The Downy Mildews - Genetics, Molecular Biology and Control
A. Lebeda I P.T.N. Spencer-Phillips I B.M. Cooke
The Downy Mildews - Genetics,
Molecular Biology and Control
Reprinted from European Journal of Plant Pathology, Volume 122, No. 1, 2008
A. Lebeda
Palacký University in Olomouc
Czech Republic
ISBN 978-1-4020-8972-5
P.T.N. Spencer-Phillips
University of the West of
England, Bristol, UK
B.M. Cooke
University College Dublin
Ireland
e-ISBN 978-1-4020-8973-2
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Cover photo captions
1. Abaxial side of Lactuca serriola leaf with Bremia lactucae sporulation
2. Bremia lactucae conidiosporangia
3. Nitric oxide (NO) probe DAF-FM DA distributed within epidermal cell of susceptible Lactuca sativa (cv. Cobham Green)
penetrated by haustoria of B. lactucae
4. Immunolocalization of alpha-tubulin by FITC-conjugated secondary antibody showing rearrangement of microtubules in
epidermal cell of L. sativa (UCDM2) during plasma membrane invagination due to B. lactucae penetration
5. 2D-DIGE gel of the pea leaf proteome showing changes in abundance of proteins following infection by Peronospora viciae
(red = increased; green = decreased; yellow = no change)
Printed on acid-free paper
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European Journal of Plant Pathology
Volume 122 I Number 1 I September 2008
Special Issue: The Downy Mildews - Genetics, Molecular Biology and Control
Edited by: A. Lebeda P.T.N. Spencer-Philipps B.M. Cooke
I
I
Foreword 1
Population diversity and epidemiology
Evolution and taxonomy
Comparative epidemiology of zoosporic plant
pathogens
M.J. Jeger I M. Pautasso 111
Progress and challenges in systematics of downy
mildews and white blister rusts: new insights from
genes and morphology
H. Voglmayr 3
Genetic variation and molecular biology
Classical and molecular genetics of Bremia lactucae,
cause of lettuce downy mildew
R. Michelmore I J. Wong 19
Species hybrids in the genus Phytophthora with
emphasis on the alder pathogen Phytophthora alni:
a review
T. Érsek I Z.Á. Nagy 31
Proteomic analysis of a compatible interaction
between Pisum sativum (pea) and the downy mildew
pathogen Peronospora viciae
R.C. Amey I T. Schleicher I J. Slinn I M. Lewis I
H. Macdonald I S.J. Neill I P.T.N. Spencer-Phillips 41
Host-parasite interactions
Tapping into molecular conversation between
oomycete plant pathogens and their hosts
M. Tör 57
Diversity of defence mechanisms in plant–oomycete
interactions: a case study of Lactuca spp.
and Bremia lactucae
A. Lebeda I M. Sedlářová I M. Petřivalský I J. Prokopová 71
Natural history of Arabidopsis thaliana and oomycete
symbioses
E.B. Holub 91
Structure and variation in the wild-plant
pathosystem: Lactuca serriola–Bremia lactucae
A. Lebeda I I. Petrželová I Z. Maryška 127
Development of detection systems for the sporangia
of Peronospora destructor
R. Kennedy I A.J. Wakeham 147
Chemical and biological control
Fungicide modes of action and resistance in downy
mildews
U. Gisi I H. Sierotzki 157
Activity of carboxylic acid amide (CAA) fungicides
against Bremia lactucae
Y. Cohen I A. (E.) Rubin I D. Gotlieb 169
Beta-aminobutyric acid-induced resistance
in grapevine against downy mildew: involvement
of pterostilbene
A.R. Slaughter I M.M. Hamiduzzaman I K. Gindro I
J.-M. Neuhaus I B. Mauch-Mani 185
Effects of garlic (Allium sativum) juice containing
allicin on Phytophthora infestans and downy mildew
of cucumber caused by Pseudoperonospora cubensis
D. Portz I E. Koch I A.J. Slusarenko 197
Instructions for Authors for Eur J Plant Pathol are available at
http://www.springer.com/10658.
Eur J Plant Pathol (2008) 122:1
DOI 10.1007/s10658-008-9324-z
Foreword
Aleš Lebeda & Peter T. N. Spencer-Phillips &
B. M. Cooke
Received: 29 April 2008 / Accepted: 29 April 2008
# KNPV 2008
The downy mildews—genetics, molecular biology
and control
This book contains chapters presented by keynote
speakers at The Second International Downy Mildews
Symposium, held in July 2007 at Olomouc, Czech
Republic. The conference focused on the evolution,
taxonomy, biology, genetic variation, host resistance,
population diversity, epidemiology, chemical and
biological control of downy mildews and some
related plant-pathogenic oomycetes. Regular oral
A. Lebeda (*)
Faculty of Science, Department of Botany,
Palacký University in Olomouc,
Slechtitelu 11, 783 71 Olomouc-Holice,
Czech Republic
e-mail: ales.lebeda@upol.cz
P. T. N. Spencer-Phillips
School of Biosciences, University of the West of England,
Coldharbour Lane,
Bristol BS16 1QY, UK
e-mail: peter.spencer-phillips@uwe.ac
B. M. Cooke
School of Biology and Environmental Science,
College of Life Sciences, University College Dublin,
Belfield, Dublin 4, Ireland
e-mail: Mike.Cooke@ucd.ie
presentations were published in a separate volume
entitled: Advances in Downy Mildew Research, Vol.
3 (A. Lebeda and P.T.N. Spencer-Phillips, Eds.,
Palacký University in Olomouc, 2007, 278 pp., ISBN
80-86636-19-4).
Knowledge of downy mildew pathogens, and the
plant diseases they cause, has advanced substantially
since publication of the book: The Downy Mildews,
edited by D.M. Spencer (Academic Press, 1981).
Over the last 25 years, many aspects of downy
mildew biology have been investigated, and progress
in some areas has been summarized in three volumes
of the text: Advances in Downy Mildew Research
(2002, 2004 and 2007). However, expansion of
knowledge about downy mildews provided an opportunity to organize the Second International Symposium, and to publish this volume which contains 14
invited review and original research papers presented
by keynote contributors. The result is a book
comprehensively updating understanding across a
wide spectrum of the most important advances,
designed to provide a balanced overview of the
subject area.
We hope that it will be of use to students,
academics and researchers in plant pathology, mycology, crop protection, taxonomy, plant biology and
ecology, as well as to others involved in the
agricultural and horticultural advisory services and
industry.
DO09324; No of Pages
Eur J Plant Pathol (2008) 122:3–18
DOI 10.1007/s10658-008-9341-y
Progress and challenges in systematics of downy mildews
and white blister rusts: new insights from genes
and morphology
Hermann Voglmayr
Received: 31 August 2007 / Accepted: 29 May 2008
# KNPV 2008
Abstract Recent advances in the classification of
downy mildews and white blister rusts are presented
from ordinal to species level. Using molecular data
(mainly LSU of nuclear ribosomal DNA and ITS
rDNA data, but also cox2, beta-tubulin and NADH
genes), ordinal, family and generic circumscriptions
have been reconsidered and changed during the last
years; species circumscription and concepts are also
changing. These rearrangements also lead to a
reevaluation of the traditional morphological characters used for classification. The recent changes have
various implications for applied sciences (phytopathology, molecular biology) mainly at the species
level; besides name changes for some taxa, revised
species circumscriptions and improved species identification using genetic markers have important consequences on host ranges, source inocula and risk
assessment of phytopathologically important taxa.
However, there are also some substantial unresolved
problems which need to be addressed in the future
with new data and methods. These include the
systematic position of some rarely sampled taxa, the
phylogenetic relationships of the main downy mildew
lineages to each other, more detailed molecular
H. Voglmayr (*)
Department of Systematic and Evolutionary Botany,
Faculty Centre Botany, University of Vienna,
Rennweg 14,
1030 Vienna, Austria
e-mail: hermann.voglmayr@univie.ac.at
studies on speciation processes to develop appropriate
sound species concepts and circumscriptions, and the
development of a molecular bar coding system for
downy mildews enabling reliable species identification. Applying molecular methods has the potential to
greatly enhance our knowledge on the overall
biodiversity of downy mildews.
Keywords Albugo . Molecular phylogeny .
Peronosporales . Peronosporaceae . Phytophthora .
Species concept . White blister rusts
Introduction
Downy mildews and white blister rusts are members
of the class Oomycetes (Peronosporomycetes), a
comparatively small lineage with estimates of <1,000
species (Kirk et al. 2001). Due to their morphological,
physiological and ecological similarities to fungi, the
Oomycetes are traditionally treated within mycology;
however, ultrastructural, biochemical and molecular
phylogenetic data confirm that they are not related to
true fungi (kingdom Fungi), but belong to the
kingdom Chromista (Straminipila) which also contains
the chromistan (heterokont) algae (Dick 2001, 2002;
Kirk et al. 2001). Downy mildews are an important
group of obligate biotrophic plant parasites, which
have a great economic impact on numerous crops
(e.g. Plasmopara viticola on Vitis vinifera, Pseudoperonospora humuli on Humulus lupulus, Perono-
4
spora tabacina on Nicotiana spp.). With the commercial large-scale production of some crop and ornamental plants, some downy mildew diseases have
recently become of major concern, e.g. downy mildew
disease of Ocimum basilicum (basil; e.g. Belbahri et
al. 2005), Eruca sativa (rocket; e.g. Larran et al. 2006
and references cited therein), or Rubus spp. (arctic
bramble and boysenberry; e.g. Hukkanen et al. 2006
and references cited therein). In several of these cases,
classification of the causal agent is still unclear, which
demonstrates our ignorance of biodiversity of downy
mildews. With the rapid development of new research
techniques and questions in applied and theoretical
plant pathology, the interest in phylogeny of downy
mildews has increased over the last years. The
availability of a substantial number of additional new
characters less prone to subjective interpretations in
general led to a change of paradigms in classification,
as phylogenetic hypotheses could be vigorously tested
for the first time, which led to a shift from a phenetic
to a phylogenetic classification. In addition, phylogenetic analyses of DNA sequence data also enabled a
re-evaluation of morphological features in an evolutionary background and a re-investigation of species
boundaries and host specificity. Significant progress
has been made towards a phylogenetic classification,
which is presented here. However, several important
questions are still unresolved, and these will be briefly
discussed.
General considerations on systematics
and taxonomy
Before considering the changes, progress and challenges in downy mildews systematics in detail, some
general considerations will be briefly outlined.
There are often conflicts between taxonomists and
applied biologists, originating from different and
sometimes incongruent expectations on taxonomy
and systematics. Although there have been different
methodological approaches in taxonomy during its
history, it is nowadays commonly accepted that
phylogenetic relationships should be the primary
basis of a taxonomic system (Lecointre and Le
Guyader 2006). Therefore, the taxonomist seeks
consistency of a taxonomic system with theories on
phylogeny and evolution. Consequently, classification should be in line with well-supported phyloge-
Eur J Plant Pathol (2008) 122:3–18
netic hypotheses. With the increase of knowledge,
reinterpretation of phylogenetic relationships leads
necessarily to name changes. In addition, taxonomy
and classification has to be consistent with the
current rules of the International Code of Botanical
Nomenclature. Application of the latter sometimes
necessitates the change of well-established names,
which are often felt unnecessary, cumbersome or
complicated.
Non-taxonomists are primarily interested in using
scientific names without the need for a deeper
knowledge of taxonomy itself. Therefore, stability of
names is highly desirable. Consequently, taxonomy
should be easily applicable outside the taxonomic
community, e.g. for purposes of identification. As
another important requirement, taxonomy should be
also appropriate for legal measures such as quarantine
lists of species.
Of course, ideally taxonomy should fit the needs of
both taxonomists and non-taxonomists, and this is
often possible if taxonomic decisions are made
cautiously. In downy mildews, it has been possible
up to now to avoid undesirable name changes for
most phytopathologically important species. However, taxonomic changes are sometimes unavoidable to
meet the standards of a consistent phylogenetic
classification. This is no end in itself, but enables
progress also in other research disciplines. Evidently,
the non-taxonomist should be interested in reliable
species concepts and boundaries, which are prerequisites for the development of reliable PCR-based
identification systems, a record of the correct host
ranges, and the application of pest control and
quarantine measures.
Phylogenetic placement of Peronosporaceae
(downy mildews) and Albuginaceae (white blister
rusts)
Based on morphological and ultrastructural data,
Oomycetes were subdivided into three subclasses,
the Saprolegniomycetidae, Rhipidiomycetidae and
Peronosporomycetidae, the latter including Pythium,
Phytophthora and some other genera together with
downy mildews and white blister rusts (Dick et al.
1984; Dick 1995). This subdivision was largely
confirmed by subsequent molecular phylogenetic
analyses (e.g. Dick et al. 1999; Riethmüller et al.
Eur J Plant Pathol (2008) 122:3–18
1999; Hudspeth et al. 2000; Petersen and Rosendahl
2000). However, the classification at lower ranks
remained uncertain and changed quite substantially
following molecular phylogenetic analyses (Table 1,
Fig. 1). Whereas previously considered closely related
to Peronosporaceae (Fig. 1a), the Albuginaceae were
placed outside the Pythiaceae-Peronosporaceae lineage in cox2 (Fig. 1b), LSU/SSU (Fig. 1c) and ITS
rDNA (Cooke et al. 2000) sequence analyses.
Therefore, Albuginaceae should represent an ancient,
evolutionarily–derived lineage of uncertain phylogenetic affinities (compare Fig. 1b,c). Early origin of
Albuginaceae is in line with high sequence divergence (Riethmüller et al. 2002) and its unique
5
conidial and oospore morphology (Riethmüller et al.
2002; Hudspeth et al. 2003; Thines and Spring 2005;
Voglmayr and Riethmüller 2006). Consequently,
Hudspeth et al. (2003) pleaded for exclusion of
Albuginaceae from Peronosporales and for elevation
to ordinal level, whereas Thines and Spring (2005)
created even a new subclass, Albuginomycetidae.
As opposed to Albugo, DNA data confirmed a
close phylogenetic relationship of downy mildews to
the genus Phytophthora (e.g. Petersen and Rosendahl
2000; Cooke et al. 2000; Riethmüller et al. 1999,
2002; Hudspeth et al. 2003; Voglmayr 2003; Göker et
al. 2007). There is strong molecular evidence that the
genus Peronophythora, sometimes considered an
Table 1 Comparison of some ordinal, family and generic classifications of downy mildews, white blister rusts and relatives
Waterhouse (1973)
Kirk et al. (2001)
Riethmüller et al. (2002)
Göker et al. (2007)a,
Thines and Spring (2005)b
Peronosporales
Peronosporaceae
Basidiophora
Bremia
Bremiella
Peronospora
Plasmopara
Pseudoperonospora
Sclerospora
Albuginaceae
Albugo
Pythiaceae
Phytophthora
Pythiogeton
Pythium
Sclerophthora
Trachysphaera
Peronosporales
Peronosporaceae
Basidiophora
Benua
Bremia
Bremiella
Paraperonospora
Peronospora
Plasmopara
Pseudoperonospora
Albuginaceae
Albugo
Pythiales
Pythiaceae
Halophytopthora
Peronophythora
Phytophthora
Pythium
Trachysphaera
Pythiogetonaceae
Pythiogeton
Sclerosporales
Sclerosporaceae
Peronosclerospora
Sclerospora
Verrucalvaceae
Sclerophthora
(no order name)
Peronosporaceae
Basidiophora
(Benua)
Bremia
Paraperonospora
Peronophythora
(Peronosclerospora)
Peronospora
Phytophthora
Peronospora
Plasmopara
Pseudoperonospora
Sclerospora
Albuginaceae
Albugo
Pythiaceae
Lagenidium
Pythium
(Pythiogeton)
(Trachysphaera)
(Sclerophthora)
Peronosporales
Peronosporaceae
Basidiophora
Benua
Bremia
Graminivora
Hyaloperonospora
Paraperonospora
Perofascia
Peronosclerospora
Peronospora
Peronospora
Plasmopara
Plasmoverna
Protobremia
Pseudoperonospora
Sclerospora
Viennotia
(family not formally classified)
Phytophthora
Albuginales
Albuginaceae
Albugo
Pustula
Wilsoniana
For the Pythiaceae and Verrucalvaceae, only important genera (those with phytopathogenic and/or phylogenetic relevance for downy
mildews) are listed (for complete genus lists, see the respective publications); from Peronosporaceae and Albuginaceae, all genera are
considered. Taxa in parentheses: not included in the phylogenetic analyses. Phytophthora excluded from Peronosporaceae but placed
in Peronosporales without formal family classification; other traditional Pythiaceae not classified.
a
For downy mildews
b
For white blister rusts
6
Eur J Plant Pathol (2008) 122:3–18
Fig. 1 Phylogenetic hypotheses among the Oomycetes, with
special reference to obligate parasites and their closest relatives
(Pythium, Phytophthora). a Topology reflecting the hierarchical
classification of Dick (2001). b Topology obtained from cox2
sequence data (Hudspeth et al. 2003). c Topology obtained
from SSU (Petersen and Rosendahl 2000) and LSU rDNA
(Riethmüller et al. 2002) sequence data. Red branches:
Peronosporomycetidae, grey branches: Saprolegniomycetidae,
yellow branches: Rhipidiomycetidae. The three groups of
obligate parasites (Albuginaceae = white blister rusts, Peronosporaceae = downy mildews, Sclerosporaceae = graminicolous
downy mildews) are given in colour and bold; circumscription
follows Kirk et al. (2001; see also Table 1). The molecular
analyses are largely congruent except for the position of the
Rhipidiomycetidae. Due to the isolated phylogenetic position of
Albuginaceae, subclass Albuginomycetidae has been proposed
(Thines and Spring 2005)
intermediate between Phytophthora and downy mildews, is not the closest relative to downy mildews
and should rather be classified within Phytophthora
(Riethmüller et al. 2002; Voglmayr 2003; Göker et al.
2007).
same dataset using different methods of phylogenetic
reconstruction (Göker and Stamatakis 2006), downy
mildews did not appear monophyletic, with the
Phytophthora infestans group (Phytophthora 1) being
embedded within the downy mildews clade (Fig. 2b).
It is questionable whether this problem can be solved
with comparably few DNA sequences alone; more
detailed investigations on genome organisation and
ultrastructure may provide better insights.
Monophyly versus polyphyly of downy mildews
The downy mildews (Peronosporaceae), in the traditional sense, are a morphologically diverse group,
which is mainly united by obligate parasitism in
combination with more or less complex conidio- or
sporangiophores with determinate growth. In traditional morphological classifications it is generally
assumed that obligate biotrophism of the downy
mildews evolved only once.
In the first molecular phylogenetic analyses it was
uncertain whether downy mildews are monophyletic
or stem from different groups of Phytophthora, a
situation which in fact has not yet been clarified with
certainty (compare Riethmüller et al. 2002; Göker et
al. 2003, 2007; Göker and Stamatakis 2006). In a
recent multigene phylogeny involving five genes
(Göker et al. 2007), monophyly of downy mildews
was highly supported by various methods of phylogenetic reconstruction, which therefore seemed to be
corroborated (Fig. 2a). However, in an analysis of the
The paraphyly problem of Phytophthora
Phylogenetic classification using the monophyly
criterion may raise severe problems for the genus
Phytophthora, which is paraphyletic in most analyses
in respect to downy mildews (e.g. Cooke et al. 2000,
2002; Göker et al. 2007; Fig. 2). Therefore, it remains
open if a phylogenetic classification can be achieved
without either merging downy mildews with Phytophthora or generic splitting of Phytophthora into several
genera. It is foreseeable that these alternatives will not
receive broad acceptance, representing a dilemma for
classification. However, internal support for the tree
backbone is usually low even in multigene analyses
(e.g. Göker et al. 2007; Fig. 2), and additional data
from other gene regions and taxa need to be collected
before a robust phylogeny can be achieved.
Eur J Plant Pathol (2008) 122:3–18
7
Fig. 2 Phylogenetic hypotheses among the downy mildews
(genera in bold) inferred from DNA data. a Simplified tree
illustrating the phylogenetic hypotheses of Göker et al. (2007)
showing a single monophyletic downy mildews clade. b
Simplified tree illustrating the phylogenetic hypotheses of
Göker and Stamatakis (2006), showing polyphyly of two
separate downy mildew clades; Phytophthora 1 (P. infestans
group; arrowhead) is the closest relative of the downy mildews
clade 1 containing, amongst others, Plasmopara and Bremia;
the same tree topology was already largely revealed by Göker
et al. (2003). Bold and medium branches indicate boostrap
support equal or higher than 90% and 70%, respectively;
genera marked with an asterisk are represented by a single
taxon only. Note that in both trees the genus Phytophthora
(closest relative of downy mildews) is paraphyletic. Numbers
after Phytophthora correspond to the clade numbering in Cooke
et al. (2000)
The status of the graminicolous downy mildews
(Sclerosporaceae)
rophthora, sometimes considered closely related to
Sclerospora and Peronosclerospora, has been placed
within Phytophthora in a recent molecular phylogenetic analysis (Thines et al. 2008), however, without
significant internal support.
In addition to the graminicolous downy mildews in
the strict sense (i.e. Sclerospora, Peronosclerospora),
the species from the genera Plasmopara and Bremia
parasitising Poaceae were also recently reclassified
and transferred to newly described genera (Göker et
al. 2003; Thines et al. 2006, 2007). Whether these are
close relatives to Sclerospora and Peronosclerospora
cannot be resolved with the current molecular data
(Göker et al. 2007; Thines et al. 2008).
Phylogenetic relationship of the graminicolous
downy mildews within the Peronosporaceae is much
less clear. To date, only few accessions have been
analysed. In some recent publications (Göker et al.
2007; Thines et al. 2007, 2008), graminicolous
The controversial classification (Table 1, Fig. 1) of the
graminicolous downy mildews (i.e. Sclerospora and
Peronosclerospora) has been recently clarified with
molecular phylogenetic data. Classification as a
separate family and order (Dick et al. 1984), as well
as the classification within the Saprolegniomycetidae
(Dick et al. 1989), did not receive support from
molecular data (Fig. 1). In all molecular phylogenetic
investigations published so far (Riethmüller et al.
2002; Hudspeth et al. 2003; Göker et al. 2003, 2007;
Thines et al. 2007, 2008), graminicolous downy
mildews were unequivocally placed within Peronosporaceae. Therefore, separation of graminicolous
downy mildews from downy mildews and classification within a separate family Sclerosporaceae has
become obsolete (Figs. 1 and 2). The genus Scle-
8
Eur J Plant Pathol (2008) 122:3–18
downy mildews appear at varying basal positions
within the downy mildew clade. However, this
position lacks significant support.
Generic concepts in downy mildews
Generic concepts in downy mildews were (and still
are) mainly based on conidio-/sporangiophore morphology in combination with conidial/sporangial
morphology. Dichotomous versus monopodial
branching of conidio-/sporangiophore, shape of the
terminal branches and presence of conidia or
sporangia were the primary features used for genus
classification. However, interpretation of these morphological features was not always unequivocal and
dependent on the observer, which sometimes
resulted in conflicting generic concepts and delimitation (e.g. Pseudoperonospora: Skalický 1966 vs.
Waterhouse 1973). Although the segregation of the
genera Paraperonospora (Constantinescu 1989) and
Benua (Constantinescu 1998) resolved some taxonomic problems of the morphological classification
scheme of genera, generic classification remained
problematic.
With the availability of molecular phylogenies, it
soon became apparent that current generic classification and circumscription contained numerous problems and had to be adapted if standards of
phylogenetic classification were applied (for a summary,
see Table 2). Based on molecular and morphological
features, the genera Hyaloperonspora and Perofascia
were segregated from the large genus Peronospora
(Constantinescu and Faheti 2002). The genus Bremiella,
containing three species (Constantinescu 1991a), was
shown to be polyphyletic, and all species were
clearly embedded within Plasmopara (Riethmüller et
al. 2002; Göker et al. 2007; Voglmayr and Thines
Table 2 Recent taxonomic changes at the generic level of white blister rusts and downy mildews based on molecular and
morphological data
Pre-2000
Recent (post-2000)
References
Albugo
Albugo (sensu stricto)
Pustula
Wilsoniana
Basidiophora
Thines and Spring (2005)
Thines and Spring (2005)
Thines and Spring (2005)
Riethmüller et al. (2002), Voglmayr et al. (2004),
Göker et al. (2007)
Göker et al. (2007)
Voglmayr et al. (2004)
Thines et al. (2006)
Riethmüller et al. (2002), Voglmayr et al. (2004),
Göker et al. (2007), Voglmayr and Thines (2007)
Riethmüller et al. (2002), Voglmayr et al. (2004),
Göker et al. (2007)
Hudspeth et al. (2003), Thines et al. (2008)
Göker et al. (2007)
Constantinescu and Faheti (2002), Göker et al. (2003, 2004)
Constantinescu and Faheti (2002)
Voglmayr et al. (2004)
Voglmayr and Constantinescu (2008)
Constantinescu et al. (2005)
Thines et al. (2007)
Voglmayr et al. (2004)
Göker et al. (2003)
Riethmüller et al. (2002), Göker et al. (2007)
Riethmüller et al. (2002), Göker et al. (2007)
Basidiophora
Benua
Bremia
Bremiella
Benua
Bremia (B. lactucae)
Graminivora (B. graminicola)
Plasmopara
Paraperonospora
Paraperonospora
Peronosclerospora
Peronospora
Peronosclerospora
Peronospora
Hyaloperonospora (P. parasitica s.l.)
Perofascia (P. lepidii)
Core Plasmopara
Novotelnova (Pl. savulescui)
Plasmoverna (Pl. pygmaea s.l.)
Poakatesthia (Pl. penniseti)
Protobremia (Pl. sphaerosperma)
Viennotia (Pl. oplismeni)
Pseudoperonospora
Sclerospora
Plasmopara
Pseudoperonospora
Sclerospora
The pre-2000 classification is according to the Dictionary of the Fungi (Kirk et al. 2001); the references refer to the post-2000
classifications
Eur J Plant Pathol (2008) 122:3–18
2007). On the other hand, the genus Plasmopara itself
was shown to be a non-monophyletic genus, and
several genera were segregated (see Table 2). Fortunately enough, it was nomenclaturally possible to
maintain the use of the name Plasmopara for the bulk
of species including the phytopathologically important
ones (for details, see Constantinescu et al. 2005). The
genus Bremia, consisting of two species (one on
Asteraceae, one on Poaceae), was also shown to be
polyphyletic, and the genus Graminivora was segregated for the grass parasite (Thines et al. 2006). Therefore,
the number of accepted genera has gradually increased
during the last years (one merged versus eight new
genera). However, except for Hyaloperonospora, as
none of these new genera is phytopathologically
important, these changes have had little effect for
phytopathologists.
Morphology revisited: shortcomings, new features
and interpretations
The results of the molecular phylogenetic investigations also stimulated reevaluation of the morphological features traditionally used for classification and
the search for new, previously neglected characters
(Spring and Thines 2004; Voglmayr et al. 2004;
Constantinescu et al. 2005; Thines 2006; Voglmayr
and Riethmüller 2006). Already Hall (1996) stressed
the necessity of thorough morphological investigations, besides other methods, to obtain a more
satisfactory classification. Downy mildews and white
blister rusts show a low morphological complexity,
and comparatively few characters are available for
classification. However, taking the whole spectrum of
these few available characters into account, it is
astonishing that even fewer selected characters had
been used in traditional morphological classifications
(i.e. some features of conidio-/sporangiophore morphology and conidial/sporangial morphology; for a
synopsis, see Waterhouse 1973). In addition, these
few characters often had not been critically studied
and evaluated for more than a few species, and often
data were just taken non-critically from the original
descriptions. The morphology of the few common
and phytopathologically important downy mildew
species was comparatively well known, but the vast
majority of species were much less studied. Therefore, morphological classification and interpretation
9
was mainly based on the few phytopathologically
important species, neglecting much of the morphological diversity.
With the advent of molecular systematics, the
morphological features used for classification were
reinterpreted in a phylogenetic context, making previous interpretations of conidio-/sporangiophore morphology too simple, partly incorrect or unsuitable for
generic classification (e.g., Riethmüller et al. 2002;
Voglmayr et al. 2004; Thines et al. 2006). Nevertheless, importance of known but previously neglected
features became apparent, as is the case for haustorial
morphology. Presence of different haustorial types in
downy mildews was recorded by Fraymouth (1956),
but these were never used for classification. Recently,
several haustorial types could be shown to be
diagnostic for several lineages (e.g. Hyaloperonospora: Constantinescu and Faheti 2002; Plasmopara,
Bremia and their close relatives: Riethmüller et al.
2002; Göker et al. 2003; Voglmayr et al. 2004; Thines
et al. 2006; Pseudoperonospora: Voglmayr et al.
2004).
In addition, the importance of searching for
additional features to be used in classification was
emphasised by Spring (2004) and Spring and Thines
(2004). Ideally, these should be preserved in herbarium specimens to enable investigation of as many
representatives as possible, which would be difficult
if living specimens were necessary. As herbarium
specimens can be used, Spring (2004) and Spring and
Thines (2004) argued for investigations of ultrastructural features by scanning electron microscopy (SEM)
to reveal additional features. Thines (2006, 2007b)
presented the results of detailed morphological investigations of downy mildews including SEM studies of
conidio-/sporangiophores and sporangia. He produced
a morphological character matrix and analysed it
within the constraint of recently published molecular
phylogenetic investigations to reveal and evaluate
morphological synapomorphies. He concluded that
the classical features used for classification like
conidio-/sporangiophore branching or sporangial germination are not diagnostic for phylogenetic lineages
above the generic level. However, the fine structure of
the ultimate branchlets (revealed by SEM) and
haustorial shape were considered to be phylogenetically informative.
The genus Albugo sensu lato has also recently been
extensively re-investigated using light microscopy
10
and SEM. Thines and Spring (2005) emphasised that
different sporangial ornamentation types revealed by
SEM were diagnostic for the Albugo clades revealed
by molecular data, and segregated the two genera
Wilsoniana (for species on Caryophyllidae) and
Pustula (for species on Asteraceae). Voglmayr and
Riethmüller (2006) confirmed the SEM data of
Thines and Spring (2005) and added detailed light
microscopical and SEM data of the oospores, which
were also shown to be diagnostic for the different
molecular phylogenetic lineages of Albugo sensu lato.
Constantinescu and Thines (2006) investigated and
clarified sporangiogenesis in Albuginaceae; they
confirmed the presence of sporangial dimorphism
(primary and secondary sporangia) for all species
investigated.
Biochemical characters
Apart from DNA sequence data, few characters at the
biochemical level have been used for classification of
downy mildews. Recently, fatty acids were recorded
as potentially promising for the characterisation of
downy mildew species (e.g., Spring et al. 2003;
Spring, 2004; Spring and Thines 2004; Spring et al.
2005). In the case of Albugo sensu lato, Spring et al.
(2005) recorded significant differences between three
species and considered fatty acid pattern characteristic
for the species. Investigations on closely related
species are still missing, and therefore additional data
are necessary to evaluate applicability and resolution
limits of fatty acid composition for taxonomic
differentiation.
Species concepts in downy mildews
The species concept is probably the most controversial
issue in downy mildew systematics, partly due to
experimental difficulties to test it and partly due to its
profound implications for researchers outside the
systematics research community. A well-written account of the history and implications of the different
species concepts is given in Hall (1996). In downy
mildews, several species concepts were applied, which
resulted in highly different numbers of accepted
species depending on the criteria used. The main
Eur J Plant Pathol (2008) 122:3–18
problem in species delimitation in downy mildews is
that there are numerous indications that, due to their
obligate parasitism, they often have narrow host
ranges and therefore represent genetically distinct
species. On the other hand, host specificity is not
always paralleled by morphological distinctness.
Therefore, if morphology is used as a primary criterion
for species definition, only a few species can be
defined and accepted in many lineages, resulting in
genetically heterogeneous species.
Historically, two approaches were commonly
followed, which were both mainly based on host
ranges: the splitting approach of Gäumann (1918,
1923) versus the lumping approach of Yerkes and
Shaw (1959). Gäumann (1918, 1923) advocated a
narrow species concept in Peronospora, based on his
results of cross-inoculation studies and minute morphological differences. Each species was usually
assumed to be confined to one host genus or even a
few host species (one host-one species concept; see
Hall 1996). Conversely, Yerkes and Shaw (1959)
argued that host specificity is neither sufficient nor
suitable for the recognition of a species without clearcut morphological differences. As a consequence,
they lumped the numerous Peronospora species on
Brassicaceae and Chenopodiaceae each into a single
species (Peronospora parasitica and P. farinosa,
respectively), resulting in a wide one host familyone species concept.
Both the splitting as well as the lumping approach
have sincere shortcomings. Using the narrow species
concept, identification of morphologically similar
species is often difficult or impossible without correct
identification of the host. In addition, high host
specificity has rarely been conclusively demonstrated,
weakening the primary underlying assumption of the
narrow species concept.
In a wide species concept, there is the problem that
genetically distinct or even unrelated entities may be
classified in the same species, raising incorrect
assumptions on biology and host ranges. This is
especially problematic if host jumps are common and
parasitism on the same host family has evolved
multiple times, resulting in polyphyletic species.
