
genotype/phenotype for isopyrazam was similar to that of boscalid, but in four out of the
10 different sdh genotypes the EC50 value did not significantly increase compared to the
base line sensitivity (sdh b: H277Y, H277L, sdh d: D123E, H133P/T) (Figure 3).
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H. Sierotzki et al.
Figure 4: Changes in the frequency of sdh-genotypes in Alternaria alternata in different plots after 2
fungicide applications (explanations see text).
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The remaining genotypes displayed a significantly higher EC50 compared to the base
line isolates; however, the resistance factors were clearly lower for isopyrazam compared
to boscalid.
In trial plots, populations treated with a SDHI fungicide possessed between 14.6 and
17.1% of mutations in the sdh genes. In comparison, populations from plots treated with
a fungicide other than a SDHI possessed between 4 and 8.4% of mutations. Interestingly,
the pattern of mutations varied for the different SDHI-fungicides used in this experiment.
Boscalid, when used solo, predominantly selected for the mutation sdh b H277Y, while
isopyrazam and the boscalid/pyraclostrobin mixture selected in addition for the sdh c
H134R mutation. The latter two treatments resulted in a bigger diversity of mutations
(Figure 4). As expected, the frequency of sdh gene mutations was lowest in the untreated
check plots and in plots treated with the cyprodinil/difenoconazole mixture.
Conclusions
So far, no SDHI-resistant strains of any cereal pathogen species were found in the field
populations monitored. The range of baseline sensitivity in P. teres, R. secalis and M.
graminicola populations is rather wide with a factor of more than 100. The distribution
curves for the EC50 values in the pathogen populations indicate a continuous variation
between most and least sensitive isolates. There is no indication of any isolate possessing
a reduced sensitivity, i.e. no resistant isolates with a 10 times higher EC50 value over the
least sensitive member of the baseline population.
However, resistance due to target site mutations occurring in field populations is
known for other pathogen species, such as A. alternata (Avenot and Michailides, 2007
and 2010; Avenot et al., 2009), B. cinerea (Stammler et al., 2007), D. bryoniae and C.
cassiicola (Miyamato et al., 2009). The analysis of A. alternata strains from the field trial
in pistachios in California showed that some mutations confer cross resistance between
isopyrazam and boscalid. Initial tests show that this is also true for other SDHI’s recently
becoming public (data not shown). Some mutations seem to be selected specifically by
certain SDHI’s (Avenot and Michailides, 2010). Therefore, SDHI‘s might transiently
perform differently on field populations depending on which mutation is dominating.
However, population dynamic effects are assumed to favor mutations and combinations
of mutations that provide strongest resistance to all SDHI‘s, as was in fact demonstrated
by the selection pattern in the A. alternata trial.
Based on the described data and large experience with other single site inhibitors
(e.g. QoIs, MBCs), the interent risk of resistance development was defined as „medium
to high“ for the SDHI fungicides (SDHI working group of FRAC). The combined
resistance risk (fungicide X pathogen risk) is estimated as medium to high for M.
graminicola, as medium for P. teres and R. collo-cygni , as low to medium for R. secalis
and low for P. recondita. For these reasons, it is recommended to apply isopyrazam
always in mixtures or alternations with an appropriate partner fungicide from a different
mode of action group that is sufficiently active at the applied doses against current field
populations of the target pathogen (www.frac.info).
In the coming years, more SDHI based fungicides will be introduced to the market,
therefore the intensity and area of selection for resistance in European cereals are
expected to increase drastically. Since several other fungicide mode of action classes
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H. Sierotzki et al.
suffer from resistance (e.g. QoIs) or sensitivity shifts (e.g. DMI’s) in field populations, it
is crucial to undertake all possible practical measures to either prevent or delay the onset
of resistance to SDHI fungicides.
References
Avenot, H.F., Michailides, T.J. (2007): Resistance to pyraclostrobin, boscalid, and Pristine
(pyraclostrobin + boscalid) in Alternaria alternata isolates from California pistachio.
Phytopathology, 97, 5.
Avenota H., Sellamb A., Michailides T. (2009): Characterization of mutations in the membrane-
anchored subunits AaSDHC and AaSDHD of succinate dehydrogenase from Alternaria
alternata isolates conferring field resistance to the fungicide boscalid. Plant Pathology, 58,
1134–1143.
Avenot, H.F., Michailides, T.J. (2010): Progress in understanding molecular mechanisms and
evolution of resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in
phytopathogenic fungi. Crop Protection, 29, 643-651.
Broomfield, P.L.E., Hargreaves, J.A. (1992): A single amino-acid change in the iron-sulphur
protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago
maydis. Current Genetics, 22, 117-121.
Ito, Y., Muraguchi, H., Seshime, Y., Oita, S., Yanagi, S.O. (2004): Flutolanil and carboxin
resistance in Coprinus cinereus conferred by a mutation in the cytochrome b560 subunit of
succinate dehydrogenase complex (complex II). Molecular Genetics and Genomics, 272,
328-335.
Keon, J.P.R., White, G.A., Hargreaves, J.A. (1991): Isolation, characterization and sequence of a
gene conferring resistance to the systemic fungicide carboxin from the maize smut
pathogen, Ustilago maydis. Current Genetics, 19, 475-481.
Miyamotoa T., Ishii H., Sekoc T., Koboria S., Tomita Y. (2009): Occurrence of Corynespora
cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan. Plant
Pathology, 58, 1144–1151.
Newcombe, G., Thomas, P.L. (2000). Inheritance of carboxin resistance in a European field
isolate of Ustilago nuda. Phytopathology, 90, 179-182.
Stammler, G., Brix, H.D., Glättli, A., Semar, M., Schoefl, U. (2007): Biological properties of the
carboxamide boscalid including recent studies on its mode of action. Proceedings XVI
International Plant Protection Congress Glasgow, 40-45.
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27
Resistance to QoIs and SDHIs in Populations
of Botrytis cinerea
Abstract
Respiration inhibitors are widely used to control fungal disease on crops worldwide. Most are
QoIs (e.g. strobilurins) and SDHIs (e.g. carboxamides), inhibiting the cytochrome b of
mitochondrial complex III and the succinate dehydrogenase of mitochondrial complex II,
respectively. The aim of this study was to characterize the mechanisms responsible for resistance
in Botrytis field strains less susceptible to one or both of these modes of action. Multidrug
resistance (MDR) was identified in strains with low to moderate resistance to several respiration
inhibitors. Highly specific resistance to QoIs was correlated with a single mutation of the cytb
target gene. Length polymorphism of this gene may also have occurred due to an evolutionary
process controlling selection for resistance. Resistance to SDHIs was characterized by six
phenotypes, with various patterns of resistance and cross-resistance to carboxamides (SDHIs).
Several mutations, three specific to Botrytis, were identified within the sdhB and sdhD genes
encoding the iron-sulphur protein and an anchor protein of the succinate dehydrogenase complex.
Another, as yet uncharacterized mechanism of resistance was also recorded. This diversity of
resistance mechanisms makes resistance management difficult and must be taken into account
when developing strategies for Botrytis control.
Introduction
Within agricultural fungicides, those affecting respiration are widely used, and many of
them are effective against a wide range of plant pathogenic fungi. Among them,
inhibitors of mitochondrial complex III (syn. cytochrome bc1 complex) which bind to
cytochrome b at the Qo site (an outside quinol oxidizing pocket) are synthetic analogues
of natural strobilurins (e.g. azoxystrobin, kresoxim-methyl) and have been introduced in
the mid-1990s to control many fungal diseases, and more particularly, powdery and
downy mildew on grapevine (Fernandez-Ortuno et al., 2008). Inhibitors of succinate
dehydrogenase (syn. mitochondrial complex II) or SDHIs constitute another distinct
mode of action (White and Georgopoulos, 1992). From the chemistry point of view,
many of them are carboxamides exhibiting a common “cis-crotoanilide” structure. They
were derived from an α-β unsaturated carboxylic acid, the double bond of which is
incorporated into benzene or conjugated to an electron releasing atom such as O, N or S,
leading to several subgroups. In addition to these “cis-crotoanilide” molecules, several
N-methylpyrazole carboxamides with a single bond between the significant methyl and
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Origin of samples
Fifty isolates were collected in the Champagne (France) and Palatine (Germany)
vineyards between 2007 and 2008. They were kept as mono-conidial cultures on a
medium containing 20 g/L malt, 5 g/L yeast extract and 12.5 g/L agar.
Molecular procedures
DNA from the isolates was extracted using a sarcosyl-based protocol. PCR-amplification
was performed for the cytb, sdhA, sdhB, sdhC and sdhD genes (Leroux et al., 2010), and
gene sequence was produced for at least 3 isolates of each phenotype. A CAPS test,
using the SatI restriction enzyme, described to recognize the G143A change within cytb,
enabled to identify this mutation in routine tests for all isolates.
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Resistance to QoIs
Strains highly resistant to QoIs were found only within the species Botrytis group II.
These strains exhibited high resistance factors (>75) for all tested strobilurins and were
therefore named QoIR. No cross-resistance was observed to the QiI antimycin A or other
respiration inhibitors (e.g. fluazinam or tolylfluanid) (Table 1). Examination of cytb
revealed the G143A change within the protein sequence for all the resistant strains tested.
This change occurs in the Qo binding site of ubiquinone and strobilurins and is the main
resistance mechanism found in more than 30 phytopathogenic fungi (Fernandez-Ortuno
et al., 2008). In Botrytis, this resistance may have been selected unintentionally when
targeting powdery or downy mildew control.
1205 bp group I
G143
intron G143 A143
cytb
PCR
Qo13ext/Qo14ext
Figure 1. Molecular polymorphism of the gene encoding cytochrome b (cytb) in strains of Botrytis group I
and group II and discrimination of the various genotypes after the CAPS test.
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Resistance to SDHIs
Since 2007, strains resistant to the SDHI boscalid were found in the Champagne and
Palatine vineyards, at rather low but increasing frequencies. Thorough examination of
isolated strains revealed at least six distinct phenotypes, based upon their resistance
levels towards the various subgroups of SDHIs and their patterns of cross-resistance
(Table 2):
- Car R1: moderate resistance to boscalid, weak resistance to 3’ benzylcarboxin (oxa-3b)
and hypersensitivity (i.e. negative cross resistance) to benzamides
- Car R2: moderate resistance to boscalid, weak resistance to oxa-3b and absence of
negative cross resistance to benzamides
- Car R3: moderate resistance to boscalid, normal sensitivity to oxa-3b
- Car R4: moderate resistance to boscalid and to oxa-3b
- Car R5: strong resistance to boscalid and moderate resistance to oxa-3b
- Car R6: strong resistance to boscalid and normal sensitivity to oxa-3b
Interestingly, weak cross-resistance was noticed for most furan-carboxamides for all
CarR phenotypes, however it did not occur with furmecyclox, although this compound
had a very high intrinsic activity, especially towards Botrytis group I strains. Indeed, its
chemical structure is related to amine SBIs (e.g. fenpropimorph) and the primary mode
of action of this molecule might be inhibition of sterol Δ14 reduction.
When sequencing genes encoding the four subunits of succinate dehydrogenase for
various CarR strains, full correlation between phenotype of genotype was found, with the
exception of CarR2. Alterations were detected mainly in the SdhB subunit (iron-sulphur
protein) and occurred either in the second Fe-S cluster (codon 225 or 230 in CarR3,
CarR4 and CarR5) or in the third cluster (codon 272 in CarR1, CarR2 and CarR6) (Table
2; Figure 2). These mutations may alter the binding of SDHIs within the ubiquinone
binding pocket (Horsefield et al., 2006, Huang et al., 2006). CarR2 isolates exhibited
either alteration in SdhB (H272R), SdhD (H132R) or no alteration in any of the four Sdh
subunits. Recently, strains with P225F in SdhB were detected, but the phenotype of these
strains is not yet available.
Consequently, target alteration seems to be the main mechanism for resistance to
SDHIs, but a wide range of resistance alleles have been selected in Botrytis group II
populations leading to a large variety of phenotypes with various biological properties
(resistance factors, cross-resistance and fitness). This may complicate resistance
management to SDHIs, especially if molecules from different subgroups are used in the
field, and may impose quantitatively and qualitatively different selection pressures.
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a
oxa-3a: 3’-phenylcarboxin; oxa-3b: 3’-benzylcarboxin; oxa-3c: 3’-butoxycarboxin; oxa-3d: 3’-
hexyloxycarboxin; oxa-4a: 4’-phenylcarboxin; oxa-4b: 4’-butoxycarboxin.
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Figure 2: Schematic structure of mitochondrial complexes II and III indicating the main amino-acids of
the different subunits involved in binding of inhibitors and in resistance.
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Multidrug resistance
Strains exhibiting multiple resistance to distinct modes of action have been detected in
the Champagne vineyards since the early nineties and represent now more than 50% of
the population. The resistance mechanism is related to multidrug resistance (MDR), i.e.
the overexpression of either ABC- (MdR1 strains) or MFS- (MdR2 strains) transporters
that excrete drugs from fungal cells (Kretschmer et al., 2009). A third phenotype (MdR3)
cumulates the two previous resistance mechanisms. According to our data, QoIs and
SDHIs are concerned by MDR, but generally with weak resistance factors (Tables 1 and
2), which may limit the impact of this kind of resistance under field conditions. This
mechanism may not affect all molecules within a fungicide family, depending probably
upon their lipophilicity.
Conclusion
Our survey conducted in French and German vineyards showed that at least two different
resistance mechanisms (i.e. target alteration and efflux-pump overexpression) are
affecting respiration inhibitors in B. cinerea. These observations confirm that this fungal
pathogen induces a high risk of resistance development resulting in rather complex
resistance and disease management programmes, given the limited number of available
active ingredients likely to be effective.
References
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Leroux, P. and Walker, A.S. (2009): Les fongicides affectant les processus respiratoires : modes
d'action et phénomènes de résistance. In. 9ème conférence internationale sur les maladies
des plantes. Tours, 8-9 décembre 2009: AFPP.
White, G., Georgopoulos, S. (1992): Target sites of carboxamides. In: Köller W., ed. Target sites
of fungicides action. CRC Press, Boca Raton, FL, USA, 1-29.
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28
Mutations in the Target Protein Conferring
Resistance to SDHI Fungicides
Abstract
Sensitivity monitoring studies for SDHI fungicides showed that sensitivity of most tested target
pathogens was within the baseline range. Cases of resistance were only found in a few fungal
species. Sequence analysis of the corresponding gene of the target enzyme of SDHI fungicides,
succinate dehydrogenase, indicated that a number of spatially distinct mutations in the subunits B,
C and D can lead to a loss of sensitivity. In regard to the expected introduction of new SDHI
fungicides in multiple arable crops, fruits, vines, vegetables, ornamentals and turf, appropriate
resistance management strategies including extensive monitoring studies are recommended for
the respective target pathogens.
Introduction
The target protein of SDHI fungicides (SDHIs) is the succinate dehydrogenase (SDH),
which consists of four subunits (A, B, C, D), i.e. a hydrophilic flavoprotein (A), an iron
sulfur protein (B) and two lipophilic transmembrane subunits (C and D) which are
necessary to anchor the protein to the mitochondrial membrane. Inhibitors of SDH act via
the ubiquinone binding site formed by the subunits B, C and D. Plant pathogenic fungi
with sensitivities lower than the baseline sensitivity identified in monitoring studies or
generated in the laboratory were investigated for mutations in the SDH gene.
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sclerotiorum and H133 of SdhD in A. alternata are involved in the iron coordination of
heme b. Mutations at these positions may result in structural rearrangement indirectly
affecting the topology of the binding site and hence the binding affinity of SDHIs. Aa
S73P in SdhC and the S89P and D123E in SdhD of C. cassiicola and A. alternata,
respectively, are also not integral parts of the binding site and the mutations may
therefore as well have indirect effects on the topology of the binding-site and on the
binding of SDHIs.
Botrytis cinerea:
Bc(wt) 213ECILCACCSTSCPSYWWNSEEYLGPAILLQSYRWLADSRDQKKEERKAALDNSMSLYRCHTILNCSRTCP
Bc(P225L) 213ECILCACCSTSCLSYWWNSEEYLGPAILLQSYRWLADSRDQKKEERKAALDNSMSLYRCHTILNCSRTCP
Bc(P225T) 213ECILCACCSTSCTSYWWNSEEYLGPAILLQSYRWLADSRDQKKEERKAALDNSMSLYRCHTILNCSRTCP
Bc(P225F) 213ECILCACCSTSCFSYWWNSEEYLGPAILLQSYRWLADSRDQKKEERKAALDNSMSLYRCHTILNCSRTCP
Bc(H272Y) 213ECILCACCSTSCPSYWWNSEEYLGPAILLQSYRWLADSRDQKKEERKAALDNSMSLYRCYTILNCSRTCP
Bc(H272R) 213ECILCACCSTSCPSYWWNSEEYLGPAILLQSYRWLADSRDQKKEERKAALDNSMSLYRCRTILNCSRTCP
Alternaria alternata:
Aa(wt) 218ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKKAERQDALNNSMSLYRCHTILNCSRTCP
Aa(H277Y) 218ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKKAERQDALNNSMSLYRCYTILNCSRTCP
Aa(H277R) 218ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKKAERQDALNNSMSLYRCRTILNCSRTCP
Corynespora cassiicola:
Cc(wt) 219ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKTAQRQDALNNSMSMYRCHTILNCSRTCP
Cc(H278Y) 219ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKTAQRQDALNNSMSMYRCYTILNCSRTCP
Cc(H278R) 219ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKTAQRQDALNNSMSMYRCRTILNCSRTCP
Didymella bryoniae:
Db(wt) ...ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKKAERQDALNNSMSLYRCHTILNCSRTCP
Db(H->Y) ...ECILCACCSTSCPSYWWNQEEYLGPAVLLQSYRWIADSRDEKKAERQDALNNSMSLYRCYTILNCSRTCP
Figure 1: Aa sequences (partial) of SDH-B in species with documented field isolates resistant to SDHI
fungicides. Bold letters show the aa which have been found to be exchanged in resistant isolates.
Underlined are amino acids found in SDHI-resistant isolates.
Alternaria alternata:
Aa (wt) 72SSLNRITGITLSGSLYLFGIAYLIAPYTGWHLETQSMVATVAAWPAAVKTGLKAFYAFPFFFHSFNG
Aa(H134R) 72SSLNRITGITLSGSLYLFGIAYLIAPYTGWHLETQSMVATVAAWPAAVKAGLKAFYAFPFFFRSFNG
Corynespora cassiicola:
Cc(wt) 72SSFNRITGVALSGGLYLFGFAYLAAPTLGWHLETQSMVAAVAAWPVAAKVAAKISIAMPFFFHSLNG
Cc(S73P) 72SPFNRITGVALSGGLYLFGFAYLAAPTLGWHLETQSMVAAVAAWPVAAKVAAKISIAMPFFFHSLNG
Figure 2: Aa sequences (partial) of SDH-C in species with documented resistance to SDHI fungicides in
field isolates. Bold letters show the aa which have been found to be exchanged in resistant isolates.
Underlined are the amino acids found in SDHI-resistant isolates.
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G. Stammler et al.
and H152R caused high resistance factors to all the SDHI fungicides that are expected to
be launched in the next years
Corynespora cassiicola:
Cc(wt) 55VPDPSPSHGSYHWSFERAISAGLIPLTIAPFAAGSLNPVTDSILCALLVIHSHIGFEACVIDYFPAKR
Cc(S89P) 55VPDPSPSHGSYHWSFERAISAGLIPLTIAPFAAGPLNPVTDSILCALLVIHSHIGFEACVIDYFPAKR
Sclerotinia sclerotiorum:
Sc(wt) 82VPKPSPSHGSYHWTFERLIAVGLIPLTVAPFVSGSLNPATDAILCAAILIHSHIGFESCVIDYIPRKR
Sc(H132R) 82VPKPSPSHGSYHWTFERLIAVGLIPLTVAPFVSGSLNPATDAILCAAILIRSHIGFESCVIDYIPRKR
Alternaria alternata:
Aa (wt) 83VPDPDYAHGSYHWSFERIVSAGLIPLTIAPFAAGSLNPLTDSILCALLVVHSHIGFESCIIDYFPSKR
Aa(D123E) 83VPDPDYAHGSYHWSFERIVSAGLIPLTIAPFAAGSLNPLTESILCALLVVHSHIGFESCIIDYFPSKR
Aa(H133R) 83VPDPDYAHGSYHWSFERIVSAGLIPLTIAPFAAGSLNPLTDSILCALLVVRSHIGFESCIIDYFPSKR
Figure 3: Aa sequences (partial) SDH-D in species with documented resistance to SDHI fungicides in
field isolates. Bold letters show the aa which have been found to be exchanged in resistant isolates.
Underlined are the amino acids found in SDHI-resistant isolates.
References
Avenot H.F., Sellam, A., Karaoglanidis, G., Michailides T.J. (2008): Characterization of
mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with boscalid
resistance in Alternaria alternata from California pistachio. Phytopathology, 98, 736-742.
Avenot, H.F., Sellam, A., Michailides, T.J. (2009): Characterization of mutations in the
membrane-anchored AaSDHC and AaSDHD of succinate dehydrogenase from Alternaria
alternata isolates conferring field resistance to the fungicide boscalid. Plant Pathology, 58,
1134-1143.
Glättli, A., Stammler, G., Schlehuber, S. (2009): Mutations in the target proteins of succinate-
dehydrogenase inhibitors (SDHI) and 14α-demethylase inhibitors (DMI) conferring
changes in the sensitivity – structural insights from molecular modelling. 9th International
Conference on Plant Diseases, Tours, France, 670-681.
Keon, J.P.R., White, G.A., Hargreaves, J.A. (1991): Isolation, characterization and sequence of a
gene conferring resistance to the systemic fungicide carboxin from the maize smut
pathogen, Ustilago maydis. Current Genetics, 19, 475-481.
McGrath, M.T. (2008): Fungicide sensitivity in Podosphaera xanthii and efficacy for cucurbit
powdery mildew in NY, USA, in 2003-2006. Journal of Plant Pathology, 90, 2.90.
Miyamoto, T., Ishii, H., Seko, T., Tomita, Y., Kobori, S., Ogawara, T. (2008): Occurrence of
boscalid-resistant isolates of cucumber Corynespora leaf spot fungus (C. cassiicola).
Japanese Journal of Phytopathology, 74, 37-38.
Skinner, W., Bailey, A., Renwick, A., Keon, J., Gurr, S., Hargreaves, J. (1998): A single amino
acid substitution in the iron sulphur protein of succinate dehydrogenase determines
resistance to carboxin in Mycosphaerella graminicola, Current Genetics, 34, 393-398.
Stammler, G., Brix, H.D., Nave, B., Gold, R., Schoefl, U. (2008): Studies on the biological
performance of boscalid and its mode of action. Modern Fungicides and Antifungal
Compounds V, Eds: Dehne, H.W., Deising H.B., Gisi, U. Kuck, K.H. Russell, P. Lyr, H.,
DPG, Braunschweig, Germany. 45-51.
Stammler, G. (2008): Mode of action, biological performance and latest monitoring results of
boscalid sensitivity. Abstracts of the 18th Symposium of Research Committee on Fungicide
Resistance. Matsue, JPN, Phytopathological Society of Japan, 30-43.
Stevenson K.L., Langston D., Sanders B.F. (2008): Baseline sensitivity and evidence of
resistance to boscalid in Didymella bryoniae. Phytopathology, 98, 151.
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29
Development of QoI Resistance in
Ramularia collo-cygni Populations
Abstract
Ramularia collo-cygni first appeared as a major pathogen of barley in Scotland during 1998 and
was found to be easily controlled by all available QoI fungicides. This type of fungicide was
therefore recommended for Ramularia leaf spot control. However, during 2003 there was a sharp
decline in the efficacy of QoI fungicides. This study confirms the types of mutations and time
line for the development of QoI fungicide resistance in R. collo-cygni populations.
Introduction
Ramularia leaf spot caused by the fungus Ramularia collo-cygni is now one of the most
damaging diseases of barley in northern Europe. The use of QoI fungicides showed poor
control of R. collo-cygni in spring barley as early as 2003 (Figure 1). Studies on plant
Figure 1: A fungicide dose response curve comparing three commercial QoI fungicides with
epoxiconazole (Opus).
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
pathogens have identified three amino acid substitutions, e.g. glycine to alanine at codon
143 (G143A), phenylalanine to leucine at codon 129 (F129L) and glycine to arginine at
codon 137 (G137) that confer resistance to QoIs. The aim of this study was to establish if
mutations in the cytochrome b gene were associated with QoI resistance and if DNA
diagnostics can be used to detect and quantify levels of QoI-resistant alleles in archived
field populations of R. collo-cygni.
Ramularia collo-cygni isolates (n=19) collected from different countries were initially
sequenced to obtain a 675 bp fragment of the cytochrome b gene. This fragment
encompasses codons 76-282 of the protein. The sensitivity to azoxystrobin was
determined for a selection of Scottish isolates using an agar-based bioassay. A range of
DNA-based assays (e.g. PCR-RFLP, allele-specific real-time PCR and Pyrosequencing)
was developed to detect and quantify QoI-resistance conferring alleles in isolates and
archived field samples (1996-2007) from the long-term Rothamsted Hoosfield spring
barley experiment.
The bioassay showed that R. collo-cygni is highly resistant to QoI fungicides and was
able to grow in the presence of at least 4 ppm of azoxystrobin (Figure 2). A143 alleles
were detected in all QoI resistant isolates, whereas G143 alleles were only found in
sensitive isolates using Pyrosequencing (Figure 3).
Untreated
100 ppm
20
0.8
0.16
0.032
0.0064
Figure 2: Fungicide sensitivity bioassay showing two sensitive isolates collected in 1999 from the SAC
Bush Estate (B 1.1 and B 1.2) and a resistant isolate (S 1.1) collected in 2007 from the same location.
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Figure 3: Pyrograms of a QoI-resistant (A) and a QoI-sensitive (B) Ramularia collo-cygni isolate.
Detection is accurate for allele frequencies between 5 and 95 %.
% Resistant alleles
80
20 70
pg of DNA (Log)
60
50
40
1 30
20
10
0,05 0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Time in years
Figure 4: Ramularia collo-cygni DNA levels and percentage of A143 alleles present in archived samples
using allele-specific real-time PCR.
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1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
M
Figure 5: PCR-RFLP of amplified cytochome b gene fragments from R. collo-cygni populations present
in archived Rothamsted Hoosfield spring barley samples. Digestion with AluI. G143 alleles visualised as
PCR product of 212 bp, while 99 and 113 bp fragments indicate the presence of digested A143 alleles; M
= 50 bp marker.
Conclusion
Detection of QoI-resistant isolates with A143 cytochrome b alleles were detected in the
UK during the 2001-2002 growing season, within 4-5 years of the introduction of this
highly effective fungicide group. This study also showed that R. collo-cygni followed a
similar pattern for resistance development (i.e. rapid replacement of the QoI-sensitive
population) to that of the wheat leaf blotch disease caused by the fungus Mycosphaerella
graminicola, which is closely genetically related to R. collo-cygni.
Acknowledgements
This work was financially funded by the Scottish Government Rural and Environment
Research and Analysis Directorate Work package 1.4 (Barley Pathology). Rothamsted
Research receives grant-aided support from the Biotechnology and Biological Sciences
Research Council.
Reference
Fraaije BA, Cools HJ, Fountaine JM, Lovell DJ, Motteram J, West JS, Lucas JA. 2005: QoI
resistant isolates of Mycosphaerella graminicola and the role of ascospores in further
spread of resistant alleles in field populations. Phytopathology, 95, 933-941.
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30
Phakopsora pachyrhizi: The Performance of
Soybean Rust Fungicides over Years and
Regions in Brazil
C.V. GODOY
Embrapa Soybean, Caixa Postal 231, 86001-970, Londrina, Paraná, Brazil
Abstract
The efficiency of fungicides for the control of soybean rust, caused by Phakopsora pachyrhizi,
was analyzed from 2003-04 to 2008-09 using uniform field trials (UFTs) carried out in several
soybean regions in Brazil. One premix of strobilurin and triazole (cyproconazole 24 g ai ha-1 +
azoxystrobin 60 g ai ha-1) and two triazoles (tebuconazole 100 g ai ha-1 and tetraconazole 50 g ai
ha-1) were selected from UFTs to illustrate the changes of performance observed among seasons.
The disease severity between R5 (beginning seed development) and R6 (full seed) stage was
compared with that of the untreated check to determine the percentage of disease control. Until
2007-08, tebuconazole performed as well as the mixture and better than tetraconazole in 2006-07,
when the weather conditions were favorable for disease outbreaks. A failure of control was
observed in one trial in 2007-08, in Dourados, Mato Grosso do Sul, and reported in trials in other
Cerrado regions. In 2008-09, tebuconazole performed similarly to tetraconazole, with median
around 50% of control while the mixture was above 80%. This lower efficiency of triazoles,
observed since 2007, has been associated with the selection of less sensitive population of the
fungus due to the intensive use of straight triazoles. Since then, the use of straight triazoles has
been decreasing and the use of triazole-strobilurin mixture was intensified as the major strategy
to reduce the risk of resistance.
Introduction
C.V. Godoy
causing premature defoliation and early maturity. Yield losses up to 80% have been
reported (Bromfield, 1984; Yorinori et al., 2005).
In general, optimum climatic conditions for the crop are considered equally
favorable for the establishment and development of the rust. The fungus infects the plants
at temperatures ranging from 18 to 26.5oC and needs a minimum dew period of 6 hours
(Melching et al., 1989). Continuous leaf wetness, promoted either by dew or rain,
favours disease development after its establishment, with rainfall being considered an
important factor in determining epidemic levels in the field (Tschanz, 1984; Del Ponte et
al., 2006). The conditions in large parts of Brazil are conducive for year-round survival
of the pathogen (Pivonia and Yang, 2004), not only on other (alternative) hosts, but also
on soybean sown during winter in the central Cerrado region.
Several strategies have been adopted in Brazil to manage this disease which include
the use of cultivars belonging to the early ripening group planted in the beginning of the
crop season; the adoption of the free host period, a period of 60 to 90 days from July to
September during which farmers are restricted from planting soybean except under
strictly controlled conditions; the use of resistant cultivars, when available, and
fungicides applied preventatively or when first symptoms become visible.
Fungicides used for the soybean rust control belong mostly to the QoI (strobilurin)
and the DMI (triazole) compounds inhibiting the mitochondrial respiration or sterol
biosynthesis, respectively. Until 2007, straight triazoles were widely used to control rust
but, since then, a lower efficiency has been reported in the fields.
More than 60 different commercial fungicides are currently labeled for soybean rust
control in Brazil, and many of them have been evaluated annually since 2003-04 in a
network of standardized uniform field trials (UFTs), coordinated by Embrapa Soybean, a
research unit of the Brazilian Agricultural Research Corporation.
The objective of this work was to illustrate the changes in triazoles performance
over years, using results from UFTs.
The data base for this work consisted of UFTs conducted in Brazil during the 2003-
04 to 2008-09 growing seasons (Godoy, 2005a,b; Godoy et al., 2007; Godoy et al., 2009)
in the main soybean regions. The trials used standard protocols with mostly two
fungicide applications made at recommended rates and timed at specific growth stages,
starting at bloom stage (R1/ R2) and repeated 21 days later, around R5 (beginning pod)
(Fehr et al., 1971). The experimental design was a complete randomized block with four
replications. Each replicate plot was at least six rows wide and 6 m long, with the middle
four rows treated with fungicide. Applications were made with a CO2-pressurized
backpack sprayer equipped with a spray wand calibrated to deliver 150 or 200 l ha-1, with
fungicides applied at label-recommended rates.
Disease and yield data were obtained from the centre two rows. General crop
management was adapted to local commercial production practices, except that most
trials were sowed later in the season to increase the probability of epidemic development
owing to get the inoculum from the earlier plantings. Soybean rust epidemics developed
naturally in all trials. Plot-level disease severity index was estimated with the aid of a
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diagrammatic scale (Godoy et al., 2006) as the percentage of leaflet area covered with
uredinia associated chlorosis. Disease severity represented the mean of assessments from
20 leaflets in each of three canopy layers (lower, middle, and upper) taken at four
locations within each plot. Defoliation was taken into account attributing 100% of
disease severity when it was observed in some parts of the canopy. Soybean rust
epidemics developed naturally in all trials. Disease assessments were made just prior to
fungicide application and again between R5 (beginning seed) and R6 (full seed) stage of
soybean development (Fehr et al., 1971). To illustrate the changes of fungicides
performance a mixture of cyproconazole 24 g ai ha-1 + azoxystrobin 60 g ai ha-1 (DMI +
QoI) and two triazoles, tebuconazole 100 g ai ha-1 [DMI (1)] and tetraconazole 50 g ai ha-
1
[DMI (2)] were chosen from all the fungicides evaluated since they were present in
most of the growing seasons. The data base for triazoles includes all commercial brands
that present a similar efficiency in UFTs. The percentage of disease control was related to
the untreated check, present in all trials. Trials with a final disease severity inferior to
20% in the untreated check were excluded from the data base using the premise that
experiments where disease pressure was very low would not be useful in determining
fungicide efficiency.
Box-plots were used to show the distribution of percentage of control among
treatments and growing season.
Results
The number of trials per treatment group varied among seasons and products (Table 1)
due to the variation of UFT numbers per growing season and the presence of generic
brands for tebuconazole and tetraconazole. Tetraconazole was not present in UFTs in
2005-06 and 2007-08.
Table 1: Number of trials for the mixture of cyproconazole + azoxystrobin (DMI + QoI), tebuconazole
[DMI (1)] and tetraconazole [DMI (2)] in the growing seasons from 2003-04 to 2008-09.
2003-04 9 25 13
2004-05 15 25 25
2005-06 3 16 -
2006-07 16 75 27
2007-08 8 8 -
2008-09 41 122 82
The median of the disease control for 2003-04 and 2004-05 growing seasons was
above 80% and all the fungicides showed a similar pattern in performance (Figure 1A, B).
In 2005-06, DMI (1) was as efficient as DMI + QoI (Figure 1C), with a lower
performance compared to the previous growing season. The 2006-07 growing season was
very favorable for disease outbreaks and DMI (1) performed better (median 65%) than
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C.V. Godoy
DMI (2) (median 47%) (Figure 1D). Up to this season fungicides were grouped
according to the efficiency and DMI (1) was the only fungicide grouped together with the
mixture of triazoles- strobilurins (Tecnologias, 2006). In the 2007-08 growing season,
although the median of disease control was similar for DMI + QoI and DMI (1), in one
specific trial, DMI (1) was not efficient (Dourados, Mato Grosso do Sul) (Figure 1E).
This was the only trial conducted in the Cerrado region in this growing season. In 2008-
09 the median of efficiency of DMI (1) (51%) was similar to that of DMI (2) (52%) and
lower than that of the mixture (above 80%) (Figure 1F).
100 A 100
D
80 80
Control (%)
Control (%)
60 60
40 40
20 20
0 0
DMI+QoI DMI (1) DMI (2) DMI+QoI DMI (1) DMI (2)
Fungicides Fungicides
100 B 100
E
80 80
Control (%)
Control (%)
60 60
40 40
20 20
0 0
DMI+QoI DMI (1) DMI (2) DMI+QoI DMI (1)
Fungicides Fungicides
100 100
C
F
80 80
Control (%)
Control (%)
60 60
40 40
20 20
0 0
DMI+QoI DMI (1) DMI+QoI DMI (1) DMI (2)
Fungicides Fungicides
Figure 1: Percentage of soybean rust control related to the untreated check for mixture of cyproconazole
+ azoxystrobin (DMI + QoI), tebuconazole [DMI (1)] and tetraconazole [DMI (2)] in 2003-04 (A), 2004-
05 (B), 2005-06 (C), 2006-07 (D), 2007-08 (E) and 2008-09 (F) growing seasons. ■ - Median, □ - 25%-
75%, І - 1%-99%, o – outliers, * - extremes.
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Discussion
The efficiency of fungicides varied among seasons, and this was expected since
application timing in the Brazilian UFTs was crop-phenology-based with a fixed number
of 2 applications. A weaker efficiency of all fungicides can normally be expected in
growing seasons favorable to disease outbreaks.
The UFTs from 2003-04 to 2006-07 were reviewed in a meta-analytical synthesis
(Scherm et al., 2009) which showed that for soybean rust control in general, triazole
fungicides applied alone performed better than strobilurins alone, but there was a wide
range in efficiency among individual triazoles. Generally, mixtures of strobilurins with
triazoles improved disease control and yield compared with either class alone, although
straight tebuconazole and prothioconazole often performed as well as the mixture. The
triazolinthione prothioconazole that belongs to a new class of azole compounds was
never recommended alone and was not present in the Brazilian market until 2010.
Until 2007, because of the high level of efficiency and the presence of generic
brands in the market, straight triazole compounds, especially tebuconazole, were used
quite frequently in sequential applications and recommended for application under
curative conditions for rust control. The weaker efficiency of straight triazole compounds
was observed in some regions for the first time at the end of the 2007-08 growing season,
mainly in central Cerrado regions. In this season, only one UFT was carried out in the
Cerrado region, and tebuconazole showed no disease control. In 2008-09 this weaker
efficiency was again observed in UFTs, especially later in the season. Up to 2008-09, in
the Southern region of Brazil, the triazoles performed as well as mixtures of triazoles-
strobilurins but were recommended only at the beginning of the growing season and
never as sequential application, following the general FRAC recommendations for the
use of SBI fungicides. In the Cerrado regions, the recommendation since 2008-09 was to
avoid straight triazoles and to use preferably mixtures of triazoles-stroblurin for soybean
rust control (Consorcio Antiferrugem, 2010).
The weaker fungicide efficiency was associated with the selection of less sensitive
populations of the fungus to triazoles. Several factors contributed to this fact. The
average latent period (the time from infection to the next generation of inoculum) of P.
pachyrhizi is about 7-9 days (Marchetti et al., 1976, Alves et al., 2006) and the disease
tends to become obvious at bloom stage (around 45 days after sowing). At the same time
also first fungicide applications are started, suggesting that at the maximum 8-10 cycles
of infection can take place in a single crop season and they are all likely to be exposed to
fungicide selection. Another factor favoring selection is that in Brazil the window for
soybean sowing reaches from October to December and a double crop season is possible
in some regions. Therefore, a generation overlap is possible, all being exposed to
fungicide droplets which are easily spread by the wind from one field to another.
It was expected that the intercrop period, where the host free period is adopted,
would contribute do reduce this less sensitive population but UFTs from 2009-10 (data
not shown) do not confirm this hypothesis. The efficiency of triazoles in 2009-10 was
even weaker than 2008-09 and this was observed in all soybean production regions.
A sensitivity monitoring program was started by Bayer CropScience in 2005 and up
to the end of the season 2007-08, sensitivity monitoring data, generated with soybean
rust samples from different Brazilian states did not show a significant increase in
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C.V. Godoy
tebuconazole EC50 values. However, samples collected later in the season presented
higher EC50 values, especially in the Cerrado regions (Mehl, 2009). Higher EC50 values
were again observed in 2008-09, later in the season predominantly in central Cerrado
regions (Calegaro et al., 2009). However, the EC50 values observed in sensitivity
monitoring tests do not always explain the erosion of triazole efficiency observed in the
field (Koga et al., 2010) and further studies must be carried on.
Triazoles have been the leading agents for the control of fungal diseases of plants,
man and animals for over 30 years. For plants, despite their long-term widespread use,
resistance developed slowly, in cereal pathogens, for example, loss of efficacy in practice
is still rare (Cools et al., 2006). The reduced performance of triazoles, observed seven
years after P. pachyrhizi introduction in Brazil reached a level at which rust control with
straight triazoles becomes unsatisfactory. Since 2007, the use of straight triazoles has
been decreasing and the use of triazole-strobilurin mixture was intensified as the major
strategy to reduce the risk of resistance.
Acknowledgments
The author thanks all the collaborators of UFTs and the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) for a research fellowship. This
research was funded by CNPq (Edital CNPq/MAPA/SDA no 064/2008) and Embrapa
project 02.07.01.002.00.
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sobre a ferrugem da soja. Ferrugem asiática da soja, 37-59. Ed. Zambolim, L. Suprema
Gráfica e Editora Ltda. Viçosa MG, Brazil.
Bromfield, K.R. (1984): Soybean Rust. Monograph 11, American Phytopathological Society, St.
Paul, MN, USA.
Calegaro, P., Geraldes, J., Kemper, K., Pereira, R., Santos, C., Singer, P. (2009): Resultados do
monitoramento de resistência de Phakopsora pachyrhizi a fungicidas em soja. Reunião do
Consórcio Antiferrugem Safra 2008-09. Godoy, C.V., Seixas, C.D.S., Soares, R.M. (Eds).
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Cools, H.J., Fraaije, S.H., Kim, S.H., Lucas, J.A. (2006): Impact of changes in the target P450
CYP51 enzyme associated with altered triazole-sensitivity in fungal pathogens of cereal
crops. Biochemical Society Transactions, 34, 1219-1222.
Del Ponte, E.M., Godoy, C.V., Li, X., Yang, X.B. (2006): Predicting severity of Asian soybean
rust epidemics with empirical rainfall models. Phytopathology, 96,797-803.
Fehr, W.R., Caviness, C.E., Burmood, D.T., Pennington, J.S. (1971): Stage of development
descriptions for soybeans, Glycine max (L.) Merrill. Crop Science 11, 929–931.
FRAC (2010): General recommendations for the use of SBI fungicides. Available:
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Godoy, C.V. (2005a): Ensaios em rede para controle de doenças na cultura da soja. Safra
2004/05. Série Documentos 266, Embrapa, Londrina, Brazil, 183 pp.
208
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Godoy, C.V. (2005b): Resultados da rede de ensaios para controle químico de doenças na cultura
da soja. Safra 2003/04. Série Documentos 251, Embrapa, Londrina, Brazil, 88 pp.
Godoy, C.V., Koga, L.J., Canteri, M.G. (2006): Diagrammatic scale for assessment of soybean
rust severity. Fitopatologia Brasileira, 31, 63-68.
Godoy, C.V., Pimenta, C.B., Wruck, D.S., Siqueri, F.V., Feksa, H.R., Santos, I., Nunes Junior, J.,
Ito, M.A., Meyer, M.C., Martins, M.C., Almeida, N.S., Andrade, P., Souza, P.I.M., Abud,
S., Furlan, S.F. (2007): Eficiência de fungicidas para controle da ferrugem asiática da soja,
Phakopsora pachyrhizi, na safra 2006/07. Resultados sumarizados dos ensaios em rede. In:
Anais do Simpósio Brasileiro de Ferrugem Asiática da Soja. Série Documentos 281,
Embrapa, Londrina, Brazil, pp. 55–73.
Godoy, C.V., Silva, L. H.C.P., Utiamada, C.M., Siqueri, F.V., Lopes, I.O.N., Roese, A.D.,
Machado, A.Q., Forceline, C.A., Pimenta, C.B., Nunes, C.D.M., Cassetari Neto, D.,
Jaccoud Filho, D.S., Fornarolli, D. A., Wruck, D.S., Ramos Junior, E. U., Borges, E.P.,
Juliatti, F.C., Feksa, H.R., Campos, H.D. Nunes Junior, J., Silva, J.R.C., Costamilan, L.M.,
Carneiro, L.C., Sato, L.N., Canteri, M.G., Ito, M.A., Iamamoto, M.M., Ito, M.F., Meyer,
M.C., Costa, M.J.N., Dias, M.D., Martins, M.C., Lopes, P.V., Souza, P.I.M., Barros, R.,
Balardin, R.S., Igarashi, S., Silva, S.A., Furlan, S.H., Carlin, V.J. (2009): Eficiência de
fungicidas para controle da ferrugem asiática da soja, Phakopsora pachyrhizi, na safra
2008/09. Resultados sumarizados dos ensaios cooperativos 2009. Circular Técnica 69.
Embrapa, Londrina, Brazil. 12pp.
Koga, L.J., Lopes, I.O.N., Godoy, C.V. (2010): Sensitivity monitoring of Phakopsora pachyrhizi
population to triazoles in Brazil. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H. Kuck, P.E.
Russell, H. Lyr. Modern Fungicides and Antifungal Compounds VI. DPG-Verlag,
Braunschweig, Germany, 211-216.
Marchetti, M.A., Melching, J.S., Bromfield, K.R. (1976): The effects of temperature and dew
period on germination and infection by urediospores of Phakopsora pachyrhizi.
Phytopathology, 66: 461-463.
Mehl, A. (2009), Phakopsora pachyrhizi: sensitivity monitoring and resistance management
strategies for DMI and QoI fungicides. In: V Congresso Brasileiro de Soja, Mercosoja
2009, Goiânia. Londrina : Embrapa Soja, 2009. v. CD-Rom.
Melching, J.S., Dowler, W.M., Koogle, D.L., Royer, M.H. (1989): Effects of duration, frequency,
and temperature of leaf wetness periods on soybean rust. Plant Disease, 73, 117-122.
Pivonia, S., Yang, X.B. (2004): Assessment of the potential year-round establishment of soybean
rust throughout the world. Plant Disease, 88, 523-529.
Scherm, H., Christiano, R.S.C., Esker, P.D., Del Ponte, E.M., and Godoy, C.V. (2009):
Quantitative review of fungicide efficacy trials for managing soybean rust in Brazil. Crop
Protection, 28, 774–782.
Sinclair, J.B., Hartman, G.L. (1999): Soybean rust. Compendium of soybean diseases, 25;26.
Hartman, G.L., Sinclair, J.B., Rupe , J.C. (Eds) 4th ed. APS Press, Saint Paul, MN.
Tecnologias de produção de soja - região central do Brasil 2007. (2006): Londrina: Embrapa
Soja: Embrapa Cerrados: Embrapa Agropecuária Oeste. 225p.
Tecnologias de produção de soja - região central do Brasil 2009 e 2010. (2008): Londrina:
Embrapa Soja: Embrapa Cerrados: Embrapa Agropecuária Oeste. 262p.