However, due to its easier applicability, the approach
to classify all accessions of a given host family within
a single species was widely followed by phytopathologists and molecular biologists.
Eur J Plant Pathol (2008) 122:3–18
Impact of molecular data on downy mildew species
concept and circumscription
Recently, molecular phylogenetic investigations have
enabled the evaluation of the species problem using
new perspectives and have led to the shift from a
morphological to a phylogenetic species concept. A
biological species concept directly addressing mating
barriers has never been applied to downy mildews
due to sincere methodological difficulties, and it is
unlikely that these can be overcome. Therefore,
reproductive isolation can only be indirectly assessed,
e. g. by genetic distance of sequence data. The impact
of molecular data is manifold: (1) numerous additional characters are available for recognition and distinction; (2) presence and amount of reproductive
isolation can be assessed; (3) presence and amount
of genetic distances provide indirect but strong
evidence for host specificity and host ranges; (4)
molecular data are less variable and prone to subjective
interpretation than morphological data; (5) molecular
data provide a sound basis for species identification
even if morphological data are missing or incomplete;
(6) pathotypes or races, the basic entities for experiments in applied sciences, can be properly attributed to
a species and their phylogenetic relationships can be
assessed. Therefore, in the absence of sound morphological characters, the species concept is increasingly
based on molecular evidence of reproductive isolation,
which is a general tendency within mycology. Consequently, morphologically similar cryptic species are
often recognised as distinct species if reproductive
isolation and genetic distinctness can be demonstrated.
However, evaluation of species boundaries by molecular data require thorough sampling throughout the
distribution area to assess genetic variability as well as
reproductive isolation, and at best several molecular
markers should be used for corroboration of species
boundaries.
Due to easy amplification and variability, the ITS
rDNA region has been used in most investigations
addressing the species concept in downy mildews and
white blister rusts (e.g. Choi et al. 2003, 2005, 2006,
2007a, b, c, d; Voglmayr 2003; Göker et al. 2004;
Scott et al. 2004; Cunnington 2006; Spring et al.
2006; Voglmayr et al. 2006; Landa et al. 2007; García
Blázquez et al. 2008). However, the mitochondrial
cox2 region may also be a promising candidate to
11
resolve species boundaries and for species identification (e.g. Choi et al. 2006, 2007d). Interestingly, the
current evidence from molecular phylogenetic investigations often supports a narrow species concept as
advocated by Gäumann (1918, 1923), although there
are sometimes marked differences in detail. In the
following, the results of recent molecular investigations will be briefly summarised for the different
genera.
Hyaloperonospora
According to Constantinescu and Faheti (2002), about
140 species names were published attributable to this
genus. In their separation of Hyaloperonospora from
Peronospora, Constantinescu and Faheti (2002) only
recognised six morphologically distinct species, and
accessions from most hosts of Brassicaceae were
placed in Hyaloperonospora parasitica. However,
subsequent molecular phylogenetic investigations
demonstrated that the latter was a paraphyletic
assemblage with respect to the other five Hyaloperonospora species, and that many more species should
be accepted based on the high genetic distances
observed (Choi et al. 2003; Göker et al. 2003, 2004;
Voglmayr 2003). Usually, these genetically distinct
entities deserving species rank have a narrow host
range and are confined to host genera or even species;
however, in some cases accessions from the same host
do not form a monophylum (e.g. from Armoracia
rusticana; see Göker et al. 2004). Therefore, it is
problematic when species are determined solely on
host association, as this is often but not always
conclusive. The case study of Hyaloperonospora is
also relevant for investigations at the molecular level
of plant–pathogen interactions, as numerous studies
are performed with the plant model organism Arabidopsis thaliana and its Hyaloperonospora parasite.
The parasite is usually named H. parasitica, but it is
genetically quite distinct from H. parasitica sensu
stricto which is confined to Capsella bursa-pastoris
(Göker et al. 2004); therefore, the name H. arabidopsidis should be used for the Arabidopsis parasite.
Peronospora
The molecular phylogenetic analyses dealing with the
genus Peronospora (e. g. Voglmayr 2003; Choi et al.
12
2007c; García Blázquez et al. 2008) also provide
evidence for a narrow species circumscription. Species parasitising e.g. Chenopodiaceae do not form a
monophyletic lineage (Voglmayr 2003; Choi et al.
2007c), but are interspersed with species from other
host families and can often also be separated
morphologically (Choi et al. 2007c). Interestingly,
the type species of Peronospora, P. rumicis, a parasite
of Rumex spp. (Polygonaceae), is embedded within a
group of species infecting Chenopodiaceae (Choi et
al. 2007c), which may indicate a recent host jump.
Choi et al. (2007c) demonstrated that Peronospora
effusa (spinach downy mildew) is genetically homogeneous world-wide, but distinct from P. farinosa
sensu stricto. Reasons for this genetic homogeneity
may include the recent introduction to most of its
present growth area as well as pathogen transmission
by seeds, enabling rapid dispersal from a small
geographic source area via international seed trade.
Choi et al. (2008a), investigating five Peronospora
species from different species of Chenopodium,
recorded significant molecular and morphological
differences, and they concluded that these are welldistinct species and should not be merged with P.
farinosa. Therefore, the approach of Yerkes and Shaw
(1959) to merge all species on Chenopodiaceae under
P. farinosa appears to be inappropriate, as P. farinosa
according to Yerkes and Shaw (1959) is evidently a
polyphyletic assemblage. However, further studies
using additional variable genes are required to reveal
and evaluate the phylogenetic species, as the ITS
region does not give significant support or resolution
for many nodes of the backbone (Voglmayr 2003;
Choi et al. 2007c).
Traditionally, from de Bary (1863) onwards, only
two Peronospora species have been recognised on
Fabaceae, P. trifoliorum and P. viciae, which were
observed to differ in their oospore ornamentation.
Gäumann (1923) and subsequent authors again
described numerous new species from different
host species, resulting in >100 Peronospora binomials described from 25 host genera of Fabaceae
(Constantinescu 1991b). The results of Voglmayr
(2003) indicated that Peronospora on Fabaceae does
not form a monophyletic lineage, although most
accessions including those from Vicia and Trifolium
were united in a single monophyletic clade. However,
the clades did not correspond to the classical two
species recognised from Fabaceae and showed that
Eur J Plant Pathol (2008) 122:3–18
more than two species are involved. Cunnington
(2006) confirmed high genetic distances between
accessions from different hosts, which were traditionally included in P. viciae. In the most extensive
study on Peronospora parasites of Fabaceae, GarcíaBlázquez et al. (2008) showed that numerous hostspecific lineages are present on Fabaceae. Although
their study did not include Peronospora species
from other host families, there is evidence that they
do not even form a monophyletic lineage (Voglmayr
2003), a situation comparable to the Peronospora
species on Chenopodiaceae. Although there are
numerous nomenclatural problems left in the Peronosporas from Fabaceae which need additional
studies (García-Blázquez et al. 2008), the results
are roughly concordant with the classification of
Gäumann (1923).
Molecular phylogenies using ITS data not only
often confirmed a rather narrow species concept in
Peronospora, but also helped to clarify species
attribution. Scott et al. (2004) identified the parasite
of oilseed poppy (Papaver somniferum) from Tasmania, which was previously listed as Peronospora
arborescens, as P. cristata, and both species were
shown to be genetically distinct. Conversely, Landa et
al. (2007) reported Peronospora arborescens as the
causal agent of downy mildew of P. somniferum from
Spain. Therefore, apparently both P. arborescens and
P. cristata can infect P. somniferum and are of
different importance in various regions of the world.
Pseudoperonospora
Conversely to the other examples listed above, in
Pseudoperonospora molecular data provided evidence for conspecificity of species from different host
families. Choi et al. (2005) performed a study
including Pseudoperonospora humuli (from Humulus
spp., Cannabinaceae) and P. cubensis (from Citrullus
vulgaris, Cucumis spp. and Cucurbita spp., Cucurbitaceae). Distinction as separate species was mainly
based on their occurrence on two non-related host
families, but almost identical ITS sequences and
absence of significant morphological differences
indicate conspecificity. Therefore, Choi et al. (2005)
synonymised Pseudoperonospora humuli with P.
cubensis. These data confirm recent and possibly
multiple host shift from Cannabinaceae to Cucurbitaceae. However, for detailed insights into the evolu-
Eur J Plant Pathol (2008) 122:3–18
tionary processes involved, the ITS data offer too
little resolution, and molecular markers with higher
resolution should be applied.
ITS length differences as potential markers
for species
In addition to phylogenetic analyses of ITS data, the
structure of the ITS itself has also recently received
increasing attention. This appears to be especially
promising for the Plasmopara-Bremia clade, which
was shown to have an ITS region of variable length
up to about 3,200 bp (Choi et al. 2007b; Komjáti et al.
2007; Thines et al. 2005; Thines 2007a), which is
remarkable compared to the usually about 800 bp in
downy mildews (Voglmayr 2003; Göker et al. 2004).
Size increase mainly concerns the ITS2 region and
originates from repetitive elements, the number and
length of which appear to be taxon-specific (Choi et
al. 2007b; Thines 2007a, c). The number and
sequence of repetitive elements is usually conserved
within different lineages. Choi et al. (2007b) investigated Bremia accessions from different hosts and
identified nine repetitive elements showing high
sequence heterogeneity between the accessions from
different hosts, which indicates that they may represent distinct species. Thines (2007a) investigated
repetitive elements for several species and recorded
a highly variable number. He concluded that these
repetitive elements could be useful for investigation
of speciation and radiation processes. Based on
sequence variability observed in the ITS2 region,
Spring et al. (2006) could separate and characterise
two groups of pathotypes of Plasmopara halstedii
from sunflower.
Repetitive elements are also present in the ITS2
region of some Hyaloperonospora species (Voglmayr
2003; Göker et al. 2004); however, they are confined
to a few species for which they may be diagnostic.
Thines (2007a) recorded similarities to the repetitive
elements of the Plasmopara-Bremia clade and suggested that this may be indication of a closer
relationship for these two lineages.
Repetitive elements are also observed within
Peronospora sensu stricto, where they have been
reported within the ITS1 region (Voglmayr 2003).
The number of repetitive elements has recently been
investigated and analysed in detail for Peronospora
13
on Trifolium (García Blázquez et al. 2008). The
number of additional copies of a region about 70 bp
long ranged from one to 11 and was, with few
exceptions, diagnostic for different host-specific lineages. Therefore, different lengths of ITS may, with
limits, be suitable for species identification. However,
little is yet known on the mechanisms governing the
copy number of repetitive elements, and taxon
specificity needs additional detailed investigations.
Discovery of morphologically distinct species
by molecular data
Molecular phylogenetic data also stimulated closer
morphological examinations at the species level,
which showed that the non-critical but widely applied
species identification solely on host association can
be misleading. Several new species could be described which were morphologically well distinct but
remained remarkably unnoticed (e.g., Voglmayr et al.
2006; Choi et al. 2007a). Plasmopara on Geraniaceae
provides an excellent example for this. Traditionally,
two morphologically distinct species, Plasmopara
pusilla on European and P. geranii on North
American Geranium species, were accepted and
recognised (Constantinescu 2004). Detailed molecular
and morphological investigations confirmed distinctness of another previously described but neglected
species and revealed two additional undescribed
species which are morphologically distinct, widespread and rather common (Voglmayr et al. 2006).
This indicates that biodiversity of downy mildews is
still imperfectly known even in regions which are
considered well-studied.
Unresolved questions and future perspectives
Detailed phylogenetic relationships and evolutionary
scenario within downy mildews
It remains still unresolved whether downy mildews
are monophyletic and how the major groups of downy
mildews are related to each other. Clarification of
phylogenetic relationships will necessitate additional
molecular markers, new methods of phylogenetic
inference as well as improved taxon sampling. The
graminicolous downy mildews and representatives of
14
Eur J Plant Pathol (2008) 122:3–18
remote, little sampled areas (Africa, South America,
East and Southern Asia) are especially underrepresented in the present studies, and additional taxa need
to be sampled. Albuginales are also little-investigated,
and recent investigations on the Albugo candida
complex indicate a high level of, as yet, undescribed
biodiversity (Choi et al. 2007d, 2008a, b, which could
also be true in other lineages. In other cases such as
the downy mildews with pyriform haustoria Plasmopara, Bremia and relatives, additional sequence data
are needed to confirm more robust phylogenetic
relationships, a precondition for a sound delimitation
of genera. In addition, the investigations need to be
embedded in a wider taxonomic context. Phytophthora especially needs to be included, which is a key
genus for the evolution of downy mildews. In the
recent multigene phylogeny of Phytophthora by Blair
et al. 2008), obtained from a data matrix of 8,700 bp
from seven genes, various Phytophthora groups were
highly supported; however, internal support for the
tree backbone was still only moderate to low in
maximum parsimony and likelihood analyses, despite
the large data matrix, which may indicate that it may
be difficult to obtain highly supported gene phylogenies for Phytophthora and its relatives. To test this,
these sequence data should be complemented and
analysed with corresponding sequence data of representative downy mildews as well as from the genus
Pythium. Sound phylogenetic hypotheses are a precondition for detailed insights into the processes of
character evolution, adaptive radiation and speciation
of downy mildews on different host groups. The
species-rich genera Peronospora and Plasmopara
especially require extended, representative taxon
sampling as well as additional molecular markers to
provide a sound phylogenetic framework. It is still
little investigated whether polyploidy is involved in
speciation, and detailed studies on nuclear genome
size may provide information on genome evolution
(Voglmayr and Greilhuber 1998).
relationships of graminicolous downy mildews, detailed relationships of genera). The availability of
whole genome sequences of several species of
Phytophthora should accelerate our understanding
of molecular evolution in plant pathogenic oomycetes
(Lamour et al. 2007). However, for progress in
comparative genome analysis, it is critical that high
quality genome sequence assemblies and gene models
are developed, which is a next, urgently needed step
(Lamour et al. 2007). When such high-quality data
become available for Phytophthora, they could
provide the basis for new evolutionary and phylogenetic investigations on downy mildews. As Phytophthora is the closest relative of downy mildews, the
genetic models of host specificity and host jumps
could be adapted to and tested in downy mildews.
The Hyaloperonospora-Arabidopsis pathosystem
would be an ideal candidate, as both host and
pathogen are genetically well studied, and as the
genus Hyaloperonospora contains numerous hostspecific, genetically distinct entities commonly recognised as separate species (Choi et al. 2003; Göker
et al. 2004). In addition, comparative analysis of
genome organisation could provide detailed evolutionary insights. However, it should be mentioned that
these investigations are methodologically difficult and
require the development of sophisticated techniques
as downy mildews cannot be cultivated on artificial
media. It is evident that sequencing of the whole
genome or even large quantities of the genome can
practically only be done for a few selected representatives. In addition, suitable computational methods of
phylogenetic analysis of such large data sets need to
be developed. Despite these limitations, whole genome data could contribute to more robust phylogenetic hypotheses than investigations based on one or a
few sequences regions only, and could provide help
for the selection of promising proper genome regions
for phylogenetic analyses on an extended taxon
selection.
Whole genome analysis in an evolutionary context
Applicable species definitions and the need
for taxonomic and nomenclatural revisions
For a better understanding of the evolutionary processes and of phylogenetic relationships, the integration of whole genome data is also promising,
especially where phylogenetic relationships remain
unsettled with the current molecular phylogenetic
analyses (e.g. monophyly of downy mildews, detailed
The most important issues of downy mildew systematics outside the taxonomic community concern the
species concept. Nomenclatural stability as well as
sound applicable species circumscription are eagerly
anticipated. For this, more detailed and conclusive
Eur J Plant Pathol (2008) 122:3–18
molecular studies are required to resolve the species
boundaries, especially in the species-rich genera
Peronospora, Hyaloperonospora and Plasmopara.
In addition, to achieve nomenclatural stability, numerous taxonomic and nomenclatural problems involving host range and correct typification need to be
solved. Although molecular data provide strong
evidence that a narrow species concept as advocated
by Gäumann (1918, 1923) may in many cases be
appropriate, there are numerous problems in his
classification in detail. Whereas some of his species
are apparently conspecific, others are heterogeneous.
As he did not designate type collections, lectotypification is necessary which for heterogeneous assemblages has great impact on species nomenclature. This
has been recently discussed for Peronospora on
Fabaceae (García Blázquez et al. 2008), but is true
also for other lineages of Peronospora and Plasmopara. Therefore, thorough investigations of types and
proper typification whenever necessary are tedious
but unavoidable prerequisites to receive correct and
stable circumscription for many species. In addition,
as shown above, numerous distinct species remained
undetected up to now, which require additional
thorough studies involving morphological as well as
molecular data. Even lineages containing important
plant pathogens such as the Plasmopara halstedii
group are in need of critical taxonomic and nomenclatural revision to reveal an appropriate species
delimitation.
Molecular bar coding systems for improved
and reliable species identification
Another important issue is the development of
methods for reliable and easy species identification.
As discussed above, morphology is often not the best
basis for identification due to a lack of morphological
distinctness of numerous genetically well distinct
lineages. In addition, all morphological structures
necessary for identification are not always present
on a specimen to be identified. Especially for the
plant pathology community, molecular methods are
much easier to use, and are nowadays routine
procedures, and provide reliable identification even
with the lack of morphological structures required for
identification (e.g. sporangiophores, oospores). Therefore, a species identification system based on sequence data is highly desirable. Numerous sequence
15
data are already available in sequence databases like
Genbank; however, these data usually suffer from the
lack of standards concerning correct identification,
documentation, nomenclature but also the sequence
data quality. Therefore, molecular bar coding initials
have been recently proposed and started for many
taxonomic groups of organisms to facilitate identification (see the publications of the themed issue of the
Philosophical Transactions of the Royal Society,
Biological Sciences 360, Number 1462, 2005).
Molecular bar coding requires strict quality standards
for the laboratory routine, the sequence data as well as
identification, documentation, specimen deposition
and nomenclature; for details see the homepage of
the Consortium for the Barcode of Life (http://
barcoding.si.edu/index.htm). Evidently, a bar coding
approach needs to be accompanied by thorough
taxonomic revisions to provide a proper taxonomic
framework.
Crucial for the resolution of a molecular bar coding
system is the selection of the sequence region used.
Ideally, species identification should be possible with
a single and the same sequence over a wide range of
organisms, at best with the same primers, which is
problematic for downy mildews. Therefore, a compromise between applicability for as many different
taxa as possible and sufficient taxonomic resolution
needs to be found. One possible candidate is the ITS
rDNA: it is a multicopy region easy to amplify in
most downy mildews, can detect pathogens with high
sensitivity, specific as well as universal primers are
available, and it is also a region of choice in
Phytophthora, the closest relative of downy mildews.
In addition, numerous data on ITS are already
available for downy mildews which is important for
testing the discriminatory power of the data. However, amplification and sequencing can be troublesome
in lineages affected by substantial size increases of the
ITS region (see above), limiting universal applicability. Alternatively, sequences from the mitochondrial
DNA should be considered. It is usually much easier
to amplify from historic material than nuclear DNA
due to a higher copy number, and therefore herbarium
collections can be used. A promising candidate for
bar coding is cox2, which offers high resolution in
some closely related species groups (e.g. Choi et al.
2006, 2007d), also mtDNA sequence stretches less
variable than cox2 should be considered, e.g. ribosomal mtDNA and cox1 or cox3. Additional detailed,
16
comparative studies are necessary before the most
appropriate sequence region can be selected. However, the development of a molecular bar coding system
is now within reach.
References
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S. (2008). A multi-locus phylogeny for Phytophthora
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DOI 10.1007/s10658-008-9305-2
Classical and molecular genetics of Bremia lactucae,
cause of lettuce downy mildew
Richard Michelmore & Joan Wong
Received: 30 November 2007 / Accepted: 3 March 2008 / Published online: 3 April 2008
# KNPV 2008
Abstract Lettuce downy mildew caused by Bremia
lactucae has long been a model for understanding
biotrophic oomycete–plant interactions. Initial research involved physiological and cytological studies
that have been reviewed earlier. This review provides
an overview of the genetic and molecular analyses
that have occurred in the past 25 years as well as
perspectives on future directions. The interaction
between B. lactucae and lettuce (Lactuca sativa) is
determined by an extensively characterized gene-forgene relationship. Resistance genes have been cloned
from L. sativa that encode proteins similar to
resistance proteins isolated from other plant species.
Avirulence genes have yet to be cloned from B.
lactucae, although candidate sequences have been
identified on the basis of motifs present in secreted
avirulence proteins characterized from other oomycetes. Bremia lactucae has a minimum of 7 or
8 chromosome pairs ranging in size from 3 to at least
8 Mb and a set of linear polymorphic molecules that
range in size between 0.3 and 1.6 Mb and are
inherited in a non-Mendelian manner. Several methods indicated the genome size of B. lactucae to be ca.
50 Mb, although this is probably an underestimate,
comprising approximately equal fractions of highly
R. Michelmore (*) : J. Wong
The Genome Center and Department of Plant Sciences,
University of California,
Davis, CA 95616, USA
e-mail: rwmichelmore@ucdavis.edu
repeated sequences, intermediate repeats, and lowcopy sequences. The genome of B. lactucae still
awaits sequencing. To date, several EST libraries have
been sequenced to provide an incomplete view of the
gene space. Bremia lactucae has yet to be transformed, but regulatory sequences from it form
components of transformation vectors used for other
oomycetes. Molecular technology has now advanced
to the point where rapid progress is likely in
determining the molecular basis of specificity, mating
type, and fungicide insensitivity.
Keywords Bremia lactucae . Lettuce . Virulence .
Resistance . Oomycete
Introduction
Bremia lactucae is an obligate oomycete pathogen
belonging to the Peronosporales. Members of the
Peronosporales exhibit a gradient in modes of
parasitism from saprotrophy through necrotrophy
and varying degrees of biotrophy (Ingram 1981;
Göker et al. 2007). Bremia lactucae represents one
of the most highly specialized downy mildews at the
biotrophic end of this spectrum. Like all members of
the Peronosporaceae, it is an obligate biotroph (i.e. it
can only currently be cultured in association with its
host). However, the asexual spore germinates directly
rather than via zoospores that are used by most other
members of the Peronosporaceae. Bremia lactucae also
20
directly penetrates through the plant cuticle and
epidermal cells rather than entering the leaf through
stomata. Both of these attributes indicate that it is one
of the most highly evolved downy mildews. Recent
molecular phylogenetics supports the advanced taxonomic position of B. lactucae (Voglmayr et al. 2004).
Bremia lactucae has a long history as a model for
understanding biotrophy in the Oomycetes (Maclean
et al. 1974; Andrews 1975; Ingram et al. 1976;
Maclean and Tommerup 1979; Ingram 1981; Woods
et al. 1988). Its biotrophic mode of nutrition involves a
close interaction with its host, in which the plant
plasmalemma is invaginated around simply lobed
haustoria. Compatible interactions result in minimal
macroscopic disturbance until sporulation. Although it
is an obligate pathogen, B. lactucae can readily be
cultured in the laboratory on lettuce seedlings; it is a
tractable genetic system and many of the necessary
tools for manipulating it in the laboratory in conjunction with its host (Lactuca spp.) have been developed.
The classical genetics of specificity in lettuce downy
mildew is one of the best understood of any gene-forgene plant–pathogen interaction. Simultaneous studies of host and pathogen showed that specificity is
determined by numerous gene-for-gene interactions
(Crute and Johnson 1976; Farrara and Michelmore
1987). The molecular determinants on the host side
are becoming increasingly well worked out (Meyers
et al. 1998a, b; Shen et al. 2002; Kuang et al. 2004).
However, the molecular biology of B. lactucae has
lagged behind.
Bremia lactucae causes lettuce downy mildew, the
most important disease affecting lettuce worldwide.
Lettuce ranks as one of the top ten most valuable
crops in the USA with an annual value of over $2.26
billion (US Department of Agriculture 2003, 2006).
Lettuce is grown as extensive monocultures, often
with several crops per year. Such intensive production
makes the crop susceptible to major epidemics and
lettuce suffers from several economically important
pests and diseases, particularly downy mildew. These
are currently controlled by a combination of genetic
resistance, cultural practices, and chemical protection
including the application of over 1.5 million pounds
of insecticides and fungicides per year (US Department of Agriculture 2003). Several of these compounds are being withdrawn from agricultural use due
to environmental concerns over their safety or have
been rendered ineffective by changes in B. lactucae
Eur J Plant Pathol (2008) 122:19–30
(Crute et al. 1987; Schettini et al. 1991; Brown et al.
2004). Breeding for resistance to B. lactucae is a
major activity of most lettuce improvement programmes, and there is an increasing need for
information and methods to accelerate the development of new disease-resistant cultivars. Downy
mildew resistance (Dm) genes provide high levels
of resistance but have only remained effective for
limited periods of time due to changes in pathogen
virulence. Much of the breeding effort is currently
focused on introgressing new genes from wild species
in response to pathogen changes. New strategies are
needed to provide more durable forms of resistance.
The purpose of this review is to summarize what is
now known of the classical and molecular genetics of
B. lactucae and its interaction with lettuce, as well as
to consider future developments that are imminent
due to the application of genomics approaches.
Classical genetics of resistance
The interaction between lettuce and B. lactucae is one
of the most extensively characterized gene-for-gene
plant–pathogen relationships (Crute and Johnson
1976; Farrara et al. 1987; Hulbert and Michelmore
1985; Michelmore et al. 1984; Norwood and Crute
1984; Norwood et al. 1983; Ilott et al. 1987, 1989).
The genetics of resistance has been facilitated by
simultaneous studies of avirulence. At least 27 major
Dm genes or resistance (R) factors are now known
that provide resistance against specific isolates of B.
lactucae in a gene-for-gene manner (Farrara et al.
1987; Bonnier et al. 1994; Maisonneuve et al. 1994;
Jeuken and Lindhout 2002). Many other sources of
resistance have been identified but have not yet been
extensively characterized genetically (e.g. Farrara and
Michelmore 1987; Gustafsson 1989; Bonnier et al.
1994; Lebeda and Zinkernagel 2003; Beharav et al.
2006). As more Dm genes are characterized from
these and other sources, it is likely that many hundred
Dm genes with specificity to B. lactucae will be
identified.
Most of the currently identified Dm genes confer
high levels of resistance. This may be a consequence
of these genes being the ones identified and used by
breeders. Some Dm genes, e.g. Dm6, confer incomplete resistance phenotypes (Crute and Norwood
1978). Partial phenotypes do not necessarily imply
Eur J Plant Pathol (2008) 122:19–30
quantitative inheritance or more durable resistance.
The phenotype of the interaction depends on the
gene and environment. Heterozygotes of some Dm
genes, e.g. Dm18, also confer incomplete resistance
(Maisonneuve et al. 1994). In addition, different
isolates of B. lactucae can exhibit different levels of
incompatibility to the same Dm gene (Ilott et al.
1989). At lower temperatures, resistance conferred
by several Dm genes becomes less effective;
temperature shift experiments suggested that the
determinants of specificity are present in most host
cells and expressed throughout pathogen development (Judelson and Michelmore 1992). There are
also resistance genes of minor effect that confer
incomplete or field resistance (Eenink et al. 1983;
Jeuken and Lindhout 2002). Many genes of minor
effect will probably be identified in the future by
quantitative trait locus (QTL) analysis using molecular markers.
The known Dm resistance phenotypes are located in
at least five clusters in the lettuce genome (Hulbert and
Michelmore 1985; Farrara et al. 1987; Bonnier et al.
1994). The major cluster contains over nine genetically
separable Dm specificities, as well as resistance to root
aphid. Another large cluster contains several Dm
genes, resistance to the root-infecting downy mildew
Plasmopara lactucae-radicis, and the hypersensitive
reaction to Turnip mosaic virus (Witsenboer et al.
1995).
Molecular genetics of resistance
One downy mildew resistance gene, Dm3, has been
cloned through a combination of map-based cloning
and candidate gene approaches (Shen et al. 1998,
2002; Meyers et al. 1998a). Dm3 encodes a nucleotide binding site and leucine-rich repeat (NBS-LRR)
protein, similar to genes cloned from other species for
resistance to downy mildews and other pathogens
(McHale et al. 2006). Dm3 is large, containing nearly
double the number of LRRs compared to proteins
characterized in other species. Dm3 is a member of
the large RGC2 (Resistance Gene Candidate2) multigene family that can vary in copy number from 12 to
over 30 (Meyers et al. 1998a, b; Kuang et al. 2004).
Sequence analysis of paralogues from several species
indicated that this large cluster evolves by a birth-anddeath mechanism (Michelmore and Meyers 1998;
21
Kuang et al. 2004). Genes in the RGC2 family exhibit
two distinct patterns of evolution. Type I genes are
extensive chimeras resulting from frequent sequence
exchange between paralogues, and individual genes
are rare in nature. Dm3 is a Type I gene and only
rarely present in nature (Kuang et al. 2006). Type II
genes occur more frequently in nature, and sequence
exchanges only rarely occur between individual
lineages (Kuang et al. 2004). Trans-specific polymorphism was observed for different groups of Type II
orthologues, suggesting balancing selection. Different
evolutionary forces have impacted different parts of
RGC2 genes. The RGC2 cluster is not highly
recombinogenic; it exhibits a recombination frequency 18 times lower than the genome-wide average
(Chin et al. 2001). This is consistent with reduced
pairing during meiosis between haplotypes due to
structural heterozygosity.
The meiotic spontaneous mutation rates differ
between the Dm genes (Chin et al. 2001). Spontaneous mutations in Dm1, Dm3 and Dm7 occurred at the
rate of 10−3 to 10−4 per generation. No spontaneous
mutations were detected for Dm5/8. Spontaneous
mutations at the Dm3 locus but not the Dm7 locus
were frequently associated with large deletions resulting from unequal crossing-over. One spontaneous loss
of Dm3 resistance was observed to be the result of a
gene conversion event between the LRR-encoding
regions of similar paralogues (Chin et al. 2001).
Given that a lettuce plant is capable of producing
several thousand seeds per generation, such mutation
rates suggest that in every generation an average of
one progeny with a novel haplotype at a resistance
locus is produced per plant.
PCR using degenerate oligonucleotides designed to
sequences encoding conserved NBS domains has
resulted in the identification of over 20 distinct
families of resistance gene candidates (RGCs; Shen
et al. 1998; McHale and Michelmore, unpublished).
These are being mapped relative to phenotypic
resistances to provide a comprehensive view of the
genomic distribution of resistance genes, including
many Dm genes.
The clustered genomic distribution of Dm genes
suggests that they are similar genes. This has been
confirmed for the major cluster of Dm genes. An
interfering hairpin RNA (ihpRNA) construct containing fragments encoding the LRR of Dm3 was used to
induce post-transcriptional gene silencing of the
22
RGC2 family (Wroblewski et al. 2007). This showed
that the resistance specificity encoded by the genetically defined Dm18 locus is the combination of two
resistance specificities, only one of which was
silenced by ihpRNA derived from Dm3. Analysis of
progeny from crosses between transgenic, silenced
tester stocks and lettuce accessions carrying other
resistance genes previously mapped to the RGC2
locus indicated that two additional resistance specificities to B. lactucae, Dm14 and Dm16, as well as
resistance to lettuce root aphid (Pemphigus bursarius),
Ra, are encoded by RGC2 family members. This
strategy is now being extended to other clusters of
resistance genes for which RGC sequences and
phenotypic resistances co-segregate.
Numerous haplotypes and homologues at the major
cluster of resistance genes that contains Dm3 have been
identified. Fifty-one different haplotypes were identified in 74 accessions studied using molecular markers
diagnostic of the RGC2 cluster (Sicard et al. 1999).
The copy number of RGC2 paralogues at a locus can
vary from 12 to >30 (Kuang et al. 2004). No
accessions have been observed that completely lack
RGC2 genes even though they do not carry detectable
Dm specificities. The large number of different
haplotypes is consistent with there being a minimum
of several hundred distinct Dm genes in Lactuca
species and indicates that wild germplasm will be a
rich source of new resistance genes that can be
introgressed and pyramided using molecular markers.
There is also a growing understanding of the
signalling pathways and downstream genes and
proteins that are involved in plant resistance (Jones
and Dangl 2006). However, there are little specific
data on genes involved downstream of Dm genes in
the interaction with B. lactucae. Homologues of genes
from other species known to be involved in pathogen
interactions are present in ESTs from Lactuca spp.
(http://compgenomics.ucdavis.edu), and therefore it is
likely that similar processes are involved in lettuce as
in other plants. Ultrastructural and biochemical
studies indicate that the hypersensitive response is
typical but includes the induction of phytoalexins characteristic of the Compositae (Maclean and Tommerup
1979; Bennett et al. 1996; Bestwick et al. 1998;
Lebeda et al. 2008). As the molecular understanding
of B. lactucae develops, it will be interesting to
determine how the pathogen has adapted to deal with
these defences.
Eur J Plant Pathol (2008) 122:19–30
Mating system
Bremia lactucae is diploid for the majority of its lifecycle and predominantly heterothallic (Michelmore and
Ingram 1980; Michelmore and Sansome 1982). Both
the asexual life-cycle of 1 to 3 weeks and the sexual
cycle of several months’ to many years’ duration can
be readily induced in the laboratory. The asexual cycle
allows the facile clonal propagation of individual
phenotypes. Its heterothallic nature allows controlled
crosses between isolates of known phenotypes for the
investigation of the genetics of (a)virulence.