Tschanz, A.T. (1984): Soybean rust epidemiology: Final Report. Asian Vegetable Research and
Development Center, Shanhua, Taiwan.
Yorinori, J.T., Paiva, W.M., Frederick, R.D., Costamilan, L.M., Bertagnolli, P.F., Hartman, G.E.,
Godoy, C.V., and Nunes Junior, J. (2005): Epidemics of soybean rust (Phakopsora
pachyrhizi) in Brazil and Paraguay from 2001 to 2003. Plant Disease, 89, 675-677.
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C.V. Godoy
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31
Sensitivity Monitoring of Phakopsora
pachyrhizi Populations to Triazoles in Brazil
Abstract
Asian soybean rust, caused by Phakopsora pachyrhizi, was reported in 2001 in South America
and spread quickly to Brazilian producing areas. It is considered one of the main foliar diseases
of the crop. Fungicides used for the control belong to QoI (strobilurins) and DMI (azole)
compounds. A weaker efficacy of straight azole compounds was observed in some regions in the
end of 2006/07 cropping season. To determine if the problem observed was due to resistance
development, a sensitivity monitoring test was carried out in 2008/09 and 2009/10 to detect
possible changes in the EC50 values of the fungal population. The tests were carried out according
to FRAC methodology. The azoles tested were cyproconazole, metconazole, tebuconazole and
prothioconazole. Leaves samples infected with P. pachyrhizi were sent from several Brazilian
states. The spores collected were inoculated in detached treated leaves, with four replicates.
Disease severity was evaluated 15 days after inoculation. The EC50 values were estimated by
Proc Probit. Differences in EC50 values among the populations were statistically significant
(P<0.05). The results showed oscillation occurrence of EC50 values in the P. pachyrhizi
population from different locations through the cropping seasons.
Introduction
Generally, the rust pathogen group is classified as a low risk group in regard of resistance
development (Brent, 1999). However, Phakopsora pachyrhizi Syd. & P. Syd., that causes
Asian rust on soybean (Glycine max (L.) Merr.) presents multiple risk factors, such as
high genetic variability, abundant sporulation, short latent period and a large numbers of
hosts (Yamaoka et al., 2002; Hartman et al., 2005; Slaminko et al., 2008). Besides the
characteristics of the fungus, in Brazil soybean is planted in large areas and the planting
window reaches from October to December. Double cropping is possible in some regions.
The average of fungicide applications per crop season is between two and three
(Consorcio antiferrugem, 2010).
Up to the 2006/07 crop season, due to the high level of efficacy, the low price and
the availability of generic brands in the market, straight azole fungicides (DMIs) were
used to control soybean rust. Lower efficiency of these fungicides was observed in some
regions at the end of that crop season.
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Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
The objective of this work was to investigate this lower efficiency of azoles to
control soybean rust using sensitivity monitoring tests.
Monitoring bioassays were carried out using a detached leaf method, developed by Bayer
CropScience and approved by FRAC (PHAKPA, 2006) to evaluate the EC50 values of P.
pachyrhizi populations. Soybean leave samples infected with rust were collected in nine
Brazilian states in 2008/09 (36 populations) and in seven states in 2009/10 (46
populations). The fungus was stored at 5⁰C for a maximum of 2 days until the bioassays
started.
The azoles evaluated were those with a registration for single applications or high
efficiency in rust control according to earlier field experiments (Godoy et al., 2007).
In the 2008/09 crop season, the rates tested were 0; 0.125; 0.25; 0.5; 1.0; 2.0; 4.0;
8.0; 16.0; 32.0 ppm for cyproconazole, metconazole, tebuconazole and prothioconazole.
In 2009/10, the prothioconazole rates were reduced to 0; 0.031; 0.065; 0.125; 0.25; 0.5;
1.0; 2.0; 4.0; 8.0 ppm because during the first cropping season it had turned out that
already at 0.25 ppm 100% control was observed resulting in a too narrow distribution of
EC50 values.
The experimental design was completely randomized with four repetitions; each
repetition consisted of a Petri dish with three leaflets. Urediniospores from infected
leaves were harvested using a vacuum collector. In each sample the percentage of
germination was assessed. After inoculation, the leaflets were incubated at 25 ± 2 ºC
applying a 12/12h light/dark cycle and a relative humidity of > 60%. Disease severity
was evaluated by using a diagrammatic scale (Godoy et al., 2006) 15 days after
inoculation. The EC50 values were estimated by Proc Probit, SAS®, version 9.1.3. EC50
estimates were determined using the data of the four replicates of each fungicide
concentration instead of adopting the mean percentage of control.
Results
Although the estimates of EC50 obtained from the mean percentage of control had
provided a model with highest quality fit, the adjusted models using the repetitions
provided lower variances and, consequently, more narrow confidence intervals for EC50,
indicating that their estimates were more accurate.
Differences in EC50 values (using commercial formulations) among P. pachyrhzi
populations were statistically significant (P <0.05).
Figures 1 (2008/09) and 2 (2009/10) demonstrate the mean, the lower and the upper
limits for EC50 values for all the populations sampled during the crop seasons. The mean
values of EC50 for cyproconazole, metconazole, tebuconazole and prothioconazole during
the first and second year of monitoring are shown in Table 1.
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Figure 1: EC50 values (▪) and 95% fiducial inferior and superior limits distribution for cyproconazole (A),
metconazole (B) and tebuconazole (C) for P. pachyrhizi populations in different regions in Brazil.
Growing season 2008/09.
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C
EC50 (ppm)
Jan
Mar
Mar
Mar
Mar
Feb
Feb
Feb
Feb
Mar
Mar
Mar
Mar
Mar
Mar
Mar
Mar
Mar
Mar
Mar
Feb
Feb
Feb
Mar
Mar
Mar
Mar
Jan
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Figure 2 continuing
Figure 2: EC50 values (▪) and 95% fiducial inferior and superior limits distribution for cyproconazole (A),
metconazole (B), tebuconazole (C) and prothioconazole (D) for P. pachyrhizi populations in different
regions in Brazil. Growing season 2009/10.
Table 1: Mean, minimum and maximum EC50 values (ppm) from Figures 2 and 3.
Discussion
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Acknowledgements
The first author thanks CNPq and FRAC-Brasil for the fellowship.
References
Brent, K.J. (1999): Resistência a fungicidas em patógeno de plantas cultivadas: como manejá-la?
[S.l.: s.n.] (FRAC Monograph Nº 1).
Consorcio Antiferrugem (2010): Tabela de custo. Available:
http://www.consorcioantiferrugem.net/?Conhe%E7a_a%26nbsp%3Bferrugem%26nbsp%3
B:Tabela_de_custo
Dekker, J. (1985): The fungicide resistance problem: will it grow worse? EPPO Bulletin, 15,
337-344.
Godoy, C.V., Koga, L.J., Canteri, M.G. (2006): Diagrammatic scale for assessment of soybean
rust severity. Fitopatologia Brasileira, 1, 63-68.
Godoy, C.V., Pimenta, C.B., Miguel-Wruck, D.S., et al (2007): Eficiência de fungicidas para
controle da ferrugem asiática da soja, Phakopsora pachyrhizi, na safra 2006/07. Resultados
sumarizados dos ensaios em rede. Londrina: Embrapa Soja. (Embrapa Soja. Circular
Técnica 42).
Hartman, G.L., Miles, M.R., Frederick, R.D. (2005): Breeding for resistance to soybean rust.
Plant Disease, 89, 664-665.
Köller, W., Scheinpflug, H. (1987): Fungal resistance to sterol biosynthesis inhibitors: a new
challenge. Plant Disease, 71, 1066-1074.
Niklaus, J., Grünwald, N.J., Sturbaum, A.K., et al (2006): Selection for fungicide resistance
within a growing season in field populations of Phytophthora infestans at the center of
origin. Phytopathology, 96, 1397-1403.
Slaminko, T. L., Miles, M. R., Frederick, R. D., et al (2008): New legume hosts of Phakopsora
pachyrhizi based on greenhouse evaluations. Plant Disease, 92, 767-771.
Yamaoka, Y., Fujiwara, Y., Kakishima, M., et al (2002): Pathogenic races of Phakopsora
pachyrhizi on soybean and wild host plants collected in Japan. Journal of General Plant
Pathology, 68, 52-56.
PHAKPA detached leaf monitoring method BCS (2006): Available:
http://www.frac.info/frac/Monitoring_Methods/Monitoring_Methods.htm
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32
Analysis of Azole Fungicide Resistance in
Mycosphaerella fijiensis, Causal Agent of Black
Sigatoka
Abstract
Mycosphaerella fijiensis causes black Sigatoka, economically the most important disease in
bananas and plantains. Disease control is mainly achieved through the application of specific
fungicides, including azoles. However, their intensive use has favoured the appearance of
resistant strains. In this study, we examined the variation in sensitivity to azole fungicides in field
isolates of M. fijiensis collected in Ecuador, Brazil, Costa Rica, Africa, and Southeast Asia using
a 96-well microtitre plate assay. The isolates tested showed a wide range in sensitivity to the
azole fungicides cyproconazole, propiconazole and imazalil. A clear pattern of cross-sensitivity
was found among the isolates for cyproconazole and propiconazole. Sequence analysis of the
CYP51 gene amplified from sensitive and resistant strains, showed the presence of several point
mutations located around the putative substrate binding site in the encoded protein. The most
common mutations detected were Y136F, A313G, Y463D, Y463H, Y463N, and Y463S. This
study provides important preliminary information for the understanding of the mechanisms of
azole resistance in this fungus and in the future will help to optimize the use of azoles in the
control of black Sigatoka.
Introduction
P. Chong et al.
most important disease affecting bananas and plantains worldwide. The fungus
originated in Southeast Asia from where it spread to Africa and Latin America. Black
Sigatoka produces extensive necrotic lesions in the leaves, decreasing the photosynthetic
capacity of the plant, which results in lower yield and quality (Stover and Simmonds,
1987; Marin et al., 2003).
Azole fungicides have been used for controlling black Sigatoka as early as 1987, and
since 1991 propiconazole became widely used against this disease. Currently, several
other azole fungicides are being used in spraying programs against M. fijiensis including
difenoconazol, bitertanol and epoxiconazol. Fungicide resistance has been described in M.
fijiensis for the majority of the systemic fungicides that are applied, including strobilurins,
benzimidazoles and azoles (Romero and Sutton, 1997; Canas-Gutierrez et al., 2006,
Amil et al., 2007).
Beside the more general information given by the FRAC Banana Working Group
(www.frac.info), only few studies have as yet examined sensitivity of M. fijiensis to
azole fungicides other than propiconazole, and hence there is mostly only limited
knowledge of the degree of cross-resistance with other azoles. Moreover, the effect of
mutations in the target site of these compounds in M. fijiensis (the CYP51 gene) is
unknown. Given the importance of the disease in global banana production there is an
urgent need to understand the relationships between the genetic background of the
different pathogen populations and their fungicide sensitivity profile.
Here, we determined the in vitro sensitivity of 40 field isolates of M. fijiensis to the
azoles propiconazole, cyproconazole and imazalil and examined the presence of
mutations in the coding sequence of the CYP51 gene.
M. fijiensis strains
Forty strains of M. fijiensis originating from Africa, South-East Asia and Latin America
and two M. eumusa strains from Thailand and Vietnam were used in this study (Table 1).
Strains were grown in Potato-Dextrose Agar (PDA) (BBL, Becton Dickinson
Cockeysville, USA) at 27 °C.
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Table 1: List and origin of strains of M. fijiensis or M. eumusa (*) used in this study.
Origin Strain
Brazil AM_1, AM_5, AM_25, AM_30, AM_47, AM_68, AM_134, AM_141
Costa Rica Z4_7, Z4_11, Z4_14, Z4_16, Z8_12, Z8_15, Z8_17, Z8_18, Ca1_5, Ca5_16,
Ca6_11, Ca10_13, ZTSC_59, ZTSC_77, ZTSC_79, ZTSC_87
Ecuador E22, GS_4, GS_10, RN_3, RN_5, RS_13, SaR_2, SaR_5
Indonesia X845
Philippines X846
Taiwan X847
New Caledonia X848
Burundi X849
Gabon X851
Tanzania X852
Thailand X870*
Vietnam X874*
Cameroon C_86
CYP51 sequencing
The CYP51 gene along with 333 bp of promoter sequences and 85 bp of terminator
sequences was amplified using primers CYP51_Mfijien_F1 (5’-
AAGGTCATATCGCAGG-3’) and CYP51_Mfijien_R1 (5’-GAATGTTATCGT-
GTGACA-3’). The PCR mix preparation was standard and the PCR program comprised
34 cycles of five min denaturation at 94°C followed by 30 sec at 94°C, 30 sec of
annealing at 55°C and 90 sec of extension at 68°C. An additional extension step of 7 min
at 72°C was performed at the end. DNA sequencing was performed by Macrogen Inc
(Seoul, Korea) directly on the PCR products. In order to sequence the entire length of the
amplified PCR product, four sets of primers were used in the sequencing reactions:
CYP51_Mfijien_F2 (5’- ACAGAAACATCACC-TCC -3’, CYP51_Mfijien_F3 (5’-
ATTGCTTCACTTTCATCC-3’), CYP51_Mfijien_F4 (5’-CTCTACCAC GATCTCG
AC-3’) and CYP51_Mfijien_R2 (5’-GATATGGATATAGTTGTC – 3’). The quality of
the obtained sequences were manually inspected and once verified assembled in contigs
using CLC DNA Workbench software (CLC bio, Denmark). Finally, multiple alignments
of the translated amino acid sequences allowed the identification of mutations that could
be responsible for the observed fungicide resistance.
Results
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P. Chong et al.
mg/L for tolerant strains and ≥ 0.79 mg/L for the resistant strains. A similar pattern was
found for imazalil with ≤ 1.90 mg/L for sensitive strains, 2.00 to 9.30 mg/L for tolerant
strains and ≥ 10.00 mg/L for resistant strains (Table 2). A clear pattern of cross-
sensitivity among the isolates was present for propiconazole and cyproconazole but not
for imazalil.
Discussion
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study showed the presence of mutations in the CYP51 gene, which correlated to
propiconazole resistance (Canas-Gutiérrez et al., 2009). Here, we examined the
sensitivity of 40 M. fijiensis isolates to three different azole fungicides, sequenced the
entire CYP51 gene and promoter region and correlated the genotypic and phenotypic data.
The different levels of sensitivity found allowed the classification of the strains in
three groups: sensitive, tolerant and resistant. Cross-resistance was observed between
propiconazole and cyproconazole, but not between any of these two fungicides and
imazalil.
A high degree of polymorphism in the CYP51 gene has been reported in many fungi
in relation with azole resistance, including the closely related wheat pathogen
Mycosphaerella graminicola (Fuckel) J. Schröt. (Leroux et al., 2007). In this study,
sequencing of the CYP51 gene from the 40 M. fijiensis strains also showed many
mutations that cause changes in the deduced amino acid sequences of the encoded
proteins. Several mutations were only present in resistant strains, but polymorphisms
were also found in sensitive strains. These may relate to natural genetic variation in this
locus and probably do not to contribute to resistance development.
The highest degree of variation in the coding region of the CYP51 gene was found in
the strains originating from Costa Rica. Most of these mutations were already reported to
contribute to propiconazole resistance (Canas-Gutiérrez et al., 2009). However, three
new mutations were identified in this study, namely A138G, A381G and G462A. These
amino acid changes are located in regions of the protein structure that is involved in
substrate recognition and thus could contribute to azole resistance (Canas-Gutiérrez et al.
2009). Further studies will extend resistance monitoring and will determine the molecular
characterization of identified resistant M. fijiensis strains.
Acknowledgements
PC is thankful to the Escuela Superior de Politécnica del Litoral, Guayaquil, Ecuador for
a travel fellowship. RA received a sabbatical fellowship from the National University of
Colombia, Sede Medellín. We acknowledge Syngenta Crop Protection AG, Basel,
Switzerland for providing azole fungicides.
References
Amil, A.F., Heaney S.P., Stanger C, Shaw, M.W. (2007): Dynamics of QoI Sensitivity in
Mycosphaerella fijiensis in Costa Rica During 2000 to 2003. Phytopathology, 97, 1451-
1457.
Canas-Gutiérrez, G.P., Angarita-Velásquez M.J., Restrepo-Flórez J.M., Rodriguez, E., Moreno,
C.X., Arango, R. (2009): Analysis of the CYP51 gene and encoded protein in
propiconazole-resistant isolates of Mycosphaerella fijiensis. Pest Management Science, 65,
892-899.
Canas-Gutierrez, G.P., Patino, L.F., Rodriguez-Arango, E., Arango, R. (2006): Molecular
Characterization of Benomyl-resistant Isolates of Mycosphaerella fijiensis, Collected in
Colombia. Journal of Phytopathology, 154, 403-409.
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P. Chong et al.
Leroux, P., Albertini, C., Gautier, A., Gredt, M., Walker, A.S. (2007): Mutations in the CYP51
gene correlated with changes in sensitivity to sterol 14-demethylation inhibitors in field
isolates of Mycosphaerella graminicola. Pest Management Science, 63, 688-698.
Marin, D.H., Romero, R.A., Guzman, M., Sutton, T.B. (2003): Black Sigatoka: An increasing
threat to banana cultivation. Plant Disease, 87, 208-222.
Montoya, J.E., David, L.E., Brito, T.J., Sánchez, D.A., Beltrán, E.R., Arango, R.E. (2006): Use
of a microtiter plate dilution assay to measure activity of antifungal compounds against
Mycosphaerella fijiensis Morelet. Revista Facultad Nacional Agronomia Medellín, 59,
3425-3433.
Romero RA, Sutton TB (1997): Sensitivity of Mycosphaerella fijiensis, causal agent of black
Sigatoka of banana, to propiconazole. Phytopathology, 87, 96-100.
Stover, R.H., and Simmonds N.W. (1987): Bananas, 3rd Ed., Tropical Agriculture Series.
Longman, Burnt Mill, Harlow, England.
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33
Evolution of Resistance to Fungicides in
Populations of Mycosphaerella graminicola:
Emergence of New Phenotypes Highly Resistant
to DMIs
Abstract
Sterol 14α-demethylation inhibitors (DMIs) have been widely used in many European countries
and erosion of efficacy, correlated with significant shifts in sensitivity of M. graminicola
populations, has been recorded for most of them. Recently, strains highly resistant to DMIs have
been isolated from French, English and Irish populations. The aim of our study was to determine
the phenotypical characteristics of these M. graminicola field isolates and to identify the possible
resistance mechanisms. Target alteration, linked to one or several changes in the gene Cyp51,
encoding sterol 14α-demethylase, was the basic resistance mechanism in all DMI-resistant strains.
Changes in Cyp51, combined with the overexpression of drug efflux transporters probably result
in multidrug resistance in the more resistant phenotypes. At last, some isolates moderately or
highly resistant to DMIs harbour an insertion in the Cyp51 promoter and/or new combinations of
already known mutations in the target gene. This work gives an updated overview of the M.
graminicola field strains resistant to DMIs: these recent findings should be taken into account to
adjust resistance and efficacy management strategies.
Introduction
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forms (i.e. thione/thiol) and has a specific chemical structure, suggesting that it does not
interact with the Cyp51 iron haem.
In pathogens of plants and humans, DMI resistance may be determined by (1)
alterations in Cyp51, decreasing the affinity of DMIs for their target site (2) Cyp51
overexpression, resulting in high levels of sterol 14α-demethylase and (3) an increase in
the efflux of DMIs, due to the upregulation of ABC (ATP-binding cassette) or MFS
(major facilitator superfamily) transporters in the membrane. Most of these efflux pumps
can transport various unrelated compounds and their overproduction may lead to multiple
drug resistance (MDR). A combination of these mechanisms, leading to the polygenic
control of DMI resistance, is commonly found in clinical isolates of Candida albicans
(Akins, 2005).
In M. graminicola, DMI resistance in European countries resulted mostly from
changes in Cyp51, at least until 2007 (Cools et al., 2005, Leroux et al., 2007). However,
a continuous shift in sensitivity to DMIs has been observed recently, consistent with
additional mechanisms, and this “quantitative” or “multiple-step” resistance is thus
considered to be polygenic (Cools and Fraaije, 2008, Chassot et al., 2008). Eight
categories of strains (TriR1-TriR8) displaying reduced sensitivity to DMIs have been
characterised in previous studies and were classified into two main groups, TriLR
(TriR1-TriR5) and TriMR (TriR6-TriR8) (Leroux et al., 2007; Figure 2). This
classification is based on in vitro responses to various families of DMIs including
pyridines (e.g. pyrifenox), imidazoles (e.g. prochloraz, triflumizole) and triazoles (e.g.
difenoconazole, epoxiconazole, fluquinconazole, propiconazole, tebuconazole,
triadimenol), and changes in the target encoded by Cyp51. Monitoring in 2008 and 2009
resulted in the identification of new strains, more resistant to DMIs than those found
before 2007. The aim of this study was then to characterize these new isolates, for their
phenotypic and genotypic characteristics.
Origin of samples
Nineteen isolates of Mycosphaerella graminicola were collected in 2009 in France and
UK after isolation from diseased wheat leaves. Irish isolates are a kind gift from
Professor O’Sullivan (Teagasc). Isolates collected before 2007 were used as reference
isolates representing the various phenotypes TriR1-TriR8 (Leroux et al., 2007). All
isolates were kept as mono-conidial cultures on a medium containing 20 g/L malt, 5 g/L
yeast extract and 12.5 g/L agar, at 17°C in the dark.
Bulk populations were produced by our large scale French monitoring and enabled
assessing the frequency of the various phenotypes in populations.
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Resistance Evolution
dark. EC50 values and resistance factors (RFs) were determined as described previously
(Leroux and Walker, 2011). MDR modulators amitriptyline, verapamil and
chlorpromazine were tested in addition of various fungicides for all resistant phenotypes.
Frequency of resistance in bulk populations were determined using discriminant
doses of fungicides (Leroux et al., 2007), including high doses of epoxiconazole,
prothioconazole, prochloraz and pyrifenox that detect only novel TriR strains.
Molecular procedures
DNA from the isolates was extracted using a sarcosyl-based protocol. PCR-amplification
was performed for the cyp51 gene as previously described (Leroux et al., 2007). Cyp51
promoter insertion was checked using the protocol from (Chassot et al., 2008).
2009 2009
Control before Control after
treatment treatment
Figure 1: Occurrence of field trials with the presence of new TriR strains in populations in Spring (left)
and Summer (right) 2009 in France.
225
a
226
Resistance factors (RFs)b
EC50
Fungicide (mg.L-1) MDR- MDR- MDR
Tri Tri Tri Tri Tri Tri Tri Tri Tri
TriS
Pyrifenox 0.0013 23.1 27.7 126.9 36.2 30.8 40.0 123.1 192.3 153.8 192.3 246.2 230.8
A.-S. Walker, M. Gredt and P. Leroux
Fenarimol 0.035 5.7 5.7 6.3 17.1 17.1 22.9 22.9 7.6 14.3 114.3 114.3 28.6
Prochloraz 0.0018 6.7 15.0 27.8 6.7 1.5 0.8 2.3 66.7 22.2 111.1 122.2 166.7
Triflumizole 0.0036 27.8 30.6 59.7 333.3 194.4 722.2 111.1 68.8 833.3 13889 1111 416.7
Bromuconazole 0.017 14.7 23.5 33.8 47.1 41.2 47.1 176.5 79.4 117.6 253.3 264.7 88.2
Cyproconazole 0.049 4.3 8.5 18.3 11.2 7.6 13.1 40.8 16.3 34.7 112.2 61.2 40.8
Difenoconazole 0.00025 20.0 1.6 2.8 32.0 32.0 60.0 60.0 6.3 6.0 800.0 600.0 28.0
Epoxiconazole 0.0020 5.0 8.5 18.3 25.5 11.0 23.0 75.0 29.9 60.0 225.0 210.0 60.0
Fenbuconazole 0.0020 12.5 1.5 5.9 50.0 50.0 75.5 175.0 18.6 15.0 650.0 500.0 40.0
Fluquinconazole 0.0031 6.5 14.2 34.7 20.3 14.5 22.6 64.5 75.8 103.2 387.1 387.1 112.9
Flusilazole 0.0057 12.3 19.3 21.9 31.6 40.4 43.9 140.4 61.4 105.3 438.6 350.9 122.8
Hexaconazole 0.0045 5.6 4.4 5.3 8.9 6.7 8.9 17.8 8.9 11.1 55.6 44.4 26.7
Metconazole 0.0020 10.0 8.0 7.9 15.5 10.0 17.5 50.0 23.6 30.0 110.0 150.0 40.0
Propiconazole 0.0037 12.2 20.5 29.1 35.1 27.0 54.1 94.8 70.9 54.1 459.5 270.3 162.2
Tebuconazole 0.011 18.2 1.8 6.4 74.5 51.8 90.9 363.6 12.0 18.2 636.4 500.0 45.5
Triadimenol 0.57 7.4 3.3 6.6 27.2 21.1 26.3 >43.9 9.6 26.3 >43.9 >43.9 26.3
Prothioconazole 0.040 3.8 5.3 10.2 7.8 7.0 7.3 12.5 16.3 25.0 25.0 25.0 12.5
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
Results for TriR1-R3 strains were not included because these phenotypes were not detected any more
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
Resistance Evolution
Table 2: Effects of drug transporter modulators on the effect of fungicides on germ-tube elongation in
Mycosphaerella graminicola field isolates (ND: Not Determined).
Fungicide Q value:
ratio [EC50 fungicide alone] / [EC50 fungicide+modulator]
Modulator TriS-TriLR-TriMR MDR-6 MDR-7
Prochloraz
Verapamil 1.0 3.0 3.3
Chlorpromazine 1.0-1.2 ND 5.0
Amitriptyline 1.0-1.5 ND 5.0
Epoxiconazole
Verapamil 0.8-1.0 3.0 2.7
Chlorpromazine 0.9-1.3 ND 5.0
Amitriptyline 0.8-1.3 ND 5.0
Tolnaftate
Verapamil 1.0-1.3 4.7 4.0
Chlorpromazine 1.0-1.1 ND 6.7
Amitriptyline 1.3-2.0 ND 5.0
Boscalid
Verapamil 1.0 ND 2.5
Chlorpromazine 0.8-1.0 ND 2.5
Amitriptyline 1.0-1.2 ND 2.2
The last three categories of phenotypes (MDR-6, MDR-7 and MDR-10) exhibit very
high cross-resistance for most tested DMIs; they therefore named as TriHR strains (Table
1; Figure 2). Positive cross-resistance was also noticed with tolnaftate, a squalene
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Conclusion
Finally, M. graminicola populations keep evolving and new resistant phenotypes can
regularly be described, surely because DMIs fungicides are not likely to be abandoned in
wheat disease management. At least three resistance mechanisms, i.e. target alteration,
target overexpression and drug transporters overexpression, seem to be responsible for
these increasing RFs observed in resistant isolates and can also cumulate in a single
strain, maybe without any evident fitness penalty.
Some of the new phenotypes (TriR5+, TriR8+, TriR9 and TriR11), classified among
the TriMR group, may not exhibit high resistance risk and could be controlled by
optimized chemical strategies. The situation may be different for TriHR-MDR strains.
Further work is of course needed to understand more accurately how the MDR
mechanism is responsible for azole, and other unrelated modes of action, resistance in M.
graminicola. Especially, concerned drug transporters need to be identified, as well as the
genome alterations responsible for their overexpression. Going to more practical
concerns, an estimation of their fitness and of the field efficacy losses they could
generate need to be understood if one wants to propose efficient preventive anti-
resistance strategies. More precisely, as these strains exhibit positive cross-resistance
between all DMI subgroups, SDHIs and QoIs, all three families being largely used on
wheat, qualitative and quantitative selective pressure of the various molecules need to be
estimated.
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Resistance Evolution
Y137F
Y137 Mut 459, 460, ∆ 459-460
F 461
? ?
TriLR TriR6 TriR5a Tri R5+ Tri R5b TriR7
TriMR
mdr I381V S524T S524T A379G mdr
TriHR
MDR-6 TriR10 TriR9a TriR9b TriR8 MDR-7
D134G mdr ?
References
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34
New Findings on the Sensitivity of
Mycosphaerella graminicola to DMI Fungicides
Abstract
A slight reduction in sensitivity of Mycosphaerella graminicola towards different demethylation
inhibitors (DMIs) was observed over Europe in the last years. The impact of mutations in the
cyp51 gene on DMI sensitivity has been analyzed, in particular the effect of amino acid
exchanges V136A, A379G, I381V and mutations and deletions in the YGYG region (at positions
459-462) of cytochrome P450 sterol 14α-demethylase. In 2009, combinations of amino acid
exchanges such as V136A+S524T and V136A+I381V+S524T were found more frequently than
in the years before. Comparison of isolates comprising the mutation V136A with isolates
containing V136A+S524T showed no or only a marginal effect of S524T on the sensitivity (ED50)
to epoxiconazole. Additional mutations such as D107V, D134G, V136C, V136G, S208T, N284H
and G412A were found in 2009. The new mutations and combinations of mutations indicate that
CYP51 of M. graminicola is still an evolving enzyme. However, sensitivity assays of field
isolates with epoxiconazole showed a wide range of ED50 values for all cyp51-haplotypes
indicating that the contribution of the mutations in cyp51 gene to the sensitivity response is
limited and that additional mechanisms may be involved. It was shown that inhibitors of efflux
transporters increased the sensitivity of tested isolates to DMIs to some extent, demonstrating that
efflux transporters are one of the factors influencing DMI sensitivity. Nevertheless, analysis of
field performance of epoxiconazole against M. graminicola in European field trials indicated no
loss of performance of registered field rates of epoxiconazole in the last decade.
Introduction
Sensitivity towards demethylation inhibitors (DMI) was investigated over the last few
years in extensive monitoring programs, particularly for Mycosphaerella graminicola.
DMIs act on the cytochrome P450 sterol 14α-demethylase (CYP51) causing a depletion
of ergosterol in the fungal cell membrane which increases membrane permeability. This
effect facilitates cell lysis and consequently leads to an inhibition of pathogen growth.
Amino acid exchanges in this protein have been described to lead to changes in the
sensitivity to DMIs in different plant pathogens including M. graminicola (Cools and
Fraaije, 2006; Leroux et al., 2007; Brunner et al., 2008; Stammler et al., 2008). In
addition to amino acid exchanges in the gene product of cyp51 encoded by the respective
haplotype, other mechanisms such as overexpression of cyp51 and efflux transporters are
also discussed to influence DMI sensitivity (Walker et al., 2011). This publication
describes the results of sensitivity monitoring conducted during recent years, the
___________________________________________________________________________________________________________
Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
G. Stammler et al.
Fungal isolates
Strains of M. graminicola were isolated from samples with typical disease symptoms
from wheat fields in different European regions. Two isolates carrying the S524T
mutation were kindly provided by Steven Kildea from the Teagasc Institute (Carlow,
Ireland).
Sensitivity analysis
Sensitivity of single pycnidia isolates towards epoxiconazole was determined by
microtiter assays at different concentrations (0, 0.003, 0.03, 0.1, 0.3, 1.0, 3.0 mg/l) of
epoxiconazole in YBG medium (1% yeast extract, 1% Bacto peptone, 2% glycerol)
according to the method described by Stammler et al. (2008). ED50 values were
calculated by Probit analysis. Studies on efflux transporter inhibition were done with 1.6
mg/l cyclosporine A as a well-described modulator, which showed no negative effects on
growth or viability of the tested isolates at the chosen concentration. The ED50 values for
epoxiconazole were determined in specific isolates in the presence and absence of
cyclosporine A.
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concentrations used in the microtiter assays. A slight shift of the curves to higher ED50
values was observed. Isolates with very low ED50 values have disappeared.
100
2001 (n=38)
2003 (n=500)
80 2004 (n=1105)
2005 (n=561)
frequency in [%]
2006 (n=599)
60 2007 (n=544)
2008 (n=473)
2009 (n=708)
40
20
0
0-0.003 0.003-0.01 0.01-0.03 0.03-0.10 0.10-0.30 0.30-1.00 1.00-3.00 > 3.00
ED50-class [ppm]
Figure 1: Frequency distribution of ED50 values of M. graminicola isolates from 2003 to 2009.
Table 1: Classification of cyp51 haplotypes (R3 to R11) according to Walker et al., (2011) updated and
simplified.
S524T with limited impact on DMI sensitivity but higher frequency in 2009
Isolates of M. graminicola of the R5 type (i.e. with V136A) were compared with isolates
of the R9 type (i.e. V136A + S524T) regarding their ED50 values for epoxiconazole,
prothioconazole, tebuconazole, metconazole and prochloraz (Figure 2). For
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G. Stammler et al.
epoxiconazole, the S524T had only slight effects but isolates of the R9 type provided
lower ED50 values than isolates of the R8 type, which dominated the population in many
wheat growing areas (Stammler et al., 2008). For prothioconazole, R9-type isolates had
the highest ED50 values in this collection. ED50 values of R5 and R9 types for
tebuconazole and metconazole were relatively low, which may be due to the concomitant
V136A mutation, since isolates with V136A were always very sensitive to these
compounds. However, the S524T isolates (R9) showed also a slight increase in ED50
values for these two compounds as compared to R5. For prochloraz, ED50 values of the
R5 and R9 types were higher than for the other isolates tested, since V136A causes some
loss in sensitivity to this compound; the S524T isolates additionally gave rise to
somewhat higher ED50 values.
0,1
0,01
0,001
0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 5,5
epoxiconazole prothioconazole tebuconazole metconazole prochloraz
Figure 2: Impact of the S524T mutation on the sensitivity of M. graminicola to various DMIs. ED50
values of isolates of R5 and R9 types are compared. Effects on sensitivity to epoxiconazole are marginal
and ED50 values of R9-type isolates are not higher than for R8-type isolates.
Quantitative analysis of samples in 2008 and 2009 from fields in UK showed that
the S524T type was present at low frequency in 2008 samples but increased in 2009
samples (Figure 3).
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2008 2009
T524 T524
S524 S524
T524
S524
T524
S524
T524
T524
T524
T524 S524
S524 S524
T524
S524 T524
T524
S524
T524 T524 S524
S524 S524
S524
Figure 3: Frequency of the S542T mutation in populations at different trial sites in UK in 2008 (left) and
in UK and Ireland in 2009 (right). The black part of the pies shows the frequency of alleles with threonine,
the grey part the frequency of alleles with serine at position 524.
0,1
ED50 [mg/l]
0,01
0,001
0 WT
1 R3
2 R3+Mod
3 R4
4 R55 R5+Mod
6 R67 R78 R89 R8+Mod
10 11
Figure 4: ED50 values of single isolates for epoxiconazole, sorted by R-type. Two isolates with high ED50
values of the R3, R5 and R8 types each were analyzed for epoxiconazole sensitivity in absence (black dots)
and presence (white dots) of the efflux transporter inhibitor cyclosporine A. ED50 values decreased when
cyclosporine A was added. Corresponding ED50 values are highlighted by white arrows.
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G. Stammler et al.
Nges=377
100
90
80
75 76 72
70 70 72
66 69 69
66 66 67
% Septoria efficacy
63 64 64
63 62 63
60
N=6
N = 19
N = 16
50
N = 38 N = 31
40
N = 18 N = 51
N = 10 N = 15 N = 24 N = 19
30 N = 19 N = 23
N = 23
N = 32 N = 33 99 mean
20
median
median
10
0
1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
# of trials
5/15 9/24
with double 3/10 7/32 4/19 8/33 4/16 3/19 0/18 1/6 0/19 0/23 2/38 0/31 5/23 1/51
applications
Figure 5: Efficacy of 125 g a.i./ha epoxiconazole (1994-2005 Opus Top®, 2006-2009 Opus®) on M.
graminicola in highly infested ( ≥ 25%) field trials in Germany . 1-2 applications, evaluation 29-59 days
after last application. Efficacies expressed as box and whisker plots (box 50%, whisker 90% of the data).
References
Cools, H.J., Fraaije, B.A. (2006): Evolution of azole resistance mechanisms in UK populations of
Mycosphaerella graminicola. Aspects of Applied Biology 78, Fungicide Resistance: Are
we winning the battle but losing the war? 21-27, Ed. AAB, Warwick, UK.
Leroux, P., Walker, A.S., Albertini, A., Gredt, M. (2007): Mutations in the cyp51 gene correlated
with changes in the sensitivity to sterol 14α-demethylation inhibitors in field isolates of
Mycosphaerella graminicola. Pest Management Science, 63, 688-698.
Brunner, P.C., Stefano, F.L., McDonald, B.A. (2008): Evolution of the CYP51 gene in
Mycosphaerella graminicola: evidence for intragenic recombination and selective
replacement. Molecular Plant Pathology, 9, 305-316.
Stammler, G., Carstensen, M., Koch, A., Semar, M., Strobel, D., Schlehuber, S. (2008):
Frequency of different CYP51-haplotypes of Mycosphaerella graminicola and their impact
on epoxiconazole-sensitivity and -field efficacy. Crop Protection, 27, 1448-1456.
Walker A.S., Gredt, M., Leroux, P. (2011): Evolution of resistance to fungicides in populations
of Mycosphaerella graminicola: Emergence of new phenotypes highly resistant to DMIs.
In: Modern Fungicides and Antifungal Compounds, Vol. VI, Eds: Dehne, H.W., Deising,
H.B., Gisi, U., Kuck, K.H., Russell, P.E., Lyr, H., DPG, Braunschweig, Germany. 223-229.
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35
Sensitivity of European Mycosphaerella
graminicola Populations to DMI Fungicides
Abstract
Sensitivity studies over the past 10 to 15 years show that populations of Mycosphaerella
graminicola have adapted to the selection pressure exerted by the use of DMI fungicides. The
sensitivity shift affects all DMI’s but its dynamics may be compound specific. The data also
suggest that the shift has continued. Recently, a lot of information on cyp51 genotypes and their
sensitivity to DMI’s has been presented. However, the cyp51 gene seems to be dynamic, and new
genotypes are emerging with altered sensitivity patterns towards DMI’s. Despite some
differences in sensitivity pattern of certain cyp51 genotypes to particular DMI fungicides, a
general cross resistance can be assumed based on the same mode of action for all DMI’s.
However, the selection process may favor certain genotypes depending on the major DMI used
for disease control. The large variation in sensitivity within each genotype suggests that, in
addition to mutations in the cyp51 gene, other mechanisms must play an important role for
sensitivity towards DMI’s. Sensitivity shifts within genotypes will be described and discussed.
Introduction
H. Sierotzki et al.
2008). Nevertheless, mutations like F137Y, I381V, alone or in combination with A379G
and V/C136A have significant influence on the sensitivity of M. graminicola towards
DMI’s (Chassot et al., 2008).
M. graminicola populations are highly divers and undergo abundant sexual
recombination. Recently, it has been shown that intergene recombination within the
cyp51 gene is possible (Brunner et al., 2008). Based on random natural mutagenesis, this
process might lead to a higher frequency of new combinations of mutations within the
cyp51 gene than expected. The impact of this adaptation on field performance of DMI
fungicides is discussed controversially and is highly dependent on the intrinsic activity of
each particular DMI.
This contribution describes the changes in the cyp51 gene in relation to the in vitro
sensitivity and possible influences by other factors.
Fungal isolates
Mycosphaerella graminicola isolates were obtained from leave samples collected at
different time points in the years 2004 to 2009 in European countries. Sensitivity towards
fungicides was tested according to the methodology available on: www.frac.info. The
fungicides (cyproconazole, prothioconazole, prochloraz and tebuconazole) were obtained
from Sigma Aldrich as technical material. EC50 values were calculated with an agstat
programme package (Syngenta internal). Statistical analysis and box plot representations
were made using the SigmaPlot programme (Systat Software, Inc. 2008).
Molecular analysis
Total genomic DNA was extracted as described in Chassot et al., (2008). Cyp51
genotyping for mutations was performed by Q-PCR using 40 bulked lesions from leaf
samples: I381V, A379G, V136A which determine the genotypes IV, V and VI.
Genotypes I to III together were calculated from the total amount of cyp51 gene in a
given sample. The cyp51 gene of single spore isolates was sequenced using the BigDye
terminator v3.1 kit (Applied Biosystems) in a ABI Prism 3130 Genetic Analyser.
Primer design was done using Primer Express v2.0 Software. 3 ARMS PCR systems
for the mutant alleles A136/C136, G379 and V381 and for the wild type alleles V136,
A379 and I381, respectively were developed. For the 136 SYBR Green assays the
following allele-specific forward primers were used: V136_f (Wt): GTC TTT GGC AAG
GAT GTG AT; A136_f (Mut1): GTC TTT GGC AAG GAT GTG AC and C136_f
(Mut2): GTC TTT GGC AAG GAT GTG TG; the allele-unspecific forward primer
136_f: CTG TCT TTG GCA AGG ATG TG and the allele-unspecific reverse primer
136_r: GGC GAT CAA GGT CAC GTA GG. The 379 SYBR Green assays were
performed with allele-specific forward primers A379_f (Wt): AAG AAA CCC TTC
GTA TTC ACA C; G379_f (Mut): AAG AAA CCC TTC GTA TTC ACA G; allele-
unspecific forward primer 379_f: AAA GAA ACC CTT CGT ATT CAC G and allele-
unspecific reverse primer 379_r: AGT GCT CGT CCA TGC GG. Finally, for the 381
SYBR Green assays the allele-unspecific forward primer 381_f: ATC TGC GAC CGA
GTC CTG and the allele-specific reverse primers I381_r: TGC GCA GAA TGG AGT
GGG T and V381_r: TGC GCA GAA TGG AGT GAA C and the allele-unspecific
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reverse primer 381_r: TTG CGC AGA ATG GAG TGG A were designed. The reactions
were performed using 6.25 µl Power SYBR Green PCR Master Mix (AB Applied
Biosystems), 1.25 µl (500 nM) common primer (5 µM), 1.25 µl (500 nM) ARMS primer
(5 µM), 1.25 µl Water and 2.5 µl DNA (undiluted or 1:10 diluted). The run conditions on
the ABI 7900HT SDS were 10 min at 95°C, and 40 cycles for 15 sec at 95°C, 15 sec at
60°C and 15 sec at 77°C.
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H. Sierotzki et al.
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both compound pairs, sub-clusters were recognized. In each sub-cluster cross resistance
was again observed. Isolates of the different cross resistant sub-clusters were further
analyzed for their cyp51 genotype. For the pair cyproconazole / prothioconazole, all
isolates in the cluster were aligned more or less according to the sensitivity profile for
each genotype, i.e. genotype III isolates were most sensitive followed by genotype IV, V
and VI; genotype IV(T) isolates were least sensitive. For the pair cyproconazole /
tebuconazole, two sub-clusters were found; the less sensitive sub-cluster contained
genotype III, V and VI isolates (from most to least sensitive ones), whereas the more
sensitive sub-cluster contained genotype IV and IV(T) isolates (Figure 2, D). For the pair
cyproconazole / prochloraz, isolates of genotypes VI and V were more sensitive
compared to genotypes IV and IV(T). Isolates of the latter genotype were least sensitive
to prochloraz and might be strongly selected by this fungicide. A few isolates with a
combination of I381V and S524T were found; they were about as sensitive as genotype
V isolates.
Figure 2: A – C: Cross resistance pattern between cyproconazole and other DMI fungicides for
Mycosphaerella graminicola isolates collected in European countries in 2009. Dotted circles indicate
assumed sub-populations for the pairs cyproconazole / prochloraz and cyproconazole / tebuconazole.
Figure 2 D: Cross resistance between cyproconazole and tebuconazole for single cyp51 genotypes from
Ireland and France.
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H. Sierotzki et al.
Among the 19 different mutations in cyt51 (Stammler et al., 2008), also deletions at
positions 459 to 461 were found in recent isolates. In populations analyzed since 2006,
no sensitivity differences were found for isolates of the same genotype with or without
deletion. Most mutations do not seem to have a direct impact on sensitivity of the isolates
(such as L50S, S188N, K513N or deletions), but might be needed as a prerequisite for
the appearance of other mutations. The recently found intergenic recombination
breakpoints in the cyp51 gene (Brunner et al., 2008) might be of special importance for
the evolution of mutations, since new combinations of mutations could appear more
frequently than expected based on random mutation rate. This could lead to an increasing
number of genoptypes, which will be more and more difficult to assign to distinct
sensitivity profiles.
Figure 3: Shift in sensitivity to cyproconazole for single genotype isolates of Mycosphaerella graminicola
collected between 1999 and 2009.
Conclusions
Comparison of sensitivity profiles in populations over the years revealed that sensitivity
shifts due to changes of genotype composition were bigger than those within genotypes.
Therefore, a change of sensitivity in a population is mainly due to a different
composition of cyp51 genotypes. However, tests with rare isolates showing a strongly
reduced sensitivity (isolates found in 2004) indicated that ABC transporters might also be
involved in sensitivity changes. The frequency of such isolates is generally low, probably
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due to a fitness penalty, but recent reports indicate a local increase of such isolates
(Walker et al., 2011). The impact on field performance is not yet elucidated.
References
Brunner P.C., Stefanato F.L., Mcdonald B.A. (2008): Evolution of the CYP51 Gene in
Mycosphaerella graminicola: Evidence for Intragenic Recombination and Selective
Replacement. Molecular Plant Pathology, 9 (3), 305–316.
FRAC: www.frac.info, 2010
Chassot, C., Hugelshofer, U., Sierotzki, H., Gisi, U. (2008): Sensitivity of CYP51 Genotypes to
DMI Fungicides in Mycosphaerella graminicola. In: H. W. Dehne, H.B. Deising, U. Gisi,
K. H. Kuck, P. E. Russell, H. Lyr (Eds.), Modern Fungicides and Antifungal Compounds
V, DPG, Braunschweig, Germany. 129–136.
Leroux, P., Albertini, C., Gautier, A., Gredt, M., Walker, A.-S. (2007): Mutations in the CYP51
gene correlated with changes in sensitivity to sterol 14α-demethylation inhibitors in field
isolates of Mycosphaerella graminicola. Pest Manag Sci., 63, 688–698.
Stammler, G., Kern, M., Semar, A., Glaettli, A., Schoefl U. (2008): Sensitivity of
Mycosphaerella graminicola to DMI Fungicides related to mutations in the target gene
cyp51 (14α-Demethylase). In: H.W. Dehne, H.B. Deising, U. Gisi, K. H. Kuck, P. E.
Russell, H. Lyr (Eds.), Modern Fungicides and Antifungal Compounds V, DPG,
Braunschweig, Germany. 137–142.
Fraaije, B.A., Cools, H.J., Motteram, J., Gilbert, S.-R., Kim, S.-H., Lucas J.A. (2008): Adaptation
of Mycosphaerella graminicola Populations to Azole Fungicides in the UK. In: H. W.
Dehne, H.B. Deising, U. Gisi, K. H. Kuck, P. E. Russell, H. Lyr (Eds.), Modern
Fungicides and Antifungal Compounds V. DPG, Braunschweig, Germany. 121–127.
Walker A.-S., Gredt, M., Leroux, P. (2011): Evolution of Resistance to Fungicides in French
Populations of Mycospahaerella graminicola: Emergence of New Phenotypes Highly
Resistant to DMI’s. In: H. W. Dehne, H.B. Deising, U. Gisi, K. H. Kuck, P. E. Russell, H.
Lyr (Eds.), Modern Fungicides and Antifungal Compounds VI. DPG, Braunschweig,
Germany. 223-229.
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H. Sierotzki et al.
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36
Mycosphaerella graminicola: Relevance of in
vitro Sensitivity and CYP51 Mutations for the
Field Performance of DMI Fungicides
Abstract
In Mycosphaerella graminicola, the presence of different point mutations in the sterol-14-
demethylase (cyp51) target site of DMI fungicides has been frequently reported, which has led to
the description of several genotype and phenotype groups. In order to characterize their practical
relevance, in vitro sensitivity and genotype structures of M. graminicola populations were
investigated along with field performance of DMI fungicides by correlating sensitivity data and
corresponding cyp51 alterations in single strains with DMI efficacy results at trial sites. Isolates
with low sensitivity were selected for greenhouse studies. Prothioconazole applied at label rate
fully controlled all strains in the plant test, and no reduced performance was observed at trial sites
with different pathogen sensitivity. Thus, the generated lab, greenhouse and field data suggest
that in vitro sensitivity data or molecular characterization of target site mutations cannot
sufficiently predict field performance of the respective DMI product. Varying correlations
between in vitro, molecular and in planta results point to the likely presence of additional
resistance mechanisms or plant/pathogen/fungicide interactions, which may have a different
relevance for in vitro than in vivo efficacy of DMI fungicides.
Introduction
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Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
evolution often results in rapidly decreasing field efficacy of QoI fungicides when
applied as solo products.
Although DMI fungicides interfere also with a single protein, and the presence of
point mutations in the sterol-14-demethylase (cyp51) target gene has been reported in M.
graminicola for more than five years (Fraaije et al., 2007; Leroux et al., 2007),
sensitivity changes over the last decade have developed much slower, following a
‘shifting-type’ pattern. An increasing number of genotype and phenotype groups of M.
graminicola has been described, generally based on the correlation of in vitro sensitivity
with cyp51 SNPs at positions 136, 379, 381 or 524, and amino acid mutations/deletions
in the YGYG-region at positions 459-462. The relevance of cyp51 alterations for the
development of azole sensitivity changes has been confirmed, but not all DMIs are
equally affected. Many current DMI solutions continued to perform very well on the
European scale.
The consequences of these findings for growers are still under discussion, especially
in regard to different spray regimes and DMI products applied at regional level. The aim
of this study is to analyze the relation between reduced in vitro sensitivity and/or
genotype structure of M. graminicola populations and observed field performance of
important DMI fungicides. In addition, sensitivity data and determination of
corresponding cyp51 mutations in single strains sampled in 2009 are shown along side
with DMI efficacy results at selected sites. Special focus was given to selected Irish and
British strains.
In vitro sensitivity data were obtained using a microplate test as described by Suty and
Kuck (1996), with slight modifications according to FRAC (www.frac.info). Cyp51
alterations were determined in single isolates by pyrosequencing focusing on the SNPs
G379A and I381V, which are the key mutations for phenotypes TriR6 to TriR8 as
classified by Leroux et al. (2007). Design and test-result evaluation of molecular,
greenhouse, and field studies followed standard protocols from Bayer CropScience.
Results
In 2009, broad scale in vitro sensitivity studies with single strains of M. graminicola
originating from different European countries showed a homogeneous sensitivity
spectrum for prothioconazole with mean EC50 values (MEC50s) ranging from 0.2 mg/l
(highest sensitivity) observed for the British population to 0.9 mg/l for the German
population (Table 1). With tebuconazole, MEC50s ranged from 0.8 mg/l in Sweden to 3.2
mg/l in the UK. Compared to all other countries studied, a different genotype structure
became visible in the Irish population, as considerably fewer genotypes carrying the
cyp51 mutation I381V were detected.
From the Irish population, three out of four isolates selected for greenhouse efficacy
studies with prothioconazole did not carry the cyp51 mutations G379A and/or I381V
(Table 2), which were reported to cause the most substantial in vitro sensitivity changes
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towards DMI fungicides and leading to phenotypes TriR6, TriR7 or TriR8 (Leroux et al.,
2007). However, the mutation S524T was detected in these isolates. All four isolates
exhibited similar sensitivity to prothioconazole with EC50 values ranging from 0.15 mg/l
to 0.40 mg/l, whereas isolate Ire4/09 exhibited lower in vitro sensitivity towards
tebuconazole compared to the other isolates.
Table 1: Cyp51 genotypes and in vitro sensitivity to DMI fungicides of M. graminicola single isolates
originating from different European wheat growing regions in 2009.
Country no. of PTZ TBZ TriR6 + TriR7 strains TriR8 strains other
isolates
MEC50 MEC50 (SNP I381V) (SNPs G379A+I381V) genotypes
[mg/l] [mg/l] [%] [%] [%]
Austria 28 0.8 2.1 32 61 7
Denmark 44 0.5 2.9 45 39 16
France 120 0.6 1.6 43 41 16
Germany 64 0.9 2.8 56 27 17
Ireland 55 0.3 1.0 24 14 62
UK 243 0.2 3.2 46 34 20
Sweden 48 0.8 0.8 65 6 29
PTZ: prothioconazole, TBZ: tebuconazole, MEC50: mean EC50, SNP: single nucleotid polymorphism
Table 2: In vitro sensitivity to DMIs and cyp51 alterations of selected Irish strains of M. graminicola
collected in 2009.
Isolate no. SNP SNP SNP EC50 [mg/l] SD EC50 EC50 [mg/l] SD EC50
G379A I381V S524T PTZ PTZ TBZ TBZ
Ire1/09 - - + 0.18 0.22 0.06 0.19
Ire2/09 - - + 0.36 0.27 0.06 0.08
Ire3/09 - - + 0.40 0.13 0.14 0.06
Ire4/09 + + - 0.15 0.17 1.12 2.53
PTZ: prothioconazole, TBZ: tebuconazole, SD: standard deviation, SNP: single nucleotid
polymorphism
Table 3: Disease development and prothioconazole in vivo efficacy against selected Irish strains of M.
graminicola (in planta greenhouse test, Proline® EC250 application 1days pre-inoculation, 200 g ai/ha).
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From isolates collected in 2009 showing high prothioconazole EC50 values, isolates
UK0509-4 and UK1009-15 were chosen for greenhouse efficacy tests and compared with
isolates expressing a baseline in vitro sensitivity. Smaller AUDPC values were observed
in untreated with both UK0509-4 and UK1009-15 compared to the reference isolate.
However, Proline® applied at full rate provided similar efficacy against both isolates
under curative conditions (Table 4). Thus, the different in vitro sensitivity of the two
isolates had no impact on the control under the tested in planta conditions.
Table 4: Disease development and prothioconazole in vivo efficacy against selected strains of M.
graminicola showing large in vitro sensitivity differences (in planta greenhouse test, Proline® EC250
application 3days post-inoculation, 200 g ai/ha).
Isolate no. EC50 [mg/l] AUDPC 20dpi AUDPC 20dpi treatment efficacy
PTZ untreated Proline® EC250 [%]
UK0509-4 27 218 21 90
UK1009-15 13 101 12 88
DE0509-2 0.4 356 41 88
UK0209-11 0.4 296 25 92
AUDPC: Area Under Disease Progress Curve, dpi: days post inoculation, PTZ: prothioconazole
Figure 1 summarizes the severity of Septoria leaf blotch in untreated and DMI
treated plots at different European trial sites in 2009 and documents the efficacy of
tebuconazole, prothioconazole, or a mixture of the two fungicides. Results were split into
two groups according to the in vitro sensitivity of the populations to prothioconazole
(MEC50 values ranging either between 0.2 mg/l and 0.9 mg/l, or between 0.9 mg/l and
1.5 mg/l). In both sensitivity scenarios, prothioconazole, applied either solo or in mixture,
provided similar and excellent control of M. graminicola, which was superior to straight
tebuconazole. Interestingly, somewhat lower disease severity could be observed at sites
showing lower in vitro sensitivity of the populations.
100 100
75 75
severity [%]
severity [%]
50 50
27
25 25
17
10
2 3 5 3 3
0 0
untreated TBZ PTZ P+T untreated TBZ PTZ P+T
Figure 1: Disease severity in untreated and DMI treated plots incited by M. graminicola isolates with
different in vitro sensitivity to prothioconazole at European trial sites in 2009.
Left: Isolates with mean EC50 values ranging from 0.2 mg/l-0.9 mg/l (5 trials). Right: from 0.9 mg/l-1.5
mg/l (4 trials). TBZ: tebuconazole, 250 g/ha; PTZ: prothioconazole, 200 g/ha; P+T: PTZ+TBZ, 125+125
g/ha.
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In eight of the nine trial sites, 85% of all studied single isolates carried the cyp51
mutation I381V, partly in combination with G379A, which lead to phenotype TriR8
(Table 5).
Table 5: Cyp51 genotype profile (frequency in percent) of prothioconazole in vitro sensitivity classes of
M. graminicola at 8 European trial sites in 2009.
In line with the disease severity results, the relative yield response of
prothioconazole containing products was higher in both sensitivity groups than those of
straight tebuconazole (Figure 2). The products do not show reduced relative yield at sites
with lower in vitro sensitivity. Also solo treatments of tebuconazole resulted in clearly
visible yield increases even in the presence of 85% M. graminicola I381V mutants.
140 140
rel. yield response [%]
130 130
121
120 120 119
111 111
110 110
107 109
100 100
TBZ PTZ PTZ+TBZ TBZ PTZ PTZ+TBZ
Figure 2: Relative yield response in DMI treated plots incited by M. graminicola isolates with different in
vitro sensitivity to prothioconazole at European trial sites in 2009.
Left: Isolates with mean EC50 values ranging from 0.2 mg/l-0.9 mg/l (4 trials). Right: from 0.9 mg/l-1.5
mg/l (4 trials). TBZ: tebuconazole, 250 g/ha; PTZ: prothioconazole, 200 g/ha; PTZ+TBZ, 125+125 g/ha.
Discussion
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genotypes compared to most other European countries. However, the correlation was not
seen in Sweden in 2009, which may be caused by tebuconazole not being registered for
use in cereals in this country. On the other hand, there was no correlation between I381V
genotypes and prothioconazole sensitivity.
Further in vitro studies with selected isolates showed that the mutation S524T within
new genotypes does not affect prothioconazole or tebuconazole sensitivity. These
findings were confirmed in greenhouse studies, as Proline® (prothioconazole) applied at
label rate on potted wheat plants fully controlled such isolates. In addition, isolates with a
sensitivity far outside the baseline (EC50 >10 mg/l) were fully controlled by Proline® in
the greenhouse even under curative conditions. Interestingly, disease development
caused by such isolates was reduced compared to reference isolates.
Isolates originating from different European trial sites were classified according to
their in vitro sensitivity. Disease severity at plots treated either with tebuconazole (250
g/ha), prothioconazole (200 g/ha) or with a mixture of both (125 g/ha + 125 g/ha) was
clearly reduced at all trial sites. Both prothioconazole-containing solutions performed at a
very high efficacy level independent of the population sensitivity. Prothioconazole was
superior to tebuconazole when applied alone. Thus, there is hardly any correlation
between the presence of cyp51 (TriR6-TriR8) genotypes, in vitro sensitivity and field
performance of prothioconazole-containing products, especially in regard to yield
response.
The presented lab and field data lead to the assumption that field performance of
certain DMIs my not be predicted adequately just on the basis of in vitro sensitivity
and/or molecular characterization of the isolates dominating the population of a cereal
growing area. Varying correlations between in vitro, molecular, and in planta study
results point to the likely. The presence of additional resistance mechanisms and
plant/pathogen/fungicide interactions may be equally important for DMI efficacy.
References
Fraaije, B.A., Cools, H.J., Kim, S-H., Motteram, J., Clark, W.S., Lucas, J.A. (2007): A novel
substitution I381V in the sterol 14-demethylase (cyp51) of Mycosphaerella graminicola
is differentially selected by azole fungicides. Molecular Plant Pathology, 8, 245-254.
Leroux, P., Albertini, C., Gautier, A., Gredt, M., Walker, A-S (2007): Mutations in the cyp51
gene correlated with changes in sensitivity to sterol 14-demethylation inhibitors in field
isolates of Mycosphaerella graminicola. Pest Management Science, 63, 688-698.
Suty, A., Kuck, K.H. (1996): Sensitivity of Wheat Leaf Spot Septoria tritici to Tebuconazole.
Proceedings of the 1996 Brighton Crop Protection Conference, Volume 2, 689-694.
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37
Outliers or Shifts? Sensitivity of the Irish
Mycosphaerella graminicola Population to
Triazole Fungicides
Abstract
Currently wheat production in Ireland is reliant upon a limited number of triazole-based
fungicides to prevent yield losses resulting from infection by M. graminicola. Since 2003
Teagasc, the Irish Agriculture and Food Development Authority, has been determining the
sensitivity of Irish M. graminicola populations to the fungicides most commonly used for its
control. Annually the sensitivities to epoxiconazole, prothioconazole and tebuconazole of M.
graminicola isolates collected at 14-15 sites have been routinely assessed using both an agar plate
and a microtitre plate assay. From 2005–2008, the population remained sensitive to
epoxiconazole and prothioconazole, with stable, unimodal distributions of sensitivity to both
fungicides. In 2005 a shift in sensitivity of the population to tebuconazole was observed, with the
sensitivity to tebuconazole displaying a bimodal distribution. During the summer of 2008, a shift
towards reduced sensitivity to both epoxiconazole and prothioconazole was observed in isolates
from one of the sampling sites. This shift was associated with the appearance of a new sensitivity
class within the Irish population: similar strains were subsequently detected throughout the
country during the spring of 2009.
Introduction
Septoria tritici blotch (STB) caused by the fungal pathogen Mycosphaerella graminicola
is currently the most economically destructive disease of wheat in Ireland and throughout
North-Western Europe. As the majority of commercially grown wheat varieties in Ireland
have only moderate resistance to M. graminicola infection and subsequent STB
development, routine applications of fungicides have become the norm to ensure
profitably. The emergence and rapid spread within the Irish M. graminicola population of
resistance to benzimidazole and QoI fungicides have reduced the number of available
chemistries capable of providing adequate control of STB (Kildea, 2009). Following the
emergence of resistance to the QoIs, monitoring of the sensitivity of Irish M. graminicola
population to the most commonly used fungicides has been undertaken by Teagasc, the
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Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
S. Kildea et al.
Irish Agriculture and Food Development Authority. These studies, while including
sensitivity assessments to benzimidazoles, QoIs and chlorothalonil, have concentrated on
triazole fungicides. With Irish crops at present receiving up to four triazole fungicide
applications, the selection pressure placed upon the Irish M. graminicola population is
immense. The work presented outlines changes in triazole sensitivity that have occurred
during the period 2005 - 2009.
Wheat leaf samples infected with M. graminicola were collected from 14-15 commercial
crops at 20 m intervals, both in early March and mid July of each year. The crops were
located throughout Ireland, principally in commercial wheat-growing regions. The same
fields were sampled each year when possible or if not neighbouring fields were sampled.
Leaves were air-dried for 24 h and subsequently stored at -20°C until required. To isolate
M. graminicola, approximately 30-40 diseased leaves from each crop sample were rinsed
under tap water for 1-2 h, surface-sterilised in 70% ethanol for 20 sec followed by 10%
sodium hypochlorite for 1½ min and rinsed three times in sterile distilled water. Once
surface-sterilised, the leaves were gently dried using a paper towel and placed on
antibiotic-amended water agar. Leaves were incubated under 12 h cycles of near
ultraviolet (NUV) / darkness at 18°C for 48 h, by which time cirri began to emerge.
Using a dissecting microscope and a sterile needle, individual cirri were picked up
(single cirrus per leaf sample) and streaked onto antibiotic-amended potato dextrose agar
(PDA). Once streaked, plates were sealed with parafilm and incubated under the above
conditions for seven days. Following 7 days’ incubation, isolates were cultured for an
additional 4 days on antibiotic-free PDA after which they were ready for fungicide
sensitivity assessment. Where possible, 20 individual isolates of M. graminicola from
each crop were obtained.
Using a microtitre plate assay adapted from Pijls et al. (1994) the sensitivity of the
isolates to the triazole fungicides epoxiconazole, prothioconazole and tebuconazole was
determined. Wells of sterile, flat-bottomed microtitre plates were filled with 150 µl
potato dextrose broth (PDB) amended with technical grade epoxiconazole,
prothioconazole or tebuconazole (dissolved in 100% methanol) to give test
concentrations of 30, 10, 3.3, 1.1, 0.37, 0.123, 0.04 and 0 mg/L (concentrations were
adjusted to 100, 30, 10, 3.3, 1.1, 0.37, 0.123 and 0 mg/L for prothioconazole from July
2008 onwards). Spore suspensions, 1x105 spores/ml of each isolate were prepared in
PDB and 50 µl of this suspension was added to the wells of the plate. In each plate, 10
isolates were assessed; the first column was left blank and the standard M. graminicola
isolate, Epo 6/9 (isolated from an experimental plot in 2004) was added to column 12 of
each plate. Plates were replicated three times, sealed and placed under NUV/darkness at
18°C for 10 days. Growth of the fungus was assessed as a measure of light absorbance
(405 nm) using a Tecan Saffire microplate reader. The effect of the different fungicide
concentrations on each isolate was calculated as the percentage inhibition with respect to
the untreated control and used in the curve fitting programme XLfit to calculate EC50
values.
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Results
During the reporting period 2005-2009, over 1,500 Irish M. graminicola isolates were
2005-2008 (N=1310)
Epoxiconazole
2009 (N=352)
50
45
40
% isolates
35
30
25
20
15
10
5
0
-1.6 -1.2 -0.8 -0.4 0 0.4 0.8 1.2 1.6 2
LogEC50
2005-2008 (N=1362)
Prothioconazole
2009 (N=343)
50
45
40
% isolates
35
30
25
20
15
10
5
0
-1.6 -1.2 -0.8 -0.4 0 0.4 0.8 1.2 1.6 2
LogEC50
2005-2008 (N=1426)
Tebuconazole
2009 (N=179)
50
45
40
% isolates
35
30
25
20
15
10
5
0
-1.6 -1.2 -0.8 -0.4 0 0.4 0.8 1.2 1.6 2
LogEC50
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S. Kildea et al.
Discussion
With the profitability of wheat production in Ireland heavily reliant on the activity of
prothioconazole and epoxiconazole to inhibit M. graminicola, the shift in sensitivity to
both chemicals during the 2008/2009 season is a worrying development. This shift in
sensitivity was first detected in a single crop in 2008 (>60% of isolates retrieved from the
crop exhibited decreased sensitivity to both epoxiconazole and prothioconazole),
however spread to all but one of the crops sampled in 2009 with varying frequencies of
detection (<5% - >60%). Determining the basis for this decrease in sensitivity and
whether it has had an effect on the efficacy of either epoxiconazole or prothioconazole in
controlling STB are ongoing.
Acknowledgements
This research has been funded by Teagasc, partially under the Walsh Fellowship scheme.
References
Kildea, S. (2009): Fungicide resistance in the wheat pathogen Mycosphaerella graminicola. PhD
Thesis, Queen’s University, Belfast.
Pijls, C.F.N., Shaw, M.W., Parker, A. (1994): A rapid test to evaluate in vitro sensitivity of
Septoria tritici to flutriafol, using a microtitre plate reader. Plant Pathol., 43, 726-732.
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38
Sensitivity of Mycosphaerella graminicola to
DMI Fungicides across Europe and Impact on
Field Performance
Abstract
European isolates of Mycosphaerella graminicola isolated in 2009 were analyzed for their
sensitivities to the demethylation inhibitors (DMIs) epoxiconazole, prochloraz, metconazole,
tebuconazole and prothioconazole. It was found that the sensitivity pattern between isolates and
DMIs were heterogeneous and that the correlation of the sensitivities was rather low. An analysis
of more than 1000 BASF field trials from 1994-2009 revealed that the relative field performance
of different DMI fungicides may have changed over the years. These findings underline the
necessity to maintain and recommend a diversity of DMIs for resistance and disease management
of M. graminicola.
Introduction
Sensitivity analysis
Strains of M. graminicola were isolated from single pycnidia of leaf samples with typical
disease symptoms collected in wheat fields of different European regions in 2009. Before
isolation, leaf samples were incubated in a moist chamber overnight. Sensitivities of
isolates towards epoxiconazole, prochloraz, metconazole, tebuconazole and
prothioconazole were determined by measuring fungal growth in microtiter plates at
different concentrations according to the method described by Stammler et al. (2008).
ED50 values were calculated by Probit analysis and assigned to one of three categories
(low, moderate and high ED50 value). Categories were defined by containing one-third of
the isolates.
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Figure 1: 119 isolates from different European countries analyzed for their sensitivity to 5 DMIs (EPX =
epoxiconazole, PZ = prochloraz, MTZ = metconazole, TEB = tebuconazole and PTH = prothioconazole).
ED50 values for each individual DMI were assigned to one of three classes, defined as 1/3 of isolates either
with low, moderate or high ED50. White squares are for low, gray for moderate and black for high ED50
class.
110
n.s.
100
(epoxiconazole = 100%)
90
n.s.
80
70
n.s
60 .
50 y = 2617
– 1.271 * year **
40
30
20
10
0
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
Figure 2: .For single trials (n=1051), the efficacy of DMI products was compared in relation to
epoxiconazole (=100%). The graph summarizes the mean values per year. The linear regression of relative
efficacy data over the years was calculated based on all single trial data:
n.s.: slope of regression not significantly different from zero (dotted lines)
**: slope of regression differs significantly from zero (p < 0.01, solid lines).
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Conclusion
Findings from the present sensitivity study and the field trial analysis indicate that a
diverse DMI portfolio is important for solid resistance management and reliable
fungicide efficacy in disease control. This is of particular importance as apart from DMIs,
only preventive contact fungicides or single-site inhibitors are available for the effective
control of M. graminicola.
References
Brunner, P.C., Stefano, F.L., McDonald, B.A. (2008): Evolution of the CYP51 gene in
Mycosphaerella graminicola: evidence for intragenic recombination and selective
replacement. Molecular Plant Pathology, 9, 305-316.
DEFRA (2007): Assessing the performance of currently available azole fungicide products
against Septoria tritici in the light of changes on sensitivity of the S. tritici population in
the UK. Final report of DEFRA project PS 2711 / CSA 7236.
Fraaije, B.A., Cools, H.J., Motteram, J., Gilbert, S.R., Kim, S.H., Lucas, J.A. (2008): Adaptation
of Mycosphaerella graminicola populations to azole fungicides in the UK. In: H.W. Dehne,
H.B. Deising, U. Gisi, K.H.Kuck, P.E. Russell, H. Lyr (Eds.): Modern Fungicides and
Antifungal Compounds Modern Fungicides and Antifungal Compounds V. DPG-Verlag,
Braunschweig, 121-127.
FRAC (2009): www.frac.info
Stammler, G., Strobel, D., Semar, M., Klappach, K. (2006): Diagnostics of fungicide resistance
and relevance of laboratory data for the field. Aspects of Applied Biology, 78, 29-36. Ed.
AAB, Warwick, UK.
Stammler G., Carstensen, M., Koch, A., Semar, M., Strobel, D., Schlehuber, S. (2008):
Frequency of different CYP51-haplotypes of Mycosphaerella graminicola and their impact
on epoxiconazole-sensitivity and -field efficacy. Crop Protection, 27, 1448-1456.
Stammler, G., Semar, M., Strobel, D., Koch A., Schlehuber, S. (2011): New findings on the
sensitivity of Mycosphaerella graminicola to DMI fungicides. In: H.W. Dehne, H.B.
Deising, U. Gisi, K.H.Kuck, and P.E. Russell, H.Lyr (Eds.): Modern Fungicides and
Antifungal Compounds VI. DPG-Verlag, Braunschweig. 231-236.
Walker A.S., M. Gredt, Leroux, P. (2011): Evolution of resistance to fungicides in French
populations of Mycosphaerella graminicola: Emergence of new phenotypes highly
resistant to DMIs. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H.Kuck, and P.E. Russell, H.
Lyr (Eds.):Modern Fungicides and Antifungal Compounds VI. DPG-Verlag, Braunschweig.
223-229.
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39
Fungicide Use and Resistance in Broad Acre
Cropping in Australia
Abstract
The use of fungicides in broad acre cropping in Australia is reviewed. The statistics suggest that
AU$250m worth of fungicides are used annually in Australia resulting in $2billion worth of
protection. The main pathogens kept at bay are the cereal bunts and smuts, stripe rust,
Stagonospora, various Pyrenophora species, the legume Ascochyta pathogens and Botrytis.
Fungicide use is tightly regulated. Fewer actives are available than in Europe and the minimum
as well as maximum dose is regulated. Despite average cereal yields being 1-2 tonne/hectare,
fungicides doses are very similar to those used in Europe where average yields are 5-12 t/ha.
Due to the smaller number of active ingredients which is available in Australia the risk of
pathogens acquiring fungicide resistance is higher. A new project to determine base-line
fungicide sensitivity levels has been initiated to determine if resistance is present. Some hints of
fungicide resistance have been uncovered.
Introduction
Fungicide use in broad acre cropping in Australia has markedly increased in intensity in
the last decade (Murray and Brennan, 2009 a,b). Nearly all crops receive seed treatments;
some crops are receiving multiple foliar sprays. The total expenditure on fungicides
(~AU$ 250m) is greater than on insecticides ($150m) but lower than with herbicides
(AU$ 900m). The control achieved with these fungicides is very considerable. The
reports cited above estimate that a total saving of $2000 million is accrued. This equates
to $8 savings for every dollar spent. The tables below show the current losses from each
disease. The diseases caused by Pleosporales (tan spot, Stagonospora and net-blotch)
dominate the listing probably reflecting the widespread adoption of no-till agriculture in
the last 20 years (Solomon et al. 2006). The tables also list the value of crop saved due to
the use of fungicides. The value of protection against seed-borne disease is expected but
it is also chastening to note how much we depend on fungicides to control rusts.
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R. Oliver et al.
A limited range of fungicides are registered; regulations differ between states but
registrations specify crop, pathogen and the permissible dose rate (Table 3). The vast
majority are DMIs. The QoI fungicides are only permitted in mixtures (azoxystrobin with
cyproconazole; pyraclostrobin with epoxiconazole) and on cereals only. Neither
prochloraz and chlorothalonil, nor morpholines nor members of groups A or 1 (b-tubulin)
fungicide are registered on cereals. Major high risk pathogens include Botrytis and
downy mildew on legumes, cereal powdery mildews, tan spot, net blotch and
Stagonospora nodorum blotch.
Table 3: Major fungicides registered in Australia (data from DAFWA and APVMA).
Group Active Ingredient Foliar or Seed Registered for
Triadimefon F
Propiconazole F
Tebuconazole F and S
C3 – DMI Flutriafol F and S
Epoxiconazole F
Difenconazole S Wheat, Barley, Oats
Triadimenol S
Prothioconazole + tebuconazole F
Cyproconazole + Azoxystrobin F
C3 + K11
Pyraclostrobin + Epoxiconazole F
G7 Carboxin S
YM5 Chlorothalonil F
A1 Thiabendazole S
Legumes
A1 Carbendazim F
YM3 Mancozeb F
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Isolates of various pathogens were obtained from the field and from historical collections.
Fungicide sensitivity levels used standard techniques, with glutamate as the sole carbon
source for strobilurin resistance (Wood and Hollomon, 2003; Cools et al., 2006) .
Results
Baseline sensitivities are reported in Table 4. These data can be used in the future to
determine if any drifts in sensitivity are occurring.
Fungus
Botrytis fabae
Stagonospora
Pyrenophora
graminis f.
Didymella
Ascochyta
Active
sp. hordei
P. teres f.
Blumeria
maculata
nodorum
repentis
Botrytis
pinodes
cinerea
Ingredient
tritici-
rabiei
Tebuconazole 5 15 0.01 1 0.6 1 NT NT
Azoxystrobin 50 15 1 1 17 1 NT NT
Epoxiconazole 5 5 0.01 0.1 0.8 0.3 1 0.5
Chlorothalonil 15 >500 0.1 0.3 9 1 NT NT
Thiabendazole 2.5 15 1 15 48 1 1 1
Prochloraz NT 100 0.01 0.02 29 0.05 0.5 1
Triadimefon NT 1 NT NT NT NT NT NT
Pyraclostrobin NT NT NT NT NT 0.3 NT NT
Propiconazole NT NT NT NT 0.8 NT NT NT
Discussion
Preliminary data has been obtained for 9 pathogens and 6 fungicides. These baselines
data have included many isolates which date back to the years prior to the widespread
use of fungicides and therefore represent a true baseline. Also target sites gene sequences
have been recorded for 2 species and 3 genes.
Some hints of resistance have been found in barley powdery mildew and some
reduced sensitivity found in other pathogens. In the upcoming season (May to November
2010) we will continue to survey fungal isolates for both genetic and phenotypic signs of
resistance.
Dose rates
In Australia, the dose rates are specified as either one or two permitted doses. In contrast,
in the UK and Europe generally, maximum doses are specified. Average cereal yields in
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R. Oliver et al.
Australia are 1-2 tons/ha considerably less than the 6-10 tons/ha that are typically
achieved in Europe. One might expect therefore that the doses of fungicides used in
Australia would (like herbicides) be considerably less that those used in Europe. Table 4
shows some data compiled from APVMA and other web sites; relatively few products
are used in both jurisdictions. With the exception of Bravo (chlorothalonil) most doses
are lower in Australia. However whereas the Australian column lists the permitted doses
the UK column lists the maximum doses. Data from Cropmonitor and Farmstat suggest
that the average dose in the UK it typically half the maximum permitted dose; in
Denmark, doses are typically 1/3 the permitted dose. This would suggest that applied
doses do not differ systematically between crops in Australia despite the 5-10 fold
difference in crop biomass.
The effect of dose rate on the incidence of fungicide resistance has been a subject of
sometimes intense debate. The current HGCA advice is to use the “minimum effective
dose”. This suggests that there is significant potential to reduce doses in Australia
without compromising disease control. This would reduce costs and, according to HGCA
advice, reduce resistance risk.
Table 5: Comparison of permitted dose rates in UK and Australia (data from APVMA and HGCA).
Acknowledgements
References
Murray, G.M., Brennan, J.P. (2009): Estimating disease losses to the Australian barley industry,
Austral. Plant Pathol., 39, 85-96.
Murray, G.M., Brennan, J.P. (2009): Estimating disease losses to the Australian wheat industry,
Austral. Plant Pathol., 38, 558-570.
Solomon, P.S., Lowe, R.G.T., Tan, K.C., Waters, O.D.C., Oliver, R.P. (2006): Stagonospora
nodorum: Cause of Stagonospora nodorum blotch of wheat, Mol. Plant Pathol., 7, 147-156.
Wood, P.M., Hollomon, D.W. (2003): A critical evaluation of the role of alternative oxidase in
the performance of strobilurin and related fungicides acting at the Q(o) site of Complex III.,
Pest Management Science, 59, 499-511.
Cools, H.J., Fraaije, B.A., Kim, S.H., Lucas, J.A. (2006): Impact of changes in the target P450
CYP51 enzyme associated with altered triazole-sensitivity in fungal pathogens of cereal
crops, Biochem. Soc. Trans., 34, 1219-1222.
http://www.hgca.com/document.aspx?fn=load&media_id=5988&publicationId=4406
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40
Location-Specific Fungicide Resistance Profiles
Aid Peach Growers in Managing Fungicide
Resistant Strains of Monilinia fructicola
Abstract
Brown rot caused by Monilinia species can be a devastating preharvest and postharvest disease of
peach and other stone fruits. Management is largely dependent on preharvest application of site-
specific fungicides, such as methyl benzimidazole carbamates, demethylation inhibitors, and
quinone outside inhibitors. The site-specific nature of these chemical classes poses increased risk
of resistance development. In fact, resistance to all three chemical classes has been described in
the southeastern United States. Traditionally, producers are asked to follow best-management
general guidelines of fungicide resistance management. However, knowledge of location-specific
resistance profiles in Monilinia will provide information for improved resistance management,
likewise resulting in improved brown rot control. We developed an agar-based assay that allows
county agents (University-employed agents of technology transfer and consultants to producers)
to determine resistance profiles outside a research laboratory within 3 days. The kit allowing to
perform the assay is shipped to agents and can be stored for up to 4 weeks. Basically, agar disks
amended with a discriminatory dose of a fungicide are sliced off lipbalm tubes, placed into a
Petri dish and inoculated in the disk-center with spores of the fungus using toothpicks. Mycelial
growth is assessed after 3 days of incubation. We are currently testing the usefulness of a web
application designed to analyze, process, and store the visual assessments with the goal to
provide an immediate fungicide resistance management response.
commercial peach cultivar is susceptible to this disease, hence host resistance is not a
management option at this time.
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resistance profiles are different depending on the location and spray history of the
orchard (Figure 1). For example, growers who sprayed DMI fungicides selected most for
DMI resistance, and growers who sprayed QoI fungicides selected most for QoI
resistance. To our surprise, growers who alternated DMI and QoI fungicides did not
select less for DMI and QoI resistance, which is an important and novel aspect to
consider when designing resistance management strategies (Figure 1). The resistance
management program was instrumental in preventing producer losses in 2009, a very wet
year in which brown rot developed rapidly.
Figure 1: Frequency of DMI- and QoI-resistant strains from commercial peach orchards following
lipbalm disk assay assessment. Orchards were sprayed with various fungicide programs, including DMI
fungicides only, DMI and QoI fungicides in alternation, QoI fungicides only, or other spray programs for
preharvest brown rot control in South Carolina and Georgia, 2008.
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Figure 2: Diagram of the web-application in support of the fungicide resistance monitoring program.
Acknowledgements
Technical Contribution No. 5817 of the Clemson University Experiment Station. This
material is based upon work supported by NIFA/USDA, under project number SC-
1000642.
We thank P. Karen Bryson, Rohan Rajevan, and Hetal Kalariya for technical assistance.
References
Amiri, A., Brannen, P.M., Schnabel, G. (2010): Reduced sensitivity of Monilinia fructicola field
isolates from South Carolina and Georgia to respiration inhibitor fungicides. Plant Disease,
94, 737-743.
Amiri, A., Brannen, P. M., Schnabel, G. (2009): Validation of the lipbalm tube assay for
evaluation of fungicide sensitivity in field isolates of Monilinia fructicola. Plant Health
Progress doi:10.1094/PHP-2009-1118-01-RS.
Amiri, A., Scherm, H., Brannen, P.M., Schnabel, G. (2008): Laboratory evaluation of three rapid,
agar-based assays to assess fungicide sensitivity in Monilinia fructicola. Plant Disease, 92,
415-420.
Brannen, P. M., Schnabel, G. (2005): Brown rot. In: Southeastern Peach Growers' Handbook.
Edited by D. Horton and A. L. S. Johnson. Cooperative Extension Service, University of
Georgia, Athens. .
Luo, C.X., Schnabel, G. (2008): The cytochrome P450 lanosterol 14-alpha demethylase gene is a
demethylation inhibitor fungicide resistance determinant in Monilinia fructicola field
isolates from Georgia. Applied and Environmental Microbiology, 74, 359-366.
Luo, C.X., Cox, D.K., Amiri, A., Schnabel, G. (2008): Occurrence and detection of the DMI
resistance-associated genetic element 'Mona' in Monilinia fructicola. Plant Disease, 92,
1099-1103.
Williams-Woodward, J.L. (2004): 2003 Georgia Plant Disease Loss Estimates. Cooperative
Extension Service, University of Georgia, College of Agricultural & Environmental
Sciences.
Zehr, E.I., Toler, J.E., Luszcz, L.A. (1991): Spread and persistence of benomyl-resistant
Monilinia fructicola in South Carolina peach orchards. Plant Dis., 75, 590-593.
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41
CAA, Phenylamide and QoI Resistance
Assessment in Plasmopara viticola Oospores
Abstract
The development of appropriate anti-resistance strategies should be based on reliable data on the
sensitivity to fungicides used for the control of P. viticola populations which were collected early
in the season before the start of grapevine development. Sensitivity tests were carried out by
assessing the germination of oospores, the overwintering structures of the pathogen, incubated at
increasing concentrations of metalaxyl-M and mandipropamid or at a single discriminatory
concentration of azoxystrobin. EC50 values (for metalaxyl-M and mandipropamid) and the
percentage of resistant oospores (for QoI) were determined to estimate resistance in field
populations. No resistance was detected for metalaxyl-M and mandipropamid, but different levels
of resistance were found for QoIs. The use of PA, CAA and QoI fungicides in mixture resulted in
reduced EC50 and percentages of resistant oospore, whereas higher rates of resistant oospores
were associated with the application of QoIs as solo formulations.
Introduction
Grapevine downy mildew, caused by Plasmopara viticola (Berk. et Curt.) Berlese and
De Toni, is one of the most devastating diseases for Vitis vinifera L. The pathogen is able
to infect all green parts of the plant including bunches, leading to losses both in quality
and quantity of the yield. Chemical control is the most effective way to prevent the
occurrence of severe epidemics. Among single-site fungicides used against P. viticola,
QoIs (Quinone outside Inhibitors), PAs (Phenylamides), CAAs (Carboxylic Acid Amides)
and cyanoacetamid-oximes (cymoxanil) are frequently used in vineyards (Gisi and
Sierotzki, 2008). Different active ingredients with cross resistance behaviour in each
group are available in the first three classes.
The resting structures of P. viticola, the oospores, are produced at the end of the
grapevine growing season; they germinate in the following spring providing the
inoculum for primary infections. The oospores not only have a key role in the epidemics,
but also represent a source of genetic diversity for the pathogen, since they are
differentiated by sexual reproduction.
Investigations carried out a few years ago showed that azoxystrobin, a QoI fungicide,
affects P. viticola oospore differentiation (Vercesi et al., 2002). Recently, a biological
assay quantifying the percentage of oospores resistant to QoIs (RO) in the tested
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© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
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populations has been developed and good correlation was found between RO and the
frequency of alleles associated with QoI resistance determined by allele-specific real-
time PCR (Toffolatti et al., 2007; 2008). On the other hand, few information is available
on the effects of PAs and CAAs on the sexual structures of the oomycetes. A general
reduction in the number of oospores was observed following the application of metalaxyl
on potato leaf discs inoculated with Phytophthora infestans (Mont.) de Bary (Hanson and
Shattock, 1998) and dimethomorph on grapevine leaves inoculated before or after the
fungicide treatment (Bissbort et al., 1997). Moreover, the application of metalaxyl
reduced also oospore germination in sensitive strains.
The aims of the present study are the application of the biological assay based on
oospore germination for QoIs, PAs and CAAs, and monitoring the sensitivity of P.
viticola populations for all three fungicide classes following different treatment strategies.
Sampling
Sampling was carried out in 11 vineyards located in Piedmont (MO), Lombardy (GV,
SO), Emilia-Romagna (3P, SG, LAV) and Apulia (BBH, BAF, BAI, BAM and BAE)
(Table 1). Five vineyards were monitored for 2 (BAI) and 3 (MO, 3P, BBH and BAE)
consecutive years, respectively. The vineyards have been treated with PAs, CAAs and
QoIs in mixtures with partners belonging to different resistance groups. In a few cases,
QoIs have been applied as solo formulations (Table 1). From 2007 until 2009, leaves
showing downy mildew symptoms were randomly collected in vineyard during October.
Four nylon bags, each containing 50 leaf fragments rich in oospores were prepared per
vineyard and overwintered at 5°C and constant water content.
Table 1: Sample, locations of vineyards and number of treatments with QoIs, PAs and CAAs applied from
2007 to 2009.
QoI PA CAA
Sample Locations
2007 2008 2009 2007 2008 2009 2007 2008 2009
MO Calosso (AT) 3 3 3 0 0 0 1 1 1
GV Pietra dè Giorgi (PV) 0 - - 2 - - 0 - -
SO Sondrio 1 - - 3 - - 3 - -
3P Cotignola (RA) 0 0 0 0 0 0 0 0 0
SG Cotignola (RA) 0 - - 0 - - 0 - -
LAV Lavezzola (RA) - 2 3 - - 0 - - 4
BBH Rutigliano (BA) 2 4*+2 5*+2 1 0 0 1 1 1
BAF Rutigliano (BA) 2* - - 2 - - 1 - -
BAI Sannicandro (BA) 2* 0 0 3 3 3 0 0 0
BAM Sannicandro (BA) 0 - - 0 - - 3 - -
BAE Noicattaro (BA) 3* 4*+2 5* 1 0 0 3 0 0
*Solo treatments
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Biological assays
The sensitivity assays were carried out at the end of January as described by Toffolatti et
al. (2007). Oospore germination was assessed on 1% water agar (Agar Noble, Difco) and
on water agar amended with a discriminatory concentration of 10 mg/L of azoxystrobin
or with increasing concentrations of metalaxyl-M (0.1-0.5-1-10-100 mg/L)or
mandipropamid (0.01-0.1-1-10-100 mg/L). The active ingredients, technical grade, were
dissolved in DMSO. The final concentration of the solvent in the medium was lower than
0.1 %. 1200 oospores were spread in three replicates on each agar plate and incubated in
the dark at 20 °C. The number of macrosporangia differentiated by the oospores was
checked with a stereomicroscope (Leica Wild M10) 7, 10 and 14 days after incubation.
Data analysis
Oospore germination (G) was calculated as the average germination percentage of the
three replicates. The percentage of oospores resistant to QoIs (RO) was calculated as
RO (Gaz 100) Gwa , where Gaz and Gwa are the germination rates of the oospores
on water agar amended with azoxystrobin and on pure water agar, respectively.
Differences among the mean values of RO in the same vineyard in different years were
analysed by ANOVA. Since the conditions for parametric analysis of variance were not
satisfied, non-parametric ANOVA (Kruskall-Wallis test) was performed for the mean
values of the germination rates of all samples (Gav) and transformed in ranks, in order to
evaluate the differences at different metalaxyl-M and mandipropamid concentrations.
Multiple comparisons of the means were performed by REGW-F. The distributions of
Gav and RO were represented by box plot analysis. EC50 values, i.e. fungicide
concentration resulting in 50 % inhibition of oospore germination, were estimated by
probit analysis for the percentage inhibition of germination (GI) as
GI 100 (Gx 100) Gwa , where Gx is the germination rate at each fungicide
concentration (x). Analyses were carried out by SPSS software.
Results
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Figure 1: Germination rates (G) of the oospores incubated on water-agar (0) and at increasing
concentrations of metalaxyl-M (a) and mandipropamid (b).
The EC50 values calculated for metalaxyl-M were lower than 1 mg/L ranging from
0.03 to 0.74, the higher value was recorded in 2008-2009 in MO vineyard (Table 3).
Compared with the untreated vineyards (mean: 0.23; median: 0.16; IQR: 0.25), the
treated sites were characterized by similar values though with a slightly narrower
distribution (mean: 0.19; median: 0.17; IQR: 0.13) (Figure 2a). All samples showed very
low EC50 values for CAA, ranging from 0.001 to 0.009 mg/L (Table 2). Moreover, no
differences between treated and untreated vineyards were detected (Figure 2 b). Apart
from a single outlier, showing an EC50 of 0.013 mg/L, the distribution of the values was
similar in both the treated (mean: 0.003; median: 0.0015; IQR: 0.004) and the untreated
vineyards (mean: 0.003; median: 0.001; IQR: 0.007).
A reduced percentage of resistant oospores, lower than 10 %, generally
characterized the samples collected from vineyards which were untreated (GV, 3P, SG
and BAM) or treated with QoIs in mixture (MO in 2007-2008, SO, LAV and BBH in
2007) (Table 3). A significant increase of RO occurred at the end of 2009 in both MO
and BBH, probably as a consequence of immigration of resistant strains from
surrounding vineyards and from solo treatments, respectively. The application of solo
treatments in BAI vineyard resulted in higher RO values in 2007 (76 %) compared to
2009 (47 %), when no QoI were applied. No differences in RO during the three years of
monitoring were found in BAE vineyard, which has been treated with QoI both as solo
and mixture formulations. Treated vineyards differed from untreated (mean: 10.4;
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median: 4; IQR: 9) in their distribution of RO (Figure 2c): while mixture application was
associated with a lower range of variability (mean: 19; median: 4.5; IQR: 33.21), solo
sprays increased the frequency of individuals characterized by high resistance rates
(mean: 40; median: 30.4; IQR: 51.8).
Table 3: Average percentages of resistant oospores (RO) and EC50 values recorded in each vineyard.
Figure 2: EC50 and RO of the samples collected in vineyards which were untreated or treated with PAs (a),
CAAs (b) and QoIs (c).
Discussion
The results obtained by the germination assays showed a progressive decrease in oospore
germination following an increase in the doses of metalaxyl-M and mandipropamid. The
dose-response effect between both CAA and PA fungicides and the oospore germination
suggests that sensitivity tests with the sexual spores of P. viticola are a reliable method.