When hyphae of opposite mating type come into
physical contact, asexual sporulation is suppressed,
clusters of gametangia are elaborated at the point of
contact, synchronous meioses occur in the oogonium
and periclinal antheridium, and haploid gametes are
transferred from the antheridium to the oogonium to
effect fertilization (Michelmore and Ingram 1981;
Michelmore and Sansome 1982). Each mating type
can probably produce both oogonia and antheridia, as
do Phytophthora species. Differences in maleness and
femaleness have not been investigated.
Heterothallism seems to be determined by two
haplotypes at a single locus, with the B1 compatibility
type being conferred by a homozygous recessive
condition and the B2 mating type by a heterozygous
condition. The two mating types segregate in approximately 1:1 ratios in sexual progeny (Michelmore and
Ingram 1981; Norwood et al. 1983; Michelmore et al.
1984; Sicard et al. 2003). However, the current data
do not preclude a more complicated situation such as
double heterozygotes and balanced lethals, as has
been proposed for Phytophthora infestans (Fabritius
and Judelson 1997). The molecular determinants of
mating type for B. lactucae await characterization as
they do for all oomycetes.
Some isolates exhibit secondary homothallism
(Michelmore and Ingram 1982). These isolates behave predominantly as B2 types in that they usually
reproduce asexually except when cultured in combination with B1 isolates, whereupon they produce
abundant oospores. However, they also produce
oospores at low frequency when cultured alone,
particularly at high inoculum densities. This is due
to the generation of B1 components at low frequency,
as shown by the isolation of stable B1 and B2 as well
as self-fertile derivatives by single–spore analysis
(Michelmore and Ingram 1982). This self-fertility
Eur J Plant Pathol (2008) 122:19–30
may be due to trisomy of the determinants of mating
type (Michelmore and Sansome 1982). Somatic
segregation of self-sterile lines from self-fertile
progenitors involves at least transitory heterokaryosis.
The prevalence of each mating type varies in
nature. Isolates of both mating types have been
frequently identified in Europe and New York State,
although the B 2 type sometimes predominated
(Michelmore and Ingram 1980; Lebeda and Blok
1990; Gustafsson et al. 1985; Yuen and Lorbeer 1987;
Petrželová and Lebeda 2003). This is consistent with
a sexually reproducing population and the high
diversity of virulence phenotypes observed. In contrast, the B2 mating type predominates in isolates
from cultivated lettuce in California; B1 isolates are
identified extremely rarely. In addition, the one B1
isolate analyzed from California had reduced fertility
(Ilott et al. 1987). The data for California isolates are
indicative of an asexual population that propagates
clonally. This is consistent with the more restricted
spectrum of virulence phenotypes observed and
widespread pathotypes that are stable from year to
year. However, even in the apparent absence of the
sexual cycle and the oospore as a survival stage, B.
lactucae has been able to change virulence phenotype
in response to the deployment of new Dm genes and it
is unclear how the pathogen survives crop-free
periods in California.
Genetics of avirulence
Several initial studies established that avirulence to
specific Dm genes was inherited as single dominant
unlinked loci (Michelmore and Ingram 1981; Norwood
et al. 1983; Norwood and Crute 1984; Michelmore et
al. 1984; Ilott et al. 1987). The gene-for-gene interaction between lettuce and B. lactucae was subsequently
analyzed critically, involving extensive crosses between 20 isolates of diverse worldwide geographical
origins to complement the simultaneous genetic analysis of resistance (Farrara et al. 1987; Ilott et al. 1989).
The majority of the data were consistent with the
underlying tenets of a gene-for-gene interaction.
Avirulence was usually determined by dominant alleles
at unlinked loci, although their expression could be
modified depending on the genetic background of the
host and pathogen. Some segregation anomalies could
be explained by hyperploidy and gene dosage effects. In
23
order to test for complementation between Avr loci, 125
tests involving 19 crosses were analyzed. In no case
were all progeny avirulent to a specific Dm gene when
both parental isolates had been virulent; therefore, there
was no evidence for complementation, indicating that
avirulence to individual Dm genes was conferred at the
same locus. To investigate the presence of dominant
inhibitors of avirulence, crosses were made between
avirulent and virulent isolates. The data for an inhibitor
locus epistatic to Avr5/8 were good but not unequivocal; there was no evidence for inhibitors of other Avr
loci (Ilott et al. 1989). Therefore, unlike the situation in
phytopathogenic bacteria (Abramovitch et al. 2003;
Espinosa et al. 2003; Jamir et al. 2004; Fu et al. 2007),
inhibitor loci do not seem to be common in B.
lactucae.
Genetic mapping
A preliminary genetic linkage map of B. lactucae was
constructed using the segregation of 53 RFLP loci,
8 Avr loci, and the mating type locus in a total of 70
F1 individuals from two crosses (Hulbert et al. 1988).
This map consisted of 13 small linkage groups,
including 35 RFLP loci and one Avr gene. However,
construction of a more detailed genetic map was
hindered by the ambiguous phase of the alleles in the
parents and an insufficient number of markers due to
the type of marker technology available at the time.
A more comprehensive genetic map of B. lactucae
was subsequently constructed using PCR-based
markers as well as additional RFLP loci (Sicard et
al. 2003). The more heterozygous of the two crosses
that had been used previously was expanded to 97 F1
progeny to facilitate the identification of the phase of
the parental alleles and to improve the detection of
linkage. Two parental maps and a consensus map
were constructed using a total of 347 AFLP and 83
RFLP markers, six Avr genes, and the mating-type
locus. One parental map contained 24 linkage groups
distributed over 835 cM; the second map contained
21 linkage groups distributed over 606 cM. The
consensus map contained 12 linkage groups with
markers from both maps and 24 parent-specific
groups.
There was no evidence for clustering of Avr genes.
All six mapped to different linkage groups. This is
consistent with the lack of linkage observed in
24
classical segregation analysis of 12 Avr loci (Ilott et
al. 1989). Also, the genetic data provided no evidence
for pathogenicity islands that have been identified in
bacteria (Alfano et al. 2000; Guttman et al. 2002;
Jackson et al. 1999; Sugio et al. 2005). Four Avr loci
were located at the ends of linkage groups. Telomeric
locations of Avr genes would be consistent with the
high instability of the avirulence phenotype in B.
lactucae. In the fungal pathogen Magnaporthe grisea,
four out of eight known Avr genes are close to a
telomere, and losses in avirulence were associated
with deletions (Mandel et al. 1997; Dioh et al. 2000).
Linkage of three Avr genes with distorted markers in
B. lactucae may be indicative of other mechanisms of
instability of Avr genes, such as high frequencies of
mitotic gene conversion as observed in P. sojae
(Chamnanpunt et al. 2001).
The current genetic map of B. lactucae is far from
saturated. Over 20% of the markers remain unlinked.
It is difficult to estimate the total number of
chromosomal groups and genetic genome size because of the possible redundancy between the parentspecific linkage groups. The mating type locus and
two Avr loci are flanked by molecular markers;
however, no close linkages have been identified.
The closest marker is 1 cM, and only loose linkages
have been identified for the majority of Avr genes.
Whether this represents a dearth of polymorphic lowcopy sequences or high rates of recombination close
to avirulence genes is unknown. We attempted bulked
segregant analysis (Michelmore et al. 1991) to
identify markers closely linked to several avirulence
genes; however, this was unsuccessful (Zungri and
Michelmore, unpublished).
Karyotype and chromosomal assignment
of markers
Cytological analysis of B. lactucae resolved at least 7
or 8 chromosome pairs at meiosis (Michelmore and
Sansome 1982). However, these chromosomes are too
small to be resolved clearly using conventional light
microscopy. Examination of isolates of diverse geographical origins as well as progeny from sexual
crosses by pulsed-field gel electrophoresis (PFGE)
revealed a minimum of seven chromosomes ranging
in size from 3 to at least 8 Mb and a set of linear
polymorphic molecules from 0.3 to 1.6 Mb (Francis
Eur J Plant Pathol (2008) 122:19–30
and Michelmore 1993). Genetic and hybridization
analyses confirmed the existence of two classes of
molecules.
The class of smaller molecules is sequence-related,
non-ribosomal, nuclear, highly polymorphic, variable
in number, and inherited in a non-Mendelian manner.
These small polymorphic molecules are therefore B
chromosomes or large liner plasmids. No RFLP
markers, and consequently none of the Avr genes,
were assigned to the small polymorphic 0.3–1.6 Mb
molecules. Therefore, there was no evidence that
these small variable molecules are involved in
variation in specificity of B. lactucae.
The second class of molecules is larger than 2 Mb,
is more constant in size and number and represents
the true chromosomes. A total of 25 probes were
successfully hybridized to these chromosomes (Sicard
et al. 2003). Of these, 23 had been mapped and
represented 16 of the linkage groups in the consensus
map; two were unlinked. This resulted in two
consensus linkage groups and seven parent-specific
linkage groups being assigned to chromosomes.
Linkage to RFLP markers allowed three Avr loci also
to be assigned to chromosomes. The mating–type
locus could not be assigned to any chromosome-sized
molecule. Together the genetic and physical data
suggest that there are at least 10 chromosomes in B.
lactucae.
Somatic variation
Bremia lactucae can exhibit somatic variation in
addition to the segregation of phenotypes following
sexual reproduction. RFLP analysis of 25 isolates
from diverse worldwide geographical origins revealed
different ploidy levels and somatic variants (Hulbert
and Michelmore 1987). Most European isolates were
clearly diploid. They were heterozygous at approximately 44% of their loci and had highly variable
genotypes consistent with the frequent occurrence of
the sexual cycle. In contrast, many of the isolates
from Australia, Japan, Wisconsin and Australia had
more than two alleles at multiple loci, indicating
that they were either polyploids or stable heterokaryons (hyperploid). Variation between similar
sympatric isolates indicated that they had arisen
by the somatic loss of alleles. One hyperploid
California isolate had resulted from the fusion of
Eur J Plant Pathol (2008) 122:19–30
two diploid California isolates of the same mating
type, providing the first evidence for natural somatic
fusion in the Oomycetes.
Several phenotypic changes in B. lactucae seem to
have resulted from somatic changes. The segregation
of self-sterile lines in secondary homothallic isolates
is one example (Michelmore and Ingram 1982).
Fungicide insensitivity seems to have arisen in the
most common virulence phenotype, rather than
involving sexual progeny (Crute et al. 1987; Schettini
et al. 1991; Brown et al. 2004). Recent changes in
virulence phenotype in California seem also to be
somatic (Ilott et al. 1987; Ochoa and Michelmore,
unpublished). The molecular genetic changes underlying these changes are unknown, but they are
becoming amenable to analysis with the advent of
technologies for whole genome analysis.
Genome size and complexity
The physical genome size of B. lactucae has been
estimated using several methods: comparisons of
hybridizations between cloned DNA fragments and
genomic DNA in dot blot reconstructions, DNA–DNA
reassociation kinetics assayed by hydroxyapatite chromatography, and summation of chromosomal sizes
determined by CHEF gel electrophoresis (Francis et
al. 1990; Francis and Michelmore 1993). All three
methods gave similar estimates of 50 Mb; however,
this may be an underestimate. Aspergillus nidulans and
Arabidopsis thaliana were used as controls in the
genomic reconstruction experiments and their sizes
were estimated to be 17 and 52 Mb, respectively;
genomic sequencing has now shown their genome
sizes to be 30 and 125 Mb, respectively (Galagan et al.
2005; The Arabidopsis Genome Initiative 2000).
Therefore the estimate for the genome size of B.
lactucae should probably be revised upward to
approximately 100 Mb. This is consistent with
estimates of 70 to 144 Mb, depending on the isolate,
measured by Feulgen absorbance cytophotometry
(Voglmayr and Greilhuber 1998). This size is comparable to that of Phytophthora species that range from
65 Mb for P. capsici to 240 Mb for P. infestans as well
as similar to Hyaloperonospora parasitica (75 Mb;
Govers and Gijzen 2006). Only sequencing the entire
genome of B. lactucae will provide an accurate
determination of its genome size.
25
DNA reassociation kinetics indicated that the
nuclear DNA of B. lactucae is comprised of approximately 65% repeated sequences and 35% low-copy
sequences (Francis et al. 1990). The repeat fraction is
made up of approximately 21% high-copy sequences
and 38% intermediate-copy sequences. Hybridization
analysis of random genomic 1 clones demonstrated
that the low-copy-number sequences are interspersed
with repeated sequences.
Regulatory sequences for transformation
of B. lactucae and other oomycetes
Transformation of B. lactucae has yet to be
achieved. Early work towards this goal involved
the isolation of regulatory sequences from B.
lactucae. These included the promoters and terminators from Hsp70 and a constitutively highly
expressed single-copy gene, HAM34 (Judelson and
Michelmore 1989, 1990). Although there was evidence for transient expression, no stable transformants of B. lactucae were obtained. Efforts were
therefore directed towards transformation of culturable oomycetes including P. infestans (Judelson and
Michelmore 1991). These studies ultimately resulted
in the stable transformation of several Phytophthora
species using vectors originally developed for B.
lactucae (Judelson et al. 1991, 1993). The function
of HAM34 is still unknown; it is present in P.
infestans (Win et al. 2005) but not yet evident in the
sequence of H. parasitica.
These experiments indicated that the transcriptional machinery of oomycetes differs significantly from
that of higher fungi but that sufficient similarity exists
so vectors developed using regulatory sequences from
one oomycete will likely function in other oomycetes
(Judelson et al. 1992). It is now time to reinitiate
experiments on the transformation of B. lactucae
using better selectable markers and reporter genes that
have become available, as well as novel methods for
introducing the transgenes.
(A)virulence effectors
Pathogens have evolved sophisticated mechanisms to
alter their hosts’ metabolism and interfere with host
defences (Jones and Dangl 2006). This is best
26
understood for Gram-negative bacteria that secrete
virulence effector proteins into host cells and the
extracellular space (Nomura et al. 2005). Some
effectors can trigger defences dependent on specific
resistance genes. Some can also block the resistance
response elicited by the activities of other effectors
(Abramovitch et al. 2003; Espinosa et al. 2003; Jamir
et al. 2004; Fu et al. 2007). Such effectors exhibit a
dominant inhibitor of avirulence phenotype. The
recent availabilities of sequenced genomes of phytopathogenic bacteria, bioinformatic tools, and efficient
functional screens have resulted in the identification
of numerous genes encoding candidate effectors (e.g.
Guttman et al. 2002; Petnicki-Ocwieja et al. 2002;
Greenberg and Vinatzer 2003; Chang et al. 2005). It is
now recognized that individual strains of phytopathogenic bacteria secrete ~40 effectors into their hosts.
Functional studies and the sequences of several
effectors suggest that they alter plant defence signalling (reviewed in Grant et al. 2006).
There is increasing evidence that fungi and oomycete pathogens also secrete diverse effector proteins
into their hosts (Torto et al. 2003; Birch et al. 2006;
Kamoun 2006). Initially avirulence genes have been
cloned from Phytophthora spp. and H. parasitica on a
gene-by-gene basis (Tyler 2002; MacGregor et al.
2002; Shan et al. 2004; Allen et al. 2004; Rehmany et
al. 2005; Armstrong et al. 2005). Recent studies of
avirulence and secreted proteins from H. parasitica and
Phytophthora spp. revealed a novel, highly conserved
RXLR amino acid motif (Rehmany et al. 2005). This
motif is predicted to be required for translocation from
the pathogen to the host (Bhattacharjee et al. 2006) and
it was recently shown to be required for translocation
of the avirulence protein Avr3a by P. infestans
(Whisson et al. 2007). Bioinformatic analyses have
identified hundreds of genes encoding other potentially
secreted proteins in the genome sequences of Phytophthora spp. (Birch et al. 2006; Tyler et al. 2006).
In order to identify (a)virulence effector proteins in
B. lactucae, we have generated several cDNA
libraries of B. lactucae from a variety of sources
including conidia, germlings and infected tissue. One
subtraction library was made by subtracting mockinoculated leaf material against heavily infected leaf
material. The resulting sequences had a bimodal
distribution of GC contents. On the basis of GC
content and (dis)similarity to plant or oomycete
sequences, sequences were categorized as most likely
Eur J Plant Pathol (2008) 122:19–30
to be of B. lactucae origin (38%), lettuce origin
(35%), or uncertain origin (27%). Many of the
putative B. lactucae unigenes had an average GC
content of 50%. In order to obtain more full-length
clones, we generated and sequenced a new library that
was enriched for B. lactucae sequences by hybridizing cDNA from heavily infected leaves to B. lactucae
genomic DNA using a protocol developed by J. Jones
(Sainsbury Laboratory, Norwich, UK). Sequences
from all libraries are being analyzed for candidate
effectors using several bioinformatics approaches. We
are searching for sequence similarity to genes encoding known avirulence proteins and putative secreted
proteins from other oomycetes. Candidate effector
sequences have yet to be identified; however, this is
not surprising as effector proteins may be evolving
rapidly. We are also searching for the presence of a
secretion signal peptide and the RXLR amino acid
motif. These analyses have so far yielded over 15
candidate sequences that satisfied one or more of
these criteria. These are currently being assayed for
function in lettuce using Agrobacterium-mediated
transient assays (Wroblewski et al. 2005).
The impact of genomic sequencing
Although B. lactucae was ranked as one of the highpriority plant pathogens targeted for sequencing since
2002 (American Phytopathological Society 2006),
this has yet to occur. The latest generation of
sequencing technologies combined with conventional
Sanger sequencing will provide large amounts of
sequence information for B. lactucae. Sequencing the
whole genome will provide an expedient and costefficient approach to the identification of effector
proteins and other types of molecules involved in
determining specificity and mating type. It will also
provide targets for disease control strategies as well as
provide an important reference genome.
Sequencing of multiple isolates will provide large
numbers of single nucleotide polymorphisms that,
combined with the new generation of marker technologies, will allow large-scale population analyses
for variation in both effector genes and genes
involved in other aspects of the pathogen’s biology.
It is likely that these whole-genome analyses will
reveal a variety of mechanisms of variation. It will be
particularly interesting to determine the basis of
Eur J Plant Pathol (2008) 122:19–30
insensitivity to the fungicides metalaxyl (Ridomil)
and fosetyl A (Alliette), as well as the bases for
changes in virulence phenotype.
Sequencing the genome will also provide insights
into what extent the genome of B. lactucae has
become streamlined in parallel with its total dependence on its host. Also, it will facilitate the identification of which biosynthetic capabilities appear to be
lacking and therefore can be supplemented in media
for axenic culture.
Acknowledgements The work described here has been the
result of many people’s efforts spread over the past 25 years.
We thank them all for their contributions. Financial support has
come from numerous sources including sustained support from
the California Lettuce Research Board and the USDA CREES
National Research Initiative.
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DOI 10.1007/s10658-008-9296-z
Species hybrids in the genus Phytophthora with emphasis
on the alder pathogen Phytophthora alni: a review
Tibor Érsek & Zoltán Á. Nagy
Received: 7 August 2007 / Accepted: 22 February 2008
# KNPV 2008
Abstract This review provides a summary of recent
examples of interspecific hybridisation within the
oomycetous genus Phytophthora. Species hybrids
either created in the laboratory or evolved in natural
environments are discussed in association with evolutionary issues and possible threats they may pose to
agriculture, horticulture and forestry. It is suggested
that sustainable control of such hybrids will depend
on the better understanding of temporal and spatial
aspects of genetic mechanisms and environmental
factors that lead to the hybridisation process and thus
the genetic diversity in Phytophthora populations.
Keywords Evolution . Interspecific hybridisation .
Oomycetes
Introduction
Hybridisation between individuals from two populations has long been known in the plant kingdom, but
T. Érsek (*)
Faculty of Agricultural and Food Sciences,
Department of Plant Protection,
University of West Hungary,
Vár 2, 9200 Mosonmagyaróvár, Hungary
e-mail: ters@mtk.nyme.hu
Z. Á. Nagy
Department of Plant Pathology, Plant Protection Institute,
Hungarian Academy of Sciences,
P.O. Box 102, 1525 Budapest, Hungary
its occurrence among populations of eukaryotic
microorganisms has received delayed recognition.
Indeed, eukaryotic microorganisms, such as fungi
and oomycetes, possess a variety of reproductive
mechanisms whereby they might undergo interspecific
genetic exchange. As a consequence of the combination of two distinct genomes via sexual or parasexual
processes, new allopolyploid hybrid species may
evolve.
The possibility of hybridisation between two closely
related species of plant pathogenic fungi or oomycetes
has been considered for many years. For instance, seven
decades ago Flor (1932) pointed out the potential for
hybridisation among fungi based on the appearance of
isolates of Tilletia with atypical morphological phenotypes. Decades later, Burdon et al. (1981) used
isozyme analysis to confirm that a rust virulent on
rough wheat grass and barley evolved via somatic
hybridisation between rye stem rust and wheat stem
rust i.e., between two formae speciales (f.sp.) of
Puccinia graminis. Conclusive proof of species hybridisation has been provided only recently with the arrival
of modern molecular genetic tools. Since the mid
1990s, a limited number of phytopathogenic species
hybrids have been detected in the fungal phyla,
Ascomycota and Basidiomycota (Brasier 2000; Olson
and Stenlid 2002; Schardl and Craven 2003).
Within the Oomycota in the Kingdom Staminipila
(Dick 2002), efforts to establish the occurrence of
species hybridisation have focused on the genus
Phytophthora. This genus contains approximately 80
32
species, including the infamous late blight pathogen,
P. infestans. These species are primarily soil-borne
pathogens that affect a wide range of crop plants,
shrubs and trees throughout the world. Although
fungus-like in appearance, phytophthoras differ fundamentally from true fungi in terms of cell wall
composition, reproductive biology and genetics (Erwin
and Ribeiro 1996). In this review we summarise the
evidence for species hybridisation in this important
genus. Evidence for hybridisation as a source of
genetic variability has derived from both laboratory
attempts to create hybrids, and from studies of
Phytophthora hybrids found in either natural or
agro-ecosystems.
Hybrids created in the laboratory
Species hybrid formation in nature is likely to be a
rare event and, consequently, it is difficult to detect
and study. A tractable approach to the study of the
possibility of genetic exchange and evolution derived
from species hybridisation would be to create such
organisms artificially.
Sexual crosses
An early report of this approach was by Boccas
(1981), who induced sexual crosses among isolates of
numerous heterothallic (outcrossing) Phytophthora
species. This first effort was minimally successful
and produced only one putative species hybrid among
220 progeny derived from several species crosses.
Subsequent attempts to induce and confirm species
hybridisation in other laboratories were more successful. For instance, Goodwin and Fry (1994) induced
sexual crosses of the sympatric, heterothallic species,
P. mirabilis and P. infestans. They confirmed that 79
out of 86 progeny were species hybrids based on
DNA fingerprinting and isozyme analyses. Notably,
mitochondrial DNA was uniparentally inherited,
predominantly from the P. infestans parental isolates.
Interestingly, most of these hybrids lost their ability to
attack hosts of either parental species, including
Mirabilis jalapa, the host for P. mirabilis, and potato
or tomato, common hosts for P. infestans. May et al.
(2003) more recently induced sexual crosses between
the homothallic (self-fertile) species, P. sojae and P.
vignae. They confirmed the hybrid nature of offspring
Eur J Plant Pathol (2008) 122:31–39
by RAPD and AFLP analyses. They also noted that
both of the tested F1 hybrids were pathogenic to
soybean, the host for P. sojae, and cowpea, the host
for P. vignae. However, the aggressiveness of these
hybrids was reduced and was substantially more
variable when compared to the parental isolates on
their respective hosts.
Somatic hybridisations
Somatic fusion has also been suggested as a mechanism for hybridisation in nature among Phytophthora
species that are heterothallic and that temporarily or
spatially lack compatible mating types (Brasier 1992;
Érsek et al. 1995). Although protoplast fusion
between strains of a Phytophthora species was
relatively easy to induce, the same method appeared
to be insufficient for the creation of interspecific
hybrids between P. sojae (syn.: P. megasperma f.sp.
glycinea) and P. medicaginis (syn.: P. megasperma f.
sp. medicaginis) (Layton and Kuhn 1988) or between
P. nicotianae (syn.: P. parasitica) and P. capsici (Gu
and Ko 2000). The first evidence of the formation of
such hybrids was obtained by the induced fusion of
uninucleate zoospores (Fig. 1) derived from noncompatible mating-type isolates of the closely related
heterothallic species, P. capsici and P. nicotianae
(Érsek et al. 1995). In these laboratory experiments,
the morphologies of the four resultant hybrid isolates
resembled P. capsici more closely than P. nicotianae.
All of the hybrids were pathogenic to tomato, a plant
Fig. 1 Nuclear status of regenerating cells stained with DAPI
following induced fusion of zoospores of Phytophthora capsici
and P. nicotianae, as viewed by epifluorescence microscopy.
Note enlarged cells and/or nuclei and multiple nuclei as
compared to a uninucleate cell of normal size (arrow). Bar:
10 µm
Eur J Plant Pathol (2008) 122:31–39
susceptible to both parental species. However, two of
the hybrid isolates exhibited an expanded host range
that included both radish and lemon, hosts that are
susceptible to P. capsici or P. nicotianae, respectively.
The hybrid nature of the fusion offspring was
confirmed by detection of DNA sequences specific
to each parental species. In these hybrids repetitive
DNA of P. capsici was detected readily by hybridisation with a species-specific DNA probe, whereas P.
nicotianae-specific DNA was revealed after PCR
amplification of DNA from hybrids using P. nicotianae-specific primers or random primers (Érsek et al.
1995; English et al. 1999). Bakonyi et al. (2002) also
induced zoospore fusion to create hybrids from the
morphologically distinct species, P. nicotianae and P.
infestans. Resultant fusion offspring were more
similar to P. nicotianae than to P. infestans on the
basis of morphological and molecular evidence.
Again, these hybrids expressed modified pathogenicity traits compared to parental species.
As a final example of the tractability of somatic
fusion, Érsek et al. (1997) created tri-parental hybrids
derived from P. capsici, P. nicotiane and P. citrophthora. In these studies, zoospore fusion offspring
contained DNA from each parental species; however,
all offspring failed to express pathogenicity to any of
the hosts susceptible to the parental species. Notwithstanding the failure of protoplast fusion to create
hybrids between P. nicotianae and P. capsici, Gu and
Ko (2000) successfully generated hybrids by transfer
of isolated nuclei from one species to the other.
Analysis of zoospore progeny of these nuclear
hybrids suggested the completion of events leading
to a parasexual cycle.
Studies on interspecific zoospore fusion and
nuclear transfer support the suggestion of Brasier
(1992) that non-sexual genetic exchange might
generate variability in pathogenicity or virulence
within pathogen populations, particularly when complementary mating types needed for sexual reproduction are lacking. Artificially induced hybrids also
suggest that it may be difficult to predict the effects of
hybridisation on pathogen survival and dominance
among populations in nature. With the development
of molecular and biochemical markers, however, there
have recently been noteworthy findings of naturally
occurring Phytophthora species hybrids that may
provide further insight into the mechanism of interspecific genetic exchange.
33
Naturally formed hybrids
Phytophthora alni
The potential for hybrid formation among Phytophthora species that has been established in laboratory
studies has, in recent years, been confirmed by the
detection of true or putative hybrids in natural and
agro-ecosystems. One of the best-studied examples of
Phytophthora species hybridisation in nature is that of
P. alni, a newly recognised pathogen of alder (Alnus
spp.). This pathogen was first discovered on dying
alders in southern Britain in the beginning of the
1990s, and it has since been found throughout
Europe, including Hungary (Brasier et al. 1995;
Szabó et al. 2000; Streito 2003). Phytophthora alni
killed approximately 10% of the alders in southern
Britain within a few years of its initial discovery
(Brasier et al. 1995; Gibbs et al. 1999). Recent
surveys indicate that the disease is even more severe
in riparian ecosystems in north-eastern France (Streito
et al. 2002) and in Bavaria (Jung and Blaschke 2004).
Initial studies showed that certain isolates of this
new pathogen of alder resembled P. cambivora. The
similarity to P. cambivora was notable through the
morphology of the gametangia (Brasier et al. 1995).
However, the new pathogen differed from P. cambivora in several other traits, for instance, in being
homothallic rather than heterothallic and in exhibiting
an extremely high level of zygotic abortion. These
properties, in addition to assessments of internal
transcribed spacer (ITS) sequences and genomic
polymorphisms, ultimately suggested that the alder
pathogen might be a hybrid of two species, the
heterothallic P. cambivora and a homothallic P.
fragariae-like species (Brasier et al. 1999). Neither
of these organisms is a known pathogen of alder.
Ultimately, the alder Phytophthora was formally
designated by Brasier et al. (2004) as a new species,
P. alni Brasier & S.A. Kirk.
Because the newly defined species comprises a
range of phenotypically diverse allopolyploid genotypes, P. alni was split into three subspecies, P. alni
ssp. alni (Paa), P. alni ssp. uniformis (Pau) and P.
alni ssp. multiformis (Pam) (Brasier et al. 2004). In
addition to the three subspecies, a series of emerging
variant types of P. alni have recently been recovered
(Brasier et al. 2004; Jung and Blaschke 2004). Prior
to their designation as subspecies, isolates of Paa and
34
Pau were termed standard types and Swedish variants, respectively, whereas Pam, including divergent
hybrid types, were considered to be Dutch, German
and UK variants of the pathogen (Brasier et al. 1999).
Paa occurs more commonly across much of
Europe, and isolates of this form are generally more
aggressive than those of the other two subspecies that
are also present in several countries (Brasier and Kirk
2001). Furthermore, Paa produces P. cambivora-like
ornamented oogonia and elongated two-celled antheridia. In contrast, the other two subspecies exhibit
unique reproductive structures. Isolates of Pam
produce oogonia that are typically ornamented, but
antheridia and gametangial fusions may vary in
morphology. Pau uniformly forms oogonia with a
smooth surface under ordinary conditions but develops Paa-like ornamented female organs when grown
at sub-optimal temperatures, i.e. ≤15°C, thus indicating that morphology-based differentiation of the two
subspecies might fail under variable conditions
(Fig. 2).
As opposed to typical Phytophthora species, which
are diploid (2n) organisms, the standard type isolate of
Paa was determined by acetoorcein staining to be an
approximate tetraploid (∼4n), having a chromosome
number of ca. 18–22 at the first metaphase division.
This isolate, however, is unable to complete meiosis
(Brasier et al. 1999). In contrast, ploidy levels of Pau
and Pam are intermediate between diploid and tetraploid and range from 2n+2 to 2n+4–7, respectively.
In addition to differences in ploidy, subspecies of
P. alni differ in details of molecular features. For
instance, Paa has dimorphic sites in the ITS region of
its rDNA genes, in which DNA sequences are
representative of two species, P. cambivora and a P.
fragariae-like species. In contrast with Paa, ITS
Fig. 2 Scanning electron
micrographs of the surface
of oogonia of Phytophthora
alni subsp. uniformis grown
at optimal temperature (a),
sub-optimal temperature (b)
and of P. alni subsp. alni
(c). Note the environmentdependent change in morphology of P. alni subsp.
uniformis. Bars: 20 µm
Eur J Plant Pathol (2008) 122:31–39
sequences in both Pam and Pau are homogeneous
and resemble the ITS sequences of either P. fragariae
or P. cambivora, respectively (Brasier et al. 1999,
2004). Subspecies of P. alni also differ on the basis of
AFLP profiles (Brasier et al. 1999), RAPD or
isozyme patterns (Nagy et al. 2003; Brasier et al.
2004) and diagnostic P. alni-specific PCR primer sets
(Table 1). A firm correspondence, as shown in Table 2,
has also been established between PCR markers and
expression patterns for glucose-phosphate isomerase
(Gpi) and malate dehydrogenase (Mdh) (Bakonyi et al.
2007). Since both isozymes are known to be nuclearencoded, this suggests that the above-mentioned PCRtargeted DNAs that differentiate the subspecies are
likely to be of nuclear origin.
Mitochondrial (mt) genomic variability within P.
alni has been examined to only a limited extent. In
studies by Nagy et al. (2003), RFLP analyses of
mtDNA showed several bands that co-migrated
between P. alni isolates and either P. cambivora or
P. fragariae. However, it was not clear whether the
appearance of such bands refers to the presence of
biparental mtDNA fragments in hybrid isolates or
whether it reflects intraspecific variation in the mt
genome of either parental species. Based on sexual
crosses within individual Phytophthora species, it has
been suggested that the mitochondrial genome is
transmitted uniparentally through the maternal line,
whereas the nuclear genome is inherited from both the
maternal and paternal lines (Förster and Coffey 1991;
Whittaker et al. 1994).