The assay has the advantages of avoiding the isolation and propagation of the pathogen
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and providing a valuable indication on resistance risk before grapevine growing season
starts, which allows a timely planning of the anti-resistance strategies. On the other hand,
it can be time consuming, since the macrosporangium formation occurs only from 7 to 14
days after inoculation. A more rapid and suitable alternative to the germination assay is
the detection and quantification of the mutations associated with resistance, which can be
done for CAAs and QoIs. The mechanism of resistance to QoI is well documented and
associated with a substitution of glycine by alanine at codon 143 (Gisi & Sierotzki, 2008).
Recently, a point mutation was identified at codon 1105 in the cellulase synthase, CesA3
gene leading to a susbtitution of glycine by serine in the protein conferring resistance to
CAAs in P. viticola (Blum et al., 2010). On the contrary, no information is available on
the site of mutation(s) in the target gene leading to metalaxyl resistance, which may
involve one semidominant gene affected by minor genes (Gisi and Sierotzki, 2008). Full
sensitivity to both PAs and CAAs was observed in all the samples monitored, whereas a
more diverse situation characterized the vineyards treated with QoIs: most of the samples,
collected from plots with were not treated or treated with QoI mixtures, showed a lower
frequency of resistant individuals in comparison with the populations sampled in
vineyards sprayed with solo QoI formulations.
In summary, no resistance was detected for PAs and CAAs, and no differences were
found in the EC50 values of the samples collected from untreated vineyards and those
treated with PAs and CAAs. However, samples treated with QoIs in mixture showed
reduced resistance rates whereas solo formulations resulted in higher selection pressure.
Acknowledgements
References
Bissbort, S., Albert, G., Schlösser, E. (1997): Effects of dimethomorph on the oospore formation
of Plasmopara viticola. J. Plant Dis. Protect., 104, 126-132.
Blum, M. Waldner, M., Gisi, U. (2010): A single point mutation in the novel PvCesA3 gene
confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara
viticola. Fungal Genetics and Biology, 47, 499-510.
Gisi, U., Sierotzki, H. (2008): Fungicide modes of action and resistance in downy mildews. Eur.
J. Plant Pathol., 122, 157–167.
Hanson, K., Shattock, R.C. (1998): Effect of metalaxyl on formation and germination of oospores
of Phytophthora infestans. Plant Pathology, 47, 116-122.
Toffolatti, S.L., Serrati, L., Sierotzki, H., Gisi, U., Vercesi, A. (2007): Assessment of QoI
resistance in Plasmopara viticola oospores. Pest Manag. Sci., 63, 194-201.
Toffolatti, S.L., Prandato, M., Serrati, L., Sierotzki, H., Gisi, U., Vercesi, A. (2008): Monitoring
QoI resistance in Plasmopara viticola oospore populations. In: Modern Fungicides and
Antifungal Compounds, Vol.V, Eds: Dehne, H.W., Deising, H.B., Gisi, U., Kuck, K.H.,
Russell, P.E., Lyr, H., DPG, Brauschweig, Germany. 159-165.
Vercesi, A., Vavassori, A., Faoro, F., Bisiach, M. (2002): Effect of azoxystrobin on the oospores
of Plasmopara viticola. Advances in Downy Mildew Research, Eds. Spencer-Phillips,
P.T.N., Gisi, U., Lebeda, A., Kluwer Academic Publishers, Dordrecht, The Netherlands.
195–199.
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42
Sensitivity to Boscalid of Stemphylium
vesicarium and Botrytis cinerea in Italy
Abstract
In a monitoring study analyzing the Stemphylium vesicarium sensitivity to boscalid, no sensitivity
changes compared to baseline values were observed in 72 isolates collected in commercial pear
orchards not treated with this fungicide. In orchards treated with boscalid for 4 years the same
sensitivity profile was found when 160 isolates were studied. A preliminary sensitivity study
performed with 70 Botrytis cinerea isolates from strawberries and grapes generally showed
sensitivity levels similar to that of a sensitive reference strain. However, the presence of reduced
sensitivity was found in some B. cinerea isolates originating from an experimental strawberry
field in Northern Italy.
Introduction
In 2006 boscalid was registered in Italy for the control of several pathogens on many
crops. It belongs to the carboxamide-fungicide group (inhibiting the respiratory chain at
the enzyme succinate dehydrogenase in complex II) and is an interesting fungicide
because it introduces a new mechanism of action in many crops. The specificity of its
target-site and the long-term experience with this mode of action in other indications
resulted in a medium risk of resistance classification by FRAC (www.frac.info). In fact,
occurrence of field-resistance has already been observed in some pathogens, such as
Alternaria alternata, Podosphaera xanthii and Corynespora cassiicola (www.frac.info).
In this study, the evaluation of boscalid sensitivity was carried out with Stemphylium
vesicarium, which is the most important fungal pathogen on pear in Italy, and with
Botrytis cinerea isolated from grapes and strawberries. For these two pathogens,
resistance to other fungicides has already been observed in Italy (Faretra and Pollastro,
1991; Gullino et al., 2000; Alberoni et al., 2005, 2010a), thus the introduction of a new
active ingredient with a different mechanism of action is useful for pathogen control
together with anti-resistance strategies that can prevent resistance occurrence.
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G. Alberoni et al.
Isolates
Isolates of S. vesicarium and B. cinerea were obtained from infected fruits collected in
commercial orchards and fields of northern Italy between 2007-2009 and 2007-2008
respectively. In particular 72 S. vesicarium isolates originated from pear orchards not
treated with boscalid, while 160 isolates belonged to orchards where this compound has
been used for pear brown spot control.
Forty-two B. cinerea isolates were collected in strawberry fields treated with
boscalid while 28 originated from grapes: hereof 19 isolates came from plots where
boscalid had not been used and 9 isolates from plots where boscalid had been included in
the treatment schedules.
S. vesicarium was isolated and maintained on V8 agar (20% V8 juice, 0.4% calcium
carbonate, 1.5% agar) at 23°C and at a 12-hour light-dark regime while B. cinerea was
grown on PDA at 20°C in the dark.
One boscalid-resistant reference isolate of B. cinerea was kindly supplied by BASF
(Limburgerhof, Germany) while the boscalid-sensitive reference isolate came from our
fungal collection.
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at 450 rpm. After 4 days, the growth was evaluated with a photometer (405 nm). EC50
values and relative growth were calculated as described above. The assays were repeated
twice and the mean is reported as final value.
In the monitoring study with S. vesicarium, the EC50 values of 72 isolates from untreated
commercial orchards ranged from 0.07 to 0.97 mg/l and were well comparable to
baseline values (0.11-0.81 mg/l, Alberoni et al., 2009). The same behaviour (EC50 values
between 0.07 and 0.98 mg/l) was shown by 160 isolates collected in commercial
orchards treated with this fungicide even for 4 years and up to 12 treatments in plot trials.
A first preliminary sensitivity study carried out with B. cinerea to compare both
methodologies generated generally similar EC50 values in both test systems, radial
growth and microtiter plate assay (Figure 1).
80
A 80
B
strawberries grapes strawberries grapes
70 70
isolate frequency (%)
60 60
50 50
40 40
30 30
20 20
10 10
0 0
0.01
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
1
2
4
5
>>5
0.01
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
EC50 (m g/l) >>1
EC50 (m g/l)
Figure 1: Frequency distribution of EC50 values of 70 B. cinerea isolates collected between 2007 and
2008 from strawberries and grapes. The sensitivity was evaluated through two methodologies: A mycelial
assays on radial growth; B microtiter test.
The analysis of radial growth on YBA solid medium yielded EC50 values with B.
cinerea isolates collected from grapes in the range from 0.04 to 0.87 mg/l.
Most B. cinerea isolates collected from strawberries (0.01-0.74 mg/l). These values
were close to that of the sensitive reference isolate (0.02 mg/l) and to baseline values
(Stammler and Speakman, 2006). All isolates were tested also by microtiter assay. B.
cinerea grows better in YBA liquid medium than on YBA agar medium, EC50s showed
less variability for the sensitive isolates (0.03-0.29 mg/l from grapes and 0.03-0.45 mg/l
from strawberries) and the ranges were comparable with those obtained through radial
growth.
Ten isolates from strawberries showed higher EC50 values with both methodologies,
some values seem to be even higher than those seen with the BASF resistant reference
isolate. A significantly reduced sensitivity to boscalid can therefore be assumed. The
relative growth values of these 10 isolates at the highest concentrations may suggest
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G. Alberoni et al.
Table 1: Relative growth (%) obtained at the highest concentrations with both methods (microtiter test
and mycelial growth assay) for isolates with reduced sensitivity collected from strawberries.
mycelial
microtiter test mycelial growth
Isolate ID host growth
1 mg/l 5 mg/l
1 mg/l
12-1.8 strawberries 67 63 18
12-2.5 strawberries n.t. 86 25
12-2.8 strawberries 81 66 39
12-3.1 strawberries 87 67 32
12-3.10 strawberries 48 62 47
12-3.11 strawberries 100 83 61
12-3.13 strawberries 94 92 76
12-3.6 strawberries 100 66 33
12-3.8 strawberries 69 74 54
12-3.7 strawberries 83 78 51
BASF-63233 Reference-resistant strain 80 96 96
n.t.= not tested
Conclusions
Acknowledgements
This research was supported by the Emilia Romagna Region (L.R. 28/98) and BASF
Italia Srl - Divisione Agro.
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References
Alberoni, G., Collina, M., Pancaldi, D., Brunelli, A. (2005): Resistance to dicarboximide
fungicides in Stemphylium vesicarium of Italian pear orchards. European Journal of Plant
Pathology, 113, 211-219.
Alberoni, G., Cavallini, D., Collina, M., Brunelli, A. (2009): Baseline sensitivity of Stemphylium
vesicarium, the causal agent of pear brown spot, to boscalid. Comm. in Agricultural and
Applied Biological Sciences, Ghent University. pp. 797-800.
Alberoni, G., Cavallini, D., Collina, M., Brunelli A. (2010a): Characterisation of the First
Stemphylium vesicarium isolates resistant to strobilurins in Italian pear orchard. European
Journal of Plant Pathology, 126, 453-457.
Alberoni, G., Cavallini, D., Ciriani A., Banorri, M., Collina, M., Brunelli A. (2010b): Sensitivity
to boscalid of Stemphylium vesicarium isolates collected in Italian pear orchards. Atti
Giornate Fitopatologiche, 2, 177-182.
De Miccolis Angelini, R.M., Rotolo, C., Pollastro, S., Faretra, F. (2010): Boscalid-resistant
mutants of Botryotinia fuckeliana (Botrytis cinerea) in southern Italy vineyards and their
genetic characterisation. J. Plant Path., in press.
Faretra, F., Pollastro, S. (1991): Genetic basis of resistance to benzimidazole and dicarboximide
fungicides in Botryotinia fuckeliana (Botrytis cinerea). Mycol. Res., 95, 943-951.
Gullino, M.L., Bertetti, D., Monchiero, M., Garibaldi, A. (2000): Sensitivity to
anilinopyrimidines and phenylpyrroles in Botrytis cinerea in North-Italian vineyards.
Phytopathologia Mediterranea, 39, 433-446.
Stammler, G., Speakman, J. (2006): Microtitre method to test the sensitivity of Botrytis cinerea
to boscalid. Journal of Phytopathology, 154, 508-510.
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G. Alberoni et al.
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43
Effects of Iprovalicarb on the Development and
Morphology of Various Phytophthora and
Pythium Species
Abstract
Iprovalicarb, an oomyceticide from the chemical class of carboxyl acid amides (CAA), is used
for the specific control of plant diseases caused by pathogens from the Peronosporales and
Pythiales. Pythium species or any other species outside of the oomycetes are not affected. The
mode of action of this compound is yet not known in detail, but the formation of the oomycetous
cell wall was described as a potential target. The main components of the cell wall are
polysaccharides, ß-1,3- and ß-1,6-glucans as well as cellulose, but not chitin. Because
iprovalicarb has no effect on Pythium species, for which the occurrence of both chitin and
cellulose in the cell wall is reported, in vitro experiments were conducted to investigate the
relevance of chitin in the response of oomycetes to iprovalicarb. The influence of iprovalicarb
and nikkomycin Z, a competitive inhibitor of chitin synthesis, on mycelium growth of
Phytophthora species and Pythium ultimum, alone and in combination, was evaluated.
Additionally, the effect on the structure of hyphae of Phytophthora spp. and P. ultimum was
investigated using histochemical techniques. Phytophthora species differed in their sensitivity to
iprovalicarb. The insensitivity of P. ultimum to iprovalicarb was confirmed. Nikkomycin Z did
neither inhibit the growth of Phytophthora-species nor of P. ultimum. In the presence of
iprovalicarb mycelial growth decreased significantly with increasing concentrations of
nikkomycin Z. Besides the inhibition of the mycelial growth a decrease in mycelium stability
were observed. After application of iprovalicarb Phytophthora species showed typically hyphae
with a beaded morphology associated with alterations in thickness and shape of the cell walls.
The hyphae of all Phytophthora species hardly showed any detectable labeling with WGA. In
contrast the external hyphal surface of P. ultimum was strongly labeled with WGA indicating that
chitin is a prominent cell wall component. The combination of iprovalicarb and nikkomycin Z
resulted in morphological modifications of P. ultimum; mycelial growth was irregular and
globular swellings of hyphae occurred. The presence of chitin in the cell wall may confer a
reduced sensitivity of Pythium species to iprovalicarb. However, high concentrations of
iprovalicarb cause deformation of P. ultimum hyphae, and in combination with nikkomycin Z
iprovalicarb influences growth and stability of the mycelium.
Introduction
pathogens belonging to the Peronosporales and Pythiales (Stenzel et al. 1998). The
compounds show protective, curative and eradicative effects, but the mode of action is
yet not known in detail. Mehl and Buchenauer (2002) precluded effects of iprovalicarb
on nucleid acid, protein, or lipid metabolism. Because morphological modifications of
mycelia as well as ultrastructural changes in cellulose formation occurred after
application of sublethal doses of iprovalicarb, the formation of the oomycetous cell wall
was described as a potential target (Jende 2001, Jende et al. 2002). The main structural
components of oomycete cell walls are ß-1,3- and ß-1,6-glucans as well as cellulose
(Bartnicki-Garcia and Wang 1983), highlighting the differences between oomycetes and
fungi, where chitin is the major constituent of the cell wall. However, although chitin is
not the typical cell wall carbohydrate of oomycetes, genes that encode putative chitin
synthases are present in species from different genera, including species in which chitin
has never been detected. For instance, analysis of the full genome of the plant pathogen
Phytophthora infestans has revealed the existence of a putative chitin synthase gene in
this species (Haas et al. 2009). While its cell wall seems devoid of chitin, it is required
for normal appressorium formation (Grenville-Briggs et al. 2008). Similarly, partial
sequences of putative chitin synthases have been isolated from Plasmopara viticola
(Werner et al. 2002), Phytophthora capsici and Achlya ambisexualis (Mort-Bontemps et
al. 1997). In the cell walls of Pythium ultimum the occurrence of both chitin and
cellulose was reported (Cherif et al. 1992).
Because iprovalicarb does not affect Pythium species or any other species outside of
the oomycetes, in vitro experiments addressing the relevance of chitin in the response of
various oomycetes to iprovalicarb have been performed. In this paper, the effect of
nikkomycin Z, a competitive inhibitor of chitin synthesis (Tariq and Devlin 1996), on
mycelial growth of Phytophthora species and Pythium ultimum was evaluated, alone and
in combination with iprovalicarb. Additionally, the effects on the structure of hyphae of
Phytophthora spp. and P. ultimum was investigated using histochemical techniques.
Phytophthora species and Pythium ultimum were grown on tomato juice agar (600 ml
H2Odemin, 150 ml tomato juice, 16 g agar-agar, 9.6 g potato dextrose broth, 2.4 g CaCO3)
in petri dishes, containing iprovalicarb and iprovalicarb in combination with nikkomycin
Z, respectively. Iprovalicarb was added from a stock solution to yield final
concentrations of 0, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 10, 30, 100, and 300 ppm. Similarly,
Nikkomycin Z was diluted to final concentrations of 0, 0.005, 0.05, 0.5, 5, 50, 500 µg/ml
and 200 µl were added to the petri dishes and distributed using a Drigalsky spatula
according to Bago et al. (1996). A mycelia plug from a 7 day old culture was placed in
the center with the air mycelium upside-down. The diameter of mycelial growth was
measured 4 and 8 days after incubation. Hyphal structures and cell wall compounds were
observed with a Leitz DMRB-photomicroscope and a Zeiss CLSM 300. For fluorescence
microscopy, hyphae were visualized with diethanol (0.05% in 0.1M Tris/HCL buffer, pH
8.0, polysaccharides) with filter combination BP340-380/FT400/LP430, Congo red
(0.1%, amyloid fibrils, polysaccharides, and chitin) with filter combination 543/BP 575-
640 and Oregon Green-labelled wheat germ agglutinin (WGA, Molecular Probes, Leiden
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Effects of Iprovalicarb
Results
Table 1: Effect of iprovalicarb on mycelium growth of Phytophthora species and Pythium ultimum.
EC50 and EC95 values were calculated using the dose/response model according to the Levenberg-
Marquardt algorithm (Marquardt1963).
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a a a
7 a a
a a a
0
0 0.1 1 10 100
Nikkomycin Z [µg/ml]
Without oomycide Iprovalicarb 0.1 ppm Iprovalicarb 0.3 ppm
Figure 1: Synergistic effect of iprovalicarb and nikkomycin Z on radial mycelium growth of P. infestans.
Different letters within each treatment indicate significant differences (Tukey, p ≤ 0.05).
A B C
D E F
G H I
Figure 2: Hyphal tips of Phytophthora infestans on agar without (A) and with 0.3 ppm iprovalicarb (B); P.
megaspermae on agar with 0.3 ppm iprovalicarb (C); P. ultimum on agar with 300 ppm iprovalicarb (D),
without iprovalicarb (E) and with nikkomycin Z (F); P. ultimum on agar with iprovalicarb (G, H), with
iprovalicarb + nikkomycin Z (I). Figures A, B, and D staining with diethanol; C, H, and I Congo Red; E, F,
and G with WGA-Oregon Green.
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Effects of Iprovalicarb
Conclusion
Iprovalicarb has significant effects on growth and cell wall formation of Phytophtora
species. Differences in the sensitivity of various Phytophthora species stress the impact
of the genetic variability of the primary target(s) of iprovalicarb in oomycetes within the
genus Phytophthora, including biotrophic as well as perthotrophic pathogens. The
presence of chitin in the cell wall may confer reduced sensitivity of Pythium species to
iprovalicarb. However, high concentrations of iprovalicarb cause deformation of P.
ultimum hyphae, and in combination with nikkomycin Z iprovalicarb affected growth and
stability of the mycelium due to the lack of chitin as an integral constituent. This
synergistic effect supports the conception that iprovalicarb has an impact on the
formation and deposition of cellulose and cross-linking in the cell wall of oomycetes.
References
Bago, B., Chamberland, H., Goulet, A., Vierheilig, H., Lafontaine, J.-G., Piche, Y. (1996): Effect
of Nikkomycin Z, a chitin-synthase inhibitor, on hyphal growth and cell wall structure of
two arbuscular-mycorrhizal fungi. Protoplasma, 192, 80-92.
Bartnicki-Garcia, S., Wang, M.C. (1983): Biochemical aspects of the morphogenesis in
Phytophthora. In: Phytophthora: Its Biology, Taxonomy, Ecology and Pathology (eds. D.C.
Erwin, S. Bartnicki-Garcia, P.H. Tsao), St. Paul, Minnesota. American Phytopathological
Society, 121-137.
Cherif, M., Benhamou, N., Belanger, R.R. (1992): Occurrence of cellulose and chitin in the
hyphal walls of Pythium ultimum: a comparative study with other plant pathogenic fungi.
Canadian Journal of Microbiology, 39, 213-222.
Grenville-Briggs, L.J., Anderson, V.L., Fugelstad, J., Avrova, A.O., Bouzenzana, J., Williaams,
A., Wawra, S., Whisson, S.C., Birch, P.R., Bulone, V., van West, P. (2008): Cellulose
synthesis in Phytophthora is required for normal appressorium formation and successful
infection of potato. Plant Cell, 20, 720-738.
Haas, B.J., Kamoun, S. Zody, M.C., Jiang, R.H.Y., Handsaker, R.E., et al. (2009): Genome
sequence and analysis of Irish potato famine pathogen Phytophthora infestans. Nature, 461,
393-398.
Jende, G. (2001): Die Zellwand des Oomyceten Phytophthora infestans als Wirkort von
Fungiziden. Dissertation, University of Bonn.
Jende, G., Steiner, U., Dehne, H.-W. (2002): Microscopical characterization of fungicidal effects
on the infection structures and cell wall formation of Phytophthora infestans. In: Modern
Fungicides and Antifungal Compounds III, Eds. Dehne, H.W., Gisi, U., Kuck, K.H.,
Russel, P.E., Lyr, H., Agro-Concept, Bonn, Germany. 83-90.
Marquardt, D.D. (1963): An algorithm for least-squares estimation of nonlinear parameters.
Journal of the Society for Industrial and Applied Mathematics, 11, 431–441.
Mehl, A., Buchenauer, H. (2002): Investigation of the biochemical mode of action of iprovalicarb.
In: Modern Fungicides and Antifungal Compounds III, Eds. Dehne, H.W., Gisi, U., Kuck,
K.H., Russel, P.E., Lyr, H., Agro-Concept, Bonn, Germany. 75-82.
Mort-Bontemps, M. Gay, L. Fevre, M. (1997): CHS2, a chitin synthase gene from oomycete
Saprolegnia monoica. Microbiology, 143, 2009-2020.
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Stenzel, K., Pontzen, R., Seitz, T., Tiemann, R., Witzenberger, A. (1998): SZX 722: A novel
systemic oomycete fungicide. The 1998 Brighton Conference – Pests & Diseases, 2, 367-
374.
Tariq, V.N.; Devlin, L (1996): Sensitivity of fungi to Nikkomycin Z. Fungal Genetics and
Biology, 20, 4-11.
Werner, S., Steiner, U., Becher, R., Kortekamp A., Zyprian, E. Deising, H. (2002): Chitin
synthesis during in planta growth and asexual propagation of the cellulosic oomycete and
obligate biotrophic grapevine pathogen Plasmopara viticola. FEMS Microbiology Letters,
208, 169-173.
284
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44
Powdery Mildew of Cucurbits:
Fungicide Resistance in Pathogen Populations of
Southern Spain
Abstract
Powdery mildew is a devastating disease of cucurbits worldwide and one of the most important
diseases affecting these crops in the Mediterranean basin under field and greenhouse conditions.
In this area, Podosphaera fusca is the main causal agent and one of the most important limiting
factors for cucurbit production. Despite the introduction of theoretically resistant cultivars,
growers still have important concerns about disease control, and application of chemicals
continues to be the main control means. In previous studies, high levels of resistance to QoI and
DMI fungicides have been detected in field populations of P. fusca in Southern Central Spain. In
order to explore the efficacy of other chemicals against P. fusca, in this work we determined the
levels of resistance to quinoxyfen in P. fusca populations. Two groups of P. fusca isolates
obtained from melon crops with different histories of quinoxyfen treatments, was used to
determine the baseline of quinoxyfen sensitivity. Using a leaf disc-based bioassay, minimum
inhibitory concentration (MIC) values were determined. MIC values were found to range from 1-
20 µg/ml in both groups of isolates. According to field application rate, no resistance to
quinoxyfen was found. These data indicates that quinoxyfen is a good alternative for cucurbit
powdery mildew management in Spain.
Introduction
Powdery mildew is probably the most common, widespread and easily recognizable
disease of cucurbits. The symptoms appear a few days after the infection, as white,
powdery spots form on both surfaces of leaves and sometimes also on fruits (Pérez-
García et al., 2009). In Spain the disease is caused by Podosphaera fusca, one of the
most important limiting factors for cucurbit production (Fernández-Ortuño et al., 2006).
Up to now, fungicides have played a decisive role in the management of this disease, but
their continuous use has caused the appearance of resistant strains, which show some
decreased sensitivity to specific fungicides (McGrath, 2001). In fact, the impact of
chemical control has been very much tempered by the speed with which P. fusca
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D. Bellón et al.
develops resistance, perhaps because high disease pressures require repeated fungicide
treatments (Hollomon and Wheeler, 2002).
In previous studies, we have observed high levels of resistance to the most important
classes of systemic site-specific fungicides, the QoI and DMI fungicides, in populations
of P. fusca in south central Spain (Fernández-Ortuño et al., 2006; López Ruiz et al.,
2010). In order to overcome this problem, it is necessary to develop new control
strategies that allow an appropriate disease management. Quinoxyfen is a medium-
resistance-risk fungicide (www.frac.info) widely used against powdery mildew diseases,
which seems to act by perturbing signal transduction (Hollomon et al., 1997; Wheeler et
al., 2003; Davey and McGrath, 2006). This compound was introduced in Spain to control
cucurbit powdery mildew around 2000. The aim of the present study was to determine
the levels of sensitivity to quinoxyfen in cucurbit powdery mildew populations in
Southern Central Spain and so anticipate possible shifts in fungicide sensitivity in
subsequent years.
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2009(n=25)
Acknowledgements
This work has been supported by grants from Dow Agro Sciences Ibérica and Plan
Nacional de I+D+I from Ministerio de Ciencia e Innovación, Spain (AGL2007-65430).
D Bellón was supported by a grant from Plan Nacional de I+D+I from Ministerio de
Ciencia e Innovación.
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D. Bellón et al.
References
Álvarez, B., Torés J.A. (1997): Cultivo in vitro de Sphaerotheca fuliginea (Schlecht. ex. Fr.),
efecto de diferentes fuentes de carbono sobre su desarrollo. Boletín de Sanidad Vegetal
Plagas, 23, 283-288.
Davey, J.F., McGrath, M.T. (2006): Sensitivity to the fungicide quinoxyfen of powdery mildew
isolates collected from pumpkin in New York in 2004. 2005 APS Northeastern Division
Meeting Abstracts, Phytopathology, 96, S177.
Fernández-Ortuño, D., Pérez-García, A., López-Ruiz, F.J., Romer, D., de Vicente, A., Torés, J.A.
(2006): Occurrence and distribution of resistance to QoI fungicides in populations of
Podosphaera fusca in south central Spain. European Journal of Plant Pathology, 115, 215-
222.
Green, E.A., Gustafson, G.D. (2006): Sensitivity of Uncinula necator to quinoxyfen: evaluation
of isolates selected using a discriminatory dose screen. Pest Management Science, 62, 492-
497.
Hollomon, D.W., Wheeler, I.E. (2002): Controlling powdery mildews with chemistry. In:
Bélanger, R.R., Bushnell, W.R., Dik, A.J., Carver, T.L.W., Eds. The Powdery Mildews. A
Comprehensive Treatise. APS Press, pp. 249-255. St. Paul, MN, USA.
Hollomon, D.W., Wheeler, I., Dixon, K., Longhurst, C., Skylakakis, G. (1997): Defining the
resistance risk of the new powdery mildew fungicide quinoxyfen. Resistance´97.
International Conference, Harpenden, UK, pp. 347-351.
McGrath, M.T. (2001): Fungicide resistance in cucurbit powdery mildew: experiences and
challenges. Plant Disease, 85, 236-245.
Lee, S., Gustafson, G., Skamnioti, P., Baloch, R., Gurr, S. (2008): Host perception and signal
transduction studies in wild-type Blumeria graminis f. sp. hordei and a quinoxyfen-
resistant mutant implicate quinoxyfen in the inhibition of serine esterase activity. Pest
Management Science, 64, 544-555.
López-Ruiz, F.J., Pérez-García, A., Fernández-Ortuño, D., Romero, D., García, E., de Vicente, A.,
Brown, J.K.M., Torés, J.A. (2010): Sensitivities to DMI fungicides in populations of
Podosphaera fusca in south central Spain. Pest Management Science, 66, 801-808.
Pérez-García, A., Mingorance, E., Rivera, M.E., del Pino, D., Romero, D., Torés, J.A., de
Vicente, A. (2006): Long-term preservation of Podosphaera fusca using silica gel. Journal
of Phytopathology, 154, 190-192.
Pérez-García, A., Romero, D., Fernández-Ortuño, D., López-Ruiz, F., de Vicente, A., Torés, J.A.
(2009): The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii),
a constant threat to cucurbits. Molecular Plant Pathology, 10, 153-160.
Wheeler, I.E., Hollomon, D.W., Gustafson, G., Mitchell, J.C., Longhurst, C., Zhang, Z., Gurr, S.J.
(2003): Quinoxyfen perturbs signal transduction in barley powdery mildew (Blumeria
graminis f. sp. hordei). Molecular Plant Pathology, 4, 177-186.
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45
Mechanisms of Fungicide Resistance in the
Apple Powdery Mildew Fungus Podosphaera
leucotricha
Abstract
Beside tree-cut in late winter or early spring the use of specific fungicides is the only option to
protect apples efficiently against the causal agent of powdery mildew, Podosphaera leucotricha.
Only few fungicides with different modes of action were licensed, e.g. penconazole acting as a C-
14-demethylase inhibitor and trifloxystrobin as a quinone outside inhibitor. Resistance against
fungicides may be caused by mutations in genes encoding fungicide targets, or by different
mechanisms reducing the intracellular fungicide concentration to non-critical values. Here we
present a strategy to analyze the mechanism(s) of strobilurin and azole fungicide resistance in
field populations of P. leucotricha, with special emphasis on the identification of point mutations
in the target genes and on MDR transporters.
Introduction
The ascomycete fungus Podosphaera leucotricha (Ell. & Ev.) E.S. Salmon (anamorph
Oidium farinosum Cooke) is the causal agent of powdery mildew of cultivated apple
(Malus×domestica), and may cause up to 30-40% yield loss (Krieghoff, 1995). The
mycelium of the fungus overwinters in dormant buds. During budding primary infection
of young leaves is initiated, leading to the production of conidia for further infection
cycles (Urbanietz and Dunemann 2005).
Intensive fungicide treatment is instrumental for disease control. Roßberg (2003)
noted that in Germany up to 20 fungicide treatments were usually performed to control
apple powdery mildew and apple scab. An important problem associated with these
treatments is that beside sulfur the active substances are single-site inhibitors, e.g. the
azole penconazole a C14-demethylase inhibitor [DMI]), inhibiting fungal sterol
biosynthesis and the strobilurin fungicide trifloxystrobin a quinone “outside” inhibitor,
(QoI), inhibiting mitochondrial ATP synthesis by binding to cytochrome b. In practical
applications it is not uncommon that single-site fungicides are applied successively. This
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strategy increases the risk of qualitative and quantitative fungicide resistance to occur
(Deising et al., 2002).
Qualitative resistance may result from mutations in genes encoding fungicide targets,
as shown for strobilurin fungicides (Ishii et al., 2001). In contrast, quantitative fungicide
resistance involves several mechanisms that are induced by sub-lethal fungicide
concentrations for example utilization of alternative metabolic pathways or enzymatic
degradation of antifungal compounds (Miguez, et al., 2004). Intracellular fungicide
concentrations can also be reduced by multidrug resistance (MDR) transporters such as
members of the ATP-binding cassette (ABC) transporter family or the Major Facilitator
Superfamily (MFS) transporters (Kretschmer et al., 2009; del Sorbo et al., 2000). These
transporter families contribute to fungicide resistance in various different fungi, and in
addition to different fungicides, the transport of a wide range of structurally diverse
molecules such as fluorescent dye ethidium bromide has been reported (Deising et al.,
2008; Reimann and Deising, 2005).
In this study, we analyzed the activity of plasma membrane transporters in conidia
of P.leucotricha with the fluorescent dye ethidium bromide. Furthermore, we used whole
genome amplification followed by specific amplification of at least 500bp the CYP51
gene encoding C14-demethylase, from DNA isolated from four independent P.
leucotricha field isolates. Sequencing of these fragments will allow detection of single
base-exchange explaining azole-resistance as shown in other fungi (Wyand and Brown,
2005).
For extraction of genomic DNA, conidia of P. leucotricha were shaken from inoculated
in vitro grown apple shoots into a sterile glass petri dish and mixed with sterile filtrated
protoplastation solution (20 mg/ml Lysing Enzymes of T. harzianum (Sigma,
Deisenhofen, Germany), 0.1 % (v/v) β-mercaptoethanol in 1.2 M KCl). After 4h at 30°C
the solution was transferred into a 25 ml corning-tube and was centrifuged (10 min at
800xg, 4°C). After discarding the supernatant the PeqLab fungal DNA Kit (PEQLAB
Biotechnologie GMBH, Erlangen, Germany) was used to extract DNA.
Whole genome amplification was performed using φ29 DNA polymerase
(Fermentas GmbH, St. Leon-Rot, Germany). An amount of 10ng template DNA was
mixed with random hexamer primers in water and heat-denatured at 95°C for 3 minutes.
After cooling on ice for 5 minutes dNTPs (Fermentas GmbH, St. Leon-Rot), BSA
(Fermentas GmbH, St. Leon-Rot), pyrophosphatase (Fermentas GmbH, St. Leon-Rot)
and the polymerase were added as suggested by the manufacturer. The mixture was
incubated at 30°C for 16h and subsequently heat-inactivated at 65°C for 10 minutes.
DNA was precipitated by using the Sure-Clean (Bioline GmbH, Luckenwalde) protocol.
The amplification of the gen fragment was performed by using Taq DNA polymerase
and degenerated primers (Pl.cyp51for: wygaytgtccwaattcmaarytmatg; Pl.cyp51rev:
ccagccatyagdagsgckatc) at an annealing temperature of 58°C for 50 cycles.
The visualization of the ethidium bromide efflux caused by transporter activity was
perform by fluorescence microscopy with an Eclipse600 fluorescence microscope (Nikon,
Düsseldorf, Germany), equipped with a UV-2A filter block (EX 340-380, DM 400, BA
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420; Nikon, Düsseldorf, Germany). In vivo produced conidia were shaken off from
inoculated in vitro grown apple shuts into a glass petri dish and incubated in darkness
with an aqueous solution of ethidium bromide (5 µg ml-1; Roche, Mannheim, Germany)
for 30 min. After three washes in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl,
8.5 mM Na/K phosphate, ph 7.2) conidia were suspended in 0.8% NaCl and evaluated by
UV microscopy.
Results
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Discussion
Acknowledgement
This work was funded by Sächsisches Landesamt für Umwelt, Landwirtschaft und
Geologie
References
Deising, H.B., Reimann, S., Pascholati S.F. (2008): Mechanisms and significance of fungicide
resistance. Brazilian Journal of Microbiology, 39, 286-295
Del Sorbo, G., Schoonbeek, H., De Waard, M.A. (2000): Fungal transporters involved in efflux
of natural toxic compounds and fungicides. Fungal Genetics and Biology, 30, 1-15.
Ishii, H., Fraaije, B.A., Sugiyama, T., Noguchi, K., Nishimura, K., Takeda, T., Amano, T.,
Hollomon, D.W. (2001): Occurrence and molecular characterization of strobilurin
resistance in cucumber powdery mildew and downy mildew. Phytopathology, 91, 1166-71.
Kretschmer, M., Leroch, M., Mosbach, A., Walker, A.S., Fillinger, S., Mernke, D., Schoonbeek,
H.J., Pradier, J.M., Leroux, P., De Waard, M.A., Hahn, M. (2009): Fungicide-driven
evolution and molecular basis of multidrug resistance in field populations of the grey
mould fungus Botrytis cinerea. PLoS Pathogens, 5, e1000696.
Krieghoff, O. (1995): Entwicklung einer Methode zur In-vitro-Selektion auf Resistenz gegen
Apfelmehltau Podosphaera leucotricha unter besonderer Berücksichtigung der
Rassenproblematik. Dissertation.
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Miguez, M., Reeve, C., Wood, P.M., Hollomon, D.W. (2004): Alternative oxidase reduces the
sensitivity of Mycosphaerella graminicola to QoI fungicides. Pest Management Science,
60, 3-7.
Reimann, S., and Deising, H.B. (2005): Inhibition of efflux transporter-mediated fungicide
resistance in Pyrenophora tritici-repentis by a derivative of 4'-hydroxyflavone and
enhancement of fungicide activity. Applied and Environmental Microbiology, 71, 3269-
3275.
Roßberg, D. (2003): NEPTUN 2001 – Erhebung von Daten zum tatsächlichen Einsatz
chemischer Pflanzenschutzmittel im Obstbau, im Hopfen und in Erdbeeren. Berlin,
Biologische Bundesanstalt.122.
Silander, K., Saarela, J. (2008): Whole genome amplification with Phi29 DNA polymerase to
enable genetic or genomic analysis of samples of low DNA yield. Methods of Molecular
Biology, 439, 1-18.
Urbanietz, A., Dunemann, F. (2005): Isolation, identification and molecular characterization of
physiological races of apple powdery mildew (Podosphaera leucotricha). Plant Pathology,
54, 125–133.
Wyand, R.A., Brown, J.K. (2005): Sequence variation in the CYP51 gene of Blumeria graminis
associated with resistance to sterol demethylase inhibiting fungicides. Fungal Genet. Biol.,
42, 726-35.
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46
Poor Fungicide Control of Cercospora Leaf Spot
on Sugar Beet in Switzerland:
Sensitivity Monitoring of C. beticola Isolates
S. SCHÜRCH
Agroscope Changins-Wädenswil Research Station, CP 1012, 1260 Nyon, Switzerland
Abstract
In Switzerland, protection of sugar beet against Cercospora leaf spot relies on DMI fungicides.
Lately, a single fungicide application was not sufficient any more to obtain a good control of the
disease and, in some cases, up to three treatments were necessary. This apparent decline in
efficacy may be due to a decrease in sensitivity of the target pathogen population or to an
inadequate application technique/timing. To test these hypotheses, fungicide field trials with four
widely used commercial products were set up at two locations in 2007 and 2008. There was a
tendency for products with two active ingredients to show a higher efficacy than products with a
single active substance. In vitro sensitivity tests of Cercospora beticola showed a small shift
towards lower sensitivity when comparing isolates collected before treatment with isolates
collected after treatment. In 2009, three commercial fields with a disease frequency of
approximately 90% after three fungicide applications were sampled. None of the isolates from
these fields showed a sensitivity level markedly different from that observed in 2007 or 2008.
Our in vitro tests did not show a clear loss in sensitivity despite a lack of efficacy in fields treated
meticulously at the onset of the epidemic.
Introduction
Cercospora leaf spot (CLS) is the most damaging foliar disease of sugar beet in
Switzerland. Sterol-demethylation inhibitors (DMIs) are widely used against Cercospora
beticola, the causal agent of CLS. DMI fungicides are used alone or in mixture,
sometimes with a quinone-outside inhibitor (QoI) or with fenpropimorph. CLS used to be
controlled adequately by a single fungicide application. However now, in some fields up
to three treatments are necessary. Poor field performance may be due to a decrease in
sensitivity of the target pathogen population or to an inadequate application technique or
timing. Triazole-resistant C. beticola isolates were reported in Greece (Karaoglanidis et
al., 2000). Field trials were conducted to obtain novel data on DMIs efficacy and, in
parallel, the resistance of C. beticola to the products used in the trials was determined in
vitro.
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© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
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S. Schürch
Fungicide trials were set up at two locations in 2007 and 2008 and included four
commercial products (active ingredients: cyproconazole, flusilazole, propiconazole +
difenoconazole, or epoxiconazole + pyraclostrobine). Sugar beet leaves were collected
before and after treatments. In 2009, leaves were collected from three commercial fields
showing a high disease severity despite three fungicide applications. C. beticola was
isolated from this leaf material and the growth of the isolates was tested on artificial
media (potato dextrose agar) amended with the fungicides used in the field trials.
As disease severity reached 80%, fungicide efficacy ranged from 17 to 95%. Products
containing two active ingredients showed a better efficacy (80%) than products with a
single active ingredient (55%). The in vitro sensitivity assays showed a small shift
towards lower sensitivity when the isolates collected before any treatment were
compared to isolates collected after fungicide application. Resistance factors were
calculated based on the IC50 population means and were comprised between 0.7 and 2.4.
There was a tendency for fungicide treatments to be less efficient when C. beticola
populations had a higher resistance factor. Isolates recovered from fields with poor CLS
control showed a sensitivity profile similar to that of isolates collected in the fungicide
trials of the previous years. Since the in vitro tests did not reveal the presence of
particularly resistant C. beticola isolates, further work could concentrate on improving
the application of the fungicides to the particularly waxy sugar beet leaves.
Acknowledgements
We would like to thank Ulrich Widmer (Centre Betteravier Suisse) for trials set up,
follow up and sampling, as well as Peter Frei and Nicole Lecoultre (Agroscope) for the in
vitro fungicide assays.
References
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47
Determination of the Potato Sprout Inhibitor
Chlorpropham and its Metabolite
3-Chloroaniline in Potato Samples
Abstract
A simplified method based on soaking overnight extraction coupled to HPLC - UV analysis was
developed for the simultaneous determination of the residue levels of the potato sprout inhibitor
chlorpropham (CIPC) and its metabolite 3-chloroaniline (3CA) in potato samples. The method
gave values approximately 25% higher when compared with a standard Soxhlet –GC method.
The results of spiking different layers from the potato tuber showed a high recovery of CIPC (>
95%) in all layers but the recovery of its metabolite 3CA was lower than 50% in the pith and 5%
in both cortex and skin.
Introduction
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Modern Fungicides and Antifungal Compounds VI
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Analytical grade reagents were used in this study: Chloropropham (purity 95%) was
supplied by Sigma, 3-chloroaniline (99%) was obtained from Aldrich, and propham from
Riedel- de Haën (Sigma-Aldrich). Methanol and Hexane that used were HPLC grade.
The HPLC system comprised a GILSON® 234-auto sampler, Cecil 1100 Series
pump, Phenomenex® ODS-2 250 x 4.60 mm 5µ Sphereclone column, and Thermo
Separation UV100 detector at 210 nm coupled with Dionex Peaknet software. An
isocratic method was employed with 62% (v/v) methanol as mobile phase at a flow rate
of 1.5 ml/m, 20µl sample injection volume, and chromatographic run time 15 minutes.
GC analysis was performed on a Hewlett Packard HP 5890A coupled to a Flame
Ionization Detector (FID) with HP 7633A auto sampler unit and DB-1 column (30 m,
0.53 mm i.d., 1.5 µm film thickness).
The procedure of soaking extraction method involved peeling the potato, chopping
the peel into fine pieces and mixing to obtain a homogenous sample. A 5g peel sample
was weighed into a 100 ml screw top jar, then 40 ml methanol containing the internal
standard 10 µg/ml Propham (IPC) was added as extracting solution and left to soak
overnight (~ 18 h) at room temperature. Next day, the extract was filtered and transferred
into HPLC vials through syringe (2 ml) and 0.2µm PTFE membrane syringe filter.
The soaking – HPLC method was validated and compared with Soxhlet extraction
which is the standard method at University of Glasgow. This standard method was
performed on the remainder of the peel for each tuber which was placed into a Soxhlet
thimble that contained 10 g sodium sulphate then extracted with 150 ml of hexane for 2
hours. The extract was then concentrated to 1 ml using a rotary evaporator, and 200 µl of
1000 µg/ml Propham (IPC) added and the volume was made up to 2 ml for GC analysis.
A robust method based on reversed phase HPLC with UV detection coupled with
soaking overnight extraction was developed for the separation and determination of
CIPC and 3CA in potatoes extracts. Applying optimum chromatographic conditions
achieved a best separation of chlorpropham, propham, and 3-chloroaniline at the
retention time (~ 12, ~ 6, and ~ 4 minutes respectively).
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Chlopropham Determination
The limits of detection (LOD) and quantification (LOQ) for the soaking- HPLC
method were determined by ten replicate injections (n=10) of a 0.05 µg/ml mixture of
CIPC, IPC and 3CA prepared in an extract of organic potato. LOD and LOQ of CIPC,
IPC and 3CA reported low values (0.002, 0.015, and 0.002) (0.008, 0.051 and 0.005)
mg/kg respectively.
To validate the soaking-HPLC method, it was compared with a standard Soxhlet -
GC method as shown in Figure 1. The regression line shows good correlation between
the CIPC residues in potato tubers analysed by both methods, however, the soaking –
HPLC method gave results approximately 25% higher than Soxhlet – GC standard
method. This difference can be attributed to the time of extraction and the higher polarity
of the methanol compared to hexane.
30
CIPC (mg/kg) by Soaking-
25 y = 1.25x
20
R2 = 0.97
HPLC
15
10
0
0 5 10 15 20 25
Figure 1: Shows the correlation between CIPC extract by methanol soaking extraction- HPLC analysis
and hexane Soxhlet extraction- GC analysis.
The recovery of CIPC and 3CA from spiking different layers from potato tuber
The recovery efficiency of soaking–HPLC method for CIPC and 3CA from spiking
different layers of the potato tuber produced high recovery of CIPC (> 95%) in all layers
but the recovery of its metabolite 3CA was lower than 50% in the pith and 5% in both
cortex and skin as shown in Figure 2.
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120
3CA
CIPC
100
Recovery% (n=5)
80
60
40
20
0
skin cortex pith
Figure 2: Shows the recovery of CIPC and 3CA from spiking different layers of potato tuber.
The low recovery of 3CA could be due to binding or instability of 3CA with the
potato tissues. Moreover, to the changes in the structural tissues and biological materials
for these various layers within the potato tuber tissues that could lead to difficult
extraction of 3CA as explained by others (JMPR, 2001; Still et al., 1981; Worobey et al.,
1987). From this poor recovery of 3CA found particularly from spiking potato skin and
cortex which less than 5% recovery, it can be concluded, the residue concentration of
3CA represents approximately 5% of the actual amount present in the potato tuber
treated with CIPC, and this low recovery is due to incomplete extraction. Therefore,
further work will be required to find a suitable way to improve the extraction of 3CA
from the potato tuber to obtain a higher recovery and investigate possibly losses of 3CA
from spiked potatoes.