The comparatively meagre knowledge about the mt
genome of P. alni has been broadened recently. Ioos
et al. (2006) performed phylogenetic analysis of the
mt genes, cox1 and nadh1, that revealed that mtDNA
sequences from either P. cambivora or P. fragariae
Eur J Plant Pathol (2008) 122:31–39
35
Table 1 PCR primer pairs developed for specific detection of Phytophthora alni
Primer name
Sequence (5′–3′)
Amplicon size (bo)
Specificitya
Reference
SAP1
SAP2
SWAP1
SWAP2
PA-F
PA-R
PAM-F
PAM-R
PAU-F
PAU-R
D16F
D16R
GGC ACT GAG GGT TCC TC
GGC ACT GAG GTC TAG ATT
TGG CCC TCA CAT TAA AAC TGC TGC
GGC CCT CAC CAA ATG CGA AAT GA
GGT GAT CAG GGG AAT ATG TG
ATG TCG GAG TGT TTC CCA AG
CTG ACC AGC CCC TTA TTG GC
CTG ACC AGC CAT CCC ACA TG
GAG GAT CCC TAA CAC TGA ATG G
GAT CCC TGG TTG AAG CTG AG
AGG GCG TAA GGG TGC GAA ATA
AGG GCG TAA GCC TGG ACC G
930
Paa, Pam
Bakonyi et al. (2006)
1130
Pau, Paa
450
Paa, Pau, Pam
590
Paa, Pam
750
Pau, Paa
366
Paa, Pau
a
Ioos et al. (2005)
De Merlier et al. (2005)
Paa, Pau and Pam are for Phytophthora alni subsp. alni subsp. uniformis subsp. multiformis, respectively.
did not cluster with those from the hybrid isolates,
likely to be the result of uniparental inheritance of the
mt genome. Furthermore, mtDNA sequences of P.
alni isolates from the three subspecies clustered into
only two groups, one that included Paa and Pam, and
the other, Pau. Surprisingly, the mtDNA profiles of
certain isolates that had been identified as Paa using
morphological and nuclear markers were identical to
those of Pau isolates (Ioos et al. 2006; Bakonyi et al.
2007). Such isolates may represent additional hybrid
forms that encompass the nuclear type of Paa and a
mitotype represented by Pau (Table 2).
The complexity of the nuclear and mitochondrial
genomes of Paa, Pau, and Pam suggests that P. alni
may be a species in a state of continuing evolution.
As compared to its putative parental species, P. alni
has exploited a new host (Brasier and Kirk 2001). The
source of the parental species is not clear, since P.
cambivora and P. fragariare are believed to be exotic
to Europe. The evolutionary mechanism leading to
the formation of subspecies of P. alni is also
uncertain. Based on cytological and molecular analyses, Brasier et al. (1999) favoured the view that Paa
could have arisen via somatic fusion followed by
further segregation, rather than via a sexual cross
between P. cambivora and a P. fragariae-like species.
These authors further suggested that Pau and Pam
might have then evolved through subsequent recombination events and chromosome losses in Paa that
led to reversions towards the P. cambivora-like or P.
fragariae-like parental genotypes.
Recently, Ioos et al. (2006) proposed an alternative
evolutionary model by which Paa might have arisen
via hybridisation of Pam and Pau. They suggested
Table 2 Patterns of nuclear and mithochondrial traits in subspecies of Phytophthora alni according to the results of Bakonyi et al.
(2006, 2007)
Marker type
Pam (M)a
Pau (U)
Paa (A)
PCR with primer set SAP1/SAP2
PCR with primer set SWAP1/SWAP2
RAPDs with primer OPG-02 or OPG-05
Isozyme locus Mdh-1
Isozyme locus Mdh-2
Isozyme locus Gpi
mtDNA-RFLP with MspI or HaeIII
+b
−
M
91/100
94/94
85/100
M (=A)
−
+
U
83/83
100/100
93/93
U
+
+
M+U
83/91/100
94/100
85/93/100
A (=M) or U
a
Pam (M) Pau (U) Paa (A) are for Phytophthora alni subsp. multiformis, subsp. uniformis and subsp. alni, respectively.
b
Specific amplicon is produced (+) or not produced (−).
36
that Pau might have evolved from P. cambivora,
whereas Pam might have either been generated itself
by an ancient reticulation or by autopolyploidisation.
Their hypotheses were based on analyses of a large
European-wide collection of P. alni isolates showing
that Paa possessed three alleles for each of four
nuclear genes studied, two of which were also present
in Pam, and a third one that matched a single allele in
Pau. Furthermore, the Paa isolates displayed a
mtDNA RFLP pattern identical to isolates of either
Pam or Pau, implying uniparental inheritance of the
mt genome in the suspected hybridisation process.
These results are supported by data of Bakonyi et al.
(2007) who found that the studied nuclear-encoded
traits expressed in Paa included combined expression
profiles of Pam and Pau, whereas mtDNA restriction
profiles of Paa matched that of either Pam or Pau
(Table 2).
Isolates of Pam and Pau have been recovered from
alder lesions far less frequently than have isolates of
Paa, and they have also proven to be significantly
less aggressive in colonising alder bark (Brasier and
Kirk 2001). On the basis of these observations,
Bakonyi et al. (2007) suggested that the emergence
of atypical Paa isolates with a Pau mitotype might
have occurred in bark tissue co-colonised by Paa and
Pau. In this niche, Paa and Pau isolates might have
hybridised by either somatic or gametangial interaction. In this scenario, Bakonyi et al. (2007) also
suggested the possibility that these atypical Paa
isolates may have arisen through the introgression of
mitochondria from Pau into the nuclear background
of Paa. Interactions like these must be very rare in
nature, and indeed, there has been only one such
report, in association with the causal agents of Dutch
elm disease. According to Bates et al. (1993), certain
Ophiostoma novo-ulmi isolates exhibited typical O.
novo-ulmi nuclear DNA profiles, but they also
exhibited O. ulmi-like mtDNA patterns. They attributed these patterns to somatic fusion between the two
related species.
The hybridisation event that led to the emergence of P.
alni is believed to be recent, and it may have occurred
in a European nursery, perhaps on raspberry or another
host that is common to the putative parental species
(Brasier et al. 1999; Brasier and Jung 2003). It is
assumed that P. alni arrived in Britain, the country of
first record, as a result of commercial trade of colonised
plant material. Its subsequent spread over long distances
Eur J Plant Pathol (2008) 122:31–39
is likely to have occurred via distribution and planting
of infested nursery stock (Brasier et al. 1999).
Local spread from points of P. alni introduction is not
likely to be related to the movement of oospores, since
these structures have poor survival ability in soil (Delcan
and Brasier 2001). More likely, zoospores and plant
debris containing mycelium contribute to pathogen
movement at this scale. Alders are key trees in wetlands
and riparian environments, where they stabilise river and
stream banks. In these habitats, the presence of saturated
or flooded soils, and water movement, would enhance
spread of zoospores and debris. This scenario is
supported by observations of higher disease incidence
among alders growing near rivers than those some
distance away (Gibbs et al. 1999).
Phytophthora cactorum × P. nicotianae
Species hybridisation has also been reported in
hydroponic greenhouse systems in The Netherlands.
Under such circumstances novel Phytophthora diseases have appeared on diverse ornamental species.
For instance, Man in’t Veld et al. (1998) reported the
isolation of Phytophthora that differed morphologically from known pathogenic species from Spathiphyllum and Primula plants. Isozyme and RAPD
analyses revealed that the unusual isolates represented hybrids of P. nicotianae and P. cactorum. In
addition, mtDNA restriction patterns of the hybrid
isolates were identical to those of P. nicotianae (Man
in’t Veld et al. 1998). Phytophthora nicotianae is an
introduced species in The Netherlands, and it can
infect both Spathiphyllum and Primula. In contrast,
P. cactorum is a resident species, but it does not
cause disease on these host plants. Additional hybrid
isolates were obtained from a Cyclamen sp., which is
not known to be a host of either of the parental
species (Bonants et al. 2000). Subsequent analysis of
the ITS region of rDNA and AFLP analyses
provided further evidence of the biparental origin
of the recovered isolates.
Similar hybrids have been characterised recently
from loquat trees (Eriobotrya japonica) grown in
orchards in central Taiwan (Man in’t Veld 2001). The
unlikely movement of hybrid isolates between such
distinctly different and separated agricultural and
horticultural systems suggests a potential for hybridisation between P. cactorum and P. nicotianae when
both species occupy the same habitat.
Eur J Plant Pathol (2008) 122:31–39
Phytophthora cactorum × P. hedraiandra
Man in’t Veld et al. (2007) recently reported the
involvement of P. cactorum in the formation of yet
another hybrid species after hybridisation with P.
hedraiandra. These new hybrids were shown to be
heterozygous at the dimeric malic enzyme (Mdhp)
locus, possessing the MDHP alleles of the two
parental species. They also contained dimorphic sites
in the ITS region, exactly at those positions where the
parental sequences differ. Consistent with the hybrid
hypothesis, most hybrid isolates contained the mitochondrial-encoded cytochrome oxidase I (Cox I) gene
sequences that were identical to those of P. hedraiandra, and one isolate had the gene sequences of the
other putative parent.
Phytophthora cactorum has been isolated from
numerous hosts, including Rhododendron spp. During
the past decade, however, only these novel hybrids
have been found on Rhododendron in the Netherlands, suggesting that they are replacing the resident
P. cactorum population on this host. While P.
cactorum is an indigenous species in Europe, P.
hedraiandra is believed to be a recent introduction
from North America, where it infects Rhododendron
spp. In comparison with the parental species, the
hybrid isolates exhibit expanded host ranges, including monocots (Allium spp.) as well as dicots (Idesia
and Penstemon spp.). These isolates are known to be
proliferating in the environment in the Netherlands
and in Germany (Man in’t Veld et al. 2007).
Concluding remarks
Laboratory and field studies suggest that interspecific
hybridisation in Phytophthora populations occurs
rarely. However, such rare events may prove to be
an important source of genetic diversity, in addition to
the more commonly recognised processes of mutation
and sexual or parasexual reproduction within individual species. Studies summarised in this review
suggest that Phytophthora species hybridisation may
produce unique offspring capable of exploiting an
expanded range of host plants. In addition, hybrid
offspring with increased aggressiveness may be
selected to such an extent that they become a
dominant component of Phytophthora populations
within a region.
37
The studies summarised here also suggest that
species hybridisation occurs readily between allopatric species that have not co-evolved in the same
location. To date, known Phytophthora species
hybrids represent the offspring of a native and an
exotic or two exotic species that occupy the same
habitat and niche. No reports have described similar
hybridisation among indigenous, sympatric Phytophthora species populations, even though such hybrids
can be generated in the laboratory. The reason for this
limitation is uncertain, although it is believed that
strong genetic barriers have evolved to restrict
hybridisation among sympatric oomycete and fungal
species (Brasier 2000; Olson and Stenlid 2002;
Schardl and Craven 2003).
The mechanisms of species hybridisation in Phytophthora populations are not known, but studies of
several cases have provided evidence for hybrid
populations in various states of genomic evolution.
The role of diverse ploidy levels and genomic
reorganisation in determining host range, aggressiveness, and population dynamics bears further investigation. In addition, it is noteworthy that most
Phytophthora species hybrids have acquired the
mitochondrial genome of the exotic, introduced
parental species (Man in’t Veld et al. 2007). Since
mitochondrial control of virulence was reported for
artificially made hybrids of the basidiomycete fungus
Heterobasidion annosum (Olson and Stenlid 2001),
further research is needed to examine the influence of
the acquired mitochondrial genome on host selection
by species hybrids of Phytophthora.
Interspecific hybridisation among Phytophthora
species is likely to increase with expanding world
trade that introduces plants and associated pathogens
into new regions with uniquely different environmental conditions (Brasier 2000). Opportunities for
interactions between Phytophthora species are also
enhanced as plants are managed under hydroponic
and other non-traditional agricultural conditions (Man
in’t Veld et al. 1998, 2007; Bonants et al. 2000).
Finally, over longer periods of time, human disturbance factors such as pollution, climate change, and
land use may accentuate the emergence of newly
adapted hybrids with unique pathogenicity attributes.
Limited evidence for these possibilities was provided
by Gibbs et al. (1999), who showed that pollution of
water with oxidised nitrogen can sensitise alder trees
to P. alni infection. Consequently, interspecific
38
hybrids appear to be the products of recent evolutionary events. However, some of them might have
existed for a long period of time without being
identified as hybrids, due to the lack of appropriate
tools. Natural hybridisation has been suspected but
never proven with P. meadii (Sansome et al. 1991).
As for fungi, a particular poplar rust identified
recently as a hybrid of Melampsora medusae and M.
occidentalis is represented in specimens from nearly a
century ago (Newcombe et al. 2000).
Although species hybridisation as a source of new
epidemic outbreaks should not be exaggerated, it is of
interest to regulatory officials to monitor the emergence of new hybrid genotypes. Unfortunately,
emerging hybrid populations are unlikely to be
detectable by conventional, morphology-based
approaches. Consequently, population sampling methods and molecular techniques for characterising
pathogen genetic structure require further development for effective detection and management.
Acknowledgements Part of this study was supported by the
Hungarian Scientific Research Fund (OTKA) grant K-61107.
Special thanks are due to James T. English for critically
reviewing the manuscript.
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Eur J Plant Pathol (2008) 122:41–55
DOI 10.1007/s10658-008-9313-2
Proteomic analysis of a compatible interaction
between Pisum sativum (pea) and the downy mildew
pathogen Peronospora viciae
R. C. Amey & T. Schleicher & J. Slinn & M. Lewis &
H. Macdonald & S. J. Neill &
P. T. N. Spencer-Phillips
Received: 11 October 2007 / Accepted: 27 March 2008 / Published online: 9 May 2008
# KNPV 2008
Abstract A proteomic approach was used to identify
host proteins altering in abundance during Peronospora
viciae infection of a susceptible cultivar of pea (Pisum
sativum cv. Livioletta). Proteins were extracted from
fully developed pea leaflets at 4 days post-inoculation,
before visible symptoms were apparent. Cytoplasmic
proteins and membrane- and nucleic acid-associated
proteins from infected and control leaves were examined using two-dimensional difference gel electrophoresis. The majority of proteins had a similar abundance in
control and infected leaves; however, several proteins
were altered in abundance and twelve were found to
have increased significantly in the latter. These proteins
were selected for either matrix-assisted laser desorption/
ionisation time-of-flight mass spectrometry or electrospray ionisation quadrupole time-of-flight tandem mass
spectrometry analysis following trypsin digestion, with
sequence identity being assigned to eight of the proteins.
These included the ABR17 stress-response protein, the
pathogen-induced PI176 protein, three photosynthetic
proteins, a glycine-rich RNA binding protein and two
glyceraldehyde 3-phosphate dehydrogenases (cytosolic
and chloroplastic) which can be induced by a range of
R. C. Amey : T. Schleicher : J. Slinn : M. Lewis :
H. Macdonald : S. J. Neill : P. T. N. Spencer-Phillips (*)
Centre for Research in Plant Science,
Faculty of Health and Life Sciences,
University of the West of England,
Coldharbour Lane,
Bristol BS16 1QY, UK
e-mail: peter.spencer-phillips@uwe.ac.uk
abiotic and biotic stresses in many plant species. The
possible roles of these proteins in the response of the
pea plant during P. viciae infection are discussed. This
study represents the first proteomic analysis of downy
mildew infection of pea leaves, and provides the basis
for further work to elucidate molecular mechanisms of
compatibility in P. viciae infections.
Keywords Electrophoresis . DIGE . MALDI-TOF .
Mass spectrometry . Oomycete . Protein
Abbreviations
2-D DIGE
two-dimensional difference gel
electrophoresis
dpi
days post-inoculation
ESI Q-TOF
electro-spray ionisation quadrupole
MS/MS
time-of-flight tandem mass
spectrometry
GAPDH
glyceraldehyde 3-phosphate
dehydrogenase
MALDI-TOF matrix-assisted laser desorption/
MS
ionisation time-of-flight mass
spectrometry
Introduction
Downy mildew is the most common foliar disease of the
pea crop (Pisum sativum) in the UK, with up to 55%
losses in yield observed where plant resistance is
42
ineffective (Clark and Spencer-Phillips 2000). Downy
mildew is also a significant problem in other parts of
the world where peas are grown (Amey and SpencerPhillips 2006).
Production of conidia by Peronospora viciae results
in a substantial loss of photosynthate from the host to
the pathogen, contributing to symptoms such as stunted
growth, distortion and early death of the infected plant
(Mence and Pegg 1971). This re-direction of photosynthates and the other effects are likely to be accompanied by changes in the abundance of certain host proteins.
Proteomics not only has the potential to identify these
proteins, but also to provide quantitative data which
signify their relative importance to the process.
Proteomic technologies such as 2D-DIGE (Ünlü et
al. 1997), mass spectrometry and bioinformatics are an
effective and accurate way of identifying and measuring protein differences between cell types (BeranovaGiorgianni 2003). Typically, the proteome of a control
cell type or tissue is compared to a treated or diseased
cell type or tissue. Protein differences observed
between the two samples are investigated further to
identify protein function and origin. 2D-DIGE and
mass spectrometry are being used increasingly to
identify proteins that increase or decrease in abundance
during plant-microbe interactions (Corbett et al. 2005;
Coulthurst et al. 2006). At present, little is known
about mechanisms of pathogenesis in P. viciae infections of pea, with very few host and pathogen factors
explained at a biochemical and molecular level (Clark
and Spencer-Phillips 2004). Proteomics provides a
global approach to explore changes in abundance of
specific components of the pathosystem proteome, and
hence to identify specific proteins and processes likely
to be central to the outcome of infection.
To date, few proteomic studies of oomycete pathogens of plants have been performed. Most have focused
on Phytophthora species such as P. infestans, a
devastating pathogen of solanaceous plants (Ebstrup
et al. 2005; Grenville-Briggs et al. 2005), P. nicotianae
which has a wide host range (Mitchell et al. 2002) and
P. palmivora, a serious pathogen of tropical crops
including cocoa (Shepherd et al. 2003). The latter
study examined the proteome of the asexual spores at
various stages of development and germination, and
identified a number of proteins that may be specific to
different phases of the asexual life-cycle. The study by
Mitchell et al. (2002) examined proteins from zoospores and cysts, whereas Grenville-Briggs et al.
Eur J Plant Pathol (2008) 122:41–55
(2005) examined the proteomes of mycelium, zoospores and germinating cysts with appressoria, and
Ebstrup et al. (2005) compared cysts, germinated cysts
and appressoria. This work, however, only provided
information on pathogen proteins during pre-invasion
stages of infection. In contrast, Colditz et al. (2004)
examined the proteome of the roots of the legume
Medicago truncatula during infection by the oomycete
Aphanomyces euteiches, with the majority of induced
proteins belonging to the pathogenesis-related (PR)-10
group of pathogenesis-related (PR) proteins.
Proteomics has been applied previously to the study
of pea proteins, including two host genotypes inoculated
with the powdery mildew pathogen Erysiphe pisi (Curto
et al. 2006). These authors compared the proteomes of
E. pisi-infected and non-inoculated control leaves, and
identified seven and 16 proteins with increased
abundance following infection in resistant and susceptible interactions, respectively. The proteins functioned
in photosynthesis, carbon metabolism, energy production, stress and defence, protein synthesis, and degradation, and signal transduction. Other published data
relate to abiotic stresses such as salinity (Kav et al.
2004), biotic stresses such as infection by the parasitic
plant Orobanche crenata (Castillejo et al. 2004) or a
combination of interactions such as in Glomus mosseae-inoculated pea roots treated with calcium (Repetto
et al. 2003). Numerous studies have examined the
proteomes of other legumes. These include the symbiotic relationships of Medicago with G. mosseae
(Bestel-Corre et al. 2002) and Sinorhizobium meliloti
(Djordjevic et al. 2003), and soybean infected by the
bacterium Bradyrhizobium japonicum to gain further
information about the processes involved in nodulation
(Wan et al. 2005).
The aim of the present work was to generate
fundamental information on the most abundant proteins
specifically involved in a compatible P. viciae–pea
interaction, and to compare this with data on proteins
in compatible and resistant interactions between pea
and E. pisi (Curto et al. 2006).
Materials and methods
Inoculation of pea with P. viciae
P. viciae isolate Nitouche (kindly provided by Dr
David Kenyon, National Institute of Agricultural
Eur J Plant Pathol (2008) 122:41–55
43
Botany, Cambridge, UK) was maintained on a
mixture of seven pea cultivars (P. sativum cvs
Livioletta, Kelvedon Wonder, Maro, Krupp Pelushka,
Early Onward, Solara and Progreta). For protein
extractions, plants of P. sativum cv. Livioletta were
cultivated from seed in compost (Levington F2S) in
growth chambers (Sanyo; 16 h light at 20°C, 8 h dark
at 14°C). At 10 days after sowing, the surfaces of
fully developed leaflets were rubbed gently to flatten
waxes before being inoculated with P. viciae conidia
by the method of El-Gariani and Spencer-Phillips
(2004). Control plants were inoculated with sterile
distilled water only.
22,000 g at 17°C and the resulting supernatant
containing membrane and nucleic acid-associated
proteins (fractions II and III) was removed and
stored at −80°C.
The proteins were prepared for 2-D DIGE using
the 2D Clean-up kit (Amersham Biosciences), resuspended in lysis buffer (30°mM Tris pH 8.5; 4%
w/v 3-[(3-cholamidopropyl) dimethylammonio]-1propane-sulphonate) (CHAPS); 7 M urea; 2 M
thiourea) and quantified using the 2D Quant kit
(Amersham Biosciences) according to the manufacturer’s instructions. Protein samples were stored at
−80°C until further analysis.
Protein extraction and preparation
CyDye labelling
Proteins were extracted from fully developed leaflets
of healthy pea plants and from P. viciae-infected
plants at 4 dpi according to the method of Giavalisco
et al. (2003). Their method is claimed to result in
three fractions, comprising enriched preparations of
(I) cytosolic proteins, (II) membrane-bound proteins
and (III) nucleic acid-associated proteins. Protein
fractions II and III were pooled for analysis in the
present investigation. In brief, leaves were ground in
liquid nitrogen before addition of 0.125 (v/w) inhibitor mixture 1 (100 mM KCl; 20% v/v glycerol;
50 mM Tris, pH 7.1), including Complete Protease
Inhibitor Cocktail Tablet (Roche, Germany) used
according to the manufacturer’s instructions, and
0.05 (w/w) of inhibitor mixture 2 (1 mM Pepstatin
A, 1.4 mM PMSF). Samples were centrifuged for
60 min at 22,000 g at 4°C. The supernatant containing
the soluble cytosolic protein (fraction I) was removed
and stored at −80°C. The pellet was ground further in
liquid nitrogen before addition of 0.125 (v/w) of
inhibitor mixture 3 (200 mM KCl; 20% v/v glycerol;
100 mM phosphate buffer, pH 7.1; Complete Protease
Inhibitor Cocktail, as before), one volume of buffer A
(100 mM phosphate buffer, pH 7.1; 200 mM KCl;
20% v/v glycerol; 2 mM MgSO4; 4% w/v CHAPS)
and 2% (w/w) ASB14 detergent (Calbiochem, UK).
Samples were homogenised thoroughly before the
addition of 0.025% (v/w) DNase and the resulting mix
stirred at 4°C for 45 min. Subsequently, 23% v/w buffer
B (700 mM 1,4-dithiothreitol (DTT), 7 M urea, 2 M
thiourea) was added and the homogenate was
stirred at room temperature for 45 min. The
homogenate was then centrifuged for 60 min at
Samples were labelled using fluorescent cyanine dyes
(Amersham Biosciences) according to the manufacturer’s protocols. The cyanine dyes were reconstituted
in fresh 99.8% anhydrous dimethyl formamide.
Aliquots of 50 μg of protein were labelled with
400 pmol of amine reactive CyDye for 30 min on ice
in the dark, then 1 μl of 10 μM lysine was added to
the tube and incubated on ice in the dark to halt the
reaction. The samples were made up to 100 μl with
rehydration solution (8 M urea; 2% w/v CHAPS;
0.002% w/v bromophenol blue; 0.2% w/v DTT; 2% w/
v immobilised pH gradient (IPG) buffer (pH 3–10,
Amersham Biosciences)).
2-D DIGE
Samples were subjected to isoelectric focusing (IEF)
using IPG strips (24 cm, Amersham Biosciences) in the
pH 3–10 non-linear range, with rehydration loading to
separate proteins in the first dimension according to
isoelectric point. The IPG strips were rehydrated
overnight at room temperature in the protein sample
made up to 450 μl with rehydration solution and
covered with mineral oil. The strips were transferred to
an Ettan IPGphor II (Amersham Biosciences) and IEF
was performed with a 50 μA limit/IPG strip. IEF
voltage conditions were 300 V step and hold for 3 h,
1,000 V gradient for 6 h, 8,000 V gradient for 3 h and
8,000 V step for 4 h 40 min.
Sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis was used to separate proteins in the
second dimension according to molecular weight.
Following focusing in the first dimension, each strip
44
was removed from the IEF unit and equilibrated in
15 ml equilibration buffer (50 mM Tris pH 8.8; 6 M
urea; 30% v/v glycerol; 2% w/v SDS; 0.002% w/v
bromophenol blue) amended with 150 mg DTT with
gentle shaking for 15 min at room temperature. The
strips were equilibrated further in 15 ml equilibration
buffer amended with 375 mg iodoacetamide with
gentle shaking for 15 min at room temperature in the
dark. Finally, the strips were equilibrated in 10 ml
equilibration buffer alone for 5 min at room temperature. The strips were loaded onto a 12.5% acrylamide gel (dimensions 24 cm×20 cm×1 mm) and
overlaid with 1% agarose in SDS running buffer
(25 mM Tris pH 8.3; 192 mM glycine; 0.1% SDS)
amended with 0.002% (w/v) bromophenol blue. The
gels were electrophoresed in SDS buffer at 2.5 W per
gel for 30 min, followed by 100 W until the
bromophenol blue dye front had run off the bottom
of the gels. A minimum of three biological samples
was used in these experiments, with two replicate gels
produced for fraction I and the combined fractions II
plus III of the extracted proteins from each sample. A
minimum of three additional, non-CyDye-labelled
gels were run for each sample for protein spot
picking.
Image analysis
Gels were scanned on a Typhoon 9400 imager
(Amersham Biosciences) to visualise CyDye-labelled
proteins. Cy3 scans were obtained using a 532 nm
laser and emission filter of 580 nm BP30. Cy5 scans
were obtained using a 633 nm laser and a 670 nm
BP30 emission filter. Scans were performed at
100 μm resolution with the photomultiplier tube
voltage set for a maximum pixel intensity of 60 to
80,000 pixels. All images were cropped using ImageQuant V5.2 software prior to analysis to remove areas
outside the gel. Analysis of each of the gels was
performed with DeCyder Differential In-gel Analysis
module software (V5.0; Amersham Biosciences)
using the double detection setting and an estimated
protein spot number of 2,500. Parameters for an
exclusion filter were determined and applied according to the manufacturer’s instructions, with resulting
spots confirmed individually by visual inspection.
Protein spots altering in abundance by at least twofold consistently on all gels, and one protein that
remained unchanged, were selected for analysis.
Eur J Plant Pathol (2008) 122:41–55
MALDI-TOF MS and ESI Q-TOF MS/MS analyses
Protein spots were excised from the gels using an Ettan
Spot Picker (Amersham Biosciences) and subsequently digested using an Ettan Digester (Amersham
Biosciences) with 10 μl trypsin (20 ng μl−1; Promega
Sequencing Grade Porcine Modified) in 20 mM
ammonium bicarbonate (Sigma) overnight at room
temperature. Following tryptic digestion, the peptides
were extracted in 50% acetonitrile/0.1% trifluoroacetic acid to a clean microtitre plate and transferred
to an Ettan Spotter (Amersham Biosciences). The
peptides were mixed with matrix (10 mg ml−1 αcyano-4-hydroxycinnamic acid in 50:50 v/v methanol/
acetonitrile) for spotting onto Micromass target plates
for analysis in a MALDI-TOF mass spectrometer
(Waters-Micromass, UK). Peptide mixtures were
analysed using a nitrogen UV laser (337 nm). MS
data were acquired in the MALDI reflector positive
ion mode in the mass range 800–3,500 Da. Identification of proteins from the mass fingerprints generated was performed using Proteinlynx Global Server
software (V2.0.5, Waters-Micromass, UK) for searching against the SwissProt and National Centre for
Biotechnology Information (NCBI, Bethesda, USA)
databases. Search parameters included a peptide mass
tolerance of 100 ppm, estimated calibration error of
+0.025 Da, one missed cleavage per peptide, fixed
carbamidomethylation of cysteine, and variable oxidation of methionine.
Nanoelectrospray ionization tandem mass spectra
were acquired using a Q-TOF Micro mass spectrometer
(Waters-Micromass, UK) coupled to a LC Packings
capillary liquid chromatography system. Aliquots
(15 μl) of peptide solutions prepared as before were
injected using an auxiliary solvent flow of 30 μl min−1
and desalted on a C18 PepMap Nano-Precolumn (5×
0.3 mm internal diam (i.d.), 5 μm particle size; Dionex,
Amsterdam, The Netherlands) for 4 min. Peptides were
eluted and separated using a C18 PepMap100 nano
column (15 cm×75 μm i.d., 3 μm particle size) with a
gradient flow of 200 nl min−1 and solvent system of:
auxiliary solvent, 0.1% HCOOH; solvent A, 5% v/v
CH3CN/95% v/v 0.1% v/v aqueous HCOOH; solvent
B, 80% v/v CH3CN/20% v/v 0.1% v/v aqueous
HCOOH. The solvent gradient was 4 min at 5%
aqueous solvent B, 5% to 55% B over 40 min, 55% to
80% B over 1 min, maintained at 80% B for 5 min,
then reduced to 5% B in 0.1 min and the column
Eur J Plant Pathol (2008) 122:41–55
washed with solvent A for 9.9 min before the next
sample injection. The column was connected to the
nanosprayer of the Z-spray ion source using a short
length of 75 μm i.d. capillary. Voltages used were
3,500 V for the capillary, 45 V for the sample cone and
2.5 V for the extraction cone. MS spectra were acquired
throughout the chromatographic run, while MS/MS
spectra were acquired in data-dependent mode on the
most abundant ions having charge states of 2+, 3+ and
4+ between m/z 400–2,000. The collision cell was
pressurised with 1.38 bar ultra-pure argon (99.999%,
BOC) and collision voltages depended on the m/z and
charge states of the parent ions. The mass spectrometer
was calibrated daily using MS/MS fragment ions from
[Glu1]-fibrinopeptide B (Sigma). Processed data were
submitted to ProteinLynx Global Server (V2.0.5) and
also to MASCOT (Matrix Science) for searching
against SwissProt and NCBI databases. Search criteria
were: peptide tolerance of 100 ppm; fragment tolerance
of 0.1 Da; two missed cleavages per peptide; fixed
carbamidomethylation of cysteine and variable oxidation of methionine modifications.
Results
The proteins from pea plants inoculated with P. viciae
and sterile distilled water (SDW) controls were
visualised using 2D-DIGE, with each gel comprising
two experimental samples labelled with Cy3 (control)
and Cy5 (P. viciae-infected). DeCyder software
detected between 977 and 1337 protein spots on two
representative gel images (Table 1). The total number
of spots detected on the gels varied by 19.7% between
the two replicates of fraction I (enriched cytosolic
soluble proteins), and by 26% between the replicates
of combined fractions II plus III (enriched membraneassociated and nucleic acid-associated proteins). The
proportion of proteins with decreased abundance
following P. viciae infection was 1.7% and 3.5% for
the replicates of fraction I, and 0.38% and 0.92% for
the replicates of fractions II plus III. The proportion of
proteins with increased abundance was 5.2% and
7.2% for fraction I, and 2.3% and 4.5% for fraction II
plus III. Therefore the proteins for MALDI-TOF MS
and ESI Q-TOF MS/MS were selected on the basis
that their abundance altered significantly and reproducibly on all gel replicates of the different biological
samples (two CyDye-labelled replicates plus a mini-
45
Table 1 The relative abundance of proteins detected in 2D gels
for fractions I (cytosolic proteins) and fractions II + III
(enriched in membrane and nucleic acid-associated proteins)
from pea leaves 4 dpi with conidia of P. viciae, compared to
SDW, in two separate experiments (a and b)
Fraction Relative abundance of proteins (no. of spots detected)
Decreased
Ia
Ib
II + IIIa
II + IIIb
22
37
5
9
Similar
1,245
960
1,285
924
Increased
70
77
30
44
Total
1,337
1,074
1,320
977
mum of three additional unlabelled replicate gels)
thus removing potential biological and gel artefacts
from the analysis. This resulted in 12 proteins that
increased in abundance during P. viciae infection as
indicated in Fig. 1, a representative gel from this
experiment. In contrast, no proteins decreased in
abundance significantly (greater than two-fold) and
consistently following infection. An additional protein
(spot number 6) is indicated on the gel as an example
of a protein that does not alter in abundance upon
infection by P. viciae. The relative fold abundance of
protein spots from infected versus SDW-inoculated
leaves determined by DeCyder software, and selected
individual spot images, are illustrated in Figs. 2 and 3.
The molecular weight, pI, matched peptides, sequence
coverage and score (either Proteinlynx Global Server
or MASCOT) of each protein is indicated in Table 2.
Protein 1 (Figs. 1 and 2) was identified as the
disease resistance response protein PI176 from pea
(accession number P13239). Compared to control
plants, its abundance increased by 3.4 and 6.6-fold in
fractions I and fractions II plus III respectively. The
protein has similar predicted and observed molecular
weights and iso-electric points. The three peptides
matching published sequences of PI176 represented
16.4% of its amino acid sequence. Protein 2 was
identified as abscisic acid responsive protein ABR17
from pea (accession number Q06931). The abundance
of this protein resembled that of PI176, with increases
of 2.9 and 5.8-fold for fractions I and II plus III
respectively. Theoretical and observed values for
molecular weight and pI are in accord and the seven
matched peptides provided 51% coverage of the
amino acid sequence. Protein 3 was matched following de novo sequencing by ESI Q-TOF MS/MS to a
glycine-rich RNA binding protein from Sinapis alba
46
Eur J Plant Pathol (2008) 122:41–55
Fig. 1 A typical 2D-DIGE
gel obtained from analysis
of the pea leaf proteome
during early stage infection
(4 dpi) by P. viciae, compared to proteins from control leaves treated with
SDW, for cytosolic fraction
I. Spot colour indicates the
effect of P. viciae on protein
abundance: red = increased;
green = decreased; yellow =
no change. Proteins that
increased in abundance reproducibly on all gels are
indicated by the numbers 1–
13, except for spot 6 which
represents a protein showing
no change in abundance
during infection by P. viciae
(see Fig. 2)
pI
3
10
50
4
5
MW (kD)
7
8
35
1
10
3
12
15
9
2
6
11
13
similar, with the nine matched peptides covering
34.6% of the amino acid sequence. Protein 5 was
identified as a chloroplastic precursor of GAPDH A
from pea (accession number P12858). Compared to
control samples, the protein increased in abundance
by 7.7-fold for fraction I and 8.6-fold for fractions II
plus III. Whilst 15 peptides were matched, covering
44.7% of the amino acid sequence, and the predicted
and observed pI values were in accord, the observed
molecular weight was significantly less than predicted. This suggests that processing of the precursor
(accession number P49311) and, compared to controls, had increased abundances of 6.4 and 6.5-fold
for fraction I and II plus III respectively. The
theoretical and observed molecular weight and pI
values agreed, and the single peptide covered 4.7% of
the amino acid sequence. Protein 4 matched to
cytosolic GAPDH from pea (accession number
P34922). In comparison to control plants, the abundance of the protein increased by 6.1-fold in fraction I
and by 6.3-fold in fractions II plus III. Predicted and
observed molecular weight and pI values were
30
Relative abundance
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Protein
Fig. 2 Relative abundance of proteins 1–13 that increase in pea
leaves at 4 dpi after inoculation with P. viciae, compared to
proteins from control leaves treated with SDW. Protein
abundances for fraction I (black bar, cytosolic proteins) and
fractions II plus III (gray bar, enriched in membrane and
nucleic acid-associated proteins) were calculated using
DeCyder Software (Amersham Biosciences) from two replicate
experiments. Standard errors are indicated
Eur J Plant Pathol (2008) 122:41–55
47
Fig. 3 Images of representative protein spots on 2D
gels, from two replicate
experiments (a and b). Protein 2 (ABR17) increases in
abundance during P. viciae
infection of pea leaves in
fractions I and fractions II
plus III; protein 11 (photosystem I reaction centre
subunit II) increases to a
greater extent in fraction I
than fractions II plus III;
protein 6 (tentatively identified as thioredoxin M-type,
chloroplast precursor) does
not differ in abundance between control (SDW) and
infected pea leaves
Protein
Experiment
Protein 2
(Q06931)
Fraction I
P. viciae
Control
4 dpi
Fractions II plus III
Control
P. viciae
4 dpi
a
ABAresponsive
protein
ABR17
b
Protein 11
(P20117)
a
Photosystem I
reaction
centre
subunit II
b
Protein 6
a
Thioredoxin
M-type,
chloroplast
precursor
b
Table 2 Proteins identified using MALDI-TOF MS and ESI Q-TOF MS/MS that differ in abundance in response to infection by P.
viciae at 4 dpi
Spot
number
1
2
3
4
5
6
11
12
13
Matching protein
Protein
accession no.
Observed/predicted
mW (kDa)
Observed/
predicted pI
PI176
ABR17
Glycine-rich RNA binding
protein
Cytosolic GAPDH
Chloroplastic GAPDH
Thioredoxin M-type
precursor
Photosystem I reaction
centre subunit II
ATP synthase epsilon chain
Photosystem I iron sulphur
centre
P13239
Q06931
P49311
17.0/16.9
16.9/16.6
17.0/16.4
5.1/5.1
5.2/5.1
5.5/5.5
P34922
P12858
P48384
36.0/36.6
36.0/43.3
14.0/12.5
Q9S7H1
P05039
P10793
Matched
peptides
Sequence
coverage
Score
3
7
1a
16.4
51.0
4.7a
11.2b
9.1b
NA
7.0/7.0
8.5/9.0
5.4/5.4
9
15
1
34.6
44.7
5.2
11.6b
11.9b
11.2b
14.0/23.1
8.0/9.8
3
10.1
121c
15.0/15.2
9.0/9.2
8.6/6.6
8.5/7.5
3
3
15.3
17.3
99c
89c
All proteins increased in abundance by more than two-fold (see Fig. 2), except protein 6 which was essentially unchanged between
treatments. Spot numbers relate to Fig. 1; all accession species were P. sativum, except S. alba for spot 3 and A. thaliana for spot 11
NA Not applicable
a
Protein matched by de novo sequencing using ESI Q-TOF MS/MS
b
Proteinlynx Global Server
c
MASCOT
48
may have occurred. Protein 6 was identified tentatively (only one matched peptide) as a thioredoxin
M-type chloroplast precursor from pea (accession
number P48384) and was selected as a protein that
differed by less than two-fold compared to control
plants. Indeed, in some gels (e.g. Fig. 1), its
abundance appeared unchanged following P. viciae
infection.
Protein 11 had the highest peptide match to the
photosystem I reaction centre subunit II precursor
from Arabidopsis thaliana (accession number
Q9S7H1), and also matched to a partial sequence
obtained for the same protein in pea (accession
number P20117). The predicted molecular weight
and pI values were different to those observed, both
being larger than the values observed on the gel, and
suggesting that spot 11 represents a fragment of this
protein. The greatest increase in abundance was
observed for this protein (Fig. 2), which increased
by 21.7-fold in fraction I compared to an increase of
3.3 for fraction II plus III. Three peptides from protein
12 matched to an adenosine triphosphate (ATP)
synthase epsilon chain from pea (accession number
P05039), covering 15.3% of the amino acid sequence.
The molecular weight for the protein observed on the
gel matched the predicted, but the pI differed. Protein
13 matched to the photosystem I iron sulphur centre
from pea (accession number P10793). Three peptides
were matched, covering 17.3% of the amino acid
sequence, with predicted pI and molecular weight
values matching those observed.
The abundances of the unidentified proteins 7 and
10 (Figs. 1 and 2) increased similarly in both
fractions. The abundance of protein 8 was greater in
the soluble fraction I than the membrane and nucleic
acid-associated proteins of fraction II plus III, having
a 3.3 and 2.5-fold increase in abundance compared to
control samples, respectively. In fraction II plus III,
protein 9 increased in abundance by more than twice
that observed in fraction I (9.3 and 4.3-fold respectively). Thus differences between the relative abundance of proteins isolated from the two fractions were
apparent for four of the 13 proteins (Fig. 2).
Discussion
Eight proteins whose abundance was observed to
have increased consistently by at least two-fold in
Eur J Plant Pathol (2008) 122:41–55
4 day-old P. viciae infections of pea were identified
by either MALDI-TOF MS or ESI Q-TOF MS/MS. A
further four proteins were observed to increase in
abundance consistently during P. viciae infection, yet
could not be identified using MS. The possible roles
and functions of the identified proteins during the
response to infection by biotrophic pathogens are
discussed. It is notable, however, that none of these
proteins were reported to increase in abundance
following E. pisi infection (Curto et al. 2006), which
suggests that they reflect a specific response to P.
viciae in this compatible interaction. This study
differs from previous proteomic investigations of
oomycetes in that it examines the proteome of the
plant-pathogen interaction in leaves.
It is believed widely that the majority of plant
defence mechanisms, such as basal resistance and the
hypersensitive response, are induced early during
infection via a complex network of signals that is
initiated following perception of the pathogen by host
cells (Dangl and Jones 2001; Kamoun et al. 1999b;
McDowell and Dangl 2000). The identification of
proteins with potential and proven roles in plant
defence in plants harvested at 4 dpi would correlate
with these concepts. Proteins were harvested at this
stage as well-developed colonies are present, even
though external symptoms are not apparent (ElGariani and Spencer-Phillips 2004). Indeed, conidiophore initials are first observed at 4.5 dpi in this
host-pathogen system (Clark and Spencer-Phillips
2004), with sporulation visible macroscopically by
7 dpi. Further proteomic analyses of these later stages
of infection are underway to identify additional
proteins involved in the P. viciae-pea interaction.
The difference in numbers of proteins identified as
increased or decreased in abundance between the two
fractions implies that a slightly larger number of
cytosolic proteins increase in abundance than in a
fraction enriched in membrane plus nuclear-associated
proteins (6.1% and 3.2%, respectively). However, it
should be noted that the same proteins were identified
in both fraction types, indicating that the method of
extraction may not be effective at specifically selecting
cytosolic, membrane-associated and nucleic acidassociated proteins for pea samples. Whilst it may be
more appropriate to pool all fractions in further studies
to facilitate comparison of proteomes, some proteins
(e.g. protein 11) showed significant differential increases between the fractions.
Eur J Plant Pathol (2008) 122:41–55
Of the 12 proteins observed to have abundances
significantly and consistently increased during the P.
viciae interaction with pea, eight were matched to pea
or other plant proteins in the SWISS-PROT database.
The observed and theoretical isoelectric points and
molecular weights of the proteins were mostly in
accord, except for proteins 5 and 11 where the
predicted molecular weight values were much greater
than those observed. For protein 11, this may reflect
the match to the A. thaliana protein rather than the
pea protein which has not been sequenced fully.
Additionally, this protein was identified as photosystem I reaction centre subunit II (Table 2) and therefore
should be membrane bound, but it was mostly present
in fraction I which Giavalisco et al. (2003) suggests
should contain soluble proteins. Together with the
discrepancy in the predicted and observed molecular
weights, this suggests that a protein fragment has
been identified. The match of protein 5 to a precursor
of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may indicate that the processed
protein was present here.
Even in transcriptional profiling studies, it is often
the case that not every gene that alters in expression
during a specific interaction can be identified from
databases, as shown in the yeast Saccharomyces
cerevisiae (Gygi et al. 1999) and in M. truncatula
roots following infection by the oomycete A.
euteiches (Colditz et al. 2004; Nyamsuren et al.
2003). Indeed, further studies are needed to identify
the four unidentified proteins, as they are likely to
play significant roles in this plant-pathogen interaction. Identification would provide information about
probable function and thereby help elucidate the
molecular mechanisms of pathogenicity and host
response in downy mildew infections.
Three of the proteins with increased abundance are
likely to be involved in initiating and maintaining a
defence response by pea during P. viciae infection.
Disease resistance response protein PI176 was identified originally by Fristensky et al. (1988), and is a
member of the pathogenesis-related class 10 (PR10)
family of proteins which are found exclusively in
plants. This set of proteins generally is induced by
abiotic and biotic factors such as wounding (Liu et al.
2003; Warner et al. 1992, 1993), salt stress (Moons et
al. 1997), pathogenic infections (Fristensky et al.
1988; Liu et al. 2003; Matton and Brisson 1989;
McGee et al. 2001; Pinto and Ricardo 1995;
49
Schmelzer et al. 1989; Somssich et al. 1988), drought
(Dubos and Plomion 2001), chemicals such as copper
(Utriainen et al. 1998) and plant hormones (Moons et
al. 1997; Wang et al. 1999a). PI176 is a variant of the
pea PI49 protein, differing in four amino acid
substitutions and one amino acid deletion (Fristensky
et al. 1988). There are conflicting views on the
potential biological action of the PR10 proteins.
Some studies suggest they have RNase activity
(Bantignies et al. 2000; Moiseyev et al. 1994; Park
et al. 2004; Swoboda et al. 1996), with others
(Biesiadka et al. 2002) suggesting that this is not the
case, as the crystal structure of some PR10 proteins
such as LIPR10.1A and LIPR10.1B from Lupinus
luteus does not support RNase activity. Additionally,
Biesiadka et al. (2002) noted that the L. luteus PR10
proteins studied have little or no RNase activity. This
discrepancy in the literature may reflect the large
number of PR10 protein homologues within plants.
For example, 13 different PR10 cDNA clones have
been identified in Pinus monticola (Liu et al. 2003)
and, although some redundancy is likely to exist in
the activity of these proteins, it is also possible that
some of the proteins have significantly different
functions within plants. Similar proteins in bean
(Phaseolus vulgaris) are thought to act intracellularly
as no signal peptides have been identified for the
proteins (von Heijne 1985; Walter et al. 1990), whilst
transcripts of the parsley (Petroselinum crispum)
PcPR1 gene accumulate rapidly and to a large extent
in cells adjacent to the site of pathogen infection
(Schmelzer et al. 1989; Somssich et al. 1988). From
the literature and the protein abundance pattern of
PI176 observed in this study, it appears that PI176 is
important in the intracellular response of pea to
infection by P. viciae in this compatible interaction,
and further analysis of this family of proteins is
merited.
Abscisic acid response protein 17 (ABR17) is an
additional member of the pathogenesis-related class
10 (PR10) family and has a similar pattern of
abundance to PI176. This has also been observed for
PR10 genes in other pea-microbe interactions, such as
infection by the arbuscular-mycorrhizal fungus G.
mosseae where transcript levels of the PI49 and
PI176 genes increase in pea roots up to 15 dpi (RuizLozano et al. 1999), and for their proteins in A.
euteiches infection of M. truncatula (Colditz et al.
2004). The gene for ABR17 is closely related to
50
ABR18 having significant similarities at the DNA and
amino acid levels, a trait shared by abscisic acidinducible proteins from other plant species such as
alfalfa (Medicago sativa; Luo et al. 1992) and the
barrel medic (M. truncatula; Colditz et al. 2004).
Abscisic acid is thought to play a key role in
mediating adaptive plant responses to environmental
stress, plant development, seed dormancy and germination, as well as plant defence (Luo et al. 1992;
Moons et al. 1995). It is thought that the ABR
proteins in pea increase in abundance in response to
both abscisic acid and environmental cues, with the
present study indicating a role also in response to
pathogens.
The increased abundance of both ABR and PI
proteins would indicate that ABA-mediated signalling
is important in the P. viciae–pea interaction. This
notion concurs with Nyamsuren et al. (2003) who
observed increased transcripts of these genes in A.
euteiches infections of M. truncatula, a close relative
of pea. Although ABA has been shown to play a role
in signalling, Colditz et al. (2004) showed that of six
PR10 proteins identified in M. truncatula, only three
increased in abundance in response to ABA, and none
altered in abundance in response to drought stress.
Therefore, three of the PR10 proteins increased in
abundance in response to A. euteiches alone, for the
limited range of stresses assessed. It would seem that
some proteins may play a specific role in response to
biotic stress such as pathogen invasion, perhaps
providing opportunities for the development of
pathogen-tolerant crop species. Further studies should
compare both plant-microbe interactions, especially
as A. euteiches is also a serious pathogen of pea crops
(Pfender 1989). This may determine whether the
induction of the ABA-responsive genes is a general
plant response to oomycete pathogens, resulting from
increased ABA content as a side-effect of infection
due to senescence, reduced water availability and cell
death, or perhaps a combination of several factors.
Glycine-rich proteins (GRPs) have been implicated
in numerous roles in plants. In pea, GRPs have been
associated with dormancy and have similarity to
proteins that are stimulated by auxin and numerous
abiotic stresses (Luo et al. 1991; Laberge et al. 1993;
Stafstrom et al. 1998). Structurally related GRPs are
often components of the cell walls of higher plants
(Showalter 1993), and accumulate in vascular tissues
as part of the defence mechanism against pathogens
Eur J Plant Pathol (2008) 122:41–55
and wounding (Mousavi and Hotta 2005). A second
class of GRPs, the glycine-rich RNA-binding proteins
(GR-RBP) such as protein 3 identified tentatively in
the present study, are thought to play an important role
in post-transcriptional regulation of gene expression.
Some evidence exists for altered transcript and/or
protein abundance of GR-RBPs in response to virus
infection (Geri et al. 1999; Naqvi et al. 1998), acute
hypersensitive response and salicylic acid treatment
(Naqvi et al. 1998), abscisic acid treatment (Aneeta
Sanan-Mishra et al. 2002; Baudo et al. 1999; Bergeron
et al. 1993; Carpenter et al. 1994; Gomez et al. 1988;
Kim et al. 2005) and methyl-jasmonate treatment
(Richard et al. 1999), thus providing evidence of a
role in the response of plants to pathogens. There are
no previous reports regarding the response of these
proteins to attack by oomycete pathogens, and the
tentative identification in the present study indicates
that further investigation is needed.
Cytosolic GAPDH (protein 4 in the present study)
is one of three forms of GAPDH in plants, and is
involved in the second phase of glycolysis, catalysing
the conversion of D-glyceraldehyde 3-phosphate into
3-phospho- D -glyceroyl phosphate. Evidence for
GAPDH having a role in defence is provided by
Laxalt et al. (1996) who noted that cytosolic GAPDH
transcripts accumulated in potato plants infected by P.
infestans, and treated with both the P. infestans
elicitor eicosapentaenoic acid and salicylic acid.
Interestingly, although GAPDH transcripts increased
to their highest levels at 2 dpi in potato, it was not
until 3 dpi that a corresponding increase in enzyme
activity was observed. This would indicate that there
is a substantial time-lag between induction of genes
involved in the pathogen response and the production
of their corresponding proteins. It has been suggested
that these proteins be given the term ‘stress-induced
metabolic response’ proteins (Laxalt et al. 1996). The
time-lag also suggests that the selection of 4 dpi as the
time point for extracting proteins in the present study
is suitable for investigating host-pathogen interactions
in plants capable of adjusting their metabolism to
survive various stresses in this short time period.
The possibility that the increase in GAPDH
abundance is due simply to an increase in metabolic
turnover of the protein, and not a specific stress
response, should not be overlooked. The abundance
of this protein and the other proteins, both identified
and unidentified, in other plant parts such as roots,
Eur J Plant Pathol (2008) 122:41–55
stems and tendrils of pea is also worth investigating.
Elevated levels of plant defence proteins in roots, for
example, would help prevent downy mildew infection
via soil-borne oospores. Investigations are in progress
to establish whether the increase in GAPDH abundance is also evident in pea in response to abiotic
stresses such as wounding and water-deficit.
The discovery that levels of a chloroplastic GAPDH
A were also elevated in response to P. viciae infection
(protein 5) is surprising, as to our knowledge there is no
previous evidence for this in other biotrophic infections.
It is likely that chloroplastic GAPDH plays a similar
role to cytoplasmic GAPDH in that an overall increased
metabolic state is induced during infection by P. viciae.
Alternatively, GAPDH may have a role in plant
signalling pathways, with increasing evidence indicating that plant enzymes may be multi-functional (Moore
2004). For example, GAPDH has been demonstrated to
be an inhibitory target of hydrogen peroxide in A.
thaliana with potential roles in mediating reactive
oxygen species signalling (Hancock et al. 2005).
Interestingly, mRNA and enzyme levels of GAPDH
are also induced by abscisic acid treatment (Velasco et
al. 1994), indicating that ABA signalling is important in
the plant response to pathogens, especially as ABR17
protein levels also increased following P. viciae
infection. GAPDH is also associated with the cell wall
in A. thaliana (Chivasa et al. 2002), and found in the
peribacteroid membrane of root nodules in Lotus
japonicus (Wienkoop and Saalbach 2003). The specific
function of GAPDH remains to be elucidated, and it
seems that proteins involved in making adjustments to
plant metabolism during stress are under-studied.
Three proteins increasing in abundance in response
to P. viciae infection are involved in photosynthesis.
Two are integral to photosystem I, the reaction centre
subunit and the iron sulphur centre, whilst the third is
involved in the synthesis of ATP (ATP synthase
epsilon chain). Photosystem I is a chloroplastic,
multimolecular complex that uses ferredoxin-like
iron-sulphur cluster proteins as terminal electron
acceptors. The ATP synthase epsilon chain is a small
sub-unit of the chloroplast ATP synthase, which
produces ATP from ADP in the presence of a proton
gradient across the chloroplast thylakoid membrane
(Hopkins and Hüner 2004). Whilst most studies
indicate that infections by plant pathogens result in a
reduced photosynthetic rate, transient increases in the
rate of photosynthesis during infection by biotrophic
51
pathogens have been observed (reviewed in Scholes
1992), which may reflect the increase in proteins of
the photosynthetic apparatus observed in this study. In
the biotrophic pathogen-plant interactions where
photosynthetic rate initially increased during infection, an overall reduction in photosynthesis was
eventually observed, when compared to control
plants. An analysis of the proteins observed to alter
in abundance at later stages of infection by P. viciae,
along with experimental data on photosynthetic rates,
would reveal if a similar pattern occurs. The hypothesis that a fragment of the photosystem I reaction
centre subunit II has been identified in the present
study, however, suggests that the intact protein has
been degraded as a result of infection. This would be
expected to cause a decrease, rather than an increase,
in photosynthetic activity. The possibility that photosynthetic rates in adjacent, uninfected tissues and cells
may increase to compensate for a reduction in
colonised areas also merits investigation. These
effects have been observed in Botrytis fabae infections of Vicia faba leaves, uninfected leaves on P.
vulgaris plants infected by the rust fungus Uromyces
phaseoli, and uninfected leaves of pea plants inoculated with powdery mildew spores (Lucas 1998).
Thus it would appear that pea leaves are capable of
implementing a response to P. viciae infection
through the up-regulation of various pathogen-related
and metabolic pathway proteins. Whether these
proteins act in concert rather than individually is not
known, although previous studies have shown that
plants synthesise a number of proteins in response to
stresses that may work individually and/or in conjunction with other proteins (Luo et al. 1992).
It is noticeable that no P. viciae proteins were
identified in this study, which may be because there is
very little database coverage of DNA and proteins
from this pathogen. This was also the case with the
proteomic study of A. euteiches infection of M.
truncatula (Colditz et al. 2004), where no A. euteiches
proteins were found. Reasons given included the
restricted amount of pathogen-infected root cells and
the restricted growth of A. euteiches in host cells. The
difficulty in identifying oomycete proteins was illustrated by Shepherd et al. (2003), who identified just
three P. palmivora proteins from 2D gels loaded with
proteins extracted from hyphae grown in vitro, all of
which corresponded to actin. Indeed it can be difficult
to identify genes specifically expressed during infec-
52
Eur J Plant Pathol (2008) 122:41–55
tion by oomycetes (Beyer et al. 2002 and references
therein). Despite improved techniques, up to 70% of
gene sequences cannot be identified using genomic
studies (Beyer et al. 2002), and house-keeping genes
are more easily identified than those that may be
involved in infection. Proteomic approaches will
always tend to be more successful if supported by
extensive and complete genome databases. P. infestans, Phytophthora sojae and Hyaloperonospora
parasitica genome projects are currently underway
or are near completion (Beynon, personal communication; http://www.pfgd.org/; Kamoun et al. 1999a;
Qutob et al. 2000) which may enhance the success of
proteomic analyses of oomycetes. Indeed, the peptide
mass fingerprints obtained for the unidentified proteins in this study may yield matches when used to
search these new data sets, and also any proteins
identified from analysis of the P. viciae conidial
proteome (Chuisseu Wandji et al. 2007).
To our knowledge, the data presented here represent
the first application of 2-D DIGE to investigate a plant–
oomycete pathogen interaction, and indicate the
sensitivity and accuracy of this method for identifying
proteins that are important to the plant’s response to
infection. An increased understanding of these mechanisms may accelerate progress in developing novel
control strategies for downy mildew diseases. Indeed,
the DRR49 protein from pea has been used to increase
potato tuber production in the presence of pathogeninfested soil (Chang et al. 1993), and to increase levels
of resistance to Leptosphaeria maculans in canola
(Wang et al. 1999b). It is therefore possible that the
proteins identified in the current study may be of use
in the development of novel plant lines with enhanced
resistance to biotic and abiotic stresses.
Acknowledgments
HH3216SFV.
This work was funded by DEFRA grant
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Eur J Plant Pathol (2008) 122:57–69
DOI 10.1007/s10658-008-9288-z
Tapping into molecular conversation between oomycete
plant pathogens and their hosts
Mahmut Tör
Received: 20 November 2007 / Accepted: 31 January 2008
# KNPV 2008
Abstract Several plant pathogenic oomycetes have
been under investigation using modern molecular
approaches. Genome sequencing and annotations are
underway or near to completion for some of the
species. Pathogen-associated molecular pattern molecules (PAMPs) and effector molecules perform interand intracellular tasks as adaptation factors and
manipulators of the defence network. Hundreds of
secreted putative effectors have been discovered and
conserved molecular patterns such as RXLR and EER
motifs have been identified and used for classifications. PAMPs and effectors are recognized directly or
indirectly by the pattern recognition receptors at the
cell surface including receptor-like kinases and
receptor-like proteins, and/or by nucleotide binding
site–leucine rich repeat proteins within the cytoplasm.
The current knowledge of effectors, immune receptors
and the defence network, will help us understand the
‘intricate genetic dance’ between the oomycete
pathogens and their hosts. This review concentrates
on the recent findings in oomycete-plant interactions.
Keywords PAMPs . Effector . Oomycete . Receptor .
Biotrophy
M. Tör (*)
Warwick HRI, University of Warwick,
Wellesbourne,
Warwick CV35 9EF, UK
e-mail: Mahmut.tor@warwick.ac.uk
Introduction
The oomycetes include a unique group of biotrophic
and hemibiotrophic plant pathogens including Plasmopara viticola (grapevine downy mildew), Albugo
candida (white rust), Bremia lactucae (lettuce downy
mildew), Hyaloperonospora arabidopsis (downy
mildew on Arabidopsis, formerly Hyaloperonospora
parasitica; Göker et al. 2004) and Phytophthora
infestans (potato and tomato late blight; Kamoun
2003; Hardham 2007). These pathogens establish
intimate relations with their hosts by forming haustoria
during the infection, which are well known structures
used for obtaining nutrients from the plant, redirecting
host metabolism and suppressing host defence in
biotrophy (Hahn and Mendgen 2001; Voegele and
Mendgen 2003; O’Connell and Panstruga 2006).
Bremia lactucae, P. viticola or P. infestans have a
significant economic importance in agriculture (Agrios
1997). Hyaloperonospora arabidopsis in Arabidopsis
thaliana has been developed as an important model
system to study plant–microbe interactions (Holub
2008). In addition, A. candida provides an alternative
model on Arabidopsis; however, it has not been fully
explored despite interesting characteristics such as the
suppression of R-gene mediated and non-host resistance mechanisms to allow the growth of a second
parasite like H. arabidopsis and causing a hormonal
imbalance, which induces ‘green island’ formation
(Cooper et al. 2002; Holub 2006, 2008).
58
Understanding the mechanisms of microbial pathogenesis and plant–microbe interactions has motivated plant pathologists for a long time. In nature,
plants are generally resistant to most pathogens
due to their innate ability to recognize pathogenderived molecules and to mount a series of carefully
orchestrated and highly evolved defence responses.
Much of the progress in the field of molecular
plant–pathogen interactions has been led by the
research on prokaryotic bacterial plant pathogens.
This may have been due to the fact that bacterial
pathogens have the ability to secrete effectors including avirulence proteins with their type III secretion
system into the cytoplasm of their host plant cell (Van
den Ackerveken et al. 1996; Mudgett and Staskawicz
1998; Chisholm et al 2005). However, in the last few
years, significant progress has also been made in the
understanding of interactions between eukaryotic
pathogens, including oomycetes, ascomycetes and
basidiomycetes, and their host plants (Allen et al.
2004; Catanzariti et al. 2006; Shen et al. 2007).
Results from these studies led to the establishment of
a general consensus on plant-microbe interactions,
which is that; (a) pathogens have pathogen associated molecular pattern molecules (PAMPs) and effector molecules that modulate the immune system
(Kamoun 2006; Lotze et al. 2007); (b) the plant
innate immune system is a collection of subsystems
that carry out distinct functions in the host’s defence;
(c) the cell surface receptors or pattern recognition
receptors (PRRs) and cytoplasmic receptors or
nucleotide binding site-leucine rich repeat (NBLRR) proteins play a significant role in the detection
of these PAMPs and effectors (Chisholm et al.
2006), and (d) effector molecules are virulence
factors and have the ability to suppress the immune
system of the plant (Bos et al. 2006; Jones and Dangl
2006; Fig 1).
Recent genomic studies on the oomycete pathogens including Phytophthora sojae, Phytophthora
ramorum, P. infestans and H. arabidopsis (Win
et al. 2007; Whisson et al. 2007) have revealed
hundreds of hypothetical effectors that are secreted
into the apoplast or the cytoplasm of the host plants.
This review focuses on the contributions of recent
findings to the understanding of oomycete pathogenesis
with emphasis on PAMPs, effectors and receptors rather
than repeating the existing reviews on gene-for-gene
interactions.
Eur J Plant Pathol (2008) 122:57–69
PAMPs play a significant role in pathogenesis
and trigger the innate immune system
These molecules were originally described as microbial
elicitors and could be present in pathogenic and
non-pathogenic microorganisms. They are unique
to microbes, invariant among the given class of
microorganisms and seen as foreign molecules by
plants. They are important for microbial fitness and are
able to elicit innate immune responses in a non-cultivar
specific manner. Their conserved nature makes it difficult
for the pathogen to avoid recognition through adaptive
evolution of these molecules (Hahn 1996; Medzhitov
and Janeway 2002; Ingle et al. 2006; Medzhitov 2007).
Several bacterial PAMPs including flagellin and
Ef-Tu have been identified and studied in detail (Felix
et al. 1999; Zipfel et al. 2006). The majority of PAMP
studies on oomycete pathogens have been carried out
with Phytophthora species. For example, studies on
P. parasitica var. nicotianae have identified the cell
wall elicitor protein cellulose binding elicitor lectin
(CBEL), which enables the pathogen to attach to the
host cell and contains two cellulose-binding domains
1 and 2. When recombinant CBEL is expressed in
Escherichia coli and the protein injected into tobacco
(Nicotiana tabacum) leaves, activation of the defense
gene expression and formation of necrotic lesions
have been observed. In addition, CBEL production in
planta induced necrosis and synthetic peptides derived from CBEL activated the defence response in
tobacco and A. thaliana leaves, indicating that these
molecules have the necessary molecular patterns to be
recognized by the innate immune system of plants
(Gaulin et al. 2002, 2006).
Another molecule that has the characteristics of a
PAMP is the β-glucan obtained from the cell wall of
the soybean oomycete pathogen P. sojae (formerly
known as Phytophthora megasperma f. sp. glycinea,
Sharp et al. 1984). Treatment of the soybean cell
suspension cultures with β-glucan has been shown to
induce defence reactions including an increase in the
cytosolic calcium concentration, the production of
reactive oxygen species (ROS), and the activation of
genes such as two mitogen-activated protein kinases
(MAPKs) and one MAPK kinase, which play a role in
signal transduction (Mithofer et al. 2001; Yamamizo
et al. 2006; Daxberger et al. 2007).
Pep-13 is also a cell wall product from the soybean
pathogen P. sojae and considered to be a PAMP. Initial
Eur J Plant Pathol (2008) 122:57–69
59
Fig. 1 Pathogen-associated molecular pattern molecules
(PAMPs) and effector molecules help pathogen adapt to its
niche. Oomycete pathogens including P. infestans, B. lactucae,
A. candida and H. arabidopsis signal their presence with
PAMPs such as β-glucan or Pep13. Recognition of these
PAMPs by yet unidentified cell surface receptors (or pattern
recognition receptors) activates a signalling cascade leading to
innate immune responses including a small burst of reactive
oxygen species (ROS). These pathogens have effector molecules, such as enzyme inhibitors, small cysteine rich proteins
and RXLR type cytoplasmic effectors, which are encoded by
the pathogen genome and are delivered into the apoplast or
cytoplasm of the plant cell. These effectors are usually
virulence factors and have the capability of suppressing the
plant’s immune response by modifying host proteins, interfer-
ing with signalling and inhibiting enzyme activities. However,
similar to PAMPs, these effectors (which are then termed as
AVR proteins) could also be recognized directly or indirectly
by RLK/RLP type receptors at the cell surface or NB-LRR type
receptors within the cytoplasm. Recognition of AVRs triggers a
defence response including activation of signalling pathways,
generation of a strong ROS, cell wall alterations, irreversible
membrane damage, protein modifications, DNA laddering,
hypersensitive response and the release of endogenous elicitors.