Acknowledgements
References
JMPR. (2001): Joint FAO/WHO Meeting on Pesticide Residues. Geneva, www. fao.org.
Nagayama T., Kikugawa K. (1992): Influence of frying and baking on Chlorpropham residue.
Japanese journal of toxicology and environmental health.., 38 (1), 78-83.
Park L., Duncan H., Briddon A., Jina A., Cunnington A., Saunders S. (2009): Review and
development of the CIPC application process and evaluation of environmental issues.
Project Report 2009/5 © Agriculture & Horticulture Development Board (AHDB),
www.potato.org.uk.
SANCO. (2009): Commission of The European Communities. SANCO 04967 rev 3.
Still G. G., Balba H. M., Mansanger E. R. J. (1981): Studies on the nature and identity of bound
chloroaniline residues in plants. Journal of agricultural and food chemistry, 29, 739-746.
Worobey B.L., Sun, W.-F. (1987): Isolation and identification of Chlorpropham and two of its
metabolites in potatoes by GC-MS. Chemosphere, 16 (7), 1457-1462.
Worobey, B. L., Pilon J. C., Sun W-F. (1987): Mass Spectral Characterization of a Halogenated
Azobenzene (3, 3’-Dichloroazobenzene) from Potato Peels. Journal of agricultural and
food chemistry, 35, 325-329.
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48
Azole-Resistance in Aspergillus fumigatus:
Collateral Damage of Fungicide Use?
Abstract
Invasive aspergillosis due to multi-azole-resistant Aspergillus fumigatus has emerged in the
Netherlands since 1998, with 6 to 12.8% of patients harbouring resistant isolates. The presence of
a dominant resistance mechanism, a substitution at codon 98 of the Cyp51A-gene and a 34-base
pair tandem repeat in the gene-promoter region (TR/L98H), was found in over 90% of clinical A.
fumigatus isolates. This is consistent with a route of resistance development through exposure to
demethylase inhibitors (DMIs) in our environment. Indeed, TR/L98H A. fumigatus isolates were
cultured from soil and compost, were shown to be cross-resistant to certain DMIs, and were
genetically related to TR/L98H clinical resistant isolates. Given the limited alternative treatment
options in Aspergillus diseases and the importance of DMIs in food production and prevention of
spoilage, industry and academia should join forces with respect to research and management of
this emerging problem.
Introduction
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resistant isolates originating from the environment and from patients were more related
to each other than to control isolates that were azole-susceptible (Snelders et al., 2009).
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have been recovered for over 10 years in the Netherlands, it appears that TR/L98H does
not come with a significant fitness cost.
Conclusions
Azole resistance is emerging in the Netherlands and poses important challenges for the
management of patient with Aspergillus diseases. The azoles represent the most
important drug class for the prevention and treatment of Aspergillus diseases and are the
only agents that can be administered orally. Furthermore, for several clinical entities,
such as central nervous system aspergillosis, equally effective alternative drugs are not
available. On the other hand DMIs represent an important class of compounds for crop
protection and are vital for food production and prevention of spoilage. Therefore,
industry and academia should join forces to gain insight in the relation between the use
of DMIs and resistance development to medical triazoles in A. fumigatus. It is evident
that much more research is warranted in order to understand both the environmental and
patient routes of resistance development. This research will enable us to develop
antifungal stewardship benefitting both agriculture and medicine.
References
Chen, J. (2005): Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus
fumigatus serially isolated from a patient with lung aspergilloma. J Antimicrob Chemother,
55, 31-37.
Cornely, O.A. (2007): Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with
neutropenia. N Engl J Med.,. 356, 348–359.
Einsele, H. (1998): Prediction of invasive pulmonary aspergillosis from colonisation of lower
respiratory tract before marrow transplantation. Lancet, 352, 1443.
Hof, H (2001), Critical annotations to the use of azole antifungals for plant protection.
Antimicrob. Agents Chemother., 45, 2987–2990.
Howard, S.J. (2006): Multi-azole resistance in Aspergillus fumigatus. Intern J Antimicrob Agents,
28, 450–453.
Howard, S.J. (2009): Frequency and evolution of azole resistance in Aspergillus fumigatus
associated with treatment failure. Emerg Infect Dis., 15, 834-836.
Lass-Flörl, C. (2009): The changing face of epidemiology of invasive fungal disease in Europe.
Mycoses, 52, 197-205.
Mellado, E. (2007): A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-
resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob
Agents Chemother., 51, 1897-1904.
Müller, F-MC . (2007), Cross-resistance to medical and agricultural azole drugs in yeasts from
the oropharynx of human immunodeficiency virus patients and from environmental
Bavarian vine grapes. Antimicrob. Agents Chemother., 51, 3014–3016.
Oestreich A. (1997): Biological monitoring of the fungicide epoxiconazol during application.
Arch Environ Contam Toxicol., 33, 329-335.
Patterson, J.E. (1997): Hospital epidemiologic surveillance for invasive aspergillosis: patient
demographics and the utility of antigen detection. Infect Control Hosp Epidemiol., 18, 104-
108.
Sarfati, J. (2006): Recombinant antigens as diagnostic markers for aspergillosis. Diagn Microbiol
Infect Dis., 55, 279-291.
Snelders, E. (2008): Emergence of azole resistance in Aspergillus fumigatus and spread of a
single resistance mechanism. PLoS Med., 5, e219.
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49
Impact of Physicochemical Parameters on
Fungicide Activity
Abstract
Analysing the pattern of physicochemical properties of all fungicides registered in the latest
edition of “The Pesticide Manual” suggests the following properties of a fungicide for good
performance: a relatively high lipophilicity (logP between 2.5 and 4.5), a water solubility in the
range of 1 mg/L to 100 mg/L, and a molecular weight below 500 g/mol. Both the
physicochemical properties and the formulation similarly affect the bioavailability and the
systemic behaviour of a fungicide. Unfortunately, some important factors of influence for an
optimized biological activity like spray deposit properties, cuticle penetration kinetics and
adjuvant effects on leaf uptake are either poorly understood or extremely complex with no simple
linear relationship to any physicochemical parameter of the fungicide. The partitioning behaviour
of fungicides in the plant seems to be key for good performance. Besides water solubility and
melting point the resulting lipophilicity is probably the most important property of a fungicide
related to foliar uptake and translocation. The average lipophilicity for best performing
fungicides is relatively high (logP ca. 3.5), too high for a quick translocation in xylem, but best
suited for an even distribution in the leaves. Limitations in bioavailability of fungicidal actives
can often be compensated by the formulation. The formulation type and well selected adjuvants
can significantly change the bioavailability of a fungicide towards a more protective or a more
curative activity.
Introduction
The most common application method for fungicides is the spray application. The
fungicidal efficacy depends largely on the amount of active reaching the site of
biochemical action in the plant pathogen developing on or within the leaves. In order to
understand the field performance of a fungicide the complete sequence of steps affecting
the fungicide efficacy has to be considered starting with spray formation at the nozzles,
spray retention, spray deposit formation, deposit properties, penetration of the active into
the pathogen or into the leaf followed by redistribution in the plant tissue and long
distance translocation within the plant. The physicochemical properties of the fungicide
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Modern Fungicides and Antifungal Compounds VI
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Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
and the formulation are the most important factors impacting biological performance and
they have multiple effects on each of these steps.
The first steps - spray formation, retention and deposit formation - are solely
affected by the formulation but nevertheless relevant for the fungicide performance (Baur
and Pontzen, 2007). Most important for the efficacy of a fungicide is its release and
bioavailability from the spray deposit, the sorption into the cuticle and the penetration
into the leaf tissue. These steps are strongly affected by both the formulation components
and the physicochemical properties of the fungicide. The local, intercellular
redistribution of active ingredient within the leaf tissue or the plant pathogen and also the
long distance translocation in xylem and phloem is solely dependent on the
physicochemical properties. The field performance of a fungicide is therefore the result
of a very complex interplay of multiple factors, and in comparison to leaf applied
herbicides even a bit more complex: fungicides always need a long lasting and well
balanced protective and curative/systemic performance, whereas herbicides have to be
optimised just for high penetration and translocation leaving as little as possible of active
on the leaf surface.
There are some reviews in the literature dealing with the effect of physicochemical
properties of fungicides on biological performance (Edgington, 1981; Baker et al., 1992;
Wang and Liu, 2007) and also addressing the effect of the formulation on fungicide
activity (Stevens et al., 1988; Steurbaut, 1993), but there are still significant gaps in our
knowledge of how physicochemical properties in combination with formulants affect the
bioavailability of fungicides. This contribution is not a comprehensive review but
highlights the most important factors which affect the biological performance of
fungicides.
Fungicide Screening
During the last decades millions of compounds have been screened for fungicidal activity.
The final outcome of the screening are the fungicides listed in “The Pesticide Manual”
(Tomlin, 2009). The analysis of the physicochemical parameters of these fungicides
gives a first hint of the properties that are related to good field performance. For foliar
uptake and translocation of pesticides lipophilicity and water solubility are considered to
be the most influential (Briggs et al., 1982; Stevens et al., 1988; Baker et al., 1992).
Commercial fungicides have a large range of variation in lipophilicity and water
solubility (more than 5 orders of magnitude), but most compounds show clearly preferred
ranges (Figure 1): 69% of the fungicides have a logP in the range of 2.5 – 4.5 and the
water solubility varies between 1 and 100ppm for 62% of the compounds (out of 139
organic molecules, anorganic salts not included). High fungicidal performance is
obviously associated with relatively high lipophilicity and a low water solubility.
Interestingly, the subgroup of protective, multisite fungicides (including anorganic
salts) does not significantly differ in lipophilicity, but a very low water solubility is a
common property: with the exception of thiram (16.5 ppm) all multisite compounds have
a solubility less than 10 ppm; 60% have a solubility even less than 2 ppm. As protective
fungicides do not need to penetrate, but have to form a deposit reservoir on the leaf
surface which is stable over time as long as possible, rainfastness is a very important
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prerequisite for performance. The screening process obviously favors compounds with an
intrinsic high rainfastness based on the low water solubility.
80 80
A. B.
number of fungicides
60 60
40 40
20 20
0 0
<0 0-1 1-2 2-3 3-4 4-5 >5 < -1 -1-0 0-1 1-2 2-3 3-4 >4
logP log10 (solubility mg/L)
700
800
Class 1 Class 2 800
greenhouse performance
600
Class 3
700
700
500 600
600
400 500
(activity index)
500
AI_F
AI_F
AI_F
400
300 400
300 300
200
200 200
100 100 100
0 0 0
1 2 3 4 5 6 0 1 2 3 4 5 6 7 1 2 3 4 5 6 7
logP
LOGP_HCOOH logP
LOGP_HCOOH logP ID
LOGP_AC
The question arises whether the greenhouse screening identifies compounds with
similar physicochemical properties like today’s commercial products. Figure 2 shows
three sets of compounds representing different chemical classes (and also mode of action)
which are grouped according to lipophilicity (logP estimated by HPLC, acidic) and
biological performance in greenhouse tests. The dot-clouds (each dot represents the
average performance = activity index of one single compound) are always bell-shaped
indicating an optimum range for lipophilicity. Depending on the chemical class the
average logP (median) for best performance is 2.8, 3.9 and 3.3. These figures show that
the screening procedure in the greenhouse basically results in a lipophilicity spectrum of
actives similar to that of the commercial fungicides.
There are two further physicochemical parameters which are important for a good
field performance of fungicides: molecular weight and melting point. The molecular
weight never exceeds 500 g/mol and there is an interesting trend: the average molecular
weight of commercial fungicides is steadily increasing during the last 3 decades (see also
Figure 4). This leads to an increasing limitation by reduced mobility and thus
bioavailability and the need for adjuvanted formulations. The melting point of 80% of
commercial fungicides is in the range of 58° - 191°C.
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The decisive and common selection principles in the background of the screening
process, which also define the so-called “drugability” of a compound, will be briefly
discussed in the following paragraphs.
Spray deposit formation and properties have a strong impact on the bioavailability and
performance of fungicides, but they are poorly understood. Some effects are solely
formulation based like spray droplet spreading (mainly affected by static surface tension
and leaf surface structure) or hygroscopy which can be adapted by humectants. Detailed
information on plant surface wettability and deposit formation has been reported recently
(Baur and Pontzen, 2007).
A. B.
10 µm
Figure 3: Spray deposit of a WG-formulation on apple leaf (A) and of an EC-formulation on barley
leaf (B). SEM micrographs of leaf surfaces after cryofixation.
For best fungicide performance the spray deposit on the leaves has to be stable over
time and has to release the active timely and well dosed for initial penetration and for
protective control of the plant pathogen on the leaf. Unfavourable physicochemical
properties can lead to losses of fungicide from the deposit: high water solubility can
decrease rainfastness and a vapour pressure exceeding 10-2 mPa increases risk of vapour
loss (McCall, 1988). Stability against degradation by UV-light and hydrolysis has also to
be ensured. However, most important is the physical state of the active: a solid deposit
with crystalline active causes significantly reduced bioavailability in comparison to a
liquid-like deposit with the active in an amorphous state or even largely solubilised and
having an intimate contact to the cuticle surface (Figure 3). The higher the melting point
of a fungicide the lower is solubility and dissolution rate from crystalline deposits, as the
melting point is related to crystal lattice energy - and this leads to limited penetration
(Stevens et al., 1988).
Penetration
All aerial parts of a plant are covered by the cuticle, a lipophilic composite membrane
synthesized by the epidermis of leaves, fruits and flowers. The principal component of
the cuticular membrane is cutin, an insoluble amorphous polyester of cross-linked
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hydroxy-fatty acids and hydroxyepoxy-fatty acids and mainly aliphatic waxes (Holloway,
1993). The waxes are deposited on the cuticle surface (epicuticular waxes) and they are
also embedded in the upper layer of the cuticle, the so-called cuticular proper. The
intracuticular waxes in the cutin are mainly responsible for the rate-limiting barrier of
cuticles for foliar uptake. This has been clearly demonstrated with isolated cuticles
comparing permeabilities prior to and after extraction of the waxes: the permeability of
organic compounds increases up to 9200-fold (Riederer and Schönherr, 1985).
The penetration through the cuticle is a passive, diffusion-controlled process:
dM
K D (Co Ci )
dt
The steady state flow rate of an organic solute or a fungicide is a function of
partition coefficient K (partition spray deposit/cuticle and cuticle/epidermal cell wall), a
function of diffusion coefficient D and the concentration gradient between outer and
inner cuticle surface (= driving force). The partition coefficient is strongly affected by the
lipophilicity of the fungicide, and diffusion is mainly determined by the molecule size
and shape. This is a very rough description of the factors of influence; for more details
please refer to Bauer and Schönherr, 1992 and Kirkwood, 1999.
There is only a very limited number of studies in the literature that systematically
analyse the effect of physicochemical properties of fungicides on the penetration kinetics.
However, the conclusions are always the same: There is no simple linear relationship
between uptake and any physicochemical parameter of fungicides (Price and Anderson,
1985; Baker et al., 1992; Klittich et al., 2008). Lipophilicity is probably the single most
important property of agrochemicals related to foliar uptake (Stevens et al., 1988, Baker
et al., 1992). Klittich analyzed the systemic behaviour (penetration + translocation) of 23
fungicides and came to a similar conclusion: water solubility seems to be less important
than lipophilicity (Klittich et al., 2008). The optimum range for penetration of lipophilic
compounds was estimated to be logP > 2.9 (Baker et al., 1992). This fits quite well with
the lipophilicity distribution of commercial fungicides (Figure 1A).
Regarding mobility of agrochemicals in the cuticle there is a clear dependency: the
higher the molar volume, the lower the mobility (Bauer and Schönherr, 1992; Baur et al.,
1996). And this is obviously the explanation why the molecular weight of all commercial
fungicides never exceeds 500 g/mol (Figure 4). However, within the 100-500 molecular
weight range, the effect of molecular size on fungicide uptake seems to be not of decisive
importance, because other factors predominate and because there are suitable adjuvants
which can effectively reduce the size effect of actives in transcuticular diffusion (Baur et
al., 1997).
Cuticular penetration of fungicides is also strongly affected by the formulation and
adjuvants (in-can or added as tankmix). There are two main factors to be considered:
Solubilisation of active ingredient in the deposit (Stevens et al., 1988) and changing
of penetration barrier of the cuticle by swelling agents or plasticisers (Baur et al., 1997).
During deposit formation the fungicide concentration in the water phase of a spray
droplet steadily increases until solubility limit and precipitation. This results in different
kinetics of increasing driving force for penetration depending on water application rate,
covered leaf area, water solubility of the active and environmental conditions like
humidity and wind velocity. After deposit formation, the dissolved part of active in the
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dried deposit and the (speed of) resolubilization by high humidity, dew or rain is very
important for the long lasting bioavailability and penetration.
600
550
500 running average
of molecular weight
450
400
molecular weight
350
300
250
200
150
100
50
0
1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010
Figure 4: Molecular weight of marketed fungicides: steady increase of mean molecular weight over time.
Translocation
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lipophilicity the fungicide concentration in the water phases of apoplast and symplast
increases and the active becomes available for further diffusion into neighbouring leaf
tissue. If the compound enters a xylem vessel or a sieve element of the phloem, it will be
passively translocated by the water flow of the transpiration stream in the xylem or the
assimilate flow in the phloem.
As the water flow in xylem vessels is much higher than in the sieve tubes (2 – 10
m/h in comparison to about 0.2 – 1 m/h in the phloem), long distance translocation of
nonionised compounds only takes place acropetally in the xylem. As most fungicides are
not ionised they are only xylem-mobile; a few exceptions will be discussed later.
The long distance transport in the xylem is usually a combination of mass transport
and partitioning, similar to a chromatography. The lignified cell walls of xylem vessels
and the lipid membranes of adjacent cells act as a lipophilic sorption medium. The higher
the lignin content of xylem vessels the stronger is the adsorption of pesticides (Barak et
al., 1983). Therefore, the translocation of agrochemicals is usually slower in woody
plants in comparison to young seedlings or herbaceous plants.
In order to compare the translocation of fungicides with different physicochemical
properties, tebuconazole (logP = 3.7, solubility: 36 mg/L) and iprovalicarb (logP = 3.18,
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solubility: 11 mg/L [SR], 6.8 mg/L [SS]) were applied to cut grape vine shoots for 1 and
3 days; this experimental set-up allows the uptake of the fungicides without any
penetration barrier by cuticle or root (Figure 5). Tebuconazole shows the typical
behaviour of a lipophilic compound: slow translocation and always a strong sorption to
or around the xylem vessels. Older leaves need 3 days for a homogeneous filling with the
active and the youngest developing leaves have not been reached by the active even after
3 days. The slightly less lipophilic iprovalicarb shows a quick translocation to the
youngest leaves combined with a very uniform distribution in all leaves within 1 day.
However, after 3 days iprovalicarb begins to show an uneven distribution because of an
accumulation in the intercalary (intercostal) leaf areas leaving the xylem vessels with a
lower a.i. content. This example clearly shows that lipophilicity is more important than
water solubility in determining the xylem translocation: iprovalicarb is the much quicker
compound despite a lower water solubility! Unfortunately, there is no systematic analysis
of the xylem mobility of a broad spectrum of fungicides without penetration barrier in the
literature.
Several authors analysed the optimum lipophilicity for xylem translocation of
agrochemicals after root application (Briggs et al., 1982; Sicbaldi et al., 1997) or after
leaf application (Stevens et al., 1988). These studies always end up with optimum values
of logP between 1.5 and 3.0. Compounds with higher and with lower lipophilicity show
increasingly less translocation rates. This means that the commercial fungicides with an
average logP of about 3.5 are by far too lipophilic for maximum systemic translocation.
A simple explanation could be: A long lasting even distribution of a fungicide is more
important for a good field performance than high systemic translocation rates. As
demonstrated in Figure 5, tebuconazole with its relatively high lipophilicity needs some
days to be completely distributed but then it shows a long lasting homogeneous
distribution. Compounds with logP < 3 are expected to show an increasing accumulation
at the leaf tips; and compounds with logP > 4 are increasingly immobile or they show an
extremely slow long distance translocation (but resulting in an even distribution!).
These conclusions are only valid for non-ionised compounds. In the case of weak
acids or amines the distribution coefficients become strongly dependent on the pH and
the dissociation constant of the compound. As apoplast and symplast have a different pH
(6 and 8 respectively), agrochemicals with a pKa in the range of about 4 - 7 show a
different distribution and translocation behaviour in the plant vascular system. Many
herbicides are phloem-mobile because they are weak acids. They accumulate in the
symplast and in the sieve tubes according to the ion trap mechanism (Kleier, 1988).
Fungicides do not have the physicochemical properties needed for phloem mobility
(Brudenell, 1995) and are therefore not phloem-mobile - with one exception: fosetyl-
aluminium.
However, some fungicides are amines which can be ionised at the lower pH of the
apoplast. The morpholine fungicides tridemorph and fenpropimorph and also
spiroxamine have a pKa between 6.5 and 7 leading to a lower lipophilicity accompanied
with a higher water solubility in the apoplast. This results in a better translocation in the
xylem (Chamberlain et al., 1998; Inoe et al., 1998).
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Both the formulation and the physicochemical properties have an equally important
strong impact on the bioavailability and the systemic behaviour of a fungicidal active,
finally defining its biological performance in the field. Unfortunately, the most important
factors for an optimized biological activity like spray deposit properties, cuticle
penetration kinetics and adjuvant effects on leaf uptake are poorly understood or
extremely complex with no simple linear relationship to any single physicochemical
parameter of the fungicide. The partitioning behaviour of fungicides in the plant seems to
be decisive for good performance. Besides water solubility and melting point the
resultant lipophilicity is probably the most important property of a fungicide related to
foliar uptake and translocation. The lipophicity of the best performing fungicides is
relatively high (logP ca. 3.5), too high for a quick translocation in xylem, but best suited
for an even distribution in the leaves.
A good field performance of a fungicide needs an optimized distribution regarding
time course and space. This makes the selection process of a promising fungicidal
compound more difficult and complex in comparison to a leaf applied herbicide which
has just to be optimised for high penetration and (phloem) translocation. Fungicides
always need a well optimised balance of active taken up immediately after application for
curative and systemic activity, and the active remaining in deposit on the leaves for the
long lasting preventive action and possibly further slow release for leaf uptake. This
balance can be different for the same fungicide, if it is applied in various crops or against
different pathogens. A fungicide applied only one or two times per season may need a
different distribution pattern in comparison to an active repeatedly applied in short
intervals. Therefore, the fungicide distribution pattern has to be optimized separately for
each host-pathogen combination by field trials based on the given physicochemical
properties and the fine tuning by selection of suitable formulation type or adjuvants.
This process is unfortunately still based on a more or less empirical approach. More
systematic studies are needed to characterize and further improve the knowledge on the
mode of action of adjuvants and the impact of physicochemical parameters on the
bioavailability and systemic distribution of fungicides.
Acknowledgements
The authors would like to thank Dirk Stübler and Anne Suty-Heinze for providing the
autoradiographies of Figure 5, and Pierre Genix for fruitful discussions and supplying the
data of figure 2 and 4.
References
Baker, E.A., Hayes, A.L., Butler, R.C. (1992): Physicochemical properties of agrochemicals:
their effect on foliar penetration. Pestic. Sci., 34, 167-182.
Barak, E., Dinoor, A., Jacoby, B. (1983): Adsorption of systemic fungicides and a herbicide by
some components of plant tissues, in relation to some physicochemical properties of the
pesticides. Pestic. Sci., 14, 213-219.
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50
New Approaches to Optimize Spray Efficacy
and Foliar Uptake
Abstract
The performance of state-of-the-art pesticide formulations is significantly enhanced by
optimizing the droplet size distribution, the droplet adhesion on first impact, the spreading of
droplets on leaves, the spray retention, and by improving the uptake characteristics of the active
ingredient. A series of laboratory techniques will be introduced which are able to characterize
formulations with respect to their application properties. These techniques allow the target-
oriented development of improved formulations within short times and with a limited number of
field trials. For fungicide formulations, we show how built-in adjuvants control foliar uptake and
how, in the same formulation, non-systemic actives are deposited within the waxy layer of the
leaves. Thus, with targeted deposition and controlled translocation of active ingredients, high
curative activity is achieved with good protective activity in the same formulation, leading to
high biological efficacy and excellent yield.
Introduction
H. Auweter et al.
Product
Dilution
Spraying
Adhesion
Spreading
Uptake
Figure 1: Major aspects of the delivery chain for the spray application of cereal fungicides.
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typical size of the droplet is about 300 µm. About 10 to 15 videos are taken from one
formulation and they are numerically analyzed in order to determine the volumes of
primary and reflected droplets. From these volumes, the relative percentage of droplet
adhesion is derived.
Spray retention
As a result of good droplet adhesion, the amount of spray liquid staying on the leaves
can reach high values. The consequential retention values can be up to five or six times
the corresponding values obtained with water (Ellis et al., 2004). Spray retention values
are captured quantitatively by fluorescence labelling the spray liquids with 200 ppm
fluorescein. Typically, in the case of wheat, plants of growth state 12 (BBCH) are used.
After spraying, the leaves are collected, washed with alkaline water and analysed for the
amount of spray staying on the leaves. As a reference, the same experiment is
conducted with fluorescence labelled water. The retention obtained with water is set to
100 %.
Foliar uptake
The uptake of active ingredients into plant leaves is essential for post-emergence
herbicides, for curative fungicides, and for systemic insecticides (Schreiber and
Schönherr, 2009; Wang, 2007). Therefore, we established a laboratory test to quantify
the uptake of actives into leaves. Since these experiments shall be performed with
formulations which were produced under typical laboratory or production conditions,
we refrain from using radioactively labelled actives.
For a typical uptake measurement on wheat, we use small plants with two leaves, i.e.
of growth state 12 (BBCH). Three drops of spray solution of 1 µl each are usually placed
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H. Auweter et al.
on a leaf. The plants are then further cultivated in growth chambers at 20 °C and 80 %
relative humidity and at a day/night cycle of 16/8 hours. After the desired exposure time,
the leaves are washed with water in order to remove unattached active ingredients. Then,
the wax layer is removed from the washed leaves by means of cellulose acetate film
stripping. Finally the active ingredients, having been taken up by the leaves, are extracted
with methanol and are quantitatively analyzed by HPLC-MS/MS. In the case of more
than one active, this analysis can be performed for all actives in one single run. This
experimental procedure allows the quantitative determination of each active in three
regions of the leaf, i.e. on top of the leaf, within the waxy layer of the leaf, and inside the
leaf.
Droplet size distributions are mainly controlled by the nozzle type. For example, air
injection nozzles may reduce the amount of small droplets such that the wind drift is
reduced up to 99 %. However, we show that by modifying formulations, a reduction of
fine droplets can be achieved without a significant increase of the large droplet fraction.
Thus, properly adjusted formulations are able to reduce wind drift without sacrificing
good leaf coverage.
Besides the control of droplet sizes, the adhesion of droplets on leaves is of great
importance. In Figure 2 we show, as an example, the behaviour of a pure water droplet,
of a standard formulation, and of a newly optimized formulation, impacting on a wheat
leaf. The advances in improving droplet adhesion with new optimized formulations are
quite obvious.
Good droplet adhesion is the basis for high spray retention values: spray droplets
“stick” on their first impact and “stay” on the leaf. Thus, “Stick and Stay” is an important
new approach to improve leaf coverage and, as a consequence of this, to improve
biological efficacy.
In order to investigate the behavior of formulations under various spraying
conditions we define three different application modes. For the “reference” application,
we choose a water rate of 200 l/ha, a driving speed of 5 km/h, and a flat fan nozzle (LU
120 02, Lechler) at 3.3 bar. This application is known to give high biological
performance and will thus serve as reference. The second application mode is labeled
“low drift” mode. Here we use a water rate of 120 l/ha, an air induction nozzle (IDK 120
04, Lechler) at 1.75 bar, and the driving speed is 12 km/h. These parameters result in
coarse droplets and are thus the conditions for low wind drift behavior. Finally, the third
application mode is labeled “high speed” application. Here the water rate is 70 l/ha, the
driving speed is 20 km/h, and a double fan nozzle (TD HS 03, Agrotop) at 2.95 bar is
applied. These latter application conditions are fairly progressive and the biological
performance has to be confirmed
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600
500
300
200
100
0
water standard optimized
standard formulation: partial adhesion formulation formulation
Figure 2: Single droplets of about 300 µm diameter are impacting on wheat leaves. Top: droplet of pure
water – bouncing off. Centre: droplet of a standard formulation – satellite droplets are created. Bottom:
Droplet of an optimized formulation – total adhesion. On the right, the corresponding retention values are
shown.
700
600
500
400
300
200
0 water
Standard Optimized Standard Optimized Standard Optimized
. formulation formulation formulation formulation formulation formulation
Figure 3: Normalized spray retention values for a standard formulation and for an optimized formulation.
The details of the spray conditions are explained in the text. The dots are median values, the line in the
box is the mean value, 50 % of the values are inside the boxes, and the whiskers correspond to the
minimum and maximum values, respectively.
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H. Auweter et al.
From Figure 3 we can see that, even with advanced spraying conditions, new
optimized formulations will yield very high spray retention values. This experimental
approach allows us to characterize the application behavior of formulations rapidly and
thus to give immediate feedback to the formulation chemists for further optimization.
Figure 4 shows the results of uptake experiments with wheat plants. The formulation
applied was a solvent-based formulation, containing emulsifiers and an alcohol
alkoxylate uptake enhancer. This formulation is especially designed for both, curative
and protective activity. The two effects can be clearly distinguished: on the left hand part
of Figure 4 it can be seen that the protective active ingredient is enriched in the waxy
layer, whereas, on the right hand side it can be seen that the curative active ingredient is
mostly taken up into the leaf. Both the intake of the first active into the waxy layer and
also the uptake of the second active into the leaf are already taking place within the first
24 hours. Furthermore, it is interesting to note that the actives remain in their
corresponding reservoirs for as long as 7 days, thus ensuring long-time activity and
protection.
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
WASH WAX UPTAKE WASH WAX UPTAKE
Figure 4: Distribution of the active ingredients of a solvent-based formulation: on the leaf, in the waxy
layer of the leaf, and inside the leaf. The active ingredient with protective activity mainly remains in the
waxy layer (graph on the left), whereas the active ingredient with curative activity is taken up by the plant
(graph on the right).
In summary we showed that optimizing all steps along the delivery chain is a very
rewarding approach to optimize spray efficacy. Laboratory techniques which are able to
simulate real spraying conditions are very helpful in this respect. Finally, the
advancement of foliar uptake measurements demonstrates quantitatively if active
ingredients are properly located at their determined site of action.
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Acknowledgements
We are very grateful to Anke Reinold for performing the HPLC-MS/MS analyses very
thoroughly.
References
Ellis M.C.B, Webb D.A., Western N.M. (2004): The effect of different spray liquids on the foliar
retention of agricultural sprays by wheat plants in a canopy. Pest Management Science, 60
(8), 786-794.
Schreiber L., Schönherr J. (2009): Water and Solute Permeability of Plant Cuticles. Measurement
and Data Analysis. Springer-Verlag Berlin Heidelberg 2009.
Southcombe E.S.E., Miller P.C.H., Ganzelmeier H., van de Zande J.C., Miralles A., Hewitt A.
(1997): The International (BCPC) Classification System Including a Drift potential factor.
The 1997 Brighton Crop Protection Conference – Weeds, 371-380.
Wang C.J., Liu Z.Q. (2007): Foliar uptake of pesticides – Present status and future challenge.
Pesticide Biochemistry and Physiology, 87, 1-8.
Zabkiewicz J.A. (2007): Spray formulation efficacy – holistic and future perspectives.
CropProtection, 26, 312-319.
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H. Auweter et al.
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51
Allicin from Garlic, Effective in Controlling
Several Plant Diseases, is a Reactive Sulfur
Species (RSS) that Pushes Cells into Apoptosis
Abstract
The volatile antimicrobial substance allicin (diallylthiosulfinate) is produced in garlic when the
tissues are damaged and the substrate alliin (S-allyl-L-cysteine sulfoxide) mixes with the enzyme
alliin-lyase (E.C.4.4.1.4). The effectiveness of allicin in garlic juice against a range of plant
pathogenic microorganisms in vitro and in planta in diseased tissues has been demonstrated.
Allicin is readily membrane-permeable and a pro-oxidant which undergoes thiol-disulfide
exchange reactions with free thiol groups in proteins. It was suggested that inactivation of
specific, essential SH-containing enzymes was the basis of allicin’s antimicrobial action. We
investigated the cellular mechanism of action of allicin using Saccharomyces cerevisiae as a
model fungus. The GSH/GSSG couple is quantitatively the most important redox buffer in the
cell. In order to be able to calculate the electrochemical cell potential, we measured changes in
the absolute concentrations of reduced and oxidized glutathione after allicin treatment. We tested
the hypothesis that allicin-treatment could change the cell’s overall electrochemical potential. On
the basis of our results we propose a novel mechanism for allicin’s antimicrobial action. In our
model, rather than only targeting specific proteins as previously thought, allicin would be able to
affect the state of many oxidation-sensitive proteins throughout the whole cell, by shifting the
cell’s overall redox potential to a more oxidized state, i.e. by disturbing the cell’s redox
homoeostasis. Depending upon the magnitude of the redox shift, cells are pushed either into
apoptosis, or presumably, cells in an even more oxidized state will no longer be metabolically
competent to execute apoptosis and instead necrose.
Importantly, allicin is an example of an RSS or ‘reactive sulfur species’. RSS are redox
active in vitro and are usually physiologically active in vivo. Thus, natural sulfur products from
plants and their intracellular targets might provide the basis for innovative design of novel
antibiotics, fungicides, pesticides and anticancer agents.
Introduction
successes, as evidenced by increased yields that fungicide and pesticide use, along with
good agricultural practice, have helped to achieve. Nevertheless, the emergence and
spread of fungicide resistance is a constant source of concern, and demands effective
counter-measures and new strategies. On a limited time scale the emergence of fungicide
resistance is a microcosm for the co-evolution of plants with their pathogens. Over an
evolutionary time scale plants have been attacked by pathogens and pests seeking to use
them as a potential food source. Under this selection pressure plants have developed and
optimized their own chemical defence mechanisms to combat their enemies. The
requirement for sustainable solutions in agriculture, and consumer pressures for “green”
alternatives to conventional plant protection chemicals have accompanied a boom in the
organic farming sector. This has awakened new interest in Natural Products (Firn, 2010),
and endogenous plant defence mechanisms, as pointers for novel industrial plant
protection strategies (Slusarenko et al., 2008).
Substances honed by evolution for their function in plant defence are not optimised
for industrial production or mass application to crops and there may be much cheaper
synthetic alternative available to do the same job. Nevertheless, as a starting point as
lead compounds for development and formulation to enhance desirable and reduce
undesirable properties, natural product structures can be an important starting point.
Similarly, the mechanism of action of natural products can suggest strategies for targeted
design of novel plant protection compounds. While relatively few natural products are
used for plant protection, it is a sobering fact that in human medicine more than 50 % of
drugs currently in clinical use are of natural product origin (Peterson and Anderson,
2005). These authors go on to state that “Despite this statistic pharmaceutical companies
have embraced the era of combinatorial chemistry, neglecting the development of natural
products as potential drug candidates in favor of high-throughput synthesis of large
compound libraries”.
We believe that such a potential is to be found in the various organosulfur
compounds selected in the course of evolution in garlic (Allium sativum) for its
protection against pathogens and pests (Curtis et al., 2004; Portz et al., 2008). This
potential lies not only in the compounds themselves or their derivatives, but as a lead for
a novel plant protection strategy. Our work suggests for example, that allicin can kill
fungi by perturbing their cellular redox status and pushing the cells into apoptosis.
Pushing cancer cells into apoptosis has long been a strategy to combat this disease
(Wondrak, 2009) but, to our knowledge, use of apoptosis-inducing drugs to combat
agriculturally-important pathogens has not been tried.
Garlic Substances
Allicin, a volatile phytoanticipin (VanEtten et al., 1994), is the first major volatile sulfur
compound produced by garlic when the tissues are damaged. It was identified as the
major antimicrobial substance from garlic by Cavallito and Bailey (1944). It arises when
the cellular compartmentalization separating the enzyme alliinase (E.C.4.4.1.4) from its
substrate alliin is disrupted. Alliin is an odourless allyl cysteine sulfoxide. Alliinase also
reacts with other cysteine sulfoxides to produce quantitatively minor thiosulfinates, e.g.
methylallyl thiosulfinate. Allicin is responsible for the typical smell of freshly crushed
garlic but rapidly undergoes complex condensation reactions to produce mono-, di- and
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polysulfides, all of which contribute to the bouquet of garlic odours (Block, 2010). Many
of these follow-on products are physiologically active in their own right (Figure 1).
COO-
alliinase (binary system, O
+
H3N H COO-
activated by cellular
damage)
O NH3 pyruvate
S + H2O
S
OH
(2) sulfenic
(1) acid
x2
- H2O
sulfoxide
S
S
(3) thiosulfinate
decomposition
(ageing, heating, cooking)
S Sn
S
S
n=2-7
(4)
dithiins S polysulfides
(5) (6)
S
S [E] S
disulfide (7) O
Figure 1: Part of the reaction cascade which occurs when garlic is wounded. 1 alliin (allylcysteine
sulfoxide, 2 allylsulfenic acid, 3 allicin, 4 diallyldisulfide (n = 2) to diallylheptasulfide (n = 7), 5 = 3-vinyl-
3,4-dihydro-1,2-dithiin, 6 = 2-vinyl-2,4-dihydro-1,3-dithiin, 7 E-ajoene.
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Is allicin an antioxidant?
An ‘antioxidant’ can be defined as a substance which reduces or prevents the oxidation
of another substance under oxidizing conditions. Although chemically allicin is clearly
capable of oxidizing other molecules (see above) it is often described as having
antioxidant properties, (see for example Rabinikov et al., 2000). Why? There are two
aspects to this question; the physiological and the chemical. Physiologically, mild
oxidative stress caused in the cell by low doses of allicin and other RSS, leads to the
induction of phase II protection enzymes and the cells are thus cushioned against further
oxidative insults (Munday et al., 2003). In this way, allicin appears to protect against
oxidative stress, but the action is indirect. On the other hand, allicin readily degrades to
2-propenyl sulfenic acid, which interestingly is also the allicin precursor! Sulfenic acids
have been described as ‘ultimate’ reducing agents (Vaidya, 2009). Thus, allicin can
readily produce a strong reducing agent, while itself reacting as an oxidant.
Many cell processes are specifically regulated by redox mechanism (Buchanan and
Balmer, 2005). Many biologically important molecules, for example cysteine- and
methionine-containing proteins, NAD(P)H etc., can be reversibly oxidized and reduced.
Whether such molecules are in an oxidized or reduced state depend upon the surrounding
redox potential of the cell. This can be viewed as an electron pressure, or alternatively an
melectron vacuum, pushing or pulling electrons onto or from molecules.
Figure 2: Scheme relating the electrochemical cell potential, conditioned by the proportion of
reduced:oxidized glutathione (GSH:GSSG), to cellular activities. ‘High’ and ‘low’ refer to the proportion
of GSH:GSSG.
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The redox potential of the cell has to be carefully controlled, and changes can have
far-reaching consequences. Thus, effects on cellular redox potential can globally affect
an enormous number of different molecules throughout the whole cell (Schaffer and
Buettner, 2001). Quantitatively, the most important redox buffer in the cell is the
glutathione couple (GSH/GSSG). Interconversion of the oxidized and reduced forms
gives rise to conditions within the cell which regulate what have been called ‘nano-
switches’ for particular cellular activities. For example, in a highly reduced state cells
divide but shifting them to a more oxidized state can initiate differentiation and more
extreme oxidizing conditions lead to apoptosis (Figure 2). At a certain point the cell is in
such an oxidized state that it no longer has the metabolic competence needed for
programmed cell death and it undergoes necrosis.
The potential that allicin has for killing fungi raises wider issues about its use for the
treatment or prevention of plant disease. Garlic is used extensively in the kitchen and
because of this allicin and other garlic substances have widespread public acceptance;
even though some individuals have an extreme aversion to garlic odours! However, it is
easier to imagine allicin in certain specific applications in organic agriculture than in
others, for example, as a seed disinfectant where its use could be properly controlled in a
localized, contained environment. One might even consider applications under glass in
horticulture, but large scale field applications do not seem feasible without further
development work. Furthermore, dependent upon dosage allicin is a biocide, and there
will certainly be effects on non-target organisms.
Despite these cautionary concerns, the paradigm highlighted by the mode of action
of allicin, i.e. redox perturbation leading to apoptosis, might suggest a novel approach for
developing plant protection strategies.
Acknowledgements
Financial support from the RWTH Aachen is gratefully acknowledged. The research
leading to these results has received funding from the [European Community's] Seventh
Framework Programme [FP7/2007-2013] under grant agreement No. [215009].
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References
Block, E. (2010): Garlic and Other Alliums. The Lore and the Science. Royal Society of
Chemistry. Publishing, Cambridge 454 pp.
Buchanan, B.B., Balmer, Y. (2005): Redox Regulation: A Broadening Horizon. Annual Review
of Plant Biology, 56, 187-220.
Cavallito, C.J., Bailey, H.J. (1944): Allicin, the antibacterial principle of Allium sativum. I.
Isolation, physical properties and antibacterial action. J. Am. Chem. Soc., 66, 1950-51.
Curtis, H., Noll, U., Störmann, J., Slusarenko, A.J. (2004): Broad-spectrum activity of the
volatile phytoanticipin allicin in extracts of garlic (Allium sativum L.) against plant
pathogenic bacteria, fungi and Oomycetes. Physiological and Molecular Plant Pathology,
65, 79-89.
Firn, R. (2010): Nature’s Chemicals: the Natural Products that shaped our world. Oxford
University Press, Oxford, 250 pp.
Giles, G.I., Jacob, C. (2002): Reactive Sulfur Species: An Emerging Concept in Oxidative Stress.
Biological Chemistry, 383, 275-388.
Gruhlke, M.C., Portz, D., Slusarenko, A.J. (2011): A Putative Pathway of Apoptosis-Induction by
Allicin from Garlic (Allium sativum L.) In: H.W. Dehne, H.B. Deising, U. Gisi, K.H. Kuck,
P.E. Russell, H. Lyr (Eds.). Modern Fungicides and Antifungal Compounds VI, DPG-
Verlag, Braunschweig, Germany, 331-334.
Gruhlke, M.C., Portz, D., Stitz, M., Anwar, A., Schneider, T., Jacob, C., Schlaich, N., Slusarenko,
A.J. (2010): Allicin disrupts the cell’s electrochemical potential and induces apoptosis in
yeast. Free Radical Biology and Medicine, 49, 1916-1924, doi:
10.1016/j.freeradbiomed.2010.09.019
Jacob, C., Anwar, A. (2008). The chemistry behind redox regulation with a focus on sulfur redox
systems. Physiologia Plantarum, 133, 469-480.
Munday, R., Munday, J.S., Munday, C.M. (2003): Comparative effects of mono-, di-, tri-, and
tetrasulfides derived from plants of the Allium family: redox cycling in vitro and hemolytic
activity and Phase 2 enzyme induction in vivo. Free Radical Biology and Medicine, 34,
1200-1211.
Peterson, I., Anderson, E.A. (2005): The Renaissance of Natural Products as Drug Candidates.
Science, 310, 451-453.
Portz, D., Koch, E., Slusarenko, A.J. (2008): Effects of garlic (Allium sativum L.) juice
containing allicin on Phytophthora infestans (Mont. de Bary) and on downy mildew of
cucumber caused by Pseudoperonospora cubensis (Berk. & M. A. Curtis) Rostovzev.
European Journal of Plant Pathology, 122,197-206.
Schafer, F.Q., Buettner, G.R. (2001): Redox environment of the cell as viewed through the redox
state of the glutathione disulfide/glutathione couple. Free Radical Biology and Medicine,
30, 1191-1212.
Slusarenko, A.J., Patel, A., Portz, D. (2008): Control of plant diseases by natural products:
Allicin from garlic as a case study. European Journal of Plant Pathology, 121, 313-322.
Vaidya, V., Ingold, K.U., Pratt, D.A. (2009): Garlic: Source of the Ultimate Antioxidants -
Sulfenic Acids. Angewandte Chemie International Edition, 48, 157-160.
VanEtten, H.D., Mansfield, J.W., Bailey, J.A., Farmer, E.E (1994): Two Classes of Plant
Antibiotics: Phytoalexins versus "Phytoanticipins". Plant Cell, 6, 1191-1192.
Wondrak, GT. (2009): Redox-directed cancer therapeutics: molecular mechanisms and
opportunities. Antioxidants and Redox-Signalling, 11, 3013-3069.
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52
A Putative Pathway of Apoptosis-Induction by
Allicin from Garlic (Allium sativum L.)
Abstract
The thiosulfinate allicin is the major antimicrobial principle of garlic and is an oxidized sulfur-
containing redox-active natural product. Allicin oxidizes reduced glutathione (GSH), the most
important cellular redox buffer, in a thiol-disulfide exchange reaction. The alteration in the
GSH/GSSG ratio shifts the overall cellular electrochemical potential to a more oxidized state that
is associated with the induction of apoptosis. We investigated the pathway of apoptosis induction
after allicin treatment.
We showed that allicin induces apoptosis in baker’s yeast (Saccharomyces cerevisiae) by
demonstrating that allicin treatment activates yeast caspase 1 (Yca1p) and that overexpression of
the mammalian antiapoptotic BclxL and deletion of the yeast homolog to the mammalian
Apoptosis Inducing Factor (AIF) enhance the survival rate of yeast cells after allicin treatment.
Furthermore, yeast strains deleted in a regulatory subunit of the protein kinase A (PKA) and
the RAS2 protein, necessary for entry into apoptosis, show also an enhanced survival rate after
allicin treatment. These findings suggest that allicin targets a general redox-responsive apoptosis
inducing pathway. Cytoskeletal elements serve as receptors for the redox-conditions of the cell.