These local defence responses are further amplified by
secondary signalling. Antimicrobial compounds are synthesised
within and at the neighbouring cells and accumulate at the
infection sites. RLK, receptor-like kinase; RLP, receptor-like
protein; NB-LRR, nucleotide binding site-leucine rich repeat
proteins
studies with this molecule have been carried out with
parsley cells, which are not normally a host for this
pathogen. Activation of complex defence responses
including ion influxes and effluxes, generation of
oxidative burst, elevated expression of defense-related
genes, and phytoalexin formation have been reported
(Nurnberger et al. 1994). Later, Brunner et al. (2002)
showed that the Pep-13 pattern is conserved in all
Phytophthora species including P. infestans and forms
part of the cell wall calcium-dependent transglutaminase
(TGase) enzyme. However, it is still not clear what type
of role TGase plays in the fitness of the pathogen. When
cells of a susceptible host plant such as potato have been
used in experiments with Pep-13, responses similar to
those reported in parsley cells have been observed.
Although, Brunner et al. (2002) reported the absence of
TGase-related transcripts from other oomycete patho-
gens such as H. arabidopsis and Pythium, bioinformatic
investigation into the recently available genome sequences of H. arabidopsis showed at least five copies of
TGase elicitor precursor (M. Tör, unpublished).
One of the main reasons why PAMPs have been
reported only from Phytophthora species could be the
life-style of these pathogens, being easy to grow in
axenic culture without contaminants from the host
plants. As new and refined techniques are developed
for the identification of new PAMPs, we would expect
to see more from other oomycetes particularly the
obligate biotrophs.
If the PAMPs are triggering a defence response in
both a susceptible host and resistant non-host plants,
the important question then arises as to why these
defence responses are not sufficient to stop pathogen
invasion. One possible explanation might be that the
60
PAMP-activated defence responses including ROS are
weak and the pathogen can tolerate them. Another
and more likely answer may be the ability of these
pathogens to use other molecules in the apoplast or
within the cytoplasm of the host cell to suppress or
manipulate the immune system of plants for their own
purpose.
Effectors are adaptation factors and manipulators
of the defence network
Earlier physiological, biochemical and classic genetic
studies in plant–pathogen interactions have concentrated on understanding pathogenicity determinants
and disease resistance genes. When the modern
techniques of molecular genetics were applied to
analyse the pathogen, especially bacteria, important
pathogenicity factors including strong attachment of
bacteria to the host cell and hydrolytic enzymes, such
as pectinases and cellulases, that facilitate pathogen
invasion into host tissues, were identified. Studies on
avirulence proteins in bacteria led to the discovery of
the trafficking of effectors from the pathogen into
host cells via the Type III secretion system. These
molecules were found to bind to a protein and thereby
alter the activity of that protein (Mudgett and
Staskawicz 1998). This finding helped the establishment of a common link in the mechanisms of
pathogenicity between plant and animal pathogens.
It has also brought a change in our thinking. Rather
than killing the host cell from outside, pathogens
inject effector proteins as virulence factors into the
host cell to adapt to a particular niche (Medzhitov
2007) and manipulate it for its own purpose (Xiao
et al. 2007). When these effectors are somehow
recognized by the cytoplasmic receptors, they are
termed avirulence (AVR) proteins (Jones and Dangl
2006). Furthermore, it also promoted the question
whether effector trafficking could also be observed in
eukaryotes, such as oomycete or fungal pathogens
(Birch et al. 2006; Ellis et al. 2006). The identification
of the effector proteins, their function and their
corresponding molecular targets in the host has been
a challenge for the scientists working on oomycete
pathogens.
In the past few years, genome sequencing and
annotations, genome mapping and associated genetic
Eur J Plant Pathol (2008) 122:57–69
studies have led to the categorization of effectors into
apoplastic and cytoplasmic groupings. This is not
surprising because: (a) host plants have defence-related
proteins including glucanases, chitinases and proteases
that are secreted outside the cell for protection; (b) the
pathogens have intercellular hyphae with which they
invade the apoplastic region, and it is expected that they
deliver apoplastic effectors to inhibit or escape plant
enzymes, (c) they form haustoria within individual cells
and from which they can deliver cytoplasmic effectors,
and (d) localization studies of plant receptor proteins
have provided vital clues to the whereabouts of the
recognition sites of PAMPs and effectors.
The majority of apoplastic and cytoplasmic effectors
have signal peptides and the strategies for their
identification, classification, characteristic properties
and species of origin have been well documented
(Kamoun 2006). Currently known apoplastic effectors
include enzyme inhibitors, members of the NEP1-like
protein family and small cysteine-rich proteins (Qutob
et al. 2006; Kamoun 2006). We should also expect to
find that some effectors are delivered to the apoplast
but function within the cytoplasm after being brought
into the cell through endocytosis or membrane
trafficking.
Recently, cloning of four Avr genes, Avr1b-1,
ATR13 and ATR1NdWs and Avr3a from three oomycetes, P. sojae, H. arabidopsis and P. infestans,
respectively (Shan et al. 2004; Allen et al. 2004;
Rehmany et al. 2005; Armstrong et al. 2005) has
enabled the identification of common conserved
regions including the N-terminal RXLR (for arginine
(Arg), any amino acid, leucine (Leu), Arg) and EER
(for glutamine (Glu), Glu, Arg.) motifs (Fig. 2). These
motifs along with the signal peptide have been used
in bioinformatic studies to analyse the available
sequence data and identify a class of RXLR cytoplasmic
effectors from the oomycete plant pathogens (Kamoun
2007; Morgan and Kamoun 2007). Bhattacharjee et al.
(2006) were quick to explore the resemblances of these
two motifs to that (RXLXE/D/Q) used for translocation of the malaria parasite (Plasmodium) into host
erythrocytes (Hiller et al. 2004) and they demonstrated
the function of the P. infestans RXLR motif in the
Plasmodium system. In addition, Bos et al (2006) used
the P. infestans Avr3a effector and demonstrated that
the C-terminal-half activated the R-gene mediated
resistance and suppressed INF1-induced cell death in
tobacco (Nicotiana benthamiana) plants. These findings
Eur J Plant Pathol (2008) 122:57–69
61
Fig. 2 Predicted structures of RXLR type oomycete effectors.
RXLR family effectors from P. infestans, P. sojae and
H. arabidopsis have targeting and functional domains. These
proteins have a signal peptide and an RXLR motif (for Arg,
any amino acid, Leu, Arg). Detailed analyses of the genome
sequences of P. sojae, P. ramorum, and H. arabidopsis showed
that the majority of these RXLR proteins have also an EER
motif (Win et al. 2007). Some of the effectors with and without
EER motif are shown. Hyaloperonospora arabidopsis
ATR1NdWsB (311aa) and P. infestans AVR3a (147aa) have
the EER motif, whereas H. arabidopsis ATR13 (187aa) and P.
sojae AVR1b (138aa) do not. Phytophthora infestans IPI-O1
protein has the EER motif but also has the RGD motif that
overlaps with the RXLR motif. In these effectors, the targeting
domains are involved in secretion of the effectors out of the
pathogens and translocation into the plant cell and are usually
well conserved. The C-terminal is responsible for manipulating
the host immune system
suggest that, similar to bacterial effectors, oomycete
effectors also play a role in altering signalling in the
defence network during their adaptation into the given
niche. In addition, the N-terminal region of these
effectors that contains the signal peptide, RXLR and
EER motifs has been shown to be responsible for the
delivery of the effectors in the elegant studies by
Whisson et al. (2007). Using Avr3a from P. infestans,
Whisson et al. (2007) fused the N-terminal region
containing the RXLR and EER motifs, to the GUS
protein and showed that the GUS protein could be
delivered from the pathogen into the host cell. In
addition, P. infestans failed to deliver the Avr3a or
Avr3a-GUS fusion into the host cell when the RXLR
and EER motifs were modified. However, silencing of
Avr3a in P. infestans has not been carried out, which
would be a key experiment to reveal whether this
effector has a role in pathogenicity.
Although a great deal of information has been
accumulating on these effectors, the method of their
transmission from extracellular space into the host
cytoplasm is still not clear. Morgan and Kamoun
(2007) have proposed that RXLR binding proteins,
chaperons or translocons, originating either from the
pathogen or plant, may be required for the delivery of
these effectors into the host cytoplasm.
Phytophthora infestans IPI-B and IPI-O genes are
expressed at an early stage of the infection (Pieterse et
al. 1994) and IPI-O1 belongs to the RXLR family of
effector proteins. In addition to the signal peptide,
RXLR and EER motif, IPI-O1 has an RGD tripeptide,
which overlaps with the RXLR motif (Fig. 2). The
RGD motif has been described as a cell adhesion
motif found in several mammalian extracellular
matrix proteins and has been proposed to reduce
plant defence responses by disrupting adhesions
between the cell wall and plasma membrane (Senchou
et al. 2004). Furthermore, detailed studies showed that
RGD-containing proteins could be ligands for some
of the receptor-like kinase type cell surface receptors
described below (Gouget et al. 2006).
Bacterial flagellin is a ligand for the receptorlike kinase (RLK)-type pattern recognition receptor
flagellin-sensitive 2 (FLS2) and the ligand-stimulated
receptor endocytosis is a kind of trafficking at the
plasma membrane (Robatzek et al. 2006; Robatzek
2007). It can be proposed that at least some of the
RXLR family effectors may be translocated into the
62
extrahaustorial matrix with the help of RXLR and
EER motifs and physically interact with cell surface
receptors through the RGD motif as was shown with
IPI-O1 (Gouget et al. 2006) and could be internalized
by these receptors as was observed with FLS2.
Effectors often undergo diversifying selection as
a result of an ‘arms race’ with the host organism.
This has been well documented with studies on
H. arabidopsis ATR13 and ATR1NdWsB effector
proteins and effectors from bacterial pathogens such
as Pseudomonas syringae (Allen et al. 2004,
Rehmany et al. 2005; Guttman et al. 2006). Recent
work on RXLR effectors from P. sojae, P. ramorum,
and H. arabidopsis, showed that positive selection is
mainly on the C-terminal region, which is responsible
for the function (Win et al. 2007).
Recognition and beyond
Cell biological research of oomycete–plant interactions has entered a new phase with the identification
of hundreds of putative effector molecules. Since both
PAMPs and effectors are foreign molecules to the host
plant, we need to address several questions including;
(a) whether the effectors from oomycete pathogens
mimic the host plant protein as seen with the bacterial
effector AvrPtoB (Abramovitch et al. 2006), (b)
whether all the molecules with the same motif, such
as RXLR, function as effectors, (c) which of these
effector molecules are suppressors or activators of the
immune response and which microbial patterns are
recognized by the plant’s sensor mechanism.
Although the oomycete effector delivery system is
different from that of bacteria, nematodes and aphids,
the end result, recognition of these effectors and
activation of the defence response, would probably
use similar mechanisms. It has been well established
that plants have sensors at the cell surface and within
the cytoplasm (Fig. 3). PRRs localised at the cell
surface play a significant role in connecting the cell
wall, plasma membrane and cytoskeleton. They are
also major players in the perception and transmission
of external signals. They include several classes such
as polygalacturonase inhibitor-like proteins, receptorlike proteins (RLPs) and, RLKs (Shiu et al. 2004;
Fritz-Laylin et al. 2005). Some of these PRRs have
been shown to recognize PAMPs such as Ef-Tu
(Zipfel and Felix 2005) and effector molecules such
Eur J Plant Pathol (2008) 122:57–69
as AvrXa21 (Lee et al. 2006) from bacterial pathogens.
Until now, no PRR that recognizes an oomycete PAMP
has been identified. However, reports are emerging that
Pep-13 could be recognized in parsley by an RLK-type
receptor (Altenbach and Robatzek 2007). In addition,
β-glucan elicitor (GE) from P. sojae has been used to
identify a receptor protein. However, although a GEbinding protein (GEBP) was purified from the membrane fraction of soybean root cells, no signal peptide
or transmembrane domain was identified. Nevertheless, immunolocalization assays indicated that the
GEBPs are localized in the plasma membrane of root
cells (Umemoto et al. 1997). This suggests that the
GEBP may be part of a protein complex localized to
the membrane, which somehow interacts with other
membrane bound proteins including PRRs.
An Arabidopsis RLK with a lectin domain recognizes IPI-O1, an RXLR type effector protein with
RGD motif (Fig. 2) from P. infestans (Gouget et al.
2006). This is an interesting finding since there is no
report of an Arabidopsis lectin RLK orthologue from
tomato or potato to suggest that IPI-O1 is recognized
in tomato or potato, and secondly, the effector is from
P. infestans for which Arabidopsis is a non-host. Thus,
the immunity triggered by this effector may be that of
non-host resistance, which would be a fascinating
piece of data.
The expression level of Arabidopsis wall-associated
kinase 1, an RLK-type PRR, increases when plants are
challenged with the Hiks1 isolate of H. arabidopsis
(Eulgem et al. 2007), which is a further confirmation
that some of these plasma membrane–cell wall
interacting PRRs are involved in signal transduction.
Alterations of expression levels of the PRRs in
Arabidopsis have also been reported in studies with
Nep1-like protein (Qutob et al. 2006). Similarly, we
have investigated the publicly available microarray
databases on H. parasitica–Arabidopsis interactions
and observed increased and decreased levels of
expression in some of these PRRs (N. Holton and M.
Tör, unpublished data). Arabidopsis Brassinosteroid
insensitive 1-associated receptor kinase 1 (BAK1) is
involved in the regulation of the containment of
microbial infection-induced cell death. When bak1
mutants were challenged with several compatible and
incompatible isolates of H. arabidopsis, reduced
sporulation of the pathogen was observed (Kemmerling
et al. 2007) indicating that cell surface receptors may
also play a role in compatibility.
Eur J Plant Pathol (2008) 122:57–69
63
Fig. 3 Canonical domain structures of cell surface and
cytoplasmic receptors. A Cell surface receptors show variation
in their domains. Receptor-like kinases (RLKs) have varying
types of extracellular domains such as leucine-rich repeat (LRR)
or lectin-type, followed by a transmembrane spanning region
(TM) and a cytoplasmic kinase domain. Receptor-like proteins
(RLPs) are similar to RLKs but do not have the cytoplasmic
kinase domains. Instead, they have a short cytoplasmic tail.
Polygalacturonase inhibitor proteins have an LRR domain and
are totally extra cellular. These cell surface receptors are also
known as Pattern Recognition Receptors as they have been
implicated in the recognition of PAMPs. To date, only one
Lectin RLK type receptor from Arabidopsis (Gouget et al.
2006), has been implicated to play a role in oomycete–plant
interactions. B Cytoplasmic receptors show variation at their
N-terminal. These proteins have a central nucleotide binding
(NB) region and an LRR domain at their C-terminal. The Nterminal region shows variations and either has a TIR domain,
resembling the cytoplasmic signalling domain of the Toll and
Interleukin 1 transmembrane receptors (referred to henceforth
TIR-NB-LRR genes) or has a coiled-coil domain, (referred to as
CC-NB-LRR genes). Most of the receptors that are involved in
the recognition of oomycete pathogens are cytoplasmic and
include Arabidopsis RAC1 (Borhan et al. 2004) for recognition
of A. candida, Arabidopsis RPP1, RPP4, RPP5 and RPP13 for
H. arabidopsis (Tör et al. 2003), potato R3a (Huang et al.
2005) for P. infestans and lettuce RGC2B (Shen et al. 2002) for
B. lactucae
Another mode of direct and indirect effector
detection and recognition takes place within the
cytoplasm by NB-LRR proteins (Fig. 3). Traditionally, the genes encoding these proteins have been
known as disease resistance genes or R-genes and
form one of the largest gene families within the plant
kingdom. Several members of the R-proteins that
provide resistance to oomycete pathogens have been
identified or cloned. For example, RPP and RAC
genes from Arabidopsis confer resistance to isolates
of H. arabidopsis and A. candida (Tör et al. 1994,
2003; Holub 2001; Borhan et al. 2004), DM3 and
RGC2 gene clusters in lettuce confers resistance to
B. lactucae (Shen et al. 2002; Wroblewski et al.
2007), R1 and R3a in potato provide resistance to
P. infestans (Ballvora et al. 2002; Huang et al. 2005).
Domain structures of these proteins (Fig. 3) are
known (Tör et al. 2003) and the ways in which they
activate the immune response are beginning to
emerge. Recent findings suggest that although these
NB-LRR proteins are residents of the cytoplasm, the
majority of them have a nuclear localization signal
(Meyers et al. 2003). Some of them including barley
MLA, tobacco N and Arabidopsis RPS4, have been
shown to move into the nucleus and it has been
proposed they activate defence expression by derepressing basal defence through association with a
WRKY transcription factor (Dangl 2007; Shen
and Schulze-Lefert 2007; Shen et al. 2007). Two
Arabidopsis NB-LRR proteins RPP2a and RPP2b are
required for the recognition of Cala2 isolate of
H. arabidopsis (Sinapidou et al. 2004). In this case, it
64
will be fascinating to elucidate whether both NB-LRR
proteins travel together between cytoplasm and the
nucleus to activate the immune system.
Recognition of PAMPs or effector molecules
activate the signalling cascade and major building
blocks of the defence network including transcription
factors, kinases, components of proteolysis or innate
immunity such as EDS1, SGT1, RAR1 and NDR1,
which have been identified from Arabidopsis or from
plants that are hosts to oomycete pathogens (Tör et al.
2002, 2003; Eulgem et al. 2007, Takahashi et al.
2007). With the identification of putative effectors, it
should now be possible to investigate which one of
these signalling components are the targets for
suppression.
Physiological changes as a result of the recognition
of the oomycete PAMPs and effectors include ion
influx, formation of wall apposition around haustoria,
hypersensitive response, formation of ROS, synthesis
of phytoalexins and PR proteins and production of
salicylic acid. These have been well documented
elsewhere (Hardham 2007).
Role of PAMPs and effectors in biotrophy
A common denominator for the important oomycete
pathogens is the biotrophic phase in their life cycles.
While H. arabidopsis, B. lactucae and A. candida are
obligate biotrophs and cause minimum injury to their
hosts, P. infestans and P. sojae are hemi-biotrophs
being biotrophic for the initial stage of up to 36 h after
inoculation and subsequently becoming necrotrophic
killing the host tissue to consume the cell content
(Grenville-Briggs and van West 2005). One of the
most distinguishing features of the biotrophic phase in
these pathogens, as well as some of the fungal
pathogens including powdery mildews and rusts, is
the formation of haustoria, which are used in nutrient
acquisition (Catanzariti et al. 2007, Voegele et al.
2001). Recent studies on the flux–rust interaction
identified effector molecules such as AvrL567, AvrM,
AvrP4, and AvrP123 within the haustorium, indicating that haustoria act as reservoirs for effector
molecules during the infection process (Catanzariti
et al. 2006).
Using a viral-based expression system, Qutob et al.
(2002) identified a necrosis-inducing protein (PsojNIP)
from P. sojae and proposed that this protein plays a
Eur J Plant Pathol (2008) 122:57–69
significant role in the transition from biotrophy to
necrotrophy. However, complementation of this study
by the down regulation of this gene to show that it is
involved in biotrophy has yet to be reported. Molecular
studies carried out with H. arabidopsis infecting
Arabidopsis helped the identification of several
putative pathogen genes that are expressed in planta
and are involved in membrane or cell wall biosynthesis,
amino acid metabolism, osmoregulation, phosphorylation and protein secretion (Bittner-Eddy et al. 2003) or
in housekeeping roles (van der Biezen et al. 2000).
Similar molecular studies coupled with proteomics
carried out with P. infestans showed that the amino
acid biosynthesis in both pathogen and the host
increases during the infection. In addition, energy
consumption, and elevated metabolism are required at
the initial stage of biotrophy (Guo et al. 2006;
Grenville-Briggs and van West 2005).
Since data on PAMPs and effector molecules from
oomycetes have been accumulating, their role in the
establishment of biotrophy rather than as the activator
of immunity can be re-evaluated. Although flagella on
zoospores of A. candida and P. infestans provide
motility for the establishment of biotrophy and are an
important part of the structure, no PAMP associated
with these flagella has yet been identified. Attachment
of these pathogens to the host cell wall is important in
the early stage of infection for initiation of appressoria
and haustoria. In this regard, the role of PAMPs such
as cell wall binding proteins cannot be ignored.
The major players for the establishment of biotrophy will undoubtedly be the effector molecules.
Working on the expression of RXLR and EER motifcontaining effectors from P. infestans, Whisson et al.
(2007) divided these effectors into three groups
according to the stage of infection at which they are
induced; during pre-infection, throughout infection
and during biotrophy only. Silencing of those effectors
induced pre-infection and during the biotrophic phase
would help to understand the contribution of effectors
towards biotrophy.
Although expression of some effectors such as
ATR13 from H. arabidopsis has been found to be
present in spores (Allen et al. 2004), it is not yet
known whether the effector protein is localised in the
spore (Rebecca Allen, personal communication).
When working on an incompatible H. arabidopsis–
Arabidopsis interaction, we observed that in most
cases the resistance response is triggered after the
Eur J Plant Pathol (2008) 122:57–69
formation of haustoria (Tör et al. 2002) indicating that
the pathogen is able to develop the necessary
structures such as appressoria and intercellular hyphae
and establish a limited biotrophy before recognition.
These findings, along with those from P. infestans
infections indicate that (a) some effectors are delivered
from hyphae and appressoria into the apoplast (see
above) and are used by the pathogen as pioneering
molecules to suppress the initial innate immune
response and adapt the pathogen to the surrounding
niche; and (b) other effectors that are delivered through
haustoria into the cytoplasm may be used for diverting
nutrients towards the pathogen.
The question as to whether the pathogen is solely
responsible for initiating biotrophy is one of the
central problems in the interactions between obligate
pathogens and their host plant. If the pathogen has
PAMPs and effectors to establish a compatible
interaction, what is the contribution of the host plant
in the compatibility? A great deal of information on
defence responses and disease resistance is available.
However, the knowledge on ‘susceptibility’ is very
limited. Until now, a few host genes required for
susceptibility have been isolated through mutant screens
and subsequent genetic analysis. Some examples of
these include POWDERY MILDEW RESISTANT 4
genes (Vogel and Somerville 2000; Vogel et al. 2002,
Nishimura et al. 2003) and oomycete DOWNY
MILDEW RESISTANT genes in Arabidopsis (van
Damme et al. 2005).
Concluding remarks and future prospects
In the last few years, genome sequencing of several
oomycete plant pathogens, including H. arabidopsis,
Phytophthora capsici, P. infestans, P. sojae, and
P. ramorum has been carried out and annotations are
underway (Tyler et al. 2006). Molecular genomic
studies, including large-scale expressed sequence tag
sequencing or generating genomic libraries, are also
being carried out with other oomycete pathogens
including Bremia. Arrival of new technologies such
as use of Solexa machines should be a great help in
these studies. For those species where sequence
information is available, bioinformatics studies are
being carried out to identify putative effector molecules and classify them according to their functional
locations (apoplastic or cytoplasmic), mode of
65
actions, (e.g. enzymatic or transcription factor), their
motifs or domains (RXLR, RGD). These studies
should also consider whether these effectors are
constitutive or are induced in planta. Although we
are in the middle of stock counting and cataloguing
these effectors, we have seen some excellent studies
towards functional analysis with a few of the known
effector molecules such as the RXLR family members
from different oomycete species (Allen et al. 2004,
Shan et al. 2004, Rehmany et al. 2005; Sohn et al.
2007). In general, it is assumed that the effector
response depends on the pathogen type. Therefore,
the initial studies on the known oomycete effectors can
be used as a starting point to launch large-scale, high
throughput effector analyses to uncover whether there
are common lines of communication between oomycete pathogens and their host plants.
Several laboratories around the world are adopting
the bacterial type III secretion system to study these
oomycete effectors on a large scale (Sohn et al. 2007).
This technique may be very suitable for the study of
cytoplasmic effectors, but, other techniques should
also be adopted to investigate the effectors that are
delivered to the apoplast.
Results obtained from high throughput studies that
concentrate on individual effectors should be compared and contrasted with those obtained from native,
pathogen-delivered effectors. A given pathogen
would deliver multiple effectors some of which would
act as suppressors of the others. Therefore, pathogen
delivery should not be ignored, and if necessary the
same effector should be put back into the same
pathogen with a known tag and investigated further to
obtain a clear picture.
Secretion and translocation of the RXLR type
effectors have been attributed to the N-terminal of
these effectors (Win et al. 2007). However, it would
be interesting to find out which plant proteins, if any,
at the cell surface are involved in the endocytosis or
transmission of these effector proteins into the
cytoplasm of the plant cell.
Micro-array studies have been employed to investigate the plant’s defence network and to understand
the modulation of signalling in plants by pathogens.
However, in the next few years, we should also
expect to see microarray studies on these oomycete
pathogens. A great deal of information should then be
obtained about how the host plant can modulate gene
expression in the pathogen genome. A systems
66
biology approach can then be used to look at the
interaction from both the pathogen and the host’s side.
Although effectors are receiving much attention in
current investigations, the next few years should also
bring more publications on oomycete PAMPs. These
would be particularly useful in the elucidation of nonhost resistance.
Another area of great importance is the genetic
manipulation of these oomycete pathogens. Although
Phytophthora species can be transformed and subjected to genetic manipulations, routine genetic
transformation methods have not been established
for the obligate species. In the next few years, we
expect to see the development of different stable
transformation methods for these pathogens including
Bremia, Hyaloperonospora and Albugo. Development
of the RNA interference method to silence these
effectors within the pathogen may be an alternative
way to stable transformation. It would then be
possible to study pathogen genetics.
Although development of new technologies is vital
to investigate the interactions between oomycete
pathogens and their hosts, the ultimate aim of these
studies, in the longer term, should be the development
of intelligent systems to control economically important
crop pathogens.
Acknowledgements I am grateful to colleagues in the
oomycete field for invigorating discussions. I would like to
thank Prof Eric Holub and Dr. Alison-Woods Tör for critically
reading the manuscript and three anonymous referees for their
constructive suggestions. Related research in my laboratory has
been supported by grants BB/D000750/1, BB/C509490/1 and
BB/E02484X/1 from the UK Biotechnology and Biological
Sciences Research Council.
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DOI 10.1007/s10658-008-9292-3
Diversity of defence mechanisms in plant–oomycete
interactions: a case study of Lactuca spp.
and Bremia lactucae
Aleš Lebeda & Michaela Sedlářová &
Marek Petřivalský & Jitka Prokopová
Received: 8 September 2007 / Accepted: 18 February 2008
# KNPV 2008
Abstract Plant pathogenic oomycetes, including
biotrophic downy mildews and hemibiotrophs/
necrotrophs such as Phytophthora and Pythium,
cause enormous economic losses on cultivated crops.
Lettuce breeders and growers face the threat of
Bremia lactucae, the causal agent of lettuce downy
mildew. This pathogen damages leaf tissues and
lettuce heads and is also frequent on wild Asteraceae
plants. The interactions of Lactuca spp. with B.
lactucae (abbr. lettuce–Bremia) display extreme variability, due to a long co-evolutionary history. For
this reason, during the last 30 years, the lettuce–
Bremia pathosystem has been used as a model for
many studies at the population, individual, organ,
A. Lebeda (*) : M. Sedlářová
Faculty of Science, Department of Botany,
Palacký University,
Šlechtitelů 11,
783 71 Olomouc-Holice, Czech Republic
e-mail: ales.lebeda@upol.cz
M. Petřivalský
Faculty of Science, Department of Biochemistry,
Palacký University,
Šlechtitelů 11,
783 71 Olomouc-Holice, Czech Republic
J. Prokopová
Faculty of Science, Department of Experimental Physics,
Palacký University,
Tř. Svobody 26,
771 46, Olomouc, Czech Republic
tissue, cellular, physiological and molecular levels, as
well as on genetic variability and the genetics of host–
parasite interactions. The first part of this review
summarizes recent data on host–parasite specificity,
host variability, resistance mechanisms and genetics
of lettuce–Bremia interactions. The second part
focuses on the development infection structures.
Phenotypic expression of infection, behaviour of B.
lactucae on leaf surfaces, the process of penetration,
development of primary infection structures, hyphae
and haustoria are discussed in relation to different
resistance mechanisms. In the third part, the components of host resistance and the variability of defence
responses are analysed. The role of reactive oxygen
species (ROS), antioxidant enzymes, nitric oxide
(NO), phenolic compounds, reorganization of cytoskeleton, electrolyte leakage, membrane damage, cell
wall disruption, hypersensitive reaction and plant
energetics are discussed in relation to defence
responses. In general, the extreme variability of
interactions between lettuce and Bremia, and their
phenotypic expression, results from diversity of the
genetic background. Different mechanisms of resistance are conditioned by an orchestra of defence
responses at the tissue, cell, and molecular levels. The
various events responsible for defence involve a
complex interaction of the processes and reactions
mentioned above. This review also provides an
overview on the timing of pathogen development,
host pathological anatomy, cytology and physiology
DO09292; No of Pages
72
Eur J Plant Pathol (2008) 122:71–89
of lettuce–Bremia associations. The significance of
these factors on the expression of different resistance mechanisms (non-host and host resistance,
race-specific and race non-specific resistance, field
resistance) is discussed.
Keywords Cytoskeleton . Genetics .
Host-and non-host resistance .
Hypersensitive reaction . Infection structures . Lettuce .
Lettuce downy mildew . Nitric oxide .
Phenolic compounds . Photosynthesis .
Plant energetics . Reactive oxygen species . Specificity
Abbreviations
ATP
BAP
CKs
dai
EHM
ER
H
HA
hai
HR
IH
IMD
MTs
NADPH
NO
NOS
PAL
PAs
POX
PSII
PTIO
QTL
ROS
TEM
3-MeOBAPR
adenosine triphosphate
benzylaminopurine
cytokinins
days after inoculation
extrahaustorial membrane
endoplasmatic reticulum
intercellular hypha
haustorium
hours after inoculation
hypersensitive reaction
intracellular hypha
irreversible membrane damage
microtubules
reduced nicotinamide adenine
dinucleotide phophate
nitric oxide
nitric oxide synthase
phenylalanine ammonium lyase
phenolic acids
peroxidase
photosystem II
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide
quantitative trait loci
reactive oxygen species
transmission electron microscopy
6-(3-methoxy-benzylamino)purine-9β-ribofuranoside
Introduction
Aspects of plant defence against pathogens have
been studied in different model pathosystems,
including interactions between plants and oomycetes
(Glazebrook 2005; Göker et al. 2007; Hardham 2007;
Hulbert et al. 2001; Kamoun 2006). One of the most
important pathosystems in terms of economic loss is
the Lactuca spp.–Bremia lactucae (abbr. lettuce–
Bremia) pathosystem (Crute 1992a; Lebeda et al.
2007, 2008). This paper highlights several aspects
of defence mechanisms in this pathosystem from
the viewpoint of phenotypic expression, cytology,
physiology, biochemistry and biophysics.
Lettuce is the common name used for about 100
species of the genus Lactuca; they are prevalently
distributed in Asia and Africa, but occur also in
Europe, North and Central America (Lebeda et al.
2004). Only one species, cultivated lettuce (Lactuca
sativa), is grown as a crop worldwide. Lettuce ranks
as one of the earliest domesticated vegetables
(8,000 years ago; Lebeda et al. 2007). The centre of
Lactuca spp. biodiversity is in southwestern Asia, in
the Tigris–Euphrates region, and lettuce probably
originates as a food plant from this region. Systematic breeding of cultivated lettuce started in the
19th century and nowadays it is an extremely
variable species, both morphologically and genetically (Lebeda et al. 2007).
Many diseases of lettuce have been described
(Davis et al. 1997), but only a few are important
enough to be considered in crop protection and
resistance breeding programmes (Lebeda et al.
2007). One such disease is lettuce downy mildew,
caused by the biotrophic oomycete B. lactucae
(Lebeda et al. 2002). The breeding of lettuce for
resistance to B. lactucae started in 1920s and now is
considered as a priority among the vegetable features.
Several different mechanisms of resistance to B.
lactucae have been identified within cultivated and
wild lettuce (Lebeda et al. 2001b). Because of limited
durability of race-specific resistance (Lebeda et al.
2002, 2007; Lebeda and Zinkernagel 2003a), the
search has focused on field resistance (Grube and
Ochoa 2005) and new sources of resistance in wild
Lactuca species (Lebeda et al. 2002, 2007).
This paper deals with the complexity of pathological processes induced in host Lactuca spp. plants
following infection by B. lactucae. The main aim was
to analyse critically the recent knowledge in this area,
within a wider context of plant–oomycete interactions
and with the main focus on the lettuce–Bremia
pathosystem.
Eur J Plant Pathol (2008) 122:71–89
Host–parasite specificity
General aspects of specificity in plant–oomycete
interactions have been at the centre of mycological
and phytopathological research during the 1990s
and early 2000s (Clark and Spencer-Phillips 2004;
Glazebrook 2005; Göker et al. 2007; Grenville-Briggs
and van West 2005; Hardham 2007; Holub and
Cooper 2004; Latijnhouwers et al. 2003; Lebeda and
Schwinn 1994; Lipka et al. 2005; Lucas et al. 1995;
Mauch-Mani 2002; O’Connell and Panstruga 2006).
For the lettuce–Bremia pathosystem, a detailed survey
of host–parasite specificity can be found in Lebeda
et al. (2001b, 2002). The enormous variability in the
specificity of interactions can be explained by the
lengthy coevolution of the host–parasite association
between Lactuca spp. and B. lactucae (Lebeda 2002;
Lebeda et al. 2002).