Thus, oxidation results in reduced turnover between G- and F-actin and the reduced actin
dynamic leads via the activation of an adenylate-cyclase to activation of PKA. PKA is known to
inhibit the expression of ROS-detoxifying enzymes. The resulting enhanced cellular ROS-level is
correlated with mitochondrial changes, resulting in release of AIF and cytochrome c from the
mitochondria and activation of yeast caspase leading to apoptotic cell death.
Introduction
The state of the cellular redox determined by the electrochemical cell potential relates
closely to the physiological activities of the cell, for example whether the cell is in a state
of proliferation, differentiation, apoptosis or necrosis (Schafer and Buettner, 2001, Kwon
et al., 2003, Ciriolo, 2005). Since thiols and in particular glutathione have a low
electrochemical half-cell potential compared to other the cellular redox buffers (Foyer
and Noctor, 2005). Thus, Ehc(GSH/GSSG) = -240 mV whereas
Ehc(ascorbate/dehydroascorbate) = -100 mV, and means that GSH is able to act as a
reductant for other redox components of the cell. A consequence is that the redox state of
glutathione is an important indicator of the global cellular redox environment.
While it was assumed for long time that apoptosis is restricted to animals, it is today
known that also fungi can undergo apoptosis. Saccharomyces cerevisiae was established
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as suitable model system for fungal apoptosis (Madeo et al., 1997, Fröhlich and Madeo,
2000, Madeo et al., 2004), but also in filamentous fungi apoptosis-like processes have
been observed (Chen and Dickman, 2005). Similarly, in fungi a strong correlation
between redox stress and apoptosis was shown (Chen et al., 2003).
In cancer treatment, apoptosis is an important therapeutically target (Wondrak, 2009,
Fulda et al., 2010), but also for fungicides it might be possible to use the induction of
apoptotic programme as a strategy for killing fungi.
A number of sulfur-containing natural products found in plants and fungi belong to
the class of reactive sulfur species (Giles et al., 2001) and are highly redox-active such as
allyl-sulfur compounds from the Alliaceae-subfamily or isothiocyanates in the
Brassicaceae (Jacob and Anwar, 2008). A well known example is the thiosulfinate allicin
from garlic (Allium sativum). This compound is able to react with cellular thiols and form
mixed disulfides (Rabinkov et al., 2000, Miron et al., 2010). Glutathione buffers the
cellular redox environment against redox perturbation, and these primary mixed
disulfides are cleaved by GSH in a thiol-disulfide exchange manner resulting in the
oxidized form of glutathione (GSSG). Thus, one mol of allicin reacts with four mol GSH
and is very potent in affecting cell redox. In allicin-treated yeast cells a significant
increase in GSSG-concentration was detectable (Gruhlke et al., 2010). The increase in
GSSG is observable as a shift of cellular electrochemical potential (Schafer and Buettner,
2001). After allicin-treatment the electrochemical potential was shifted to a range that is
associated with induction of apoptosis. Thus, we consider that the apoptosis-inducing
properties of allicin work via a redox dependent pathway as well in a caspase-dependent
and caspase-independent pathway.
A shift of the electrochemical potential (in mammalian cells to an Ehc of -180 mV) is
correlated with the induction of apoptosis (Schafer and Buettner, 2001). But how are this
redox-shift perceived and the signal transduced?
Human epithelial carcinoma cells treated with allicin showed a strong activation of
the protein kinase A (PKA) and treatment with a specific inhibitor of PKA, H-89,
rescued the cells from undergoing apoptosis (Park et al., 2005). To investigate whether a
PKA-dependent pathway in allicin-induced apoptosis is also involved in S. cerevisiae,
we tested a mutant yeast strain disrupted in a regulatory subunit of the yeast PKA (tpk3).
When treated with allicin, the mutant showed a much higher survival rate compared to
the wildtype. This suggests that PKA is a positive regulator of allicin-induced apoptosis
in S. cerevisiae. Although PKA was shown to be a key regulator, the question remains
what the cellular receptor for the apoptogenic stimulus is.
Because of the apparent central role of PKA, we postulate the following model for
allicin-induced apoptosis in yeast based on what is known of the signalling pathway for
H2O2-induced yeast apoptosis (Leadsham and Gourlay, 2008, Leadsham et al., 2009). In
this model, the turnover between G- (globular) and F- (filamentous) actin is regulated by
the oxidation of protein-thiols; as mentioned before, the oxidation state of proteineous
thiols is buffered by glutathione (Farah and Amberg, 2007). Actin-turnover, influenced
by oxidation, is monitored by the Cofilin and Actin associated protein (CAP) that via the
RAS2 protein activates an adenylate-cyclase, producing cyclic adenosine monophosphate
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Apoptosis-Induction by Allicin
cAMP that in turn triggers the activity of protein-kinase A (Franklin-Tong and Gourlay,
2008). In support of this hypothesis we showed that a ras2-mutant is also more resistant
against allicin treatment than the wildtype (unpublished results). That PKA serves as a
positive regulator is explained by the fact that PKA regulates the expression and
localization of the MSN2/4 transcription factor (Smith et al., 1998, Ferguson et al., 2005)
that is responsible inter alia to regulate ROS-degrading enzymes like superoxide-
dismutase or catalase (Smith et al ,. 1998). Thus, the activity of PKA can suppress ROS
detoxification systems and lead to an enhanced ROS-accumulation; a requirement both
for caspase-activation and release of the apoptosis-inducing factor from the
mitochondrial intermembrane space (Wissing et al ,. 2004, Perrone et al., 2008).
Taken together our results suggest that allicin uses a general redox-pathway of
apoptosis that perceives the redox-stimulus via cytoskeletal elements.
Acknowledgements
Financial support from the RWTH Aachen is gratefully acknowledged. The research
leading to these results has received funding from the [European Community's] Seventh
Framework Programme [FP7/2007-2013] under grant agreement No. [215009].
References
Chen, C., M.B. Dickman (2005): Proline suppresses apoptosis in the fungal pathogen
Colletotrichum trifolii. Proc Natl Acad Sci USA, 102, 3459--3464.
Chen, S.-R., D.D. Dunigan, M.B. Dickman (2003): Bcl-2 family members inhibit oxidative
stress-induced programmed cell death in Saccharomyces cerevisiae. Free Radic Biol Med.,
34, 1315--1325.
Ciriolo, M.R. (2005): Redox control of apoptosis. Antioxid Redox Signal, 7, 432--435.
Gruhlke, M.C., Portz, D., Stitz, M., Anwar, A., Schneider, T., Jacob, C., Schlaich, N., Slusarenko,
A.J. (2010). Allicin disrupts the cell’s electrochemical potential and induces apoptosis in
yeast. Free Radical Biology and Medicine, 49, 1916-1924.
333
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
Farah, M.E., D.C. Amberg (2007): Conserved actin cysteine residues are oxidative stress sensors
that can regulate cell death in yeast. Mol Biol Cell, 18, 1359--1365.
Ferguson, S.B., E.S. Anderson, R.B. Harshaw, T. Thate, N.L. Craig, H.C.M. Nelson (2005):
Protein Kinase A Regulates Constitutive Expression of Small Heat-Shock Genes in an
Msn2/4p-Independent and Hsf1p-Dependent Manner in Saccharomyces cerevisiae, 169,
1203-1214.
Foyer, C.H., G. Noctor (2005): Redox homeostasis and antioxidant signaling: a metabolic
interface between stress perception and physiological responses. Plant Cell, 17, 1866-1875.
Franklin-Tong, V.E., C.W. Gourlay (2008): A role for actin in regulating apoptosis/programmed
cell death: evidence spanning yeast, plants and animals. Biochem J., 413, 389-404.
Fröhlich, K.U., F. Madeo (2000): Apoptosis in yeast--a monocellular organism exhibits altruistic
behaviour. FEBS Lett., 473, 6-9.
Fulda, S., L. Galluzzi, G. Kroemer (2010): Targeting mitochondria for cancer therapy. Nat Rev
Drug Discov., 9, 447-464.
Giles, G.I., K.M. Tasker, C. Jacob (2001): Hypothesis: the role of reactive sulfur species in
oxidative stress. Free Radic Biol Med., 31, 1279-1283.
Jacob, C., A. Anwar (2008): The chemistry behind redox regulation with a focus on sulphur
redox systems. Physiologia Plantarum, 133, 469-480.
Kwon, Y.-W., H. Masutani, H. Nakamura, Y. Ishii, and J. Yodoi (2003): Redox regulation of cell
growth and cell death. Biol Chem., 384, 991-996.
Leadsham, J.E., C.W. Gourlay (2008): Cytoskeletal induced apoptosis in yeast. Biochim Biophys
Acta, 1783, 1406-1412.
Leadsham, J.E., K. Miller, K.R. Ayscough, S. Colombo, E. Martegani, P. Sudbery, C.W. Gourlay
(2009): Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and
apoptosis in yeast. J Cell Sci., 122, 706-715.
Madeo, F., E. Fröhlich, K.U. Fröhlich (1997): A yeast mutant showing diagnostic markers of
early and late apoptosis. J Cell Biol., 139, 729-734.
Madeo, F., E. Herker, S. Wissing, H. Jungwirth, T. Eisenberg, K.-U. Fröhlich (2004): Apoptosis
in yeast. Curr Opin Microbiol., 7, 655-660.
Miron, T., I. Listowsky, M. Wilchek (2010): Reaction mechanisms of allicin and allyl-mixed
disulfides with proteins and small thiol molecules. European Journal of Medicinal
Chemistry, 45, 1912-1918.
Park, S.Y., S.J. Cho, H.C. Kwon, K.R. Lee, D.K. Rhee, S. Pyo (2005): Caspase-independent cell
death by allicin in human epithelial carcinoma cells: involvement of PKA. Cancer Lett.,
224, 123-132.
Perrone, G. G., S.X. Tan, I.W. Dawes (2008): Reactive oxygen species and yeast apoptosis.
Biochim Biophys Acta, 1783, 1354-1368.
Rabinkov, A., T. Miron, D. Mirelman, M. Wilchek, S. Glozman, E. Yavin, L. Weiner. (2000): S-
Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-
modifying and antioxidant properties. Biochim Biophys Acta, 1499, 144-153.
Schafer, F.Q., G.R. Buettner (2001): Redox environment of the cell as viewed through the redox
state of the glutathione disulfide/glutathione couple. Free Radic Biol Med., 30, 1191-1212.
Smith, A., M.P. Ward, S. Garrett (1998): Yeast PKA represses Msn2p/Msn4p-dependent gene
expression to regulate growth, stress response and glycogen accumulation. EMBO J., 17,
3556-3564.
Wissing, S., P. Ludovico, E. Herker, S. Büttner, S.M. Engelhardt, T. Decker, A. Link, A. Proksch,
F. Rodrigues, M. Corte-Real, K.-U. Fröhlich, J. Manns, C. Candé, S. J. Sigrist, G. Kroemer,
F. Madeo (2004): An AIF orthologue regulates apoptosis in yeast. J Cell Biol., 166, 969-
974.
Wondrak, GT. (2009). Redox-directed cancer therapeutics: molecular mechanisms and
opportunities. Antioxidants and Redox-Signalling, 11, 3013-3069.
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53
A Transposon Mutagenesis Strategy to
Investigate Resistance Against Allicin in a
Garlic-Associated Pseudomonad
Abstract
Interest in plants like garlic which possess natural defence agents has increased in recent years.
Allicin from garlic is credited with widespread antimicrobial properties and compares well to
conventional antibiotics like ampicillin. Our recent isolation of a garlic-resistant Pseudomonas
species from fresh garlic cloves has therefore understandably generated considerable interest. To
better understand the nature of this resistance, we adopted a successful transposon mutagenesis
approach that involved conjugal transfer of Tn5 from a pSUP1021 plasmid-bearing E. coli strain
to the garlic-resistant pseudomonad. The isolation and characterization of Pseudomonas mutants
altered in their sensitivity to allicin will help to further understand the molecular mechanisms of
resistance.
Introduction
Worrying reports of threatened global food security and drastic worldwide environmental
changes, caused in part by anthropogenic practices, suggest the urgent need to find new
but sustainable crop protection strategies. Moreover, the general, though not always
correct perception of Natural Products as “mild” (Slusarenko et al., 2008) has continued
to direct widespread attention to biogenic plant defence materials. Consequently, there
has been a growing interest in plants like Allium sativum (garlic) equipped by nature to
defend themselves against invading pathogens and pests. Upon wounding, garlic releases
the volatile phytoanticipin allicin (diallylthiosulfinate) when the enzyme alliinase (alliin
lyase) acts on its substrate alliin (S-allyl-L-cysteine sulfoxide). Allicin is credited with
widespread antimicrobial properties that compare favourably with those of conventional
antibiotics like ampicillin. Recently however, we isolated an allicin-resistant
Pseudomonas species (DP1) from fresh garlic cloves (Slusarenko et al., 2008). The
identity of this isolate was confirmed to the genus level by using universal primers for
16S rDNA IS sequences and sequencing the variable interstitial region (Portz, 2008). To
better understand the nature of the resistance of DP1, we have begun a transposon
mutagenesis approach to identify genes associated with the resistant phenotype.
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Figure 1: Transposon mutagenesis of DP1 with a mobilizable Tn5 carrier vector. (a)The broad host range
tra-genes of the mobilizing E. coli are contained in a chromosomally integrated RP4-derivative. (b) The
trans-acting mobilization ability of RP4 enables the movement of the vector into the recipient. (c) Tn5
transposition from vector to host genome occurs (d) The vector is unable to replicate in the recipient cell
and is lost as the cell divides.
Figure 2: DP1 and E. coli S-17 cells are mixed together for conjugative DNA transfer, and then plated on
media containing kan (to select for donor cells), nal (to select for recipient cells), and nal + kan (to select
for transconjugants).
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Allicin Resistance
Figure 3: Plates showing the selection of cells on media containing kanamycin (kan), nalidixic acid (nal)
as well as one containing both antibiotics (nal + kan).
Wild type
DP1
Allicin-susceptible P.
Syringae pv. phaseolicola
DP1
Figure 4: Sensitivity screening plate. The desired DP1 mutant(s) is expected to manifest a phenotype
similar to that of the Pseudomonas syringae pv. phaseolicola.
Tn5 carries a kanamycin (kan) resistance gene and pSUP1021 has an origin of replication
that functions in E. coli but not in Pseudomonas. pSUP1021 is therefore unable to
replicate in Pseudomonas. Selection for DP1 transconjugants carrying Tn5 stably
integrated into heritable genetic element(s) was carried out on plates containing both kan
and nal (Figure 2, 3; Anderson and Mills, 1985). Finally, DP1 mutants altered in their
sensitivity to allicin/fresh garlic juice were screened for in a Petri plate assay (Figure 4).
The screening method is labour-intensive and a more efficient method to test larger
numbers of transconjugants in a shorter time for their resistance to allicin would be
desirable. If allicin-susceptible mutants are found, the Tn5 marker will be used to isolate
the mutated gene(s). In this way, we hope to understand the basis of allicin resistance in
DP1 and thus gain more understanding of the molecular mechanisms of allicin action.
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Acknowledgements
Financial support from the RWTH Aachen is gratefully acknowledged. The research
leading to these results has received funding from the [European Community's] Seventh
Framework Programme [FP7/2007-2013] under grant agreement No. [215009].
References
Anderson, D.M., Mills, D. (1985): The use of transposon mutagenesis in the isolation of
nutritional and virulence mutants in two pathovars of Pseudomonas syringae.
Phytopathology, 75, 104-108.
Portz, D. (2008): Garlic in plant protection: possible use and molecular biological studies with
special regard to the active ingredient allicin. PhD Thesis, RWTH Aachen University,
Aachen.
Simon, R., Priefer, U., Pühler, A. (1983): A Broad Host Range Mobilization System for In Vivo
Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria. Nature
Biotechnology, 1, 784-791.
Slusarenko, A.J., Patel, A., Portz, D. (2008): Control of plant diseases by natural products:
Allicin from garlic as a case study. European Journal of Plant Pathology, 121, 313-322.
338
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54
Biological Activity and Mode of Action of
Serenade®
Abstract
Serenade® is a microbial biological control agent based on Bacillus subtilis QST 713 that protects
various crops against fungal and bacterial plant pathogens. When used in integrated spray
programmes with conventional fungicides, the biofungicide Serenade® allows growers to control
fungal attacks during the pre-harvest and harvest periods by various modes of action.
The specific B. subtilis strain QST 713, Serenade’s active ingredient, was discovered in an
orchard in California. Several modes of action have been demonstrated for B. subtilis-based
biological fungicides, e.g. antagonistic potential arising from secreted secondary metabolites,
competition for space and nutrients, positive effects on plant development and also induction of
plant resistance. Serenade's bacterium B. subtilis QST 713 has been demonstrated to release
numerous anti-fungal compounds belonging to the chemical class of lipopeptides, which interfere
with the physiological integrity of the pathogens’ cell membranes. The membrane activity of the
lipopeptides is the main cause for the drastic morphological and inhibitory effects observed in a
broad range of fungal plant pathogens.
Introduction
Farmers are under increasing pressure to offer fresh fruits and vegetables with residue
levels of chemical pesticides below limits set by regulatory authorities. The so-called
secondary standards requested by traders, supermarket chains and farming organizations
are well below legally permitted standards (e.g. maximum residue level or MRL).
Serenade® is an effective fungicide and bactericide which can be applied shortly before
harvest and can be used to control plant diseases including blight, scab, grey mould, and
several types of mildew without increasing synthetic chemical residue levels in fresh
produce.
By releasing antifungal compounds and preventing pathogens from colonizing
plants, Serenade® can be used to control a wide range of plant diseases and effectively
manage resistance. In addition, its favourable toxicological and eco-toxicological profile
is an ideal tool for integrated pest management (IPM).
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I. Siepe et al.
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fengycins, are known and were studied for their antagonistic activity on a wide range of
potential phytopathogens, including fungi and oomycetes (Ongena and Jacques, 2007).
Unique features of B. subtilis QST 713 are both the amount and the diversity of the
lipopeptides produced (Figure 1).
As the lipopeptides mimic cell membrane lipids, they can integrate into biological
cell membranes, thus altering membrane curvature and membrane fluidity, which finally
results in the formation of membrane pores.
A B
Figure 2: B. cinerea on the leaf surface stained with Uvitex dye. A: untreated control, B: leaf treated with Serenade®
(8l Serenade® ASO/1000l) 24h before fungal inoculation.
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I. Siepe et al.
A comparison of the two time points demonstrates that cells of rod-like QST 713
proliferate on the leaf surface. This is clearly indicated by the typical symmetric cell
division which results in bacterial cell chains (Figure 4B).
Figure 4: Leaf colonisation by Bacillus subtilis QST 713. A: picture taken 24h after application,
B: picture taken 48h after application.
Pathogen colonisation
Additional scanning electron microscopy studies interestingly showed that QST 713 is
not only able to colonise the leaf surface but also conidia of B. cinerea (Figure 5). In this
study inoculation of B. cinerea was carried out 24 h after treatment with Serenade®.
Additional mechanisms
Studies with wheat powdery mildew surprisingly demonstrated an additional mode
of action.
48h after inoculation, fungal spores had germinated and produced primary hyphae
on the untreated leaf area (Figure 6A). Spores which were in direct contact with the spray
droplet and deposit (Figure 6B, red circle) collapse and did not germinate. Additionally,
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it was observed that spores in close proximity but not in direct contact with the spray
deposit germinated, but showed reduced growth combined with abnormal morphology.
This either indicates biological activity of Serenade® via the gas phase (effects of volatile
compounds) or local diffusion of soluble antifungal metabolites in the microfilm of water
on the leaf surface which are capable of inhibiting cells not directly in contact with the
spray droplet.
Figure 5: B. cinerea on bell pepper leaf surface. Inoculation of B. cinerea was carried out 24h after
treatment with Serenade®. Picture was taken 48h after inoculation with B. cinerea.
Figure 6: Blumeria graminis grown on wheat leaf surface for 48h: A: untreated control, B: leaf treated
with Serenade 24h before fungal inoculation. Red circle indicates spray deposit.
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I. Siepe et al.
Biological efficacy
A field trial (4 replications, randomised block design) was carried out in Taiwan in 2008
under preventive conditions to evaluate the efficacy of Serenade against
Alternaria solani on tomatoes. Four applications were applied to a tomato crop in 7-9
day intervals, starting at growth stage BBCH 23. The water volume was adjusted
according to the crop stage between 500 L/ha at the beginning and 1,000 L/ha at the end
of the trial. An artificial inoculation was made with conidial suspension of A. solani.
Evaluations were made 13 days after the final application by estimating the percentage of
diseased leaf area. Infection developed homogeneously and was high at the time of the
assessment shown in Figure 7. Three different fungicide spray programmes were
compared:
1. Chemical programme, which consisted of 2 or 3 consecutive sprays with the
synthetic fungicide Signum
2. “Sequential” spray programme, which consisted of 2 sprays of the synthetic
fungicide Signum, followed by 2 sprays with the biological fungicides
Serenade ASO
3. “Full season” biological spray programme, which consisted of 4 sprays with the
biological fungicides Serenade ASO (8 L/ha)
61
60
40
18
20
4,4
2,1 2,4
0
l/kg /ha Chemical Sequential Full season
%w/v
0,3 0,3 0,3 + 8 8
Control
Signum Signum Signum Ser. ASO
Signum Signum Signum Ser. ASO
Signum Ser. ASO Ser. ASO
Ser. ASO Ser. ASO
Application: 2-4 at 7-9 days interval
% infection, 13 DAA(4)
Figure 7: % Infection with Alternaria solani on tomatoes, disease assessment 13 days after 4th foliar spray
application, 3 artificial inoculations made 8h after foliar sprays 1, 2 and 3;
Products: Signum: WG formulation containing 26,7% boscalid and 6,7% pyraclostrobin; Ser. ASO:
Serenade ASO containing 1,34% QST 713 strain of Bacillus subtilis, 1x109 cfu/g; Comparison between 1.
chemical spray programme (“Chemical”), consisting of 2 or 3 consecutive sprays with synthetic fungicide
Signum, 2. spray programme consisting of 2 sprays of synthetic fungicide Signum followed by 2 sprays
with biological fungicides Serenade ASO, and 3. biological spray programme (“Full season”), consisting
of 4 sprays with biological fungicides Serenade ASO; Field trial conducted in 2008 in Taiwan.
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Applied products:
Signum: WG formulation containing 26.7 % boscalid and 6.7 % pyraclostrobin
Serenade ASO: containing 1.34 % QST 713 strain of Bacillus subtilis, 1x109 cfu/g
Serenade “Full season” programmes showed approx. 30 to 50 % efficacy, depending on
the rate applied. A clear dose response could be established for both formulations. The
“Chemical” programmes were comparable to the “Sequential” programmes, with
efficacy between 93 and 96%. Both the use of synthetic fungicides and the consecutive
spray of Serenade after synthetic fungicides showed excellent performance. Moreover,
the use of Serenade in sequential spray programmes, i.e. before harvest, reduces
residues of synthetic fungicides in the harvested produce (unpublished data). The field
trial showed that Serenade can be used to replace synthetic fungicides before harvest
without compromising the health of crops under high disease pressure.
References
Ongena, M., Jacques, P. (2008): Bacillus lipopeptides: versatile weapons for plant disease control.
Trends in Microbiology, 16, 115-125.
B.P. Kremer (2002): Das große Komsos-Buch der Mikroskopie Franckh-Kosmos Verlag.
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I. Siepe et al.
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55
Antifungal and Herbicidal Activity of
Rosmarinus officinalis L. and Pelargonium
odoratissimum (L.) L’Hér. Essential Oils
Abstract
The essential oil of a wild population of Rosmarinus officinalis growing in Sicily, Italy, and a
commercial sample of Pelargonium odoratissimum were analyzed by GC and GC/MS. Both
species were rich in oxygenated monoterpenes, with 1,8-cineole (33.65% of total oil) as the main
compound in R. officinalis, and citronellol (20.40% of total oil) in P. odoratissimum essential oil.
Antifungal activity was evaluated in vitro against F. oxysporum f.sp. lycopersici and F.
oxysporum f.sp. dianthi. Between the essences, Pelargonium essential oil at the maximum
concentration (1 l/ml) showed a highest inhibitory activity against F. oxysporum f. sp. dianthi
than the other formae speciales. Their herbicidal activity was tested against Portulaca oleracea
and Conyza canadensis. The essential oil of Pelargonium was more active than that of
Rosmarinus. At 0.5 and 1 l/ml concentrations it completely blocked germination of seeds of C.
canadensis and reduced germination of seeds of P. oleracea significantly. Essential oil of R.
officinalis inhibited germination of seeds of C. canadensis at concentrations between 0.250-1
l/ml but it was not active against P. olearacea. The seedling length of the weeds was reduced
significantly by both essential oils at all concentrations tested (0.5-1l/ml).
Introduction
A. Salamone et al.
Chenopodium album and Portulaca oleacea whereas the essential oil obtained from
wild-grown plants in the protected area of Montemarcello (La Spezia Province)
completely inhibited germination of seeds of these weeds (Angelini et al., 2003).
Pelargonium odoratissimum (Geraniaceae) is an ornamental plant with scented leaves.
Commercial geranium oil has proved to be a very strong antimicrobial agent (Lis-Balchin
et al., 1996). Concerning the antimicrobial activity, the essential oils obtained from
Pelargonium species showed good activity against different genera of bacteria and fungal
plant pathogens and their major components demonstrated various degrees of growth
inhibition (Dorman and Deans, 2000; Lis-Balchin and Deans, 1996) whereas, the
antifungal assays showed a feeble effect of rosemary especially against F. graminearum
(Angioni et al., 2004).
The aim of this work was to test the antifungal and herbicidal potential of R.
officinalis grown in Sicily, Italy, and a commercial sample of P. odoratissimum essential
oils.
Fresh plant material of R. officinalis L. grown in Sicily, Italy, was collected in October
2009, at the flowering stage. Aerial parts were subjected to hydrodistillation for 3 h.
Essential oil of P. odoratissimum (L.) L’Hér. was purchased from Titolchimica.
Antifungal activity
The same concentrations of essential oils as described above were used to evaluate the
inhibitory effect of in vitro assay against Fusarium oxysporum f. sp. lycopersici and F.
oxysporum f. sp. dianthi, causal agents of the tracheomycosis on tomatoes and carnation
plants, respectively. The method described by Grover and Moore (1961) was used. Petri
dishes were incubated at 22±1°C in the dark. After 3, 6 and 9 d the inhibition zone
diameter of each fungal colony was measured. The experiments were replicated 4 times
for each treatment and the fungitoxicity was calculated in terms of percent colony
inhibition in comparison with control.
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A. Salamone et al.
Table 1 continuing
Oxygenated
monoterpenes
Citronellyl formate 1278 - 3,34
Neryl formate 1286 - t
Bornyl acetate 1290 0,73 -
Lavandulyl acetate 1296 t t
Thymol 1299 t -
Geranyl formate 1305 - 1,91
Carvacrol 1305 t -
Neryl acetate 1367 - 1,82
Geranyl acetate 1387 - 4,23
Geranyl butanoate 1563 - 2,56
Sesquiterpene 7,42 t
hydrocarbons
β-Caryophyllene 1423 6,64 t
α-Humulene 1459 0,65 -
trans-β-Farnesene 1464 t -
β-Bisabolene 1511 0,13 -
β-Sesquiphellandrene 1527 t -
Oxygenated 0,86 1,52
sesquiterpenes
Hedycaryol 1542 - t
Caryophyllene oxide 1588 0,86 -
Guaiol 1603 - 0,46
10-epi-γ-Eudesmol 1629 - 0,28
γ-Eudesmol 1642 - t
β-Eudesmol 1661 - t
α-Eudesmol 1664 - t
Bulnesol 1674 - 0,78
α-Bisabolol 1690 t -
Others 0,52 17,30
1-Octen-3-ol 989 0,08 -
Dipropilene glycol 1043 - 2,84
Phenylethyl alcohol 1127 - 2,32
Isononyl acetate 1178 - 4,94
1,2,3-propanetriol
1363 - 7,15
diacetate
Eugenol 1365 t -
Methyl eugenol 1415 t -
Methyl jasmonate 1646 0,11 -
Dodecyl acrylate 1665 0,27 -
2-Bornanone 1952 - 0,05
Dibutyl phthalate 1983 0,06 t
Total identified 99,92 95,92
Compounds listed in order of elution in the HP-1 column. t, traces <0,03% . RI, retention index relative to
C8-C32 n-alkanes on the HP-1 column. R. Rosmarinus officinalis. P. Pelargonium odoratissimum. Peak area
percentages are calculated in GC on apolar HP-1 column.
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Table 2: Inhibitory effect (%) of essential oils on the mycelia growth of Fusarium species.
Essential oils 0.125 µl · ml-1 0.25 µl · ml-1 0.5 µl · ml-1 1.0 µl · ml-1
% ArcSin % % ArcSin % ArcSin % ArcSin
±S.E. %±S.E. %±S.E. %±S.E.
F. oxysporum f. sp. lycopersici
R. officinalis 2.2 0±0 24.8 29.8±1.8 16.4 23.9±0.9 15.7 23.2±1.7
P.odoratissimum 14.2 22.0±1.6 13.9 20.6±4.5 60.6 51.1±0.5 61.7 51.8±1.6
F. oxysporum f. sp. dianthi
R. officinalis 2.6 8.9±1.6 18.4 25.4±0.4 17.3 24.5±0.8 14.6 22.3±1.7
P.odoratissimum 19.3 25.9±1.6 27.5 31.6±1.0 52.1 46.2±1.2 90.9 75.9±6.1
Mycelial growth data were recorded after 6 days from seeding.
In herbicidal assays essential oil of P. odoratissimum was the most effective (Table
3), being more phytotoxic to C. canadensis, controlling its germination at all
concentrations applied, whereas to P. oleracea only high concentrations showed
significant differences to the control. The essential oil of R. officinalis showed also
stronger effects against C. canadensis, inhibiting its germination at concentrations higher
than 0.250 µl/ml significantly. However, it had little effect on germination of seeds of P.
oleracea, and only controlled germination at 1 µl/ml. Finally, both essential oils
significantly reduced the seedling length of the two weeds at all concentrations tested
(Figure 1).
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A. Salamone et al.
Table 3: Effects of the essential oils of R. officinalis and P. odoratissimum on seeds germination of C.
canadensis and P. oleracea.
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Figure 1: Seedling length (mm) (mean ± SE) of C. canadensis and P. oleracea treated with essential oil
from P. odoratissimum (A y B) and R. officinalis (C y D).
References
Angelini, L.G., Carpanese, G., Cioni, P.L., Morelli, I., Macchia, M., Flamini, G. (2003): Essential
oils from Mediterranean Lamiaceae as weed germination inhibitors. Journal of
Agricultural and Food Chemistry, 51, 6158-6164.
Angioni A., Barra A., Cereti E., Barile D., Coïsson J.D., Arlorio M., Dessì S., Coroneo V.,
Cabras P. (2004): Chemical composition, plant genetic differences, antimicrobial and
antifungal investigation of the essential oil of Rosmarinus officinalis L. Journal of
Agricultural and Food Chemistry, 52, 3530-3535.
Bowers J., Locke J.C. (2000): Effect of botanical extracts on the population density of Fusarium
oxysporum in soil and control of Fusarium wilt in the greenhouse. Plant Disease, 84, 300-
305.
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A. Salamone et al.
Dudai, N. Poljakoff-Mayber, A., Mayer, A.M., Putievsky, E., Lerner, H.R. (1999): Essential oils
as allelochemicals and their potential use as bioherbicides. Journal of Chemical Ecology,
25, 1079-1089.
Grover R.K., Moore D. (1961): Adaptation of Sclerotinia fruticola and Sclerotinia laxa to higher
concentrations of fungicides. Phytopathology, 51, 399-401.
Lis-Balchin, M., Deans, S.G., Hart, S. (1996): Bioactivity of commercial Geranium oil from
different sources. Journal of Essential Oil Research, 8, 281–290.
Lis-Balchin M., Deans S.G. (1996): Antimicrobial effects of hydrophilic extracts of Pelargonium
species (Geraniaceae). Letters in Applied Microbiology, 23, 205-207.
Salamci, E., Kordali, S., Kotan, R., Cakir, A., Kaya, Y. (2007): Chemical compositions,
antimicrobial and herbicidal effects of essential oils isolated from Turkish Tanacetum
aucheranum and Tanacetum chiliophyllum var. chiliophyllum, Biochemical Systematics
and Ecology, 35, 569-581.
Verdeguer, M., Blázquez, M.A., Boira, H. (2009): Phytotoxic effects of Lantana cámara,
Eucalyptus camaldulensis and Eriocephalus africanus essential oils in weeds of
Mediterranean summer crops. Biochemical Systematics and Ecology, 37, 362-369.
354
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56
Influence of Сotton Plant Antifungal
Compounds on Verticillium dahliae
T.A. VLASOVA
Department of Plant Physiology, Faculty of Biology, Moscow State University, Vorobjovy Gory,
119992 GSP-2 Moscow, Russia
Abstract
The initial stage of the interaction of the phytopathogenic fungus Verticillium dahliae Kleb. with
cotton plant was studied. The attention was focused on the ultrastructure of the fungal hyphae
contacting the plant root and penetrating root cells at the early stage of infection. Most hyphae
surrounding the roots and entering the root cells in both cotton cultivars studied showed the
ultrastructure typical for intact and viable fungal cells. However, in some of them, symptoms of
degradation were evident. Many vacuoles in the mycelial cells appeared to be autophagic and
contained vesicles and membrane structures. Multivesicular bodies were also often present in the
hyphae. Some fungal cells underwent complete lysis. In addition, large lipid inclusions in hyphal
cells and osmophilic inclusions like polyphosphate granules in some fungal vacuoles were found.
These features could be explained by the presence of fungitoxic compounds in the plant.
In the resistant cotton cultivar, the total phenol content and the activities of phenol-oxidizing
enzymes, peroxidase and polyphenoloxydase, were higher. The results suggest that in cotton
antifungal phenols represent factors limiting growth and distribution of V. dahliae.
Introduction
The soil-born fungal phytopathogen Verticillium dahliae Kleb. causes wilt disease in
many vascular plant species and can cause serious losses in agricultural crops. In cotton-
growing regions, wilt caused by Verticillium and Fusarium species represent significant
problems. Verticillium wilt as a typical tracheomycosis is characterized by predominant
hyphal growth in the vascular system of the plant (Garrett, 1956, Pegg and Brady, 2002).
However, the initial events in the non-vascular tissues are supposed by many authors to
be the crucial point of the establishment of wilt disease (Pegg and Brady, 2002).
The aim of the present work was to compare the initial stage of the interaction of the
fungus V. dahliae with resistant and susceptible cultivars of the cotton (Gossypium
hyrsutum L.). The attention was focused on the ultrastructure of the fungal hyphae
contacting the plant root and penetrating root cells in the early stage of wilt infection. In
vitro cultivated detached cotton roots were used in the experiments as a suitable model
object.
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Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
Persistent Identifier: urn:nbn:de:0294-sp-2011-Reinh-8
T.A. Vlasova
In vitro cultures of cotton (Gossypium hyrsutum L.) roots were inoculated with
Verticillium dahliae Kleb. Cotton cultivars C-4727 (highly susceptible to Verticillium
wilt) and Tashkent-1 (relatively resistant to wilt) were examined. Conidia of V. dahliae
strain Chl-288 (virulent to the both cultivars) were washed from cultures maintained on
potato-sucrose agar. The roots of cotton seedlings grown aseptically were detached,
dipped into a conidial suspension and placed into flasks containing Smirnov's agar
medium. The inoculated roots were studied by light and electron microscopy. For
electron microscopy, the parts of roots covered with fungal mycelium were fixed in 5%
glutaraldehyde, postfixed in OsO4, dehydrated in ethanol, embedded in Araldite and
observed in the transmission electron microscope.
Results
V. dahliae grew intensively on the root surfaces. Light and electron microscopy revealed
that rhizodermal and cortical cells were penetrated by the mass of mycelium at the sites
of fungal accumulation on the root surfaces. The hyphae in the root tissues grew both
intracellularly and intercellularly. No obvious qualitative specificity was observed in the
hyphal root penetration and growth and distribution in the root tissues of both cultivars
studied. However, a clear difference in the intensity of fungal colonization of these
cultivars was detected: In the roots of the susceptible cultivar the percentage of cortical
cells occupied by mycelium was higher than in the roots of the resistant cultivar. Most
hyphae surrounding the roots and entering the root cells in both cultivars showed the
ultrastructure typical for intact and viable fungal cells (Griffiths, 1971). However, in
some of them, certain effects of plant fungitoxic compounds were observed. This has
also been described in our previous study (Vlassova, 2000). Rather frequently the fungal
hyphae contacting and penetrating rhizodermal cells of roots of the resistant cv.
underwent complete lysis (Figure 1). For comparison, in axenic cultures of V. dahliae
hyphal lysis was a very rare event. In viable hyphae, vacuoles appeared autophagic and
contained vesicles and membrane structures (Figure 2). Multivesicular bodies were also
often present in the mycelial cells (Figure 2).
Structures, probably related to the fungal tolerance to toxic plant compounds, were
also observed in fungal cells. Interestingly, endospore-like structures were also found in
individual hyphae (Figures 1, 3). It looked like a specific proliferation, so-called
secondary growth, i.e. formation of outgrowths within the hypha. The formation of such
structures is interpreted as a defense response in fungi to the unfavorable environmental
conditions (Aube and Pelletier, 1968). In addition, large lipid inclusions were present in
many fungal cells (Figure 4). Lipids are considered as a factor mediating fungal tolerance
to toxic compounds; thus, the high lipid content might allow fungal survival in the
presence of fungitoxic substances. The occurrence of osmophilic inclusions like
polyphosphate granules in some fungal vacuoles could also be connected with the
reduction of the concentration of toxic substances in the cytoplasm. In addition, at some
sites of fungal penetration, formation of the local thickenings of the plant cell walls
(papillae) was observed (Figures 3, 4). These structures are associated with plant
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response to pathogen penetration and are discussed in connection with plant resistance
(Griffiths, 1971a, Aist, 1983). However, in our experiments, papillae were observed only
in about 50% cases and did not serve a safe barrier for fungus.
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T.A. Vlasova
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Discussion
It is well known that plant defense responses include a broad range of reactions. The
general reactions are formation of mechanical barriers (e.g. cell wall reinforcement and
papillae formation), accumulation of antimicrobial proteins (pathogenesis-related
proteins), and formation of phytoalexins (Zhou and Dai, 2006, Cai et al., 2009). Plant
phenols, many of which are phytoalexins, are important factors of the plant defense
system (Farkas and Kiraly, 1962, Chaube and Pundhir, 2005, Zhou and Dai, 2006). The
phenolic compound gossypol and its derivatives are the main phytoalexins of cotton plant
(Bell, 1969, Cai et al., 2009). To evaluate the participation of phenols in the studied
interaction some tests were undertaken (Vlassova, 1994). Among the root exudates of
both cotton cultivars, phenolic compounds were found, in accordance with the data in the
literature. Our histological tests revealed the accumulation of phenols in the outer root
cell layers and on the root surfaces as well as on the fungal cells. The depositions of
osmiophilic substances on the surfaces of plant cells and fungal hyphae shown in
electron micrographs (Figures. 2, 4) also may be interpreted as phenol depositions. The
total phenol content was higher in the non-inoculated roots of the resistant cotton cultivar,
and the difference between resistant and susceptible cultivars progressed during the
infection process. The activities of phenol oxidizing enzymes, peroxidase and
polyphenoloxydase, were compared in inoculated and non-inoculated plant examples.
The increase of oxidase activities was detected as a result of infection in roots of both
cultivars. However, in the resistant cultivar, enzyme activity was higher. Similar results
were obtained by other authors studying pathogenesis of Fusarium and Verticillium wilt
(Retig, 1974, Zhou and Dai, 2006).
The results suggest that in cotton roots phenolic compounds play important roles as
factors limiting growth and distribution of V. dahliae in the plant.
References
Aist, J.R. (1983): Structural responses as resistance mechanisms. In: The dynamics of host
defence. Acad. Press, New York, USA, 33-69
Aube, C., Pelletier, G. (1968): Formation of endospores in Verticillium albo-atrum (R. & D.).
Canadian Journal of Microbiology, 14, 606-607.
Bell, A.A. (1969): Phytoalexin production and Verticillium dahliae reactance in cotton.
Phytopathology, 59, 1199-1127.
Cai, Y., Xiaohong, H., Mo, J., Sun Q., Yang, Y., Liu, J. (2009): Molecular research and genetic
engineering of resistance to Verticillium wilt in cotton: A review. African Journal of
Biotechnology, 8, 7363-7372.
Chaube, H.S., Pundhir, V.S. (2005): Crop Disease Management. Prentice Hall of India, New
Dehli, India, 703 p.
Farkas, G.L.,Kiraly, Z. (1962): Role of phenolic compounds in the physiology of plant disease
resistance. Phytopathologische Zeitschrift, 44, 105-110.
Garrett, S.D. (1956): Biology of root-infecting fungi. Cambridge University Press, London &
New York, GB, 292 p.
Griffiths D.A. (1971): The fine structure of Verticillium dahliae Kleb. colonizing cellophane.
Canadian Journal of Microbioogy, 17 (1), 79-81.
Griffiths D.A., (1971a): The development of lignitubers in roots after infection by Verticillium
dahliae Kleb. Canadian Journal of Microbioogy, 17, 441- 444.
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T.A. Vlasova
Pegg, G.F., Brady, B.L.(2002): Verticillium wilts. CABI Publishing, New York, USA, 398 p.
Retig, N., (1974): Changes in peroxidase and polyphenoloxidase associated with natural and
induced resistance of tomato to Fusarium wilt. Physiological Plant Pathology, 4, 145.-150
Vlassova, T.A. (1994): Total phenol content and phenol oxidizing enzyme activities in detached
cotton roots inoculated with Verticillium dahliae Kleb. Acta Horticulturae, Internatonal
Symposium on Natural Phenols in Plant Resistance Weihenstephan, Germany, 13- 17
September 1993, Vol.1, 381, 287-290.
Vlassova, T., (2000): Ultrastructural characters of Verticillium dahliae interacting with cotton
roots. Botanikertagung Jena 2000, 17-22 September 2000, Tagungsband, 113.
Zhou, T.H., Dai X.F. (2006): Research on physiological and biochemical mechanisms of cotton
defense against Verticillium wilt. Molecular Plant Breeding, 4, 593-600.
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57
Application of Arbuscular Mycorrhizal Fungi to
Improve Productivity of Vicia sativa (L.)
Abstract
The present work investigates the influence of native populations of arbuscular mycorrhizal fungi
on biomass production of the crop plants Vicia sativa (L.), soils differing in humus content.
Growth and accumulation of biomass of plants was recorded at various growth phases. Data
indicate that of arbuscular mycorrhizal fungi led to an increase of biomass by up to approx. 30%.
Growth of plants was not significantly increased.
Introduction
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Modern Fungicides and Antifungal Compounds VI
© Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany, 2011
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Biological product prepared on the basis of crushed mycorrhizal roots and soil. Seeds of
plants infected before crops from calculation 100 g a biological product on 100 m2 of the
area of the earth. Crop of seeds were made in damp soil on depth by of 4-5 cm in number
of 350 grains/m 2.
Beans (Vicia sativa L.) grown in soils enriched in arbuscular mycorrhizal (AM) fungi
yield increased levels of biomass, but plants did not show corresponding increases in
growth. Interestingly, the effect of AM fungi was more pronounced in soil containing
3,8 % organic matter. As the interaction between the symbiotic fungus and the plant lead
to provision of the plant with nitrogen, phosphorus, potassium, it is likely that nutritional
effects caused increased biomass production.
Under the influence of AM fungi, V. sativa plants showed increased biomass
production by up to approx. 30% on soils containing 2.6% organic matter (Table 1).
There was only a moderate effect of AM fungi on growth, as determined as plant height,
irrespective of the humus content (Table 1). The effect of AM fungi on biomass
production is likely due to improved mineral nutrition of V. sativa plants.
Table 1: Dynamics of growth and biomass accumulation of Vicia sativa L. under the influence of a
preparation of AM fungi.
References
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58
Influence of Fungitoxic Plants on Formation of
Arbuscular Mycorrhizal Fungi and Development
of Wheat
T.P. YURINA
Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 119992 Moscow,
Russia
Abstract
The effect of fungitoxic plants (Artemisia absinthium L., Symphytum officinale L.) on the
development of wheat (Triticum aestivum L., cv. Khakasskaja) and root colonization of
mycorrhizal fungi was studied. Both biomass and root colonization increased with increasing
distance of wheat to fungitoxic weeds. We propose that the decrease in wheat development was
due to interference with the establishment of the symbiotic mycorrhizal interaction.
Introduction
Arbuscular mycorrhizal (AM) fungi are widely distributed in soils of various geographic
zones. They form symbiotic associations with many plants belonging to the poales. Due
to their obligate symbiotic lifestyle, arbuscular mycorrhizal fungi need to associate with
plants for growth and proliferation. Arbuscular mycorrhizal associations involve different
fungi in the phylum Glomeromycota and roots of a wide diversity of plants. Symbiotic
associations with the roots of plants lead to increased growth and health of many plants.
The special attention is given to Glomus species. Symbioses with Glomus species
promote development and yields of crop plants, basically by increasing the availability of
essential elements such as phosphorus, nitrogen and potassium. Process of formation of
symbiotic associations in biocenosis depends on a degree of fertility of soil, which may
be modified by application of fertilizers. Fungicides suppress or completely suppress
growth of arbuscular mycorrhizal fungi. However, not only fungicides, but also
fungitoxic plants are able to suppress the formation of mycorrhizal interactions. In this
respect, we investigated the effect of fungitoxic plants on formation of symbiotic
associations in wheat roots. Among the fungitoxic weed plants used in these studies were
common wormwood (Artemisia absinthium L.) and comfrey (Symphytum officinale L.).