Taxonomy, host range and specialization
of B. lactucae
It has become apparent that taxa in the Peronosporaceae
are polyphyletic or paraphyletic (Thines et al. 2006;
Voglmayr 2008; Voglmayr et al. 2004). As a result of
molecular phylogenetic investigations, six new genera
have been described in the Peronosporaceae, with
features revealed by scanning electron microscopy
(Thines 2007). The genera Bremia, Protobremia,
Paraperonospora, Plasmoverna, Basidiophora, Benua
and Plasmopara form a dense cluster due to the uniting
aspects of similarities in the morphology of their
haustoria (vesicular to pyriform) and ultimate branches
(Göker et al. 2007; Thines 2007; Voglmayr 2008).
Bremia lactucae, a pathogen of cultivated and wild
lettuce (Lebeda et al. 2002), has been reported to
infect plants of more than two hundred species from
about 40 genera of the Asteraceae (Crute and Dixon
1981; Lebeda et al. 2002). New host species continue
to be reported (e.g. Koike and Ochoa 2007). Based on
cross-inoculation experiments and morphological
observations, the specialization of B. lactucae into
11 formae speciales was accepted (Lebeda et al. 2002;
Skidmore and Ingram 1985). These experiments
showed a high specificity of the formae speciales,
with each almost exclusively limited to an individual
host genus (Lebeda et al. 2002). However, several
previous (Lebeda and Syrovátko 1988) and recent
(Vieira and Barreto 2006) experiments have suggested
73
the possibility that infection of lettuce (L. sativa) by
B. lactucae originates from Sonchus spp. and vice
versa. Sonchus oleraceus was the most common weed
hosting B. lactucae outside of Lactuca spp. (Lebeda
et al. 2008). From about 100 wild species described
within the genus Lactuca (Lebeda et al. 2004), only
14 are reported as natural hosts of B. lactucae
(Lebeda et al. 2002). Lactuca serriola is considered
to be the most common weed host in central Europe
(Lebeda et al. 2008; Petrželová and Lebeda 2004).
Specificity of the interactions between wild Lactuca
spp. and B. lactucae is still not completely understood. Comprehensive data are available only for
L. serriola (primary lettuce gene pool), L. saligna and
L. virosa (secondary and tertiary gene pools, respectively; Lebeda et al. 2002, 2007).
Molecular phylogenetic studies, using nuclear
large subunit rDNA sequences with D1/D2 regions
(Voglmayr et al. 2004) and internal transcribed spacer
rDNA (Choi et al. 2007), revealed the presence of
several highly supported clades within the B. lactucae
complex. These lineages match partially to formae
speciales (Skidmore and Ingram 1985). Most importantly, the genetic distance of isolates originating from
Lactuca spp. and those from other hosts was clearly
demonstrated, suggesting a lack of interbreeding.
Therefore, infected wild Asteraceae plants other than
Lactuca are unlikely to serve as a source of inoculum
for infections in Lactuca spp. populations (Lebeda
et al. 2002, 2008; Voglmayr et al. 2004). Previous
individual (Lebeda 1986) and recent population
(Lebeda 2002; Lebeda et al. 2008) studies, however,
have shown that isolates of B. lactucae from L.
serriola are significantly more pathogenic to L.
serriola than to L. sativa. Data from these phenotypic
studies demonstrate the possibility of very close
genetic affinity between Lactuca host species and B.
lactucae and vice versa, and this must be considered
in the planning of experiments focused on research of
resistance mechanisms.
Resistance mechanisms in Lactuca spp.
In most interactions, the resistance of Lactuca spp. to
B. lactucae is considered as host-resistance, according
to the phenotypic, tissue and cellular expression. Only
L. saligna appears to differ in several features, thus
raising the possible existence of non-host resistance
(basic incompatibility; Lebeda et al. 2002). Recent
74
studies with individual plants (Beharav et al. 2006;
Lebeda and Zinkernagel 2003b) and populations
(Petrželová et al. 2007) of L. saligna showed a high
degree of resistance to all B. lactucae races originating from lettuce, and also those from L. serriola
(Lebeda 1986; Lebeda and Boukema 1991). Moreover, studies at the tissue, cellular and physiological
levels (Lebeda et al. 2001b, 2002, 2006; Lebeda and
Pink 1998; Lebeda and Reinink 1994; Sedlářová and
Lebeda 2001; Sedlářová et al. 2001b, 2007a, b)
confirmed that the mechanism of resistance in L.
saligna differs significantly from the mechanisms
known in L. sativa, L. serriola and L. virosa (Lebeda
et al. 2002).
Host resistance (basic compatibility) is a better
known phenomenon in this pathosystem as it has been
studied since the beginning of the 20th century from
many perspectives. The most common three categories of host resistance are reviewed below, i.e. racespecific resistance, race non-specific resistance and
field resistance (Lebeda et al. 2001b, 2002).
Race-specific resistance with its characteristic
phenotypic expression and intensively studied genetics
can be found in cultivars of L. sativa (e.g. Lebeda et al.
2007). The specificity is determined by dominant
resistance genes and/or factors in the host (Dm genes
and/or R-factors) which are matched by pathogen
dominant factors of avirulence (Crute 1992b; for more
details see Genetics of Lactuca spp.–Bremia lactucae
interactions). Race-specificity is well documented
also in wild Lactuca spp. (Table 1) and a few closely
related genera (Lebeda et al. 2002). Recently, it was
found as a common phenomenon in wild populations
of L. serriola where enormous diversity of this type of
resistance was described (Lebeda et al. 2008; Lebeda
and Petrželová 2004).
Race non-specific (non-differential) resistance is
conferred by several genes and characterized by
effectiveness against a spectrum of B. lactucae races.
Lactuca spp. genotypes with this type of resistance
posses a certain level of non-specific resistance
according to phenotypic expression (Lebeda et al.
2002). The presence of race non-specific resistance is
not well-documented for L. sativa (Lebeda et al.
2001b). It has only been reported in some accessions
of L. serriola (PI 281876 and PI 281877) for which
the genetic background is not well known, and the
presence of some major genes and modifiers is
predicted (Lebeda et al. 2002).
Eur J Plant Pathol (2008) 122:71–89
Field resistance is a complex epidemiological
phenomenon (Lebeda et al. 2002), expressed by
reduced susceptibility of mature plants grown in the
field with natural infections of B. lactucae (Grube and
Ochoa 2005). A search for sources of field resistance
in L. sativa located a high level of this resistance in
cvs Iceberg and Grand Rapids (Crute and Norwood
1981). Recent studies suggested simple inheritance of
this trait, but the single gene models did not fit
the data obtained (Grube and Ochoa 2005). Field
resistance also is expected in wild Lactuca spp., with
direct evidence existing for some L. serriola accessions (e.g. PI 281876; Lebeda et al. 2002).
Genetics of Lactuca spp.–Bremia lactucae
interactions
Nowadays, more than 45 host race-specific resistance
genes/factors (Dm/R) and complementary pathogen
virulence (v) genes (factors) are predicted in the
lettuce-Bremia pathosystem (Lebeda et al. 2006).
Many of these are used for phenotypic screening of
B. lactucae isolates and characterization of their
virulence (Lebeda and Petrželová 2008). The number
of host resistance genes is expected to increase further
with continuation of extensive phenotypic characterization of Lactuca germplasm (Beharav et al. 2006;
Lebeda and Petrželová 2004; Lebeda and Zinkernagel
2003b) and molecular investigations (Kuang et al.
2004, 2006; Michelmore, pers. comm.). At least
15 Dm genes have been characterized in lettuce spp.
(Dm1, Dm2, Dm3, Dm4, Dm5/8, Dm6, Dm7, Dm10,
Dm11, Dm12, Dm13, Dm14, Dm15, Dm16 and R18).
These genes occur in distinct clusters within the
lettuce genome, at least five clusters being recognized
(Witsenboer et al. 1997). Some of these Dm genes
originate from L. serriola (Lebeda et al. 2002), others
were described and subjected to preliminary genetic
characterisation (Bonnier et al. 1994), and numerous
others are to be expected (Lebeda and Petrželová
2004). Some recently released R-factors (R36 and
R37), which are located in L. sativa originate from L.
saligna (Michelmore et al. 2005). Other resistance
gene candidates (RGCs) are proposed and used for
evolutionary studies to explore the diversity of
Lactuca spp. germplasm (Kuang et al. 2004, 2006).
However, the specificities of the genes need to be
established and their effectiveness against given
pathogen races must be demonstrated. This poses a
Category of
resistance
Lactuca spp. genotype
Non-host?
Race-specific
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
Race-non-specific
Field
saligna (LSA/6)
sativa (Cobham Green)
sativa (Dandie)
sativa (Valmaine)
sativa (Mariska)
serriola (PIVT 1168)
saligna (CGN 5147)
virosa (LVIR/57/1)
serriola (PI 281876)
sativa (Iceberg)
Genetical background/
resistance gene (factor)a
Response to Bremia
lactucaeb
Relative degree of infection structure development and tissue responsec
Primary
vesicle
Secondary
vesicle
Hyphae
Haustoria
Hypersensitive
reaction
Subepidermal
necrosis
?
R 0 (?)
Dm 3
Dm 5/8
R 18
R?
R?
R?
R ? (+modif.?)
nR?
−
+
+
−
−
−
−
(−)
(−)
+
3
4
4
3/4
2
2
3
3/4
4
3
2
4
4
1
2
1
1
3
4
3
0
4
2
1
2
0/1
1
1/2
2
3
0
4
1
0
2
0
0
1/2
2
3
1
1
1
2
4
4
1
4
4
1
1
0
1
0
3
2
0
2/3
2
0
Eur J Plant Pathol (2008) 122:71–89
Table 1 Generalized overview on variability in formation of B. lactucae infection structures and reactions of Lactuca spp. tissues at 48 h after inoculation in various categories of
resistance (compiled according to Lebeda et al. 2001b, 2002, 2006; Lebeda and Pink 1998; the data were obtained on leaf discs derived from adult plants)
? This category is still questionable for L. saligna (see discussion in: Jeuken and Lindhout 2002; Lebeda et al. 2001b, 2002)
a
? Not known or unspecified, R race-specific resistance factor, Dm race-specific resistance gene; modif. modifier gene(s); n more R-factors
b
Categories of phenotypic expression of Lactuca spp. response to B. lactucae: − incompatible (no sporulation); (−) incompletely incompatible (very limited sporulation occurring
mostly at the cutting edges of leaf discs); + compatible (profuse sporulation); a field resistance cannot be distinguished by screening either on cotyledons or leaf discs
c
Relative degree of occurrence of pathogen infection structures and plant tissue response compared to susceptible control (details are given in Lebeda et al. 2002): 0 none recorded,
1 very low frequency, 2 low frequency, 3 medium frequency, 4 high frequency. Significant differences in frequency and timing are specific for given genotype-race interaction,
usually L. sativa genotypes vary from other wild Lactuca spp.
75
76
barrier to the rapid engineering of durable resistance.
Currently, much effort in lettuce resistance breeding is
focused on deployment of non-durable R-genes
(Lebeda et al. 2007; Pink 2002). Thus, breeders have
to look for new sources in wild Lactuca spp. (Beharav
et al. 2006; Lebeda et al. 2002; Lebeda and
Zinkernagel 2003b), but there is still a lack of genetic
and molecular data on variation and resistance in
other wild Lactuca spp. (e.g. L. virosa, L. saligna;
Kitner et al. 2008; Lebeda et al. 2002, 2007).
The first detailed genetic studies dealing with L.
saligna resistance against B. lactucae were performed
by Jeuken and Lindhout (2002) as a QTL analysis on
plants of a L. saligna (resistant) × L. sativa (susceptible) cross. The phenotype of the F2 population
showed a continuous range of resistance categories
from completely resistant to completely susceptible,
providing evidence that both qualitative and quantitative resistance were involved. Subsequent QTL
mapping revealed a qualitative gene (R39) and three
QTL (RBQ1, RBQ2 and RBQ3) accountable for the
quantitative resistance. Some additional studies implied that resistance in L. saligna was quantitatively
expressed and might be race non-specific. The current
general view on L. saligna non-host resistance is that
it is not explained by accumulation of race-specific
resistance genes (Dm genes) but instead by resistance
mechanisms based on QTL (Jeuken and Lindhout
2002).
Pyramiding of resistance genes in lettuce cultivars
(Crute 1992b) forms a selection pressure that alters
the structure of pathogen populations (Lebeda and
Zinkernagel 2003a) and initiates the boom and bust
cycle. On the other hand, gene-flow from cultivated to
natural Lactuca spp.–B. lactucae populations and
vice versa must also be considered (Lebeda 2002;
Lebeda et al. 2008). An hypothesis of Hooftman et al.
(2007) attributed the expanding distribution of prickly
lettuce (L. serriola) in Europe to enhanced plant
fitness by hybridisation with lettuce (L. sativa).
However, this ecological study revealed that introgression of an important crop trait, downy mildew
resistance, from lettuce into L. serriola hybrids was
insignificant for plant reproductive fitness. In contrast,
effectiveness of some resistance traits introduced to
lettuce from wild Lactuca spp. (esp. L. serriola)
might be broken by pathogen populations present
in wild plant pathosystems (Lebeda 2002; Lebeda
et al. 2008).
Eur J Plant Pathol (2008) 122:71–89
Development of Bremia lactucae
Symptoms of lettuce downy mildew
Description of downy mildew symptoms on lettuce
(L. sativa) can be found elsewhere (e.g. Crute and
Dixon 1981; Davis et al. 1997). Symptoms typically
appear as areas of chlorotic tissue, mostly delimited by
the main veins, which is accompanied by profuse
sporulation on the abaxial side of leaves in compatible
interactions. In field conditions, the air-borne asexual
conidia are the most important means of disease spread
throughout the growing season. Intensity of sporulation
as well as viability of conidia is influenced substantially
by environmental factors (Judelson and Michelmore
1992; Nordskog et al. 2007) and by concentration of
primary inoculum (Crute and Dickinson 1976).
Broad variation of phenotypic expression of B.
lactucae infection was reported in both susceptible
and resistant Lactuca spp. genotypes (Table 1; for
review see Lebeda et al. 2001b, 2002, 2008). The
phenotype of non-host resistance (e.g. in some L.
saligna accessions and most Asteraceae species) is
characterized by a lack of symptoms (Crute and
Dickinson 1976; Lebeda and Reinink 1994; Lebeda
and Syrovátko 1988; Sedlářová et al. 2001b). Nevertheless, expression of macroscopic chlorosis (Crute and
Dickinson 1976), necrosis (Lebeda and Reinink 1994;
Norwood et al. 1981) or sub-epidermal necrosis
(Lebeda and Reinink 1994; Lebeda et al. 2006) was
also recorded. Expression of host resistance symptoms
varies according to the ontogenetic stage of the host, as
it was found to differ between the cotyledons and
adult plants within the same interaction (Crute and
Dickinson 1976; Lebeda and Reinink 1991; Lebeda
et al. 2006). In lettuce–Bremia interactions a wide array
of symptoms occurs, ranging from no visible symptoms to an extensive necrotization (incompatibility),
and from limited sporulation (incomplete resistance) to
profuse sporulation without any other visible symptoms (full compatibility; Lebeda et al. 2002). These
categories of symptoms are highly specific and
conditioned by race-specific resistance Dm genes in
many cases. For example, Dm7 conditions reduced
sporulation and necrosis in some genotype–race
interactions (Crute and Johnson 1976). Wild Lactuca
spp., e.g. L. serriola and L. virosa, have also been
reported to express a broad spectrum of symptoms
(Lebeda and Pink 1998; Norwood et al. 1981). There
Eur J Plant Pathol (2008) 122:71–89
are many intermediate phenotypes between the
extremes with a substantial influence of experimental
and environmental conditions (for overview see
Lebeda et al. 2001b, 2002).
Characteristics of leaf surface: influence on conidial
germination and appressorium formation
The characteristics of plant leaf surfaces, referred to
here as the indumentum and including the number
and character of trichomes, thickness and composition
of waxes, number and position of stomata, determine
success or failure of pathogen spore deposition and
subsequent ingress by infection structures. Spores
deposited on the leaf surface face several obstacles to
gaining host nutrients: cuticle, cell wall and plasma
membrane (Lebeda et al. 2001b).
The cuticle is known as a ‘two-step’ barrier
comprising an internal and external layer. The internal
cuticle on the inner periclinal walls of epidermal cells
functions primarily in water exchange regulation
(Pesacreta and Hasenstein 1999). More important
from the pathogen perspective is the external cuticle.
Its structure and function have been documented in
many plants but experimental data are lacking for
Lactuca species. Study of indumentum characteristics
revealed substantial differences among Lactuca species (Lebeda et al. 1999), but there are no data
relating the indumentum pattern to B. lactucae
germination. However, evidence does exist for a
relationship between leaf epidermal characteristics in
cultivars of potato (Solanum tuberosum) and expression of resistance or susceptibility to Phytophthora
infestans (Mahajan and Dhillon 2003).
There are several crucial steps required prior to the
start of oomycete pathogenesis similar to fungal
pathogens, i.e. adhesion of spores to the plant surface,
and the formation of germ tubes, appressoria and
penetration pegs (reviewed in Latijnhouwers et al.
2003). Attachment of germinating spores is mediated
through secretion of an extra-conidial matrix. As soon
as a germ tube emerges from the Hyaloperonospora
arabidopsidis (H. parasitica) conidium, an ‘adhesive
coctail’ composed of proteins, glycoproteins and β1,3-glucans is released (Carzaniga et al. 2001).
Recently, Hardham (2007) brought together microscopic and molecular data relating to early stages of
the infection process in Phytophthora, Pythium and
Hyaloperonospora spp.
77
The mechanisms of retention and adhesion of B.
lactucae conidia to host leaf surfaces have not been
elucidated in detail. The pre-penetration phase was
studied by Andrews (1975) who reported the possibility that B. lactucae absorbs nutrients (e.g. glucose)
from the leaf surface. Many papers deal with B.
lactucae spore germination, penetration and the
further development of infection structures (reviewed
in Lebeda et al. 2001b). Conidia start germination
mostly at 1–3 h after inoculation on both non-host
and host plants (summarized in Lebeda et al. 2002).
Germ-tube length is a highly variable parameter
(compare Fig. 1a and g; Lebeda and Pink 1998) and
does not relate directly to the host resistance, i.e. to
Dm gene expression, but seems to be specific for each
host genotype–parasite race interaction (Lebeda et al.
2001b, 2006). In general, significantly shorter germ
tubes develop on wild Lactuca spp. than on lettuce
(L. sativa) genotypes (Lebeda and Pink 1998; Lebeda
et al. 2006).
The germination of B. lactucae conidia (Fig. 1a) is
affected by environmental conditions, such as temperature (Sargent 1976; Sargent and Payne 1974). The
peripheral cytoplasm of conidia in the ‘dormant’ stage
contains lipid droplets which are dispersed during the
phase of activation (preceding germination). This
phase is followed by activation of dictyosomes and
endoplasmatic reticulum. Mobilisation of reserves for
development of the germ tube tip is accompanied by
increased lipolytic activity in mitochondria and
esterase activity in vacuoles (Duddridge and Sargent
1978).
Formation of oomycete appressoria, non-pigmented
swellings of germ tube tips that differentiate penetration
pegs, is not synchronised with germination and may be
induced by topological features of the leaf surface
(Latijnhouwers et al. 2003; Lebeda et al. 2001b).
Significant differences in the frequency of appressorial
formation were found between cotyledons (higher
frequency) and leaf discs of adult plants (Lebeda and
Reinink 1991). The influence of leaf surface character
on appressorial formation was demonstrated in cv.
Iceberg (genotype with high level of field resistance),
where frequency of appressorial formation was significantly lower than on lettuce cultivars with ineffective
race-specific resistance (Lebeda and Reinink 1991). A
comparative study showed a higher incidence of B.
lactucae appressorial development on lettuce (L.
sativa) plants compared to wild relatives (L. serriola,
78
Fig. 1 Development of Bremia lactucae (race BL16) and the
response of Lactuca spp. cells. a–c. Pathogen growth within
tissues of susceptible L. sativa (Cobham Green); a, germination
and appressorial formation (12 hai); b, colonisation of host
tissues (48 hai); c, formation of numerous haustoria (120 hai).
d. A detail of haustorium with callose deposited around its neck
(168 hai), susceptible L. sativa (British Hilde). e, f. Realignment of host MTs due to infection (immunolocalisation of αtubulin); e, microtubular ‘basket’ formed with host plasma
membrane invagination (48 hai), susceptible L. sativa
(UCDM2); f, depolymerization of cortical microtubules induced by initiation of HR (48 hai), resistant L. serriola (PIVT
1309). g. Peroxidase activity localised both in pathogen
infection structures and in the cell of susceptible L. sativa
Eur J Plant Pathol (2008) 122:71–89
(UCDM2; 24 hai). h. Signal for NO in epidermal cell of
susceptible L. sativa (Cobham Green) penetrated by haustoria
beneath the growing hypha (192 hai). i, j. Hypersensitive
reaction (HR); i, initial stages of HR with granulation of
cytoplasm (48 hai), L. virosa (NVRS 10.001602); j, necrosis, a
visible outcome of HR is caused by oxidation of phenolics
(336 hai), resistant L. serriola (PIVT 1309). Infection structures:
spore (S), germ tube (GT), appressorium (A), primary (PV) and
secondary vesicle (SV), intracellular hypha (IH), intercellular
hypha (H), haustorium (HA). The bar corresponds to 20 μm. The
micrographs were obtained by conventional light microscopy
(a–c, g, i, j), fluorescence microscopy (d–f) and confocal laser
scanning microscopy (h). Photo courtesy by M. Sedlářová
Eur J Plant Pathol (2008) 122:71–89
L. saligna, L. virosa), whereas no significant differences were found among the wild Lactuca spp.
(Sedlářová et al. 2001b).
Penetration and development of primary
and secondary vesicles
Penetration of plant surfaces by oomycetes is performed by a combination of mechanical force and
secreted chemicals, as with other fungal pathogens
(Latijnhouwers et al. 2003; Lebeda et al. 2001a).
Appressoria of B. lactucae are a prerequisite for
penetration, and exert high pressure on cell walls
allowing penetration pegs to pierce the periclinal cell
wall, and to colonize the underlying epidermal cell by
the formation of primary and secondary vesicles
(Sargent et al. 1973). To date, the turgor pressure
exerted by appressoria has not been quantified in any
oomycete species. The cuticular penetration predominates in B. lactucae, with penetration via stomata
incident in about 1–5% of germ tubes (Lebeda and
Reinink 1991).
Environmental factors are crucial for the B.
lactucae penetration process. The effect of temperature is especially important, as seen from an optimum
of 15–20°C for germination (Sargent 1976) but 12–
15°C for penetration (MacLean and Tommerup 1979).
Some details related to the timing of this process were
summarized by Lebeda et al. (2001b). The penetration
rate is a frequently studied parameter in screening
studies (e.g. Lebeda and Pink 1998; Lebeda et al.
2006; Sedlářová et al. 2001b).
Chemical degradation of the cuticle and cell wall is
the second necessity for successful penetration of
plant cells. Secretion of a wide range of degradative
enzymes has been described for fungi and oomycetes
(Lebeda et al. 2001a), including hemibiotrophic
Phytophthora and Pythium spp. (Hardham 2007).
Extensive genomic studies have initiated the characterization of genes encoding these enzymes. However,
direct demonstration of the action of cell walldegrading enzymes is a perspective for future work
(Hardham 2007).
Data on B. lactucae-derived enzymes are quite
limited, and only polygalacturonase, esterase and
protease activities have been reported (Van PeltHeerschap and Smit-Bakker 1993). Pathogen lipolytic
enzymes, mentioned above in the context of germination (Characteristics of leaf surface: influence on
79
conidial germination and appressorium formation),
enable lipid degradation in the cuticle and enable
subsequent pathogenesis (Sargent et al. 1973). Lipase
activity is also elevated during penetration and the
formation of primary vesicles (Duddridge and Sargent
1978). At this stage, the lipolytic activity is localized
in lomasomes which occur mostly at the periphery of
the expanding vesicle, as illustrated by transmission
electron microscopy (Zinkernagel 1985; Zinkernagel
and Bartscherer 1978).
Once the pathogen overcomes the barriers of the
cuticle and cell wall by the activity of cell-wall
degrading enzymes (Lebeda et al. 2001a; Sargent
et al. 1973), it gains access to the cell lumen. Bremia
lactucae forms primary infection structures within the
host epidermal cell by invagination of the host plasma
membrane. Formation of the primary vesicle (PV),
secondary vesicle (SV), and intracellular hypha (IH;
Fig. 1b) are not initially followed by destruction of the
host cell plasma membrane (Ingram et al. 1976). Only
subsequently is the plasma membrane perforated, with
colonization of sub-epidermal tissues by intercellular
hyphae (H) and haustoria (HA) ensuing (Fig. 1c).
Various aspects of this process were reviewed by
Lebeda et al. (2001b).
Early stages after inoculation are very important
for recognition and initiation of defence responses. In
lettuce–Bremia interactions, both pre-haustorial and
post-haustorial recognition occurs, based on specific
Dm/Avr gene combinations (Mansfield et al. 1997).
Incidence of PVs and SVs, as well as the timing of
their formation, may differ in interactions with an
identical phenotype (Lebeda and Pink 1998; Lebeda
et al. 2006; Sedlářová et al. 2001b). Formation of
PVs and SVs also can be found in non-host plants,
though at significantly lower frequencies than in
host plants (summarized in Lebeda et al. 2002). In
some L. saligna interactions with B. lactucae, the
SV represents the final stage of oomycete development which is considered to distinguish non-host
resistance (Lebeda and Reinink 1994; Lebeda et al.
2002, 2006; Sedlářová et al. 2001b). Quite intriguing
is B. lactucae development in interspecific hybrids
of lettuce (L. sativa) with wild Lactuca spp., where
an ‘heterosis’ effect was recorded (increased rate of
B. lactucae infection structures in F1 hybrids
compared to both parents). Details can be found
elsewhere (Lebeda and Reinink 1994; Lebeda et al.
2001b, 2006).
80
Development of hyphae and haustoria
The most important period for hyphal development
of B. lactucae is 24–48 hai, but the extent and speed
of formation of intra- (IH) and inter-cellular (H)
hyphae and haustoria (HA) is extremely variable
among non-host and host genotypes (Table 1;
Lebeda et al. 2001b, 2002). The growth of intercellular hyphae of B. lactucae in mesophyll tissue even
starts at 12 hai in some compatible interactions
(Sedlářová et al. 2001b). Although delayed, the
development of IH and H occur in most incompatible (non-host and host) interactions (Lebeda and
Schwinn 1994).
An extreme variability of incompatible reactions in
Lactuca spp. has been described (Lebeda et al. 2006).
In L. saligna (LSA/6), which is considered as a nonhost genotype (Lebeda et al. 2001b), B. lactucae did
not form any intra- and intercellular hyphae (Lebeda
et al. 2006). However, in many other incompatible
interactions both types of hyphae were formed with
significant differences in frequency (Lebeda and Pink
1998; Lebeda and Reinink 1994; Lebeda et al. 2006).
Formation of IH and H was suggested as a crucial
developmental stage for B. lactucae, and as a limiting
factor in host–parasite communication as well as
expression of various resistance mechanisms (Lebeda
et al. 2001b, 2006). Quantitative comparative studies
showed significant variation in size (length and
width) of hyphae (Lebeda and Pink 1998; Lebeda
and Reinink 1994), thus supporting the assumption
that these features relate to differences in the
physiology of resistance (Lebeda and Reinink 1991,
1994; Lebeda and Pink 1998; Lebeda et al. 2006;
Sedlářová et al. 2001b).
Pyriform haustoria, characteristic for B. lactucae
(Voglmayr et al. 2004), originate as hyphal side
branches in penetrated cells to accomplish parasitic
feeding. The haustorium remains outside the plant
protoplast and an altered interface is developed that
probably assists uptake of nutrients and the exchange
of signals between both partners (Spencer-Phillips
1997). Composition of the extrahaustorial membrane
(EHM), separating the haustorium from the host
cytoplasm, differs from the semi-permeable plasma
membrane as described in detail for Hyaloperonospora
arabidopsidis (O’Connell and Panstruga 2006). In this
respect, the EHM in both oomycete and fungal
infections (e.g. in powdery mildews; Koh et al. 2005)
Eur J Plant Pathol (2008) 122:71–89
are similar. Callose deposits may be formed around
haustoria in lettuce–Bremia interactions (for detail, see
Plasma membrane homeostasis and deposition of
callose).
The frequency and timing of haustorial formation
and the final size of haustoria are very specific
features of host–parasite interactions (Lebeda et al.
2001b). In non-host resistance of L. saligna, haustoria
form neither on IH nor directly on SV (Lebeda et al.
2006). Frequency of haustorial formation varies
specifically among Lactuca spp. genotypes carrying
different Dm genes and/or R-factors for host resistance. In compatible host–parasite interactions, the
frequency and size of haustoria is significantly higher
than in incompatible interactions (Lebeda et al. 2002,
2006; Sedlářová et al. 2001b). The ‘heterosis’ effect
mentioned for PV and SV also was also reported for
haustoria (Lebeda and Reinink 1994; Lebeda and
Pink 1998).
Components of host resistance and variability
of defence
An integrated approach is being adopted in plant science
to understand intercellular signalling, i.e. how plants
perceive and respond to external and internal stimuli.
Combination of molecular, chemical and electrical
components is essential (Birch et al. 2006; Mansfield
2005; O’Connell and Panstruga 2006; Robatzek 2007;
Takemoto and Hardham 2004; Walters and McRoberts
2006). Several chemical and physical factors that
condition shifts in plant metabolism and architecture
induced by oomycete pathogenesis, as well as their
importance for resistance of Lactuca spp. to B.
lactucae, are considered below.
Reactive oxygen species (ROS), antioxidants
and ROS-scavenging enzymes
Release of reactive oxygen (ROS), nitrogen (RNS) and
sulphur (RSS) species intermediates, combined with
transport of phytohormones, are amongst the early
chemical signals during plant–pathogen interactions.
ROS affect establishment of infection, enable redox
signal transduction (e.g. hydrogen peroxide together
with NO and SA amplifies resistance responses;
Delledonne et al. 2003) and trigger programmed cell
death (Kamoun et al. 1999). Therefore, generation of
Eur J Plant Pathol (2008) 122:71–89
ROS may serve as a marker of pathogenesis and/or
plant defence initiation and progress.
Hydrogen peroxide (H2O2), a secondary messenger
molecule, was accumulated in Lactuca spp. tissues
challenged by B. lactucae, whereas superoxide (O2−)
was not detected (Sedlářová et al. 2007a). Dramatic
changes of H2O2 correlate with race-specific resistance, especially in L. virosa where it is characterized
by early HR onset. In contrast, the supposed non-host
resistance in L. saligna (CGN 05271) is accompanied
by only minor changes in the level of H2O2, the
content of which is generally lower compared to the
other species (Sedlářová et al. 2007a).
High antioxidant status in plants was reported to
hinder transportation of ROS across the cell (Neill
et al. 2002); therefore our experiments have included
the use of antioxidant enzymes and non-enzymatic
ROS scavengers. Changes in peroxidase (POX),
catalase and polyphenoloxidase activities in lettuce
tissue, in relation to the infection process of B.
lactucae, were demonstrated by Zinkernagel (1986).
Our study focused on the dynamics and isozyme
spectrum of three ROS-scavenging enzymes, catalase,
peroxidase and superoxide dismutase, and unveiled
the importance of peroxidase (POX). However, POX
activity (Fig. 1g) was found only in the cytosolic
fraction, with a higher basic level in wild Lactuca
spp. compared to cultivated lettuce. Increase of POX
activity was linked to expression of race-specific
resistance in prickly lettuce (L. serriola) and great
lettuce (L. virosa), with a two-peak timing (6–12 hai,
the recognition phase, and from 24 hai at induction of
HR; Sedlářová et al. 2007a). The relationship between
the increase of pre-infection POX activity and level of
field resistance to B. lactucae was demonstrated in
lettuce (L. sativa) cultivars and accessions of L.
serriola (Reuveni et al. 1991). Thus POX was
proposed to serve as a marker in the selection for
field resistance to different downy mildew pathogens
(Lebeda and Schwinn 1994). From a large group of
molecules with an antioxidative action, contents of
quercetin and rutin were studied in the leaf extracts
(for details see Flavonoids, phenolic acids and PAL).
Nevertheless, the complexity of leaf phytochemistry raises the possibility that many other antioxidants
may be involved in the interplay between Lactuca
spp. and B. lactucae. This merits investigation as it
would provide a better understanding of host–parasite
interactions.
81
Nitric oxide, NO synthase and NO modulators
Nitric oxide (NO) performs a variety of phytochemical roles during pathogenesis. NO and its metabolites
mediate transcription of specific genes during pathogenesis (Neill et al. 2002); synchronized formation of
NO and H2O2 co-regulates the cell death programme
and the phenylpropanoid pathway (Dellendone et al.
2003).
In lettuce–Bremia interactions, the formation of
NO was localized in cells penetrated by either
primary infection structures or haustoria (Fig. 1h).
NO synthase (NOS) activity was followed by the
oxyhemoglobin method to detect NO production in
lettuce and wild Lactuca spp. leaf extracts up to
216 hai. A significant increase of NOS activity was
found in L. virosa early after inoculation (4–8 hai),
with a second lower peak at 168 hai. Non-host
resistance of L. saligna (CGN 05271) correlated with
low amounts of NO production and relatively smallscale increase of NOS activity (Petřivalský, unpubl.).