We tested the influence of these plants on grow and development and the degree of root
colonization of wheat (Triticum aestivum L.) under field conditions .Water extracts from
leaves and roots of the fungitoxic plants also showed fungitoxic effects on pathogenic
fungi. Spraying of wheat with water extracts of fungitoxic plants reduced rust (Puccinia
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T.P. Yurina
Plants grew in soils with low humus contents (1.5%). In field experiments wheat was
sown at distances of 30, 60 and 90 cm from weed plants. During the reproductive stage,
the degree of colonization by mycorrhizal fungi was determined by microscopy. The
degree of colonization of roots by arbuscular mycorrhizal fungi was defined as the ratio
of the number of colonized root fragments to the total number of roots fragments
analyzed.
The allelopatic effect of of fungitoxic plants on wheat when co-cultivated has been
established logt time ago. Decreased distances between wheat and fungitoxic plants in
co-cultivation resulted in reduced biomass production and root colonization (Table 1).
The most pronounced influence was visible at a distance of 30 cm. The data shown here
may suggest that the effect of the fungitoxic plants on wheat was indirect, i.e. mediated
by diminishing the mycorrhizal interaction and, thus, nutrient supply.
Positive influence of arbuscular mycorrhizal fungi on growth and development of
various agricultural crops suggests to study further and in more depth the suitability of
different symbiotic associations in agricultural practice.
Table 1: Influence of common wormwood (Artemisia absinthium ) on wheat plants (Triticum aestivum)
and degree of colonization of roots by arbuscular mycorrhizal fungi.
References
Jones M.D., Smith S.E. (2004): Exploring functional definitions of mycorrhizas: are mycorrhizas
always mutualisms? Can.J.Bot., 82, 1089-1109.
Zhu Y.G., Miller R.M. (2003): Carbon cycling by arbuscular mycorrhizal fungi in soil-plant
systems. Trends Plant Sci., 8, 407-409.
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59
Bacterial Blight Disease on Fennel Plants in
Egypt
M.A. ISMAIL
Department of Plant Pathology, Faculty of Agriculture, El Minia University, Minia, Egypt.
Abstract
A severe leaf blight was observed on fennel plants (Foeniculum vulgare, Miller) grown in the
region of Minia, Egypt. The tips and centers of the filiform leaves first became water-soaked and
then rapidly turned necrotic. A fluorescent Pseudomonas was isolated from diseased leaves on
king's medium B. Bacterial isolates were negative for oxidase, arginine dehydrolase, potato rot
and positive for production of levan and induced a hypersensitive reaction in tobacco (Nicotiana
tabacum). Identification trials suggest that these isolates are Pseudomonas syringae pv. syringae.
Five bacterial isolates were isolated from naturally infected fennel plants and pathogenicity tests
showed they had varying virulence on fennel leaves. The highest virulence was expressed by
isolate P1 whereas the least virulent isolate was P5.
The bacterium was pathogenic to caraway, black cumin, sunflower, bean and coriander but
was not pathogenic to anise, pea, fenugreek, lentil, faba bean and chick pea.
Introduction
Fennel (Foeniculum vulgare, Miller) plant is a winter annual herb belonging to the
family Apiaceae. Its cultivation is mainly concentrated in the Middle Egypt Governorates
like El–Minia and Assuit.
Unfortunately, fennel (Foeniculum vulgare, Miller) is attacked by several diseases,
including leaf blight (Cercosporidium punctum) (Rubatzky and Yamaguchi, 1997), stem
rot (Sclerotinia minor) (Koike, 1994) and bacterial disease (Pseudomonas syringae)
(Koike, et al., 1993).
The present study describes 1) isolation of the causal agent of the bacterial blight
disease of fennel plants, 2) study of some medicinal and other plants response to
infection.
M.A. Ismail
Pathogenicity tests
Pathogenicity trials of the bacterial isolates were determined by inoculating healthy
plants grown in the experimental farm of the Department of Plant Pathology of the
Faculty of Agriculture of Minia University (Abdel-Naem and Ismail, 2005). Control
plants were inoculated with sterile water. Inoculated plants were covered with moistened
plastic bags for 24 hr to keep high relative humidity. The inoculated plants were
observed for development of disease symptoms.
A
Figure1: Natural blight leaves of fennel plants (A) and blighted leaves caused by Pseudomonas syringae
pv. syringae under artificial infection (B).
Disease assessment
Disease severity was calculated according to the methods of Liu et al. (1995):
DSI = ∑d/ (d max X n) X100
Whereas: d is the disease rating possible and n is the total number of fennel plants
examined in each replicate.
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Host range
The most virulent isolate P1 of the causal pathogen was inoculated into leaves of 11 plant
species. Five plants were used in each treatment.
Table 1: Disease severity on fennel leaves after 14 days from inoculated with 5 bacterial isolates of
Pseudomonas syringae pv. syringae.
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M.A. Ismail
Host range
The most virulent isolate (P1) was leaf inoculated to different host testers. Data show that
the following plants were not affected by inoculated bacteria. The bacterium was
pathogenic to caraway, black cumin, sunflower, bean and coriander but was not
pathogenic to ◌anise,
ِ pea, fenugreek, lentil, faba bean and chick pea. On the other hand,
the results showed in this study that the bacterial isolates have a wide host range as
reported by Bradbury (1986) who reported that the pathogens are non-specific towards
different hosts.
Table 2: Reported morphological, biochemical and physiological characters of Pseudomonas syringae pv.
syringae. in comparison with those of the isolated bacteria.
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References
Abdel-Naem, G.F., Ismail, M.E. (2005): Certain biochemical changes in geranium plants due to
batereial blight infection. Minia J. of Agric. Res. & Dept., 25 (3), 481-502.
Bradbury, G.F. (1986): “Guide to Plant Pathogenic Bacteria”. 1-322 pp., CMA. International
Microbiology Institute, Ferrylane. Kew, Surrey, England.
Giesler, L.J. (2003): Bacterial blight of geranium. Extension Plant Pathology, Jay B. Fitzgerald,
Extension Horticulturalist.
Klement, Z.; Rudolph, K., Sands, D.C. (1990): Methods in Phytobacteriology Akademiai, Kiado,
Budapest, pp.568.
Koike, S.T. (1994): First report of stem rot of fennel in the United States caused by Sclerotinia
minor. Plant Dis., 78, 754.
Koike, S.T., R.L. Gilbertson, E.L. Little (1993): A new bacterial disease of fennel in California.
Plant Dis., 77, 319.
Liu, L., J.W. Klopper, S. Tuzun (1995): Induction of systemic resistance in cucumber against
Fusarium wilt by plant growth - promoting rhizobacteria. Phytopathology, 85, 695-698.
Liu, H.L.; Hsu, S.T., Tzeng, K.C. (2002): Bacterial soft rot of chrysanthemum cuttings:
characteristics of the pathogen and factors affecting its occurrence-Plant Pathology
Bulletin, 11 (3), 157-164.
Rubatzky, V.E., Yamaguchi, M. (1997): World Vegetables, 2nd ed., Chapman & Hall, New York,
pp. 447- 449.
Saleh, O. I., Stead, D. (2003): Bacterial soft rot disease of pea in Egypt. Integrated control in
protected crops, Mediterranean climate IOBC WPRS Bulletin, 26 (10), 115-120.
Stancescu, C., Severin, V. (1983): Sunflower leaf spot, a new disease in Romania. An. Res. Inst.
for Plant Prot. (I.G.P.P.), 17, 37-43.
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M.A. Ismail
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60
Response of Sunflower Hybrids to some
Antioxidants and Stem Rot Caused by
Sclerotinia sclerotiorum
M.A. ISMAIL
Dept. Plant Pathology, Faculty of Agriculture, El Minia University, Egypt
Abstract
A severe wilting of sunflower plants (Helianthus annuus L.) grown in Minia region was observed.
Four Sclerotinia sclerotiorum isolates were identified by their morphological and pathological
characteristics and were used to evaluate eight sunflower hypbrids (cv Giza 1 ) for resistance
against infection during 2007 and 2008 seasons. Wilt and basal stem rot were recorded and the
sunflower hybrids differed in reaction against the pathogen from highly susceptible to less
susceptible infection. A positive correlation between lesion length on stem base and wilt severity
was determined. Thus, lesion length seems to be a simple and direct method for assessing
resistance of sunflower against the pathogen. Induction of resistance in sunflower plants against
infection by S. sclerotiorum was strongly affected by the type of resistance elicitors (REs) used.
All REs, e.g., ascorbic acid, C6H8O6 (AA), Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl
ester, benzothiadiazole (BTH trade name Bion), salicylic acid, C6H9(OH)COOH) (SA) and
propylgalate 3,1,5- trihydroxybenzoic acid (PG) were able to induce resistance.
Introduction
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M.A. Ismail
Table 1: Average percentage of stem rot/wilt diseases affecting sunflower plants cv. Giza 1 during 2009 growing
season at various regions of Minia Governorate.
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Table 2: Pathogenicity test of the fungal isolates obtained from stem rot of sunflower plants cv. Giza 1
(mean of 5 replicates).
Figure 1: Artificial infection with basal stem rot on sunflower plants cv. Giza 1 caused by S. sclerotiorum.
Table 3: Varietal response of sunflower hybrids towards soil infestation and stem inoculation by two
isolates of S. Sclerotiorum Sc1 and Sc2.
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M.A. Ismail
Table 4: Effect of some antioxidants seed treatments on stem rot severity to sunflower hybrids caused by
S. sclerotiorum isolate Sc1 infection.
The present work showed that the antioxidants compounds (free radical scavengers)
have the ability to induce resistance in plants against S. sclerotiorum infection similar to
that caused against various plant invaders (Ismail et al., 2006). However, values of this
work through light antioxidants and resistant hybrids or cultivars are beneficial to be
involved in the integrated diseases managements programs. In addition, data indicated
that salicylic acid has indirect antifungal activity and induced protection against infection
by P. medicaginis.
References
Cole, D.L. (1999): The efficacy of acibenzolar- S-methyl, an inducer of systemic acquired
resistance, against bacterial and fungal diseases of tobacco. Crop Prot., 18, 267-273.
Görlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp,
M., Staub, T., Ward, E., Kessmann, H., Ryals, J. (1996): Benzothiadiazole, a novel class of
inducers of systemic acquired resistance, activates gene expression and disease resistance
in wheat. Plant Cell, 8, 629-643.
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Howard, F.S., David, H.G. (2005): Sclerotinia Diseases (White Mold) on safflower. Extension
Services of Nebraska, Colorado. pp.1-3.
Ismail, M.E., Abdalla, H.M., Galal, A.A. (2006): Factors affecting induced resistance in
sunflower plants against basal stem rot caused by Sclerotiorum rolfsii (Corticum rolfsii).
Minia J. of Agric. Res. Develop., 26 (3), 405-425.
Liu, L., Klopper, J.W., Tuzun, S. (1995): Induction of systemic resistance in cucumber against
Fusarium wilt by plant growth- promoting rhizobacteria. Phytopathology, 85, 695-698.
Mosa, A.A., El-Deeb, A.A., Ibrahim, M.M. (2000): Evaluation of sunflower hybrids for
resistance to Sclerotinia sclerotium. Annals Agric. Sci., Ain Shams Uni., Cairo, 45 (2),
689-702.
Nelson, B.D., Hertsgaard, D.M., Holley, R.C. (1989): Disease progress of Sclerotinia wilt of
sunflower at varying plant populations, inoculum densities and environments.
Phytopathology, 79, 1358-1363.
Sackston, W.E. (1992): On a treadmill: Breeding sunflower for resistance to disease. Ann. Rev.
Phytopathology, 30, 529-532.
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M.A. Ismail
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61
Fluorescent Characteristics and Yield Structure
of Barley Treated with Supercritical Fluid
Extracts from Reynoutria sachalinensis
Introduction
Extracts from giant knotweed, Reynoutria sachalinensis are known to protect mono- and
dicotyledone crops from phytopathogenic fungi (Herger and Klingauf, 1990). Earlier, we
have proven the stimulant effect of aqueous extract from R. sachalinensis on the
photosynthetic activity in barley plants grown in field conditions (Karavaev et al., 2008).
In this work, we have used the supercritical fluid (SCF) extracts of R. sachalinensis. The
advantages of SCF extracts are connected with the properties of supercritical CO2,
namely with it high penetrating and also high dissolving capacities. Moreover, it is easily
separated from extracted material by bringing down the pressure.
SCF extracts were prepared from leaves of giant knotweed R. sachalinensis growing in
central Russia. The extracts were prepared on a SFE-10001-2-FMC50 laboratory setup
(Thar Instruments, Inc., USA) using CO2 with 10% of ethanol as co-solvent.
The trials were conducted in the experimental field of the Russian State Agricultural
Academy in the summer of 2009. SCF extracts were dissolved in water so as to obtain
1% and 2% solutions. Plants were sprayed with the preparations at the end of tillering.
Control plants were sprayed with water.
To estimate the photosynthetic activity of the barley leaves, we recorded the slow
fluorescence induction curves as described by Karavaev et al., (2008). Early, we have
shown that the ratio (FMFT)/FT (FM = maximal value, FT = stationary level of
fluorescence) is correlated with the rate of photosynthetic O2 evolution per chlorophyll
content (Karavaev et al., 1998).
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Spraying the plants with SCF extracts from R. sachalinensis resulted in an increase in
(FMFT)/FT values in the course of three weeks after the treatment, indicating a
corresponding increase in photosynthetic activity (Table 1). The maximal stimulant
effect was recorded two weeks after the treatment; 25–40 % for 1% R. sachalinensis
extract and 15–25 % for 2% extract. This can be associated with the transport of
physiologically active compounds into the leaves, a factor that increases the number of
electron carriers between photosystems I and II.
As to yield structure, there was a significant increase in the total number of stems
per plant (1.8 for plants treated with 1% SCF extract against 1.3 for control plants) and
also in the number of productive stems. As a result, the total mass of grains/ha increased
to 134% as compared to control plants for 1% SCF extract and to 114% for 2% SCF
extract. Technological and brewing qualities of barley were also improved.
Table 1: Average (FM – FT) / FT values of slow fluorescence induction of barley leaves.
Table 2: Yield structure of barley treated with supercritical fluid extracts from R. sachalinensis.
References
Herger, G., Klingauf, F. (1990): Control of powdery mildew fungi with extracts of the giant
knotweed, Reynoutria sachalinensis (Polygonaceae). Med. Fac. Landbouww. Rijksuniv.
Gent, 55, 1007-1014.
Karavaev, V.A., Polyakova, I.B., Solntsev, M.K., Yurina, T.P. (1998): Effects of various
chemical agents on photosynthesis studied by the method of fluorescence induction. J.
Luminescence, 76, 77, 335-338.
Karavaev, V.A., Gunar, L.E., Myakinkov, A.G., Schmitt, A., Glazunova, S.A., Solntsev, M.K.
(2008): Fluorescent characteristics and yield structure of barley treated with plant extract
from Reynoutria sachalinensis. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H. Kuck, P.E.
Russell, H. Lyr: Modern fungicides and antifungal compounds V. DPG Selbstverlag,
Braunschweig, Germany. 285-289.
The work was supported by Russian Foundation for Basic Research, grant 09-04-12177-
ofi_m.
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62
Effect of Supercritical Fluid Extracts from
Reynoutria sachalinensis on Photosynthetic
Apparatus of Bean Leaves
Introduction
Seedlings of beans Vicia faba L. were grown in a greenhouse at 20-22C under natural
daylight supplemented with incandescent lamps. The SCF extracts were prepared on a
SFE-1000M1-2-FMC50 laboratory system (Thar Instruments, Inc., USA) from leaves of
giant knotweed R. sachalinensis growing in central Russia. Extracts were obtained using
pure CO2, CO2 with 10% of ethanol as co-solvent and CO2 with 2% of ethanol as co-
solvent. Before treating the test plants, the SCF extracts were dissolved in 100 ml of
water so as to obtain the 2% solution. Seedlings were sprayed with SCF extracts
solutions from R. sachalinensis two weeks after seeds sowing; control plants were
sprayed with water. Two biophysical methods based on the registration of slow
fluorescence induction (SFI) and of thermoluminescence (TL) of the leaves were used in
the work (Karavaev et al., 1998; Solntsev et al., 1998).
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Spraying the plants with supercritical fluid extracts from R. sachalinensis resulted in an
increase in (FMFT)/FT values of SFI (FM = maximal value, FT = stationary level of leaf
fluorescence), indicating the increase in photosynthetic activity of the leaves (Table 1). A
stable stimulant effect was observed within 10 days after the treatment (from 20 to 40 %
as compared to control plants). The maximum effect was observed for treatment with the
SCF extract obtained using the CO2–2% ethanol solvent.
The increase in (FMFT)/FT values of SFI was accompanied by changes in
thermoluminescence curves: the TL intensity increased at negative temperatures (band A)
indicating an increase in the photosynthetic activity of plants, but only at low
concentration of ethanol in the extract. In addition, when CO2 and CO2–2% ethanol were
used for the extraction of physiologically active compounds, the TL intensity of the high
temperature band (band C) decreased, indicating enhancement in the resistance of
chloroplast membranes to stress conditions.
We assume that the increase in the photosynthetic activity of plants after treatment
with SCF extracts is associated with stimulation of electron transport due to the
administration of quinonic compounds into leaf cells, which act as additional electron
carriers at the reducing side of the photosystem 2.
Table 1: Changes in the (FM – FT) / FT values of slow fluorescence induction of bean leaves after their
treatment with supercritical fluid extracts from Reynoutria sachalinensis.
References
Daayf, F., Schmitt, A., Belanger, R.R. (1995): The effects of plant extracts of Reynoutria
sachalinensis on powdery mildew development and leaf physiology of long English
cucumber. Plant Disease, 79, 577-580.
Karavaev, V.A., Schmitt, A., Solntsev, M.K., Yurina, T.P., Frantsev, V.V., Kuznetsov, A.M.,
Polyakova, I.B., Trubitsin, B.V., Tikhonov, A.N. (2002): Stimulation of photosynthetic
activity in wheat leaves treated with aqueous extracts from Reynoutria sachalinensis. In:
H.W.Dehne, U. Gisi, K.H. Kuck, P.E. Russell, H. Lyr (Eds.) Modern Fungicides and
Antifungal Compounds III. AgroConcept, Bonn, Germany. 379-385.
Karavaev, V.A., Polyakova, I.B., Solntsev, M.K., Yurina, T.P. (1998): Effects of various
chemical agents on photosynthesis studied by the method of fluorescence induction. J.
Luminescence, 76, 77, 335-338.
Solntsev, M.K., Ekobena, H.P.F., Karavaev, V.A., Yurina, T.P. (1998): Dynamics of the action
of various physical and chemical factors on the thermoluminescence of photosynthetic
systems. J. Luminescence, 76, 77, 349-353.
The work is supported by Russian Foundation for Basic Research, grant 09-04-12177-
ofi_m.
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63
The Action of 3-Amino-1,2,4-Triazole on Early
Developmental Stages of the Wheat Powdery
Mildew Pathogen
Abstract
The effect of 3-amino-1,2,4-triazole (3-АТА, amitrole), an inhibitor of peroxidase and catalase,
on development of Erysiphe graminis DC. f. sp. tritici March. (Syn. Blumeria graminis), a causal
organism of wheat powdery mildew has been investigated.
Introduction
Wheat seedlings (Triticum aestivum L., cv. Zarya and wheat-Aegilops line 56/99i) were
cultivated in rolls of filter paper in Knop solution at 20–25oC under natural illumination
with additional illumination up to 16-h photoperiod. The first true leaves were inoculated
and floated adaxial side up on 3-АТА and zeatin solutions in Petri dishes. The samples
for SEM were fixed with glutaraldehyde and osmium tetroxide, dehydrated in graded
alcohols, critical point-dried with CO2 and coated with gold. The specimens were
examined in a LEO-1430 VP scanning electron microscope (Carl Zeiss, Germany).
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Figure 1: Normal development of the wheat powdery mildew fungi on wheat leaves 24, 48 h and 6 days
after inoculation (SEM).
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Figure 2: Effect of 3-ATA on development of the powdery mildew fungi on leaves of wheat-Aegilops
line 68 h after inoculation. Upper photo – microcolony on untreated control; lower photos – anomalous
development of fungi on 2 mM 3-ATA (conidia germinated with great number of short growth tubes and
abortive colony, SEM).
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Table 1: Effects of 3-ATA and zeatin on development of the powdery mildew fungi (cv Zarya).
Infection structure, %
3- Total
Zeatin, Non- Normal/
ATA, pathogen Normal Abnormal
mkM germinate Colonies abnormal
mM units appressoria appressoria
d conidia
0 0 210 37,6 14,8 2,4 45,2 6,2
1 0 336 26,2 18,5 3,6 51,8 5,2
3 0 219 11,0 5,5 0,9 82,6 6,0
0 10 369 39,8 43,1 16,8 0,3 2,6
0,25 10 110 60,0 30,9 5,5 3,6 5,7
0,5 10 123 13,0 68,3 8,9 9,8 7,6
1 10 315 30,5 52,1 8,3 9,2 6,3
1,5 10 99 41,4 41,4 15,2 2,0 2,7
3 10 296 33,8 53,7 9,5 3,0 5,7
4,5 10 284 52,8 37,0 7,4 2,8 5,0
References
Babosha A.V. (2009): Regulation of resistance and susceptibility in wheat powdery mildew
pathosystem with exogenous cytokinins. J Plant Physiol., 166, 1892-1903.
Gechev T., Gadjev I., Van Breusegem F., Inze D., Dukiandjiev S., Toneva V., Minkov I. (2002):
Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant
enzymes. Cell Mol Life Sci., 59, 708-714.
Lamb C., Dixon R.A. (1997): The oxidative burst in plant disease resistance. Ann Rev Plant
Physiol Plant Mol Biol., 48, 251–275.
Okuda T., Matsuda Y., Sugawara M., Sagisaka S. (1992): Metabolic response to treatment with
cold, paraquat, or 3-amino-1,2,4-triazole in leaves of winter wheat. Biosci Biotechnol
Biochem., 56, 1911-1915.
Serezhkina G.V., Andreev L.N., Avetisyan T.V., Batova S.N., Poleva L.B. (1996): Role of
primary reactions in interactions between the parasite and host plant with special reference
to resistance of the wheat–wheatgrass hybrids to Erysiphe graminis tritici at the stage of
penetration. Izv Akad Nauk Ser Biol., 4, 422-429.
Thordal-Christensen H., Zhang Z., Wei Y., Collinge D.B. (1997): Subcellular localisation of
H2O2 in plants, H2O2 accumulation in papillae and hypersensitive response during barley-
powdery mildew interaction. Plant J., 11, 1187-1194.
Willekens H., Chamnongpol S., Davey M., Schraudner M., Langebartels C., Van Montagu M.,
Inze D., Van Camp W. (1997): Catalase is a sink for H2O2 and is indispensable for stress
defence in C3 plants. EMBO J., 16, 4806-4816.
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64
Regularities of Direction Relative to the Axis of
Wheat Leaf for Growth of Wheat Powdery
Mildew Infection Structures
Abstract
The epidermal cells of grass leaves, the objects of attack by powdery mildew pathogen, are
strongly elongated along the long axis of the leaf. Scanning electronic microscopy (SEM) was
used to investigate the regularities of growth direction of infectious structures of the wheat
powdery mildew fungus. In our experiments growth of appressoria with normal morphology was
observed predominantly along the long axis of the leaf.
Introduction
A special trait of powdery mildews is their adhesion to epidermal tissue of the host plants
and predominant development on its surface. It was shown that on leaves of resistant
barley plants the great part of infectious structures of the fungus developed with
deviations in the morphology of growth tubes and appressoria (Mishina et al., 1988;
Serezhkina et al., 1996).
As a rule, the quantity of normally formed infectious structures of the powdery
mildew pathogen correlates with the susceptibility of the plant host and with the surface
density of pathogen colonies. Epidermal cells of grass leaves, the objects of attack by the
powdery mildew pathogen, are strongly elongated along the long axis of the leaf. The
directions along and across the leaf are nonequivalent. Regularities in the direction of
growth of wheat powdery mildew on anisotropic leaf surface were investigated by
scanning electronic microscopy (SEM).
Wheat seedlings (Triticum aestivum L.) of the susceptible variety Khakasskaya and
wheat-Aegilops line 56/99i were cultivated in rolls of filter paper in Knop solution at 20–
25o C under natural illumination with additional illumination up to 16h photoperiod. The
first true leaves were inoculated with Blumeria graminis f. sp. tritici and floated adaxial
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side up on zeatin solutions in Petri dishes. The samples for SEM were fixed with
glutaraldehyde and osmium tetroxide, dehydrated in graded alcohols, critical point-dried
with CO2 and coated with gold. The specimens were examined in a LEO-1430 VP
scanning electron microscope (Carl Zeiss, Germany). The number of normal and
anomalous (elongated) appressoria of germinated conidia and that of young colonies was
counted 24, 48, and 68h after inoculation. If the angle between the long axis of the
epidermal cell (anticlinal cell wall) and the direction of appressorium growth was within
0°– 30°, the appressorium was classified as growing along the long axis of epidermal
cells. Accordingly, appressoria growing at an angle of 30°– 90° were classed as growing
across epidermal cells.
Six classes of appressoria were specified by directions and kind of growth: normal
appressoria growing along the long axis of the leaf, normal appressoria growing across
the long axis of the leaf, anomalous appressoria growing along the long axis of the leaf,
anomalous appressoria growing across the long axis of the leaf (Figure 1), appressoria of
microcolonies directed along the long axis of the leaf, appressoria of microcolonies
directed across the long axis of the leaf.
Figure 1: Normal (upper photos) and anomalous (lower photos) appressoria of B. graminis f.sp.tritici
growing along (left) and across (right) the long axis of epidermal cells.
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Table 1: The number of normal and anomalous (grown out) appressoria directed along and across the long
axis of epidermal cells (anticlinal cell wall) in the presence of zeatin on wheat-Aegilops line 56/99i , 48 h
after inoculation.
Table 1 shows that the ratio observed in the experiment, significantly differs from
the aforementioned one. Appressoria growing along the long axis were also dominated at
the stage of young colonies (Table 2).This regularity was also observed on leaves of
wheat Khakasskaya variety (data not shown).
Treatment with several zeatin concentrations increased the number of the pathogen
colonies (Table 2). The same concentrations also changed predominant direction of
growth of normal appressorium and increased the number of normal appressoria growing
in the perpendicular direction.
Germinating conidia interact with two sites of leaf surface. However the feeding
haustorium is formed only in the place of contact of the appressorium lobe. We
suggested that the direction of appressorium growth may depend on the results of the first
host-pathogen contact realized by the primary growth tube. The most efficient strategy
for the initial development of pathogens in the case of favorable primary contact should
be appressorial growth along the long axis and formation of secondary haustoria in the
same cells of the host plant. Under unfavorable primary contact, the probability to find
the cell in a suitable physiological state increases in the direction across the long axis of
the leaf. Finally, especially unfavorable conditions lead to disturbance of regulation of
growth processes of the pathogen and to the formation of anomalously elongated germ
tubes growing in the most cases across the long axis of the leaf. Mechanism of
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Table 2: The number of normal appressoria that formed colonies directed along and across the long axis
of the epidermal cells (anticlinal cell wall) in the presence of zeatin on wheat-Aegilops line 56/99i , 48 h
after inoculation.
References
Mishina, G.N., Serezhkina, G.V., Rashal’, I.D., and Andreev, L.N. (1988): Characteristics of
development of Erysiphe graminis f. sp. hordei Marshal on leaves of barley genotypes of
different resistance, Mikol. Fitopatol., 22, (4), 292–295.
Serezhkina, G.V., Andreev, L.N., Avetisyan, T.V., Batova, S.N., Poleva, L.B. (1996): Role of
primary reactions in interactions between the parasite and host plant with special reference
to resistance of the wheat–wheatgrass hybrids to Erysiphe graminis tritici at the stage of
penetration, Biol Bull., 23, (4), 346–352.
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65
Role of Resistance and Fungicide Use in
Preventing Mycotoxin Contamination of Cereal
Commodities
Abstract
In the last 30-40 years the scientific fundaments of breeding for Fusarium head blight (FHB)
resistance were elaborated. The genetic background is known for a number of genotypes at least
on QTL level, which have medium to good resistance, and we have performed a successful
breeding program. As the resistance sources were agronomically poor, it took many years to
transform them into lines that are suitable for crossings to breed commercial cultivars. By now in
many programs moderately to highly resistant materials are available; however, the main
problem is to combine FHB resistance with other important traits like yield, quality, heat and
drought resistance, winter hardiness in winter wheat etc. The severe FHB epidemics in US,
Canada, Argentina, Hungary and Romania in 2010 underline again the necessity of more resistant
cultivars. By screening existing material, moderate to good resistance can be identified in plant
materials at a low rate. FHB tests during the variety registration process are inevitable to enhance
breeding efforts. Identifying the more resistant materials by a simple test can decrease food safety
risks by about 50%. Our prediction is that the proportion of more resistant varieties will grow
slowly, but their ratio in wheat acreage will increase more rapidly.
In fungicide application the side spray is the most important achievement. Heads have to be
completely covered by fungicide spray, and spraying from each side led to 30-40% coverage.
With the best fungicides 95% reduction in deoxynivalenol (DON) contamination was possible in
small plot tests, and up to 70% reduction was achieved in farm scale applications. As compared
to several years ago, the prevention of FHB infection and DON contamination became much
more effective, so increasing food safety is not only a theoretical possibility.
Introduction
The prevention of Fusarium head blight (FHB) in the field is highly important, as the
toxin, DON, is typically produced after head infection in the field. As agronomy and
other means are providing only moderate protection, the breeding approach becomes
more important. The Commission Regulations, EC 856/2005 (2005) and EC 1126/2007
(2007) set limits for contamination of basic materials for human food and food products.
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Á. Mesterházy et al.
For animal feeds, the Commission Regulation EC 576/2006 (2006) provides values for
contamination limits. Epidemics may cause serious yield losses, often due to toxin
contamination as this makes the harvested yield unsuitable for any later uses.
In this paper two options to decrease DON contamination, i.e. breeding and
fungicide application will be discussed. Although breeding FHB resistant cultivars is in
progress, susceptible and highly susceptible cultivars are still grown. Therefore the
improvement of fungicide treatments is inevitable.
Fungicide application
According to general field experience the fungicide efficacy remained low in epidemic
years and led to only some 20-30% reduction of disease severity or toxin contamination.
For a long time fungicides were thought to lack efficacy against the causal agent of FHB.
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However, the truth is more complex. Mesterházy and Bartók (1996), Mesterházy et al.,
(2003a) and Mesterházy et al., (2003) showed that fungicide efficacy at optimal coverage
of heads by fungicide spray may reach 80-90% disease reduction for the most effective
fungicides. The correlations between the reactions of traits like FHB severity, FDK and
DON were very close, up to r = 0.90 in most experiments, so the different traits were
similarly reduced. The reasons for the good results are: (1) Coverage. Our tests were
made at optimal coverage with hand sprayed treatments from both sides to cover the
whole ear uniformly. McMullen et al., (1997, 1999) were the first to show that spraying
technology significantly influences fungicide efficacy. Hooker and Schaafsma (1984)
compared the coverage of heads at different nozzle types and they concluded that nozzles
traditionally developed for leaf control give poor coverage, and so poor efficacy. Ruden
et al., (2005) found that a deep penetration of the spray down to the rachis is an
important condition to have effective protection. (2) Translocation. The problem is that
fungicides are only partly systemic, they move acropetally, so from the leaves there is no
transport to the heads (Mauler-Machnik and Zahn, 1994; Lehoczki-Krsjak et al., 2010).
Therefore, the fungicide should be placed on the ear directly from every side; only this
can secure an effective protection. (3) Fungicide differences. In the past 20 years a large
number a fungicides have been tested. Our ranking corresponded well with that of
Maufras et al., (1994), but with much higher efficacy levels. (4) Variety resistance
differences. Cultivars with higher resistance could be protected generally more
effectively; the level of the toxin contamination was lower. For the most sensitive
cultivars we do not have a reliable control, and this cultivar group should therefore be
withdrawn from commercial production.
In this paper we summarize a three years study where we compared large scale farm
application and hand made small pot tests in 2006-2008.
Breeding trials
The breeding program started with the selection of more resistant lines from winter
wheat materials. In the program more resistant native lines, Sumai-3 and Nobeoka Bozu
spring wheat resistance sources were used. The breeding followed the scheme reported
by Mesterházy (1995) and Mesterházy et al. (2005).
The isolates, their origin, the way of increasing and testing of aggressiveness were
published earlier (Mesterházy 1995, 2002, Mesterházy et al., 2005). The inocula, like in
all other tests in this paper, were sprayed at full flowering with a 2.0 l hand sprayer (Eva,
DiMartino, Mussolente, Italy) mediated by air pressure. In these tests FHB severity was
assessed five times (10, 14, 18, 22 and 26 days after inoculation); their mean was used as
mean severity. The threshing was made at low wind and cleaning to save grains for
further test. This secured the close correlations between traits.
In the first test we report data about resistance sources prebred materials as well as
susceptible control varieties. Three row plots, each 1.5 m long, were inoculated each year
by four different Fusarium isolates (two F. graminearum No. 12377 and 46 and two F.
culmorum No. 12375 and 12551) differing in aggressiveness. Each year 100-150 lines
were tested.
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Á. Mesterházy et al.
Each year more than hundred lines of variety candidates from the official variety
registration trial were tested. Two isolates of strain 12551 and one from strain 12375 of F.
culmorum, and strain 46.06 from F. graminearum were used for inoculation. The same
control cultivars were inoculated in different parts of the trial, allowing controlling the
reproducibility of the data. In these trials two plots were sown, each plot consisting of six
rows and four isolates were used in two groups of heads.
Fungicides Rate
(l/ha)
Prospect (200 g/l carbendazime, 80 g/l propiconazole) 1.5
Falcon 460 EC (167 g/l tebuconazole, 250 g/l spiroxamin, 43 g/l triadimenol) 0.8
Prosaro (125 g/l prothioconazole, 125 g/l tebuconazole) 1.0
Tango Star ( 84 g/l epoxyconazole, 250 g/l fenpropimorph) 1.0
Eminent 125 SL (125 g/l tetraconazole 1.0
Amistar Xtra (200 g/l azoxystrobin, 80 g/l ciproconazole) 1.0
Nativo (200 g/l tebuconazole, 100 g trifloxystrobin) 1.0
Artea 330 EC (250 g/l propiconazole, 80 g/l ciproconazole) 1.0
Juwel (125 g/l epoxyconazole, 125 g/l kresoxim-methyl) 1.0
Field applications. The same cultivars as above were sown in three ha fields, altogether
9 ha. Fungicides and dose rates were the same as above. As flowering time was very
similar (1-2 days difference) the whole test was sprayed the same day in full flowering.
The spray volume was 250 l/ha, speed of the tractor was 7-8 km/h. The boom was 18 m,
the left and right sides mounted with different type nozzles. The sprayed stripe was 7 m
wide (five stripes) and 200 m long. No artificial inoculation was used. In 2006 and 2008
epidemic conditions occurred, but not more than 5-7% of the heads were infected
(incidence) in the non-protected control plots. In 2007 the flowering time was still wet,
but subsequently a long dry period followed until harvest, so the starting epidemic was
blocked. Two nozzle types were used, the traditional TeeJet XR and the Turbo FloodJet,
this latter was mounted on the boom at 50 cm distance, back and forward. As the angle of
the spray jets was 160o, the heads received spray from every side, ensuring a more
optimal coverage close to the hand sprayed versions. Evaluation was made 15 and 22
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days after fungicide treatment where the incidence was given as infected heads/m2. At
harvest yield was measured, from each plot a sample of 1 kg grain separated for FDK
and DON analyses.
DON analysis
In all tests DON analyses were made. Six grams of grain (when more replicates were
used for an isolate within plot, they were pooled, Mesterházy et al., 2003). Samples were
milled and analyzed by HPLC (HP 1090M equipped with diode array detector, Hewlett
Packard, now Agilent, USA).
Statistical analysis
The non-replicated screening tests with four different isolates were evaluated by the two-
way ANOVA model without replication. This model was used also for the DON
evaluation where samples were pooled. In the fungicide tests the three replicates could be
elevated. Except the early generation tests, randomized block design was used. For all
analyses the Excel programs (Microsoft Inc.) were used. For the three-way variance
analyses also the Excel was used, and the functions described by Weber (1967) were
applied. In the farm-scale fungicide tests the SAS statistical program were used.
Results
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70
y = 0.6121x + 0.9446
60 2
R = 0.7815, r = 0.8840, P = 0.001
50
D ON ppm
40
30
20
10
0
0 10 20 30 40 50 60 70
FDK %
Figure 1: Relation between FDK and DON data of the 2005 resistance test series. Means for four isolates,
n=117.
Fungicide tests
Small plot and field data of the three years were evaluated for all FHB traits.
However in this paper we concentrate on the reduction of the DON contamination after
fungicide treatment. In the small plot tests, 1350 DON analyses were made during the
three years, providing a rather solid data base to draw well-supported conclusions. Table
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2 shows the means for the three cultivars. The correlation coefficients between fungicide
reactions of the three cultivars are between r = 0.87 and 0.94 (P=0.1%). The reduction of
the DON contamination by the different fungicides is very similar in the cultivars tested,
with some variations at the different epidemic severities. It is important to note that the
protected, but not artificially inoculated control groups (natural infection)showed a
similar ranking as the fungicide protected and inoculated groups of heads, with
correlations all above r = 0.80 (P = 0.1%).
180
160
140
120
100
80
60
40 DON ppm
FDK %
20
FHB%
0
GK GARABOLY
GK HATTYÚ
BRILLIANT
GK KALÁSZ
ETIDA
TIMBER
BUSER
Mv MAMBÓ
QUEBON
SzD 9547 H
BR 04/005
SzD 7913
SzD 9211
HE 78.15
Mv 18-05
Mv 07-05
Mv 19-05
SWS 700.1574
FK 05040
FK 05028
Figure 2: Fusarium traits for the candidate varieties of the 3rd year; means of four isolates, 2008.
Table 2: Effect o fungicides against FHB in wheat on DON concentration (mg kg-1) across years (2006-
2008) and four isolates.
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In parallel with these tests, the large scale farm application was also carried out. The
mean DON for the TeeJet XR nozzle was 0.89 mg/kg across fungicides and for the Turbo
FloodJet nozzle 0.46 mg/kg, representing a reduction of 47%. The best fungicide was
Prosaro (0.34 and 0.24 mg DON / kg), the least efficient was Eminent with 1.35 and 0.31
mg DON / kg. Efficacy for the farm scale use for Prosaro is 77.02%, which is still lower
than that obtained in small plots.
In Figure 3 data from the hand sprayed small plot tests and the large scale farm
application are compared. The correlation coefficient (r=9551; P=0.1%) indicated that
the performance of the fungicides was very similar in the two application forms.
35
y = 28.107x - 1.6895
30 2
R = 0.9131, r = 0.9551, P = 0.1 %
DON mg/kg, small plot test
25
20
15
10
0
0.00 0.20 0.40 0.60 0.80 1.00 1.20
DON mg/kg, farm test
Figure 3: Comparison of DON contamination of the small plot and farm scale trials at different fungicides
against Fusarium head blight, 2006-2008.
Discussion
genotypes have medium FHB severity, but very low FDK and DON contamination. We
have genotypes where FHB is 32% at 5 mgkg-1 DON and another with 28% FHB and 86
mgkg-1 DON contamination. Conceivably, the FDK has much closer correlation with
FDK than with FHB severity. The reproducibility of test results was good. The existing
methodical background is suitable for breeding and also resistance tests under different
ecological conditions, with good reproducibility of the test results (Mesterházy 1995,
Mesterházy et al., 1999, 2005).
This series of studies convinced us, however, that a significant improvement of food
safety can be achieved when the existing wheat variability is exploited. Although there
may still be significant potential to reduce toxin contamination by breeding, screening of
the existing advanced materials can improve the food safety by about 50% or more, e.g.
the toxin level can be decreased by this rate. As every breeding program has some
variability, screening of local material is important. As most breeding firms do not have
any screening with artificial inoculation for this trait, the relevance of this experimental
approach is clear. The high variability among candidate varieties allows the selection of
the more resistant candidates. The data show clearly, that, like in China, Germany, the
Netherlands and other countries, natural infection surveys or artificial inoculations and
FHB tests should become an integrant part of the registration process.
In the last decades most breeding programs considered visual field symptoms. As
the toxin limits were introduced, the main trait today is toxin contamination. The
presented data clearly show that FDK correlates more closely with DON than visual field
scores do. After completing the visual evaluation 25-30 days after flowering, some 3-4
weeks are left until harvest. Late rains may induce significant additional disease
development. According to our experience about 50% of the genotypes with acceptable
FHB severity should have been discarded. As DON and FDK correlate well, a toxin
control during breeding is not necessary. It is fully sufficient to select for low FDK levels
and the best materials may then also be tested for DON contaminations before they
submission for registration to the authority.
Fungicide application
As breeding is a slow process, and years will pass until more resistant cultivars will be
produced, this time span must be bridged by fungicide application and improved
agronomy. The data clearly show that fungicide efficacy is better on medium or higher
resistant genotypes, indicating synergistic effects between resistance mechanisms of the
plant and fungicides (Deising et al. 2002). Provision of genetic material with significant
resistance levels to Fusarium spp. may reliably allow staying below the official DON
limits, which, in turn, may lead to even stricter official toxin regulations (Berek et al.,
2001).
The fungicide results of the small plot trials fully support the statements of our
earlier report (Mesterházy et al., 2003a). The efficacy of the best fungicides is 80% or
higher for DON. We have two major conclusions. There are highly significant
differences among fungicides, with efficacies varying between 25 and 90%, a roughly
four fold difference. Ninety % efficacy means that a natural epidemic until 10 mg kg-1
DON concentration can be managed. Twenty-five % means that no effective protection
can be expected from these fungicides. For this reason it would be highly desirable to
rate the fungicides like done by the ITCF in France (Maufras et al., 1994).
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The idea to improve efficacy of the fungicide treatment, i.e. optimal coverage of by
spraying the heads, was shown to be absolutely realistic (Mesterházy et al., 1996, 2003).
The challenge is now to develop field applications to come close to the efficiency levels
– and DON values – as in small plot tests.
Large scale tests were performed to upscale the innovation of the small plot
experiments. The results are clear in two respects. The traditional XR nozzle led to an
average of nearly 100% higher DON contaminations than the Turbo FloodJet nozzles.
The fungicide ranking, with 1-2 exceptions, was similar for both nozzle types. The
fungicide Eminent performed much better in field applications using the Turbo FloodJet
nozzles, but with the fungicide Amistar Xtra the difference between the nozzles was only
very small. The other fungicides gave corresponding results.
The variety traits influence fungicide reaction significantly. Among these traits, the
uniformity of flowering is important. The best varieties are those that have all tillers
flowering within 2-3 days. A longer flowering time is problematic, as no optimal
spraying time is possible. Earlier cultivars perform better as the 3-4 week protecting
power of fungicides is sufficient until harvest. For the cultivars witha very long
vegetation period of 3-4 weeks fungicide-mediated protection is too short, and a later
rainy period can cause significant damage. We observed also an interesting phenomenon,
i.e. the receptivity of the fungicide as a cultivar trait. This means, that the varieties with
the same resistance level may respond differently to fungicides (Lechoczki-Krsjak et al.,
2010). The reasons for this are unknown, but when developing a variety-specific
technology we should also consider these effects. However, more experimental
background will be needed to determine variety-specific reactions.
The coverage with fungicides, as determined by the nozzles used, was also analyzed
(data not shown). The nozzle Turbo FloodJet gave much better coverage on the front side
of the sprayed heads, but the rear was covered much less as anticipated (Hooker and
Schaafsma, 2004). For this reason further research will be needed to find another nozzle
type allowing proper application and an even better coverage.
The field application convinced us that the generally poor farm results are not only
due to the use of traditional nozzles like TeeJet XR. Excellent results in the literature are
rare and support this view (like McMullen et al., 1999), and other technological errors
could contribute to the often discouraging results. For this reason all effects should be
considered when a fungicide program is planned, and excellent fungicide efficacy should
be combined with the best nozzle type. For application,, timing, spray volume, speed of
application, wind speed and others must be considered. Of course, good agronomical
practices are also important, e.g. after harvesting maize the maize residue should be
ploughed in.
Acknowledgements
The Authors thank NKTH-KPI for financial support via projects OMFB 01286/2004 and
OMFB 00313/2006 as well as EC KBBE-2007-222690-2 MYCORED.
References
Commission Regulation (EC) No 856/2005 of 6 June (2005): Official Journal of European Union
07.06.2005, L 143/3.
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Mesterházy, Á. (2003a): Control of Fusarium head blight of wheat by fungicides. Fusarium head
blight of wheat and barley, Évszám, 363-380. Eds. Leonard, K.J. and Bushnell, W.R. APS
Press, St. Paul, USA.
Mesterházy, Á., Bartók, T. (1996): Control of Fusarium head blight of wheat by fungicides and
its effect on the toxin contamination of the grains. Pflanzenschutz-Nachrichten Bayer, 49,
181-197.
Mesterházy, Á., Bartók, T., Mirocha, C.M., Komoróczy, R. (1999): Nature of resistance of wheat
to Fusarium head blight and deoxynivalenol contamination and their consequences for
breeding. Plant Breeding, 118, 97-110.
Mesterházy, Á., Bartók, T., Lamper, Cs. (2003): Influence of cultivar resistance, epidemic
severity, and Fusarium species on the efficacy of fungicide control of Fusarium head
blight in wheat and deoxynivalenol (DON) contamination of grain. Plant Disease, 87,
1107-1115.
Mesterházy, Á., Bartók, T., Kászonyi, G., Varga, M., Tóth, B., Varga, J. (2005): Common
resistance to different Fusarium spp. causing Fusarium head blight in wheat. European
Journal of Plant Pathology, 112, 267-281.
Mesterházy, Á., Buerstmayr, H., Tóth, B., Lehoczki-Krsjak, Sz., Szabó-Hevér, Á., Lemmens, M.,
(2007): Proceedings of the 5th Canadian Workshop on Fusarium Head Blight, 2007, 51-66.
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Miedaner, T. (1997): Breeding wheat and rye for resistance to Fusarium diseases. Plant Breeding,
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Miedaner, T., Schneider, B., Geiger, H.H. (2003): Deoxynivalenol (DON) content and Fusarium
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Paul, P.A., Lipps, P.E., Madden, L.V. (2005): Relationship between visual estimates of Fusarium
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66
Patterns of Fusarium Head Blight and
Mycotoxin Contamination in Wheat
Abstract
Investigations into the spatial distribution of Fusarium spp. were carried out in different wheat
fields at a sub-meter scale. Typically, some ears in a field showed symptoms of Fusarium head
blight (FHB), while others were without any symptom. Results of the microbiological analysis of
grain samples demonstrated higher infection levels than visual disease assessments. A total of six
Fusarium species and six mycotoxins were detected. There was considerable variation in the
prevalence of species and mycotoxin contamination in the experiments. In some samples the
frequency of infected kernels (FIK) and disease severity (DS) – quantified by qPCR - showed
aggregated patterns, while in other samples FIK and DS were distributed randomly. For the
assessment of the disease situation, the heterogeneity in the incidence of wheat kernels infected
by Fusarium spp. and associated mycotoxins need a high number of samples and an adapted
monitoring strategy within fields.
Introduction
In recent years FHB, also known as head scab, has remerged worldwide as a disease of
economic importance reducing yield quantity and quality (Mc Mullen et al., 1997). FHB
is caused by a complex of several Fusarium species and may be epidemic over large
areas in some seasons. More important, however, FHB severity varies from field to field
and between local areas. Several Fusarium species are capable of producing a wide range
of mycotoxins and secondary metabolites toxic to man and farm animals. The products
comprise numerous chemical groups, namely trichothecenes, zearalenone and fumonisins,
with different toxic compounds (Moss, 1996; Sweeney and Dobson, 1998). In Western
and Central Europe, at least 17 Fusarium species have been reported to contribute to the
FHB complex in wheat (Parry et al., 1995). Fusarium species not only differ in their
ecological requirements for optimal development, but also in the spectrum of mycotoxins
they produce. Therefore, knowledge on the incidence and spatial distribution of
Fusarium species is crucial for the assessment of kernel contamination (Bai and Shaner,
1994; Desjardins, 2006; Schlang et al., 2009).
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This study was initiated to assess the level of variability in the occurrence of Fusarium
species within wheat fields at a sub-meter scale. In order to determine the variability of
Fusarium infection in 2007, wheat samples, cv. Kris, were collected from fields under
organic and conventional farming with different preceding crops. In 2008, wheat crops,
cv. Tommi, with different nitrogen and fungicide treatments were sampled. Sampling
was carried out using an area of 1 x 1 m divided into 25 sub-samples of 20 x 20 cm with
20 to 40 g of kernels. The frequency of Fusarium species infecting kernels was
quantified microbiologically using potato dextrose agar and carnation leaf agar. Fungal
DNA was extracted from milled flour using the Wizard Magnetic DNA Purification
System for Food (Promega Corporation, Madison, USA). The severity of Fusarium
infection was analysed for F. avenaceum, F. culmorum, F. graminearum, and F. poae by
using TaqMan real-time PCR according to Waalwijk et al. (2004). Mycotoxin
contamination was determined using liquid chromatography coupled with mass
spectrometry (LC-ESI/MS; Herebian et al., 2009). Data on the frequency and severity of
Fusarium infected kernels as well as on mycotoxin contamination were analysed
statistically using SADIE® (Perry, 1995) and displayed in a geographic information
system.
Results
Investigations on the frequency of infected kernels showed that in total six Fusarium
species were involved in the FHB species complex. In 2007, the frequency of infection
by Fusarium spp. within 25 sub-samples from 1 m2 varied from 29.6 % to 81.4 %. The
predominant species was F. graminearum infecting 33.6 % of kernels. However, the
spatial distribution of F. graminearum was heterogeneous on the sub-meter scale and,
based on SADIE’s index of aggregation (Ia), the frequency and intensity of kernel
colonization showed significant aggregated patterns (Figure 1). Mycotoxin determination
demonstrated up to six mycotoxins in each sub-sample: deoxynivalenol (DON),3-acetyl
deoxynivalenol (3AcDON), enniatin B (ENN B), moniliformin (MON), nivalenol (NIV),
and zearalenone (ZON) (Table 1). Kernel contamination with DON was largely random,
although a tendency to aggregated patterns was observed (Figure 1).
In 2008, the frequency of Fusarium-infected kernels of wheat, cv. Tommi, varied
from 11.8 % to 55.9 %, depending on nitrogen fertilization and leaf application of
fungicides (Table 2). Increased nitrogen fertilization did not influence FHB prevalence.
The application of fungicides at GS 33 and GS 61 reduced the frequency of Fusarium–
infected kernels by 79 and 49%, respectively. Spatial distribution of F. tricinctum was
aggregated in three out of four sampling areas, whereas the pattern of F. graminearum,
the prevalent species, was random. As fungicide application reduced the overall
occurrence of Fusarium species, the average index of aggregation slightly increased.
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Conclusions
In 2007, the spatial distribution of F. graminearum, the frequency of infected kernels and
fungal biomass showed a statistically significant aggregated patterns, which can be
explained by aggregations of the residues of previous crops, whereas deoxynivalenol
contamination of kernels was largely random. Knowledge on Fusarium infection does
not permit the prediction of mycotoxin contamination of kernels. Therefore, the
heterogeneity of Fusarium infection of kernels and the different distribution patterns
demand a high number of samples per unit of area for representative sampling.
Figure 1: Spatial distribution of frequency (A), and intensity (B) of kernel colonization by F.
graminearum, and deoxynivalenol contamination (C) of wheat (cv. Kris) within 1 m2, in 2007.
Mycotoxin contamination
MON NIV DON 3AcDON ZON ENN B
Fungicides commonly applied for the control of leaf diseases may contribute to
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reduce FHB incidence and mycotoxin contamination of wheat ears as indicated by the
results in2008. Nevertheless, there may be interactions with nitrogen supply and cultivars
regarding FHB incidence and mycotoxin contamination. At high infection risk – due to
favourable weather conditions and the use of susceptible cultivars – fungicide
applications for the control of ear infections are irreplaceable in high-input cropping.
Small-scale heterogeneity in the incidence of Fusarium-infected wheat kernels and
associated mycotoxins need a high number of samples per area and an adapted
monitoring strategy within fields for the assessment of the disease situation.
Table 2: Effect of nitrogen fertilization and fungicide treatments on the frequency and spatial distribution
of kernel infection of wheat by Fusarium species (cv. Tommi) in 2008.
Acknowledgements
The authors gratefully acknowledge financial support from the Ministry of Innovation,
Science, Research and Technology of the federal state of North Rhine-Westphalia.
References
Bai, G., Shaner, G. (1994): Scab of wheat: Prospects for control. Plant Disease, 78, 760-766.
Desjardins, A.E. (2006): Fusarium Mycotoxins: Chemistry, Genetics, and Biology. APS Press.
260 pp.
Herebian, D.; Zühlke, S.; Lamshöft, M.; Spiteller, M. (2009): Multi-mycotoxin analysis in
complex biological matrices using LC-ESI/MS: Experimental study using triple stage
quadrupole and linear ion trap-Orbitrap. Journal of Separation Science, 32, 939-948.
McMullen, M., Jones, R., Gallenbert, D. (1997): Scab of wheat and barley: A re-emerging
disease of devastating impact. Plant Disease, 81, 1340-1348.
Moss, M.O. (1996): Mycotoxins. Mycology Research, 100, 513-523.
Parry, D.W., Jenkinson, P., McLeod, L. (1995): Fusarium ear blight (scab) in small grain cereals
– A review. Plant Pathology, 44, 207-238.
Perry, J.N. (1995): Spatial analysis by distance indices. Journal of Animal Ecology, 64, 303-314
Schlang, N. (2009): Auftreten der partiellen Taubährigkeit in Weizenbeständen – räumliche
Verteilung der Fusarium-Arten und assoziierter Mykotoxine, Dissertation, University of
Bonn.
Sweeney, M.J., Dobson, A.D.W., (1998):Review: mycotoxin production by Apergillus, Fusarium
and Penicillium species, International, Journal of Food Microbiology, 43, 141-158.
Waalwijk, C., van der Heide, R., de Vries, I., van der Lee, T., Schoen, C., Corainville, G.C.,
Häuser-Hahn, I., Kastelein, P., Köhl, J., Lonnet, P., Demarquet, T., Kema, G.H.J. (2004):
Quantitative detection of Fusarium species in wheat using TaqMan. European
Journal of Plant Pathology, 110, 481-494.
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67
The Role of Innovative Formulation Technology
in the Reduction of Mycotoxin Levels on Wheat
Abstract
Osiris®, a new highly active fungicide formulation containing epoxiconazole and metconazole,
was specifically designed to combat mycotoxin-producing Fusarium ear diseases. Both active
ingredients are completely dissolved in an innovative solvent system which allows excellent
fungicidal activity. Once an Osiris-containing spray droplet touches the waxy surface of the plant
tissue, it immediately spreads widely over the surface. The extremely low surface tension results
in excellent distribution, especially inside spikelets or glumes of cereal ears. In comparison to
conventional formulations, the ear coverage and the total amount of product on the ear is
significantly improved. Besides these effects on the surface, Osiris also exhibits high uptake of
the active ingredients which results in an increased curative activity. Established Fusarium
infections are therefore controlled more effectively.
Osiris clearly demonstrated superior Fusarium activity across a large number of trials
compared with the performance of the solo active ingredients as in Opus® or Caramba®. The
quantity of mycotoxins (i.e. Deoxynivalenol) in harvested grain was significantly reduced.
Introduction
Fusarium head blight, caused by a complex of various Fusarium species (Parry et al.,
1995), is seen as a continual threat to cereal crops across Europe. The disease is of major
relevance as most Fusarium species are able to produce mycotoxins (Botallico and
Perrone, 2002). Mycotoxin contamination of grain is a serious threat to human and
animal health. Therefore, measures have to be taken to reduce their quantities in
harvested grain.
Amongst other measures, such as planting resistant varieties (Sip, 2010) and
avoiding maize as a previous crop (Bai and Shaner, 1994) and minimum tillage, the use
of fungicides can help to reduce Fusarium head blight infestations on the ear. As a result,
the levels of mycotoxin contamination of cereal grains can be significantly reduced (e.g.
Mesterhazy et al., 2003; Paul et al., 2008).
However, even if the most effective active ingredient is used, the success of a
chemical treatment depends on multiple factors such as the exact application timing close
to infection (Jones, 2000; Pirgozliev et al., 2008), homogenous spray deposits on the ears
(Parkin et al., 2006), rapid uptake and transport to the target organism and duration in
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activity. Especially in the presence of existing fungal structures, rapid movement of the
active ingredient to the fungus is essential. As the innovative formulation of Osiris
greatly supports the availability of its solo active ingredients to target the organism,
performance is greatly optimised.
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label dose (UK label registrations). Separate tank mixes of each product were prepared
and a soluble dye added to the mix before being applied to the wheat ears. The sprayed
ears were then photographed with a conventional digital camera and assessed by visual
inspection. Percentage coverage of wheat ears was obtained by digital analysis of the
images.
Ref A (epoxiconazole)
Ref B (metconazole)
Ref A + Ref B
1s 10s 60s
Figure 1. Spreading of a spray droplet on a wheat leaf 1, 10 and 60 seconds after application.
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Similar behaviour was seen on wheat ears. Due to the rapid spreading of the Osiris
droplets, some of the spray solution reached the inner glumes. We postulate that this
effect explains the outstanding curative activity of Osiris, as hyphal networks which
usually develop mainly in the inner glumes (Kang and Buchenauer, 2000) are targeted
more effectively.
Water
Ref A
Ref A
+
Ref B
Figure 2: Adherence of single spray droplets on a wheat leaf (selected frames out of high speed videos).
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adherence on wheat leaves can be transferred to the ear thus more of the product adheres
to the ear as a result of the improved formulation.
Ref C Ref E
Figure 3: Spray deposits of different fungicide spray liquids, visualised using a coloured tracer dye and
quantified by digital image analysis.
100
75
72
57 52
50 51
45
25
33
Figure 4: Deoxynivalenol (DON) content relative to untreated (average of 4 trials France 2008;
comparison of fungicides at registered dose rates; DON content in untreated on average 24.9 mg/kg (0.4-49
mg/kg).
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about the outstanding curative activity of Osiris. At the later time points of application,
infections were advanced. By penetrating the inner glumes, Osiris was able to control
them more effectively and, as a result, give superior activity.
Table 2 shows the consistency of this performance in a larger trial series across three
years and several countries. Visual efficacy assessments and especially the analysed
DON contents confirm the superior activity of Osiris in comparison to the reference
fungicides. In addition, that trial series shows the high dose rate flexibility of Osiris.
Table 2: Efficacy of Osiris against Fusarium head blight and impact on Deoxynivalenol (DON) content.
Summary of 23 field trials 2007-2009 from France, Germany and Hungary. Single application at crop
growth stage BBCH 61-69. Disease assessment as severity of ear attack at crop growth stage BBCH 75-85.
DON content in untreated on average 19.3 mg/kg (0.2- 65 mg/kg).
DON content
Rate Fusarium attack Efficacy
Product [relative to
[l ha-1] [%] [%]
untreated]
Untreated 52 100
Osiris 2.0 19 63 33
Osiris 3.0 16 69 27
Ref D 1.0 23 56 46
Ref E 1.0 16 69 31
Acknowledgement
The authors would like to thank Rosie Bryson, BASF plc, for organizing the trialwork
with Silsoe and her valuable contribution for drafting this publication.
References
Bai, G., Shaner, G. (1994): Scab of wheat - prospects for control. Plant Disease, 78 (8), 760-766.
Botallico, A., Perrone, G. (2002): Toxigenic Fusarium species and mycotoxins associated with
head blight in small-grain cereals in Europe. Eur. J. Plant Pathol., 108, 611-624.
Jones, R. (2000): Assessments of Fusarium head blight of wheat and barley in response to
fungicide treatment. Plant Disease, 84 (9), 1021-1030.
Kang, Z., Buchenauer, H. (2000): Cytology and ultrastructure of the infection of wheat spikes by
Fusarium culmorum. Mycological research, 104 (9), 1083-1093.
Mesterhazy, A., Bartok, T., Lamper, C. (2003): Influence of wheat cultivar, species of Fusarium,
and isolate aggressiveness on the efficacy of fungicides for control of Fusarium head
blight. Plant Disease, 87 (9), 1107-1115.
Parkin, C.S., Miller, P.C.H., Magan, N., Aldred, D., Gill, J., Orson, J.H. (2006): The deposition
of fungicides on ears to control Fusarium ear blight and the mycotoxin contamination of
grain. Aspects of Applied Biology.
Parry, D.W., Jenkinson, P., McLeod, L. (1995): Fusarium ear blight (scab) in small grain cereals
– a review. Plant Pathol., 44, 207-238.
Paul, P., Lipps, P., Hershman, D., McMullen, M., Draper, M., Madden, L. (2008): Efficacy of
triazole-based fungicides for Fusarium head blight and deoxynivalenol control in wheat: A
multivariate meta-analysis. Phytopathology, 98 (9), 999-1011.
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Pirgozliev, S., Ray, R., Edwards, S.,Hare, M., Jenkinson, P. (2008): Effect of timing of fungicide
application on the development of Fusarium head blight and the accumulation of
deoxynivalenol in winter wheat grain. Cereal Research Communications, 36 (2), 289-299.
Plattner, R. D. (1999): HPLC/MS analysis of Fusarium mycotoxins, fumonisins and
deoxynivalenol. Natural Toxins, 7, 365–370.
Sip, V., Chrpova, J., Veskrna, O., Bobkova, L. (2010): The impact of cultivar resistance and
fungicide treatment on mycotoxin content in grain and yield losses caused by Fusarium
head blight in wheat. Czech J. Genet. Plant Breed., 46, 21-26.
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68
The CYP51C Gene, a Novel and Useful
Phylogenetic Marker to Distinguish Different
Fusarium Species on Cereals
Abstract
Early diagnosis and control of different Fusarium species are essential for successful
management of plant disease and subsequent prevention of toxins entering the food chain. These
issues can be addressed using phylogenetic analyses and other molecular techniques, including
the design of species-specific primers and corresponding PCR assays. This study examined if the
Fusarium-specific CYP51C gene can be used to establish evolutionary relationships between
Fusarium species and enable species-specific detection. The resolving power of the CYP51C
gene was studied for 46 Fusarium isolates representing 18 different species. The resulting
phylogeny analysis showed clear and well-structured separation of the isolates tested. The
interspecific divergence was used to design species-specific primers and the corresponding PCR
assays differentiated F. asiaticum/F. vorosii, F. avenaceum/F. tricinctum, F. cerealis, F. equiseti
and F. poae from other members of the FHB complex.
Introduction
Fusarium head blight (FHB) is a devastating disease of cereals which can cause huge
losses in epidemic years. A complex of up to 17 Fusarium species is associated with this
disease in different regions of the world, influenced by different agronomic practices and
climatic conditions (Parry et al., 1995). Infection with Fusarium species reduces the
yield and quality of seeds and can result in the accumulation of various mycotoxins,
representing a serious health threat to animal and human consumers (Gutleb et al., 2002).
Sterol demethylation inhibitor (DMI) fungicides are the largest and most important
group of antifungal agents and are widely used in both agriculture and medicine. The
CYP51 gene encodes for sterol 14 α-demethylase, a key enzyme in the pathway leading
to ergosterol, phytosterol and cholesterol biosynthesis in fungi, plants and mammals,
respectively (Yoshida, 1993). Interestingly, a number of important human and plant
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pathogenic fungi carry multiple CYP51 genes. Most filamentous ascomycetes (including
Aspergillus species, Magnaporthe oryzae, Rhynchosporium secalis and Pyrenophora
tritici-repentis) carry two copies, CYP51A and CYP51B (Hawkins et al., 2009). However
a third copy, CYP51C, has been identified in Fusarium spp. and appears to be unique to
this genus (Deng, 2006).
This study investigates the potential of the CYP51C gene to be used as a
phylogenetic marker for reliable identification of Fusarium species and for its suitability
as a species-specific target in PCR.
Results
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targets and no false positive reactions were observed for any non-target Fusarium species
(Figure 2).
Figure 1: Bayesian reconstruction of the phylogeny of Fusarium isolates based on the nucleotide
sequence of the CYP51C gene.
Figure 2: PCR assays specific to different Fusarium spp.: (A) F. avenaceum/F. tricinctum; (B) F.
asiaticum/F. vorosii; (C) F. cerealis; (D) F. equiseti and (E) F. poae. Lanes: (M) DNA ladder market; 1-
negative control (no DNA); 2- F. asiaticum CH011b; 3- F. avenaceum 492; 4- F. cerealis 76F2; 5- F.
culmorum 8984; 6- F. equiseti 378; 7- F. graminearum 16D1; 8- F. langsethiae 77F2b; 9- F.
mesoamericanum NRRL25797; 10- F. poae 8452; 11- F. proliferatum CHX015; 12- F.
pseudograminearum NRRL28065; 13- F. sporotrichioides 156; 14- F. subglutinans ITEM2143; 15- F.
tricinctum 160; 16- F. venenatum AG48m; 17- F. verticillioides 8002; 18- F. vorosii 67C1.
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Discussion
The main objective of our study was to establish if the CYP51 gene is a reliable marker
for phylogenetic studies on different Fusarium species. The phylogenetic analysis based
on 46 CYP51C sequences of 18 Fusarium species resulted in the differentiation of all
different species tested.
Among diagnostic protocols for identification and detection of different Fusarium
species, PCR assays are considered to be one of the most rapid and reliable methods. The
variability of the CYP51C gene was successfully explored for PCR detection of F.
asiaticum/F. vorosii, F. avenaceum/F. tricinctum, F. cerealis, F. equiseti and F. poae.
The PCR assays developed in this study will enable monitoring of species without the
need of culturing and, therefore, facilitate studies on the spatial contribution. The assays
can be further developed into real-time PCR format enabling high-throughput
quantitative detection on wheat samples of different Fusarium spp. (Nicolaisen et al.,
2009).
Acknowledgements
D. Fernández-Ortuño was supported by a MICINN postdoctoral fellowship (EX2008-
0323) from the Secretaría General de Estado de Universidades e Investigación del
Ministerio de Ciencia e Innovación (Spain). Rothamsted Research receives grant-aided
support from the Biotechnology and Biological Sciences Research Council (BBSRC) of
the UK.
References
Deng, J. (2006): PhD Thesis: Structural, Functional and Evolutionary Analyses of the Rice Blast
Fungal Genome. North Carolina State University, North Carolina, United States.
Gutleb, A.C., Morrison, E., Mur, A.J. (2002): Cytotoxicity assays for mycotoxins produced by
Fusarium strains: a review. Environmental Toxicology and Pharmacology, 11, 309-320.
Hawkins, N., Cools, H., Shaw, M., Sierotzki, H., Fraaije, B.A. (2009): Recent evolution of
Rhynchosporium secalis populations in response to selection by fungicides. In: Meeting
Abstracts, XX Molecular Biology of Plant Pathogens, p. 14, Oxford, United Kingdom.
Huelsenbeck, J.P., Ronquist, F. (2001): MrBayes: Bayesian inference of phylogeny.
Bioinformatics, 17, 754-755.
Milne, I., Lindner, D., Bayer, M., Husmeier, D., McGuire, G., Marshall, D.F., Wright, F. (2009):
TOPALi v2: a rich graphical interface for evolutionary analyses of multiple alignments on
HPC clusters and multi-core desktops. Bioinformatics, 25, 126-127.
Nicolaisen, M., Supronienė, S., Nielsen, L.K., Lazzaro, I., Spliid, N.H., Justesen, A.F. (2009):
Real-time PCR for quantification of eleven individual Fusarium species in cereals. Journal
of Microbiological Methods, 76, 234-240.
Parry, D.W., Jenkinson, P., McLeod, L. (1995): Fusarium ear blight (scab) in small grain cereals-
A review. Plant Pathology, 44, 207-238.
Thompson, J., Higgins, D., Gibson, T. (1994): CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position specific
gap penalties and weight matrix choice. Nucleid Acids Research, 22, 4673-4680.
Yoshida, Y. (1993): Sterol biosynthesis. Cytochrome P-450, 2nd edition, pp. 93-101. Eds. Omura,
T. et al., Kodansda, Tokyo, Japan.
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69
New Insights in the Mode of Action of
Thiophanate-Methyl
Abstract
Thiophanate-methyl (TM) is a benzimidazole fungicide and has been admitted for control of
Fusarium head blight in wheat and triticale since 2009. The primary fungicidal effect of TM is
caused by the transformation product methyl-benzimidazole-2yl-carbamate, which is a β-tubulin
inhibitor disturbing cell division. In field trials, TM reduced the deoxynivalenol contents in wheat
kernels more efficiently than head blight symptom development caused by F. graminearum. TM
reduced mycotoxin biosynthesis of Fusarium spp. by up to 95% in vitro, but fungal growth
hardly decreased. Respiration of Fusarium spp. exposed to TM was also reduced by up to 80%,
depending on the TM concentration used, without a corresponding inhibition of growth.
Moreover, TM inhibited the activity of the cytochrome c oxidase in isolated mitochondria of
Fusarium spp. by about 40-60%, depending on the investigated Fusarium species and the
incubation time.
Introduction
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While the growth rate of different species of Fusarium was inhibited by less than 10%
the mycotoxin production decreased up to 80-95% (Hirschfeld et al., 2009).
The aim of the work was to investigate whether respiration or distinct components
of the respiration chain are inhibited by TM. The respiration of whole cell cultures of
Fusarium spp. as well as the activity of the cytochrome c oxidase (COX) in isolated
mitochondria of Fusarium spp. exposed to TM and MBC was measured.
Respiration measurement
Isolates of F. culmorum (Isolate-Code: 62188) and F. verticillioides (Isolate-Code: 70168)
were activated from permanent culture in soil by dispersing the soil on SNA (Spezieller
Nährstoffarmer Agar; Nirenberg, 1976). After 3-5 days of incubation in the laboratory at
room temperature and the natural change day and night regime, pieces of overgrown agar
were transferred on new SNA-plates that incubated under the same conditions for at least
7-10 days. Afterwards pieces of overgrown SNA were added to liquid Bilay`s medium
(Booth, 1971) and shaken on a horizontal shaker at 70 rpm at natural day and night
conditions and a temperature of about 25°C. After 3-5 days of incubation, a conidial
suspension was prepared and adjusted to 105 conidia/ml. One ml of this suspension was
transferred to Bilay`s medium containing 1, 2.5, 5 or 10mg/l TM or 0.1, 0.25, 0.5 and 1
mg/l MBC and incubated for another 5 days. Cultures lacking fungicides served as
controls. The respiration of Fusarium spp. was measured within these five days by use of
the sensomat scientific system (Aqualytic, Langen, Germany). At the end of the log-
phase the growth is limited either by supply of oxygen in the gas phase or nutrients in the
liquid medium. Therefore, we regard the turning point of the respiration graph of the
untreated control (UC) as the end of the logarithmic period of growth, which we use for
the evaluation of differences in the respiration between the investigated variants.
Furthermore, the ergosterol content in the Bilay`s medium was analysed by HPLC
(Hewlett Packard, Agilent Series 1100, Waldbronn, Germany) to check the biomass
increase of Fusarium spp.. Four replicates per variant were tested.
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Results
40
UC
35 1mg TM/l
2,5mg TM/l
30 5mg TM/l
10mg TM/l
25
20
CO2 in mg/l+h
15
10
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Figure 1: Amount of released carbon dioxide per hour of F. verticillioides in liquid Bilay`s medium at
certain concentrations of thiophanate-methyl (TM) (the vertical line marks the end of the logarithmic
growth phase)
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reduced the activity of the enzyme by about 23-33% with an increasing inhibition at
longer incubation times.
20,00
18,00
Ergosterole-content in µg/ml
Figure 2: Ergosterol content in the liquid Bilay`s medium of F. verticillioides at certain concentrations of
thiophanate-methyl (TM) after 5 days of cultivation.
Table 1: Cytochrome c oxidase activity of F. verticillioides exposed to TM (10µg/ml) and MBC (1µg/ml)
at certain incubation times.
Table 2: Cytochrome c oxidase activity of F. culmorum exposed to TM (10µg/ml) and MBC (1µg/ml) at
certain incubation times.
Discussion
Data presented here may suggest that there may be an additional mode of action of TM in
Fusarium spp., directly affecting respiration by inhibiting the activity of the cytochrome
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c oxidase of the fungi which could possibly cause a lack in energy supply, thus reducing
the production of energy rich secondary metabolites such as mycotoxins.
These findings are also supported by investigations of Kataria and Grover (1976) in
which the oxygen-uptake of whole cell cultures of Rhizoctonia solani was inhibited by
about 56% and the cytochrome c oxidase activity decreased by about 43% in the
presence of 70 µM TM compared with the untreated control.
Currently, investigations into the energy status of Fusarium spp. exposed to TM and
MBC are being made to analyze whether the ATP/ADP-ratio is also affected, which
would add further support for the hypothesis that TM has an additional mode of action in
Fusarium spp..
References
Booth, C. (1971): The Genus Fusarium. Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, Surrey,
England.
Buschhaus, H., Ellner, F. (2008): Impact of DONstopp (Thiophanate-methyl 700 WDG) on
mycotoxin production in vitro and in vivo. In: H.W. Dehne, H.B. Deising, U. Gisi, K.H.
Kuck, P.E. Russell, H. Lyr (Eds). Modern Fungicides and Antifungal Compounds V, DPG-
Verlag, Braunschweig, Germany, 323-329.
Hirschfeld, T., Ellner, F., Buschhaus, H., Goßmann, M., Büttner, C. (2009): Einfluss von
Thiophanat-Methyl und Methyl-Benzimidazol-2yl-Carbamat auf mykotoxinbildende
Fusarium spp., ALVA-Mitteilungen, 7, 31-35.
Kataria, H.R., Grover, R.K. (1976): Effect of Benomyl and Thiophanate-methyl on metabolic-
activities of Rhizoctonia solani Kuhn, Annales de Microbiologie, A127/2, 297-306.
Nirenberg, H. (1976): Untersuchungen über die morphologische und biologische Differenzierung
in der Fusarium-Sektion Liseola. Mitt. Biol. Bundesanst. Land- u. Forstwirtsch., Berlin-
Dahlem, 169, 1–117.
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70
Fitness of Thiophanate-Methyl-Resistant
Isolates of Botrytis cinerea
Abstract
Gray mold caused by Botrytis cinerea is one of the economically most important diseases of
grapevine. Additionally, the pathogen has a high tendency to become resistant to systemic
fungicides. Development and evolution of fungicide resistance would be lessened if the resistant
subpopulation had lower parasitic or saprophytic fitness. The fitness parameters mycelium
growth and spore production of isolates sensitive and resistant to thiophanate-methyl were
compared at two environmental conditions. At favourable development conditions the fitness
parameters did not differ. In contrast, at unfavourable development conditions mycelium growth
and spore production of resistant isolates were significantly lower than those of sensitive isolates.
Introduction
Botrytis cinerea Pers.: Fr. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) causes
the gray mold disease of a wide variety of fruits, vegetables and ornamentals. B. cinerea
is an economically devastating pathogen in grapevine and it has a high tendency to
become resistant to repeatedly applied systemic fungicides. Only a few years after
introduction of benzimidazoles like carbendazim (MBC) and the related active
compound thiophanate-methyl, B. cinerea strains resistant to benzimidazoles appeared
frequently in northern Europe (Smith, 1988). The development of fungicide resistance in
a population is largely dependent on the fitness of the resistant portion of the population.
Usually the costs of resistance are explored by testing for a variety of fitness parameters
including the mycelium growth, spore germination, spore production in vitro and
pathogenicity (Pringle and Taylor, 2002). Because fitness costs, associated with
fungicide resistance, might be more costly under conditions that are suboptimal for a
fungal species (Brown et al., 2006), fitness parameters of MBC-sensitive and MBC-
resistant isolates of B. cinerea were compared at favourable and unfavourable
development conditions.
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Ten MBC-sensitive and ten MBC-resistant isolates were selected from a monitoring,
conducted in five German vineyards in 2007. For the characterization of resistant isolates
an allele specific PCR according to Luck and Gillings (1995) was performed using
primers BCM (5’-GGTTGAGAACTCTGACGC-3’) and BCR (5’-TTAGTCAACTCTG
GAACGG-3’). Primers were used to detect the E198A-mutation leading to a substitution
of glutamic acid by alanine at position 198 of the beta-tubulin gene, which causes a high
resistance towards benzimidazoles.
The fitness parameters mycelium growth and spore production of the isolates were
tested in vitro. Experiments were conducted at different environmental conditions: (1)
favourable at 21°C on the rich nutrition medium (PDA) and (2) unfavourable at 4°C on a
medium with low nutrition availability; CZA 10 % and autoclaved leaf discs cut from
two months old grapevine plants cv. Müller Thurgau. Radial mycelial growth was
measured after an incubation time of three days at favourable and 10 days at
unfavourable conditions. Spore production was measured after incubating isolates for 14
days at 21 °C with a 14 h photoperiod (Bardas et al., 2008). Spore production was
measured on autoclaved leaf discs inoculated by soaking them in conidial suspensions
(105 spores per ml) as described by Sosa Alvarez et al. (1995). Afterwards leaf discs
were incubated for 14 days at 4 °C with a 14 h photoperiod.
Results
Figure 1: Amplification of a beta-tubulin gene fragment of Botrytis cinerea using primers BC-M and
BC-R to detect E198A-mutation. A: Marker, B-K: MBC-resistant isolates, L: reference isolate with
E198A-mutation, M: reference wild-type isolate, N: water template, A&O: 100 bp ladders.
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Figure 2: Mycelium growth of MBC-sensitive and MBC-resistant isolates of Botrytis cinerea at different
development conditions (n = 10, error bars show standard deviation).
Figure 3: Spore production of MBC-resistant and MBC-sensitive isolates of Botrytis cinerea at different
development conditions (n = 10, error bars show standard deviation).
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Discussion
The resistance of tested B. cinerea isolates was caused by the E198A-mutation on the
beta-tubulin gene. According to Ma and Michailides (2005) this is the most common
mutation leading to resistance towards benzimidazoles found in field-isolates of B.
cinerea.
The fitness experiments have shown that no significant differences exist between the
two sensitivity groups under favourable development conditions. Similar observations
were made by Ziogas and Girgis (1993), who detected no significant differences in spore
production, germination and germ-tube elongation of UV-mutants of B. cinerea resistant
to benzimidazoles compared to the wild type strain at 22°C grown on PDA. In contrast, a
difference in fitness between the sensitivity groups could only be detected under
unfavourable development conditions for the fungus. Under these conditions mycelium
growth and spore production of MBC-resistant isolates were significantly lower
compared to that of MBC-sensitive isolates. Brown et al. (2006) mentioned that the cost
of resistance to strobilurin fungicides varies with environmental conditions; being more
costly in situations which are sub-optimal for resistant strains of Blumeria graminis and
Mycosphaerella graminicola.
According to Akagi et al. (1995) the E198A-mutation in the beta-tubulin gene alters
the binding site to carbendazim by change of an ethyl sized pocket of the protein. As
described for benomyl resistant strains of Schizosaccharomyces pombe, pleiotropic
effects of other mutations on the tubulin gene leading to an altered microtubule
architecture result in a reduced development at low temperature (Roy and Fantes, 1982).
Possibly, the E198A-mutation on the beta-tubulin gene is also associated with reduced
development at sub-optimal conditions due to temperature and nutrition.
Without selection pressure of benzimidazole fungicides, fitness costs associated with
benzimidazole-resistance might reduce the fraction of resistant isolates within the
primary inoculum when the fungus is confronted with reduced nutrition availability
and/or low temperatures. This might explain the observed decrease of the fraction of
MBC-resistant isolates in Germany since the use of benzimidazoles was discontinued in
viticulture thirty years ago. According to Bardas et al. (2008) the fitness of resistant
strains has important implications for resistance management. The existence of a fitness
cost for the resistant strains could lead to a suggestion for a strategy to delay the
evolution of fungicide resistance by means of alternation or mixture with chemicals of
different mode of action.
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References
Akagi, T., Mitani, S. Ito, K.; Shigehara, I., Komyoji, T., Matsuo, N. (1995): Structure-activity
relationships of pyridylcarbamates active against both benzimidazole‐sensitive
and‐resistant isolates of Botrytis cinerea. Pesticide Science, 44, 39-48.
Bardas, G., Myresiotis, C.K., Karaoglanidis, G.S. (2008): Stability and fitness of
anilinopyrimidine-resistant strains of Botrytis cinerea. Phytopathology, 98, 443-450.
Brown J.K.M., Atkinson, M.A., Ridout, C.J., Sacristan, S., Tellier, A., Wyand, R.A. (2006):
Natural selection in plant-pathogen interactions: From models to laboratory to field.
Conference of the Spanish Society for Phytopathology, 13, 15.
Luck, J.E., Gillings, M.R. (1995): Rapid identification of benomyl resistant strains of Botrytis
cinerea using the polymerase chain reaction. Mycological Research, 99, 1483-1488.
Ma, Z., Michailides, T.J. (2005): Advances in understanding molecular mechanisms of fungicide
resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop
Protection, 24, 853-863.
Pringle, A., Taylor, J.W. (2002): The fitness of filamentous fungi. Trends in Microbiology, 10,
474-481.
Roy, D., Fantes, P.A. (1982): Benomyl resistant mutants of Schizosaccharomyces pombe cold-
sensitive for mitosis. Current Genetics, 6, 195-201.
Smith, C.M. (1988): History of benzimidazole use and resistance. Fungicide Resistance in North
America, 23-24. Ed. Delp C.J., APS Press, American Phytopathological Society, St. Paul,
Minnesota (USA).
Sosa-Alvarez, M., Madden, L.V., Ellis, M.A. (1995): Effects of temperature and wetness duration
on sporulation of Botrytis cinerea on strawberry leaf residues. Plant Disease, 79, 609-615.
Ziogas, B.N. (1993): Cross-resistance relationships between benzimidazole fungicides and
diethofencarb in Botrytis cinerea and their genetical basis in Ustilago maydis. Pest
Management Science, 39, 199-205.
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71
Fungicide Sensitivity, Fitness and Mycotoxin
Production of Penicillium expansum Field
Isolates from Apple
Abstract
The objective of this study was to determine the fungicide sensitivity of 236 Penicillium
expansum isolates obtained from apple fruit and to measure fitness components and patulin
production in fungicide-resistant isolates. Fungitoxicity tests showed the presence of several
resistance-phenotype frequencies including resistance to anilinopyrimidines (33%), DMIs (9%)
and benzimidazoles (7.5%). Smaller portions of the population were simultaneously resistant to
tebuconazole, fludioxonil and/or iprodione. Study of fitness parameters showed that the
resistance to tebuconazole and fludioxonil had a significant adverse effect on fitness parameters,
while resistance to anilinopyrimidines and benzimidazoles was not associated to fitness costs.
Additionally, patulin production was positively correlated with the reduced sensitivity to
carbendazim, negatively correlated with the reduced sensitivity to tebuconazole and fludioxonil,
while a correlation between the sensitivity to cyprodinil and to iprodione was not apparent.
Introduction
Penicillium expansum, the causal agent of “blue mold” of apple, is the most common
producer of patulin, an important mycotoxin. Chemical control is the main disease
management method, including pre- or postharvest applications of fungicides belonging
to several classes. Investigations related to the problem of fungicide resistance
development in P. expansum are rather limited worldwide and by far restricted to the
groups of benzimidazole and DMI fungicides. The concern over the use of fungicides,
and in particular the possible effect on mycotoxigenic fungal species and mycotoxin
production, was highlighted in a recent report by the European Commission Scientific
Committee in Plants (Hardy et al., 1999). Like many other organisms, mycotoxigenic
fungi may become resistant to fungicides. In this case, an important consideration is the
influence of fungicide resistance on the mycotoxin production (Markoglou et al., 2008b,
2009). The objectives of this study were to determine the sensitivity of the fungal
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Fungal isolates
Two hundred and thirty six P. expansum isolates, collected from decayed apple fruits
stored in packinghouses of N. Greece, were used in the study.
Fitness parameters
Mycelial growth rate of the 40 isolates was measured after 7 days of incubation at 20oC,
on PDA. Aggressiveness of the isolates was measured on 20 wound-inoculated fruits per
isolate, after 7 days of incubation at 20oC lesion by measuring lesion diameter. The
experiments were repeated two times.
Patulin measurement
The mycotoxigenic ability of the isolates was estimated in vitro, on Yeast Extract
Sucrose agar medium (YES), and on artificially inoculated apple fruit. The determination
of patulin produced by each isolates was performed using thin layer chromatography
(TLC) and high performance liquid chromatography (HPLC) and confirmed with
appropriate patulin standards. The chromatographic conditions was performed as
described previously (Markoglou et al., 2008a)
Results
Fungicide sensitivity
Resistance to anilinopyrimidines was widespread accounting for 33% of the population,
while 9 and 7.5% of the population was resistant to tebuconazole and carbendazim,
respectively. A small portion of the population (3.5%) was simultaneously resistant to
tebuconazole, fludioxonil and iprodione (Figure 1). Precise measurements of EC50 values
in 40 isolates showed that for all the 5 fungicides tested the distribution of the EC50
values was unimodal, except of that to carbendazim that was clearly bimodal (data not
shown).
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Table 1: Pearson correlation coefficients among sensitivities of Penicillium expansum isolates to several
fungicides, fitness parameters and patulin production on artificially inoculated apples.
Fitness parameters
Fungicide Mycelial Growth Aggressiveness Patulin production
1
tebuconazole -0.87* -0.72* -0.48*
carbendazim 0.29 0.28 0.54*
cyprodinil 0.10 0.31 0.36
fludioxonil -0.65* -0.40* -0.52*
iprodione -0.44* -0.38* -0.04
1
values followed by * are significant at P=0.01
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Conclusions
The above mentioned data indicate, for the first time, the potential risk of increased
mycotoxin contamination of pome fruits by the predominance of highly patulin producer
isolates of P. expansum resistant to benzimidazole and/or anilinopyrimidine fungicides.
The field application of anilinopyrimidines and benzimidazoles requires careful
implementation of appropriate anti-resistance strategies to preserve their effectiveness
and the safety and quality of pome fruit.
References
Markoglou, A.N., Vitoratos, A.G., Doukas, E.G., Ziogas, B.N. (2009): Phytopathogenic and
mycotoxigenic characterization of laboratory mutant strains of Fusarium verticillioides
resistant to demethylation inhibiting fungicides. DPG-BCPC 3rd International
Symposium-Plant Protection and Plant Health in Europe, Berlin, Germany.
Markoglou, A.N., Vattis, K., Dimitriadis, K., Doukas, E.G., Ziogas, B.N. (2008a): Effect of
phenylpyrrole resistance mutations on mycotoxin production by Aspergillus carbonarius
and Penicillium expansum. 9th International Congress of Plant Pathology (ICPP 2008),
Torino, Italy.
Markoglou, A.N., Doukas, E.G., Ziogas, B.N. (2008b): Phenylpyrrole-resistance and aflatoxin
production in Aspergillus parasiticus Speare. Int. Journal of Food Microbiology, 127, 268-
275.
Hardy, A., Silva-Fernandes, A., Speijers, G., Hans, R., Delcour, M.P., Kuiper, H., Fuhr, F.,
Carere, A., Richard-Molard, D., Thomas, M. (1999): European Commission Health and
Consumer Protection Directorate-General SCP/RESI/063-Final, Brussels, Belgium.
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Index
Black Sigatoka 217
ABC transporters 96, 224 Blumeria graminis f. sp. tritici 81, 135, 385
Adaptation 237 - graminis f. sp. hordei 28, 135
Adaptation to climate 17 Boscalid 9, 49, 161, 180, 188, 273
Adjuvants 307, 317 Botrytis cinerea 41, 78, 111, 155, 187, 195,
Agricultural production 11, 12 273, 341, 423
Allicin 325, 331, 335 - elliptica 196
Allium sativum 326, 332, 335 - pseudocinerea 111
Alternaria alternata 35, 78, 179, 196 Botrytis spp. 68, 75, 259
Alternaria spp., 68 Breeding for resistance 27, 390
Ametoctradin 51, 55 Bremia lactucae 151
3-Amino-1,2,4-triazole 381 Broad acre cropping 259
Amisulbrom 51 Broad-spectrum fungicide 81
Anilinopyrimidines 429 Brown rot 263
Antifungal activity 347 Bursting of zoospores 57
Antifungal compounds 339, 355
Antimycin A 189
Antioxidants 328, 371 C-14-demethylase inhibitor 289
Anti-resistance strategies 78, 267 CAA fungicides (see Carboxyl acid amides)
Apoptosis 325, 331 CAA resistance 151
Apple 289, 429 Carbendazime 390, 423
Application technologies 317 Carboxamide fungicide 49, 63, 173, 179,
Arbuscular mycorrhizal fungi 361, 363 187, 273
Artemisia absinthium 363 Carboxyl acid amides (CAAs) 95, 103, 151,
Ascochyta 259 152, 267, 279
Asian soybean rust 211 Carpropamid 36
Association genetics 31 Cellular electrochemical potential 331
Azole fungicides 123, 135, 211, 217, 289 Cellulose synthase 3, 103, 151
Azole resistance 135 Cercospora beticola, 295
Azoxystrobin 37, 48, 187,189, 203, 261, - leaf spot 295
267, 392 Cercospora spp., 68
Cereals 81, 135, 255, 259, 389, 413
CesA3 gene 152
Bacillus subtilis 339 3-Chloroaniline 297
Bacterial blight 365 Chlorothalonil 261
Bakanae disease 36 Chlorpropham 297
Banana 217 Ciproconazole 392
Barley 18, 69, 129, 179, 199, 377 Climate change 17
Barley leaf blotch 129 Coding regions 41
Bean 379 Colletotricum spp. 68
Benomyl 36 Complex II 69, 159, 171, 187
Benthiavalicarb 103 Complex II inhibitors (SDHIs) 47, 49, 63,
Benzimidazole fungicide 6, 36, 218, 251, 75, 81, 171, 179, 195
417, 423, 429 Complex III 57, 187
Benzothiadiazole 371 Complex III inhibitors (QoIs) 38, 47, 48, 57,
Bioavailability 77, 307, 315 129, 143, 155, 187, 199, 204, 211, 251,
Biodiversity 11 263, 267, 285, 289, 295
Biofungicide 339 Corn 361
Biological activity 339 Corynespora cassiicola 196
Biological effectiveness 9 Сotton 355
Biomass 361, 363 Council directive 91/414 1
Bitertanol 218 Coverage 391
Bixafen 9, 49, 69, 81 161, 188 Crop protection markets 11
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Kasugamycin 35
3-keto reductase 111 Oculimacula spp. 69
Kresoxim-methyl 187, 392 Oilseed rape 18
Oomycete-active compounds 92
Oomycetes 55, 91, 103, 151
Land grabbing 12 Oomycetes fungicides 51, 279
Late blight 32 Organic agriculture 329
Lipopeptides 339 Organophosphorus fungicide IBP 35
Lipophilicity 307 Organosulfur compounds 326
Orysastrobin 37
Magnaporthe oryzae 35
Management of crop diseases 1 Partitioning behaviour 307
Mandipropamid 51, 93, 103, 152, 269 PAs (Phenylamides) 267
Maximum residue limit (MRL) 297 PCR analysis 43
MBI-D fungicides 35 Peaches 263
MDR transporters 289 Pears 273
Mefenoxam 95 Pelargonium odoratissimum 347
Melting point 307 Penconazole 289
Metalaxyl 95, 269 Penflufen 9, 49
Metconazole 211, 223, 233, 255, 405 Penicillium expansum 429
Methominostrobin 37 Penthiopyrad 9, 49, 161
Methyl benzimidazole carbamates 263 Phaeosphaeria nodorum 29
Millet 361 Phakopsora pachyrhizi 203, 211
Phase II protection enzymes 328
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