Modulators of NO metabolism were applied to L.
sativa tissues to follow their influence on B. lactucae
development up to 48 hai (Petřivalský et al. 2007).
Sodium nitroprusside, a model NO donor, decreased
conidial germination rate at 4 hai and strongly
inhibited further pathogen growth. On the contrary,
PTIO as a specific NO scavenger, showed a strong
stimulatory effect on pathogen development at 24–
48 hai. However, no significant effect of either LNAME (competitive inhibitor of animal nitric oxide
synthases) or sodium tungstate (specific inhibitor of
plant nitrate reductase) was found. This may be
explained by either the possible contribution of
another NO-generating system in Lactuca spp., or
the lower bioavailability and chemical stability of
these substances during leaf tissue treatment (for more
details, see Petřivalský et al. 2007).
Flavonoids, phenolic acids and PAL
Phenolic compounds are abundant in plants of the
Asteraceae family (Bohm and Stuessy 2001). Screening
was conducted in the early 1980s in order to utilize
flavoids and flavonols of the genus Lactuca in
chemotaxonomy (Rees and Harborne 1984). Recently,
the phenolic compounds in lettuce have been studied in
relation to: leafy vegetable processing to avoid browning due to mechanical injury (Saltveita et al. 2005) and
82
human medications for anti-inflammatory, anti-bacterial, anti-diabetic and anti-proliferative effects (Chen
et al. 2007).
Quercetin, rutin, caffeic acid and chlorogenic acid
and several other phenolic compounds are known as
major components of lettuce extracts (e.g. Chen et al.
2007). Quercetin, one of the aglycones, was reported
from L. sativa, L. serriola and L. virosa, whilst only
traces were found in L. saligna and (Rees and
Harborne 1984). Quercetin and rutin molecules
operate as strong antioxidants. A study was conducted
to measure their amount in all four of these Lactuca
species during the course of B. lactucae pathogenesis,
determined by the accumulation of autofluorescent
phenolics near the plasma membrane of penetrated
cells (Bennett et al. 1996; Sedlářová and Lebeda
2001). Quantitative analysis of leaf extracts led to the
finding that L. sativa genotypes with non-effective
race-specific resistance significantly differ in quercetin content (approx. 10 μmol g−1 FW) from L. sativa
with effective race-specific resistance and three wild
lettuce species (less than 1 μmol g−1 FW). This may
help in the balancing of oxidative processes induced
by B. lactucae. Content of rutin varied slightly from
0.28 μmol g−1 FW in L. virosa to 0.87 μmol g−1 FW
in L. sativa (Petřivalský, unpubl.). Whilst no striking
linkage between rutin level and genotype susceptibility/
resistance was found, the external application of
rutin solution to lettuce tissues delayed B. lactucae
germination and penetration (Petřivalský et al. 2007).
The content of phenolic acids (PAs) changes
during ontogenesis of Lactuca spp.; in cotyledons,
chlorogenic acid prevails, whereas amounts of other
PAs increase with plant development (Grúz, unpubl.).
A time-course study of PAs in adult L. sativa (cv.
Mariska) showed significant changes in caffeic acid
and minor changes in chlorogenic acids after inoculation with incompatible B. lactucae race BL16. A
two-peak (6–24 and 72 hai) decrease in their level
(Grúz, unpubl.) corresponds with induction of oxidative processes (see also previously).
Preliminary studies of phenylalanine ammonium
lyase (PAL), a key enzyme of the phenylpropanoid
pathway, have not disclosed a relationship between
PAL activity and B. lactucae colonization (Sedlářová,
unpubl.). The phenylpropanoid pathway is also
known to be connected with the formation of
structural barriers to pathogen ingress by the deposition of lignin (Mauch-Mani and Slusarenko 1996).
Eur J Plant Pathol (2008) 122:71–89
Although large pools of phenolic compounds that
might serve as a source of precursors for incorporation in the lettuce cell wall were found due to B.
lactucae challenge (Bennett et al. 1996), lignification
has not been proved (Sedlářová and Lebeda 2001).
Reorganisation of the cytoskeleton
Host cells challenged by oomycetes undergo drastic
changes similar to cells targeted by fungal pathogens
(Latijnhouwers et al. 2003; Takemoto and Hardham
2004). Rapid rearrangements of cytoskeletal components (microtubules and F-actin) begin even prior to
penetration of the cell wall (during maturation of the
appressorium), and link to the relocation of cytoplasm, nuclei and other organelles within epidermal
cells in contact with the pathogen (Koh et al. 2005;
Takemoto et al. 2003). Pathogens that continue
colonisation beyond the epidermis via intercellular
hyphae induce similar alterations of architecture in
mesophyll cells penetrated by haustoria (SpencerPhillips 1997).
The multitude of binding proteins associated with
the cytoskeleton and its extraordinary dynamics
facilitate trafficking of many pathogen-derived signals. The vital role of the host cytoskeleton in nonpathogen and pathogen recognition (Takemoto et al.
2003), the binding of effector molecules (Binet et al.
2001), gene expression (Hamada 2007) and in
relation to defects in cell wall microfibril orientation
(Wasteneys 2004) are well documented. In lettuce–
Bremia interactions, actin filaments were not detected
in epidermal cells after contact with the pathogen,
whereas cortical microtubules (MTs) supported invagination of the plasma membrane and formation of
primary and secondary vesicles (Sedlářová et al.
2001a). In compatible interactions, such a unique
layout resembles a ‘microtubular basket’ (Fig. 1e),
and is characterised by a high density of MTs at the
necks of vesicles (Sedlářová, unpubl.). This suggests
a role in the deposition of callose at these locations
(Sedlářová and Lebeda 2001). In resistant plants, the
timing and extent of the destruction of MTs (Fig. 1f)
is correlated with a hypersensitive reaction and
typically affects one cell per infection site in L.
sativa, and 2–3 cells in L. virosa (Lebeda and Pink
1998; Lebeda et al. 2006; Sedlářová et al. 2001b).
Construction of Arabidopsis thaliana mutants
with GFP-tagged cytoskeleton or organelles made it
Eur J Plant Pathol (2008) 122:71–89
possible to follow subcellular changes in vivo
(Hardham 2007; Koh et al. 2005; Takemoto et al.
2003). Rapid and continuing intracellular realignment
during Hyaloperonospora arabidopsidis challenge
was shown elegantly by Takemoto et al. (2003),
including ‘focusing-to-pathogen’ of F-actin below the
penetration site and in neighbouring cells. Secretion of
plant materials around the infection site, indicated by an
aggregation of ER and Golgi bodies, did not stop
penetration by an avirulent isolate of H. arabidopsidis
and even the non-pathogen Phytophthora sojae.
A number of detailed experimental data raise the
question: what facilitates the extreme plasticity of the
plant cytoskeleton in reaction to oomycete and fungal
pathogens? As well as the high degree of conservation
of tubulin and actin throughout a variety of genomes, a
wide array of associated molecules (proteins, RNA)
was found in the cytoskeletal complexes. The rapid
rearrangement of MTs in reaction to external/internal
stimuli occurs because the nucleation sites of MTs are
based on γ-tubulin anchors which can be relocated
easily within the plant cell (Hamada 2007).
83
autofluorescent phenolics (Bennett et al. 1996).
Pathogen recognition in resistant cells results in
irreversible loss of membrane integrity and initiation
of the HR. In compatible interactions, the hyphae
growing between mesophyll cells penetrate adjacent
cell walls (Fig. 1c) to form haustoria that invaginate
host plasma membranes. A new interface, the extrahaustorial membrane (EHM), arises from the secretion of proteinaceous and carbohydrate compounds by
both partners (Koh et al. 2005; O’Connell and
Panstruga 2006).
Formation of Bremia infection structures is associated with the deposition of callose, especially in the
necks between PVs and SVs, and between SVs and
haustoria, forming sheath-like structures around haustoria (Fig. 1d; Sargent et al. 1973). The callose is of
host origin and the strongest deposition was reported
in compatible interactions (Sedlářová and Lebeda
2001). The chemical composition of callose is very
similar to components of extracellular matrices
released by H. arabidopsidis spores upon germination, namely β-1,3-glucans which has a protective
action (Carzaniga et al. 2001).
Plasma membrane homeostasis and deposition
of callose
Hypersensitive response: extent and timing
Similar adaptations, including haustorial development,
have evolved in oomycetes and fungi to enhance a
parasitic life strategy (Latijnhouwers et al. 2003). As the
integrity of the plasma membrane is a crucial prerequisite for plant cell functionality, as well as the
homestasis of cellular processes, the biotrophic pathogens deploy mechanisms to minimise disruption of the
host cell (Glazebrook 2005; Grenville-Briggs and van
West 2005; Koh et al. 2005; O’Connell and Panstruga
2006; Walters and McRoberts 2006). The plasma
membrane regulates osmotic processes (Bennett et al.
1996). Host transmembrane proteins are engaged in the
perception of pathogen-associated molecular patterns
(O’Connell and Panstruga 2006; Robatzek 2007) and
with the aid of the cytoskeleton, facilitate vesicle
trafficking (Robatzek 2007; Takemoto and Hardham
2004).
After penetration of the host cell wall, primary and
secondary vesicles of B. lactucae are formed in the
first epidermal cell by invagination of the host plasma
membrane (Fig. 1g), as described above. Initiation of
intercellular growth is usually linked to membrane
damage (Woods et al. 1988) and accumulation of
The hypersensitive reaction (HR), a form of programmed cell death (Kamoun et al. 1999), is one of the
most important features in race-specific resistance of
lettuce to B. lactucae (Lebeda et al. 2001b). On a small
scale, it has been reported to also occur in compatible
or non-host interactions (Lebeda et al. 2002). Necrosis
of affected plant cells and tissues (Fig. 1i,j) is used for
phenotypic evaluation (Lebeda and Petrželová 2008).
Contemporary methods are able to detect the onset of
cell death before visible symptoms occur, either by
measuring natural bioluminiscence as the emission of
biophotons (Mansfield 2005) or by the application of
osmotic stress to test the plasma membrane functionality (Bennett et al. 1996). It was concluded that lettuce
cells undergoing the HR experience a prolonged
oxidative stress (Bestwick et al. 2001).
In a wide range of lettuce–Bremia interactions, the
substantial differences found in timing and rate of
formation of infection structures correspond with
detailed histological investigations of HR (Lebeda
and Pink 1998; Lebeda and Reinink 1994; Lebeda
et al. 2001b, 2002, 2006; Sedlářová et al. 2001b).
Post-haustorial resistance in Lactuca spp. with race
84
specificity includes an intensive HR. Although the
extent of the HR is specific for a genotype–race
interaction, the number of cells involved in the HR
is generally higher than one in wild lettuce species
(Fig. 1i,j), especially L. virosa, where cells of underlying mesophyll tissue often also show the HR
(Lebeda et al. 2006; Sedlářová et al. 2001b). Conversely, the non-host resistance in L. saligna (CGN
05271) is expressed before haustorial formation (Sedlářová et al. 2001b), and is characterized by a lack of
the HR which might relate to the previously mentioned
adjustment of oxidative processes (see Reactive oxygen species (ROS), antioxidants and ROS-scavenging
enzymes and nitric oxide, NO synthase and NO
modulators; details in Sedlářová et al., 2007a).
Plant energetics
The life strategy of parasites is based on the need to
derive nutrients from host tissues, thus affecting plant
energetics. Economically damaging infections of crops
by powdery mildews and rusts has led to intensive
research in this area, with the aim of reducing losses in
yield. Although the chlorotic symptoms typical of
downy mildew infections are well known (Lebeda and
Schwinn 1994), relatively little research has attempted
to elucidate the consequent changes of host photosynthetic processes. Interactions with hemibiotrophic
Phytophthora species, P. capsici (Aguirreolea et al.
1995), P. citricola and P. cambivora (Fleischmann
et al. 2002, 2005), P. infestans (Restrepo et al. 2005;
Schnabel et al. 1998) and P. nicotianae (Scharte et al.
2005), and the necrotroph Pythium aphanidermatum
(Johnstone et al. 2005) have been investigated. As for
biotrophs, the effect of white blister rust (Albugo
candida) on the photosynthetic and carbohydrate
metabolism of Arabidopsis thaliana was studied by
Tang et al. (1996). They showed that infection caused
a decrease in chlorophyll and Rubisco content, as well
as an inhibition of photosynthetic rates which might
result from accumulation of soluble carbohydrates
and starch in infected leaves. Moriondo et al. (2005)
indicated that Plasmopara viticola reduced functional
green leaf area of grapevine (Vitis vinifera), with
decreased chlorophyll content, and affected stomatal
closure and transpiration in lesions and adjacent
tissues. The reduced assimilation rate was not limited
by changes in electron transport capacity and generation of ATP and NADPH.
Eur J Plant Pathol (2008) 122:71–89
Prior to Restrepo et al. (2005) reporting the suppression of a large group of photosynthesis-related
genes in susceptible potato following Phytophthora
infestans infection, photosynthesis was supposed to be
affected indirectly. Loss of photosynthetic activity has
been attributed to the reduction of photosyntheticallyactive leaf area and lower pigment content (Moriondo
et al. 2005; Tang et al. 1996), and to changes in
stomatal aperture and transpiration (Aguirreolea et al.
1995). Impairment of the photosynthetic apparatus also
was reported by other authors (Koch et al. 1994;
Fleischmann et al. 2005).
Recently, we investigated the impact of B. lactucae
(race BL16) on photosynthetic parameters of Lactuca
spp. plants. Analyses of chlorophyll fluorescence
induction curves and content of photosynthetic pigments (chlorophylls and carotenoids) revealed a
linkage between deterioration of the photosynthetic
apparatus and compatibility by 13 dai. In susceptible
genotypes of L. sativa (Cobham Green and UCMD2),
an impairment of photosystem II (PSII) photochemistry and decreased content of photosynthetic pigments were noticed due to profuse growth of B.
lactucae. In resistant L. virosa (NVRS 10.001602),
characterised by rapid pathogen elimination via the
HR (Sedlářová et al. 2001b), no significant influence
of inoculation was observed (Prokopová, unpubl.).
Our data are in agreement with results of other authors
such as Schnabel et al. (1998), who showed a
correlation between degree of resistance and changes
in PSII photochemistry. Transmission electron microscopy (TEM) studies of susceptible L. sativa (Cobham
Green) cells revealed a decrease of chloroplast area in
sections of cells following challenge by B. lactucae
(race BL16). On the other hand, the number and area
of starch granules within host cells did not change
within 13 dai (Novotný and Sedlářová, unpubl.).
Further experiments addressed the effect of treating
host tissues with cytokinins (meta-topolin, BAP and
3-MeOBAPR) before inoculation. These compounds
significantly reduced B. lactucae sporulation on
susceptible lettuce tissues, and influenced optical
parametres of the leaves (Prokopová et al. 2007;
Sedlářová et al. 2007b). All cytokinins (CKs) applied
retained the maximal quantum yield of PSII photochemistry and content of photosynthetic pigments in
infected leaf discs, whereas they slightly reduced
these parameters in non-infected controls (Prokopová,
unpubl.). CKs also increased the area and number of
Eur J Plant Pathol (2008) 122:71–89
chloroplasts and starch granules within infected tissues
alone (Novotný and Sedlářová, unpubl.). These results
relate to several physiological aspects. In plant–
parasite interactions the parasitic partner operates as
sink to which carbohydrates are relocated. CKs are
also known to enhance sinks for the transport of
solutes, e.g. from older to younger parts of a plant.
Some biotrophs themselves produce CKs to modulate
nutrient transport, tissue senescence and even host
morphology (Walters and McRoberts 2006). Either
infection or application of CKs alone disturbs the
normal functioning of processes linked to photosynthesis. In the concurrent presence of both factors, CKs
might suppress the effect of pathogenesis. Increasing
concentration of CKs in leaf tissues by external
application preceding inoculation thus represents a
competitor with pathogen-driven relocation of photosynthates. Although the feedback mechanisms of
assimilates on the enzymes of photosynthesis are
quite complex and still not completely understood,
the increase of invertase leading to carbohydrate
accumulation seems to be a principal mechanism
(Walters and McRoberts 2006).
85
lactucae population structure, virulence evolution
(Lebeda and Zinkernagel 2003a) and gene-flow
between natural and cultivated plant pathosystems
(Lebeda et al. 2008); (3) collecting and characterization of Lactuca spp. germplasm variation (Lebeda
et al. 2007), screening of lettuce germplasm for
resistance to B. lactucae (Lebeda and Petrželová
2008) and detection of new sources of resistance
(Beharav et al. 2006; Lebeda and Zinkernagel 2003b;
Petrželová et al. 2007), their genetic characterization
(Bonnier et al. 1994) and utilization in lettuce breeding
(Lebeda et al. 2007); (4) detailed studies of resistance
mechanisms within lettuce genotypes and Lactuca spp.
(Bestwick et al. 2001; Grube and Ochoa 2005; Jeuken
and Lindhout 2002; Lebeda et al. 2002, 2006;
Sedlářová et al. 2007a); (5.) molecular mapping of
genes responsible for resistance/susceptibility and
virulence/avirulence both in host plants and the
pathogen (Kuang et al. 2004, 2006; Michelmore and
Wong 2008); (6.) development of new control methods
based on screening of various ‘natural’ compounds for
their activity to suppress lettuce downy mildew (Portz
et al. 2008; Sedlářová et al. 2007b), utilization of new
generations of fungicides (Cohen et al. 2008; Gisi and
Sierotzki 2008) and induced resistance.
Future perspectives
From the data summarized in this review it is evident
that relationships between plants and oomycetes are
heterogeneous and complex. During the last two
decades, the understanding of the biology of these
associations has advanced significantly. However,
much basic information is still needed before the very
complicated mosaic of components, processes, interactions and feedbacks can be assembled to obtain a
more complete view about host–oomycete specificity.
In particular, the Lactuca spp. and B. lactucae
interactions have been recognized as reflecting a very
diverse and complicated system (Lebeda et al. 2001b,
2002), and provide a suitable model for studies of
host-parasite specificity and variability of plant
defence mechanisms (Lebeda et al. 2001b).
Several investigations worldwide on the lettuce–
Bremia pathosystem are expected to contribute substantially to complement our present fragmentary
knowledge. These include: (1) molecular and ultrastructural studies on pathogen taxonomy and phylogeny (Choi et al. 2007; Göker et al. 2007; Voglmayr,
2008; Voglmayr et al. 2004); (2) characterization of B.
Acknowledgements The work was funded by grants from the
Czech Ministry of Education (MSM 6198959215) and Grant
Agency of the Czech Republic (GP 522/02/D011). The authors
thank Dr. P.T.N. Spencer-Phillips (UWE, Bristol, UK) for critical
reading of the first draft of the manuscript, participants at the 2nd
International Downy Mildews Symposium (Olomouc, Czech
Republic, 2007) for valuable discussions, and Olympus C&S
(Prague) for supporting the arrangement and development of the
Laboratory of Confocal Microscopy in the Department of
Botany at Palacky University in Olomouc.
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DOI 10.1007/s10658-008-9286-1
Natural history of Arabidopsis thaliana
and oomycete symbioses
Eric B. Holub
Received: 3 September 2007 / Accepted: 31 January 2008
# KNPV 2008
Abstract Molecular ecology of plant–microbe interactions has immediate significance for filling a gap in
knowledge between the laboratory discipline of
molecular biology and the largely theoretical discipline of evolutionary ecology. Somewhere in between
lies conservation biology, aimed at protection of
habitats and the diversity of species housed within
them. A seemingly insignificant wildflower called
Arabidopsis thaliana has an important contribution to
make in this endeavour. It has already transformed
botanical research with deepening understanding of
molecular processes within the species and across the
Plant Kingdom; and has begun to revolutionize plant
breeding by providing an invaluable catalogue of
gene sequences that can be used to design the most
precise molecular markers attainable for markerassisted selection of valued traits. This review
describes how A. thaliana and two of its natural
biotrophic parasites could be seminal as a model for
exploring the biogeography and molecular ecology of
plant–microbe interactions, and specifically, for testing hypotheses proposed from the geographic mosaic
theory of co-evolution.
E. B. Holub (*)
Warwick-HRI, University of Warwick,
Wellesbourne CV35 9EF, UK
e-mail: eric.holub@warwick.ac.uk
Keywords Hyaloperonospora parasitica .
Albugo candida . Downy mildew . White blister rust .
Gene-for-gene . Innate immunity . Receptor-like
proteins . Arms race . LRR . CATERPILLAR genes .
Boechera . Geographic mosaic . Non-host resistance
Introduction
In the current age of molecular technology and big
science questions, the practical aims for downy
mildew research have remained essentially unchanged
for more than a century. Investment in downy mildew
research still seeks practical solutions for improved
disease control, through better use of fungicides and
to aid the breeding and deployment of downy mildew
resistant crops.
Plant breeding, however, has been transformed
over the past 20 years by molecular genetics, which
has been providing knowledge of the genes that
underlie natural variation in valued traits of species
such as Arabidopsis thaliana, tomato and rice.
Converting valued traits into knowledge of genes is
an important step for genetic engineering, but more
importantly, it provides the means to design the most
precise molecular markers attainable for markerassisted selection of valued traits in conventional
breeding programmes (McCouch 2004). From the
initial public investment in research, the knowledge
gained has potential use across breeding efforts in
different plant species. For instance, molecular genetics
92
led to the discovery of numerous pathogen receptorlike genes that provide the molecular basis for downy
mildew resistance in A. thaliana (Holub 2001, 2007).
These major discoveries in turn have been instrumental
in the early development of marker-assisted selection
for disease resistance in crops (Aarts et al. 1998a;
Botella et al. 1997; Michelmore and Wong 2008; Shen
et al. 2002; Speulman et al. 1998).
In a similar process of scientific enquiry, conservation biology could also benefit significantly from
the assimilation of molecular genetics research.
Progress has been and will continue to be achieved
in the protection of biological diversity without
knowing much detail about the genetic variation
within species. However, by analogy with the recent
advances in plant breeding, knowledge about critical
genes (e.g., that enable adaptability of a species)
could provide useful indicators for improving the
prospects for survival of animal and plant populations
that are in most need of protection.
For example, the geographic mosaic theory of coevolution has been proposed to explain how intimate
interactions among multiple species will co-evolve
across geographic landscapes and provides a major
process in organising the earth’s biodiversity
(Thompson 1999a, b; Gomulkiewicz et al. 2007).
The theory proposes a three-part hypothesis: (a) the
species interactions will have different evolutionary
trajectories in different populations thereby generating
a selection mosaic of co-evolving traits among
populations; (b) some of the interacting populations
will be hot spots for reciprocal selection of the coevolving traits, whereas cold spots will also be
generated in which selection is unilateral or not
occurring at all; and, (c) continual re-mixing of traits
will occur across the geographic landscape (e.g., gene
flow, genetic drift and local extinction of populations). Understanding the molecular basis of coevolving traits will be essential for empirical testing
of this tripartite hypothesis, for providing fundamental
advances in evolutionary ecology, and ultimately for
providing invaluable insight that will aid the more
practical domain of conservation biology.
The purpose of this review is to describe how
A. thaliana and two of its natural biotrophic parasites could be seminal as a model for exploring the
biogeography and molecular ecology of plant–
microbe interactions, and specifically, for testing
hypotheses proposed from the geographic mosaic
Eur J Plant Pathol (2008) 122:91–109
theory of coevolution. This subject is relatively
young, but stands on the shoulders of important
biologists whose contributions provide a foundation
for this review.
Divergent enquiry from early revolutions in plant
science
Natural history and molecular biology have developed as polar disciplines (Wilson 1994; see ‘Molecular wars’ chapter, pp. 218–237), determined by the
environments in which the respective practitioners
have generally explored their chosen subjects (field
versus laboratory), and contrasting methodologies.
They also differ markedly in the relative accessibility
to amateur biologists and a public audience.
Molecular biology is rooted in a revolution of
experimental science that began in the mid-1800s and
led by European scientists including the botanists
Anton DeBary in Germany and Harry Marshall Ward
in England (Ayers 2005). Their dream was ‘the
Cause’, a new approach to botany that could equal
chemistry and physics as a rigorous, experimental
science. They incorporated both field observations
and laboratory experiments in their studies of plant–
microbe interactions catalyzed by the invention of
light microscopy but pre-dating genetics. Biochemistry and biophysics would eventually provide a strong
basis for experimental botany, as with the whole of
biology. However, Gregor Mendel’s discovery of
genetics from his seminal experiments with plants
soon provided a fundamental mathematical rigour that
is unique to biology. It was therefore fitting that the
2nd International Downy Mildew conference was
held in central Europe, and specifically in Olomouc,
Czech Republic where Mendel had been a student and
near the monastery in Brno where he conducted his
now famous experiments.
Genetics is the cornerstone of molecular biology,
used to illuminate the cause and effect mechanisms of
proteins within organisms under strict laboratory
environments. In recent years, whole genome sequences have become publicly available from pioneering
use of species like A. thaliana and Phytophthora
sojae. And, this in turn has galvanized a Linnaeanlike approach by an international community of
molecular biologists to name and classify all of the
proteins encoded within an organism, one representa-
Eur J Plant Pathol (2008) 122:91–109
tive organism at a time (Clark et al. 2007; Holub
2006, 2007; Kamoun 2007; Tör 2008; Tyler et al.
2006; Win et al. 2007). This in effect is providing a
powerful online catalogue of genes that enables
further comparisons to be made among species (socalled comparative genomics). Researchers will continue to pursue their experiments largely in the
laboratory. However, molecular biology is increasingly being enriched by biometricians and mathematicians who are enabling extrapolation of what has been
learned from biochemical mechanisms in model
organisms into other less tractable species, and
predictive modelling in the emerging field of molecular systems biology which aims to advance understanding of the cause and effect associations amongst
molecules of cellular processes (Holub 2007; Jönsson
et al. 2006; Prusinkiewicz et al. 2007).
Natural history, on the other hand, is transforming
into evolutionary ecology in its modern research form
(Thompson et al. 2001). This young cross-discipline
is rooted in comparative methods and mathematical
modelling as its cornerstones, and builds on ecology
as the ultimate arena for ‘systems biology’ research
(Wilson 1994). Evolutionary ecology has progressed
from the early revolutions led by Karl Linnaeus (the
founder of taxonomy) and Charles Darwin (the
founder of evolutionary theory) into a molecular age
of being able to, for example, reassess classifications
of species through the inference of familial history (or
phylogenetics) of species with comparative analyses
of informative DNA, RNA and protein sequences.
Phylogenetic trees provide the structure for bold new
ventures led by evolutionary biologists, ecologists and
amateur taxonomists to assemble information on the
earth’s biodiversity in online encyclopaedias (www.eol.
org; www.tolweb.org; www.nbn.org.uk). These publicly accessible libraries could eventually incorporate web
links to vast amounts of genome-wide, DNA-based
information from reference species and thereby stimulate growth of new research and educational tools that
would join the polar disciplines together. This may
require a new generation of cross-disciplinary biologists and creative educators.
At the moment, evolutionary ecology provides a
powerful means for elucidating the specific and
variable characteristics of habitats to which a species
is unable to adapt and thereby render it vulnerable to
local or global extinction. However, the molecular
basis for the potential vulnerability of a species is
93
poorly understood. Phrased as a more positive
question: what naturally variable genes provide a
species with ‘genetic resilience’, by enabling it to
adapt in a changing environment or to survive in a
diversity of circumstances across its geographic range
of habitats? In a nutshell, this question encapsulates a
large gap that exists between the disciplines of
molecular systems biology and evolutionary ecology,
and defines molecular genetics of natural variation as
a key means for bridging the gap.
Converging the enquiry from polar disciplines
within plant science
Answers to this fundamental question, aimed at
improving habitat conservation and species protection, will benefit from a molecular genetic approach
to understanding how natural variation within wild
populations enables a species to survive in increasingly variable habitats. This will require linking of our
genome-wide (genomic) knowledge of organisms
with an equally in-depth knowledge of suitable
habitats. This approach should at the very least
provide an illustrative guide to the minimal levels of
genetic diversity required to sustain the survival of a
species in a natural habitat, and thereby provide
genetic indicators (e.g., disease resistance traits,
highlighted below) for the direction and scale of
public investment required for species conservation.
The Plant Kingdom provides many opportunities
to probe this question. We can see and experience the
magnificence of biodiversity in vascular plants that
currently exists across our planet, in vast habitats such
as precious tropical rainforests or in small patches of
aged meadow (Silvertown 2005). As sedentary
autotrophs, plants are supreme providers of sustenance, indispensable generators of diverse microhabitats for animals and microorganisms, and the
backbone of terrestrial communities. They, along with
their symbioses with microorganisms, make terrestrial
life possible. Thus, understanding the genetic resilience of existing plant species is vital to human
survival on Earth.
To address the question posed above, we will
require genetically-based insights from three aspects
of natural variation for a range of contrasting plant
species representing different survival strategies.
These insights include: 1. identifying the critical genes
94
that are naturally variable within and among established populations of a species; 2. mapping the allelic
variation in these genes onto a geographic distribution
of established populations; and 3. determining the
selective factors that vary among microhabitats and
drive fluctuations in natural variation in a given
species.
Establishing a genetic model for co-evolved
plant–microbe interactions
Since a major selective characteristic in a plant’s
microhabitat is often likely to be biological (e.g.,
symbiotic associations with parasitic or mutualistic
microorganisms; Thompson 1999b; Gomulkiewicz et
al. 2007), the remainder of this review considers
where the natural history of A. thaliana and its
common oomycete symbionts currently stands as an
early model which a new generation of experimental
botanists could soon be using to explore fundamental
genetically-based questions at the forefront of conservation biology.
Host specialization is a characteristic that corresponds with a high degree of speciation in fungus-like
oomycetes that cause downy mildew and white rust
diseases in a wide range of plant genera (Constantinescu
and Fatehi 2002; Göker et al. 2004; Riethmüller et al.
2002; Voglmayr 2008; Voglmayr and Riethmüller
2006). Speciation of these plant parasites caught the
attention of botanists before biology had been transformed by genetics (Lindau 1901; Gäumann 1918).
Their reports included downy mildew and white rust in
A. thaliana, which was rediscovered much later in
1988 by Professor Paul H. Williams (University of
Wisconsin-Madison) who was seeking a potential
model for research on the molecular genetics of
plant–microbe interactions (Table 1).
A visiting scientist working with Williams, Eckhard
Koch, had attempted to infect A. thaliana with downy
mildew and white rust using parasite isolates collected
from brassica species but with no success because, as
we can conclude with hindsight, the chosen isolates
were constrained by a high degree of host preference
(Fig. 1a). Williams, however, informed by his reading
of the earlier German reports, was successful in
acquiring support from the US National Science
Foundation for a solo trip to collect A. thaliana seed
and parasite material in Europe. He visited Germany,
Eur J Plant Pathol (2008) 122:91–109
France and England during May of 1988, and was
successful in finding both downy mildew and white
rust of A. thaliana in Kent (southeast England; Fig. 2)
while visiting Professor Ian Crute, who had recently
moved to a horticulture research institute at East
Malling (now East Malling Research). Williams was
unable to secure additional funding, but fortunately the
seed of opportunity had been sown with both Koch
(who found downy mildew on A. thaliana in Switzerland after completing his visit to UW-Madison) and
Crute. They were each subsequently successful in
establishing A. thaliana downy mildew and white rust
as model pathosystems for molecular genetics research
(Table 1).
With respect to the phylogenetics talks presented at
the 2nd International Downy Mildew meeting by M.
Thines and reviewed by Voglmayr (2008), I will refer to
the two oomycete symbionts that Williams rediscovered
in Europe on A. thaliana as Hyaloperonospora arabidopsis (Gäumann 1918; Göker et al. 2004; Rehmany et
al. 2000; Constantinescu and Fatehi 2002; formerly
Peronospora parasitica subsp. arabidopsis; abbreviated
below as HpA) causing downy mildew, and Albugo
candida subsp. arabidopsis (abbreviated below as AcA)
causing white rust.
Current molecular knowledge of A. thaliana–
oomycete symbioses has largely been constructed from
genetic analyses of interactions amongst a small
sample of host accessions and a highly variable but
also relatively small collection of HpA isolates (Holub
and Beynon 1997; Slusarenko and Schlaich 2003). The
HpA isolate names (e.g., Emoy2) reflect their origins,
predominantly from the UK, with two prefix letters
that indicate the geographic source (As = Aspatria,
Cumbria; Bi = Biggar, Scotland; Ca = Canterbury,
Kent; Ed = Edinburgh, Scotland; Em = East Malling,
Kent; Go = Godmersham, Kent; Hi = Hilliers
arboretum, Hampshire; Ma = Maidstone, Kent; Nu =
Nunnery Walks, Cumbria; Wa = Wageningen, Netherlands; We = Weiningen, Switzerland), and two suffix
letters indicating an accession available from A.
thaliana stock centres suitable for cultivation of the
particular isolate (Co = Columbia; Ks = Keswick-1; La =
Landsberg erecta; Nd = Niederzenz; Oy = Oystese;
Wa = Wassilewskija). Most of the HpA isolates
currently being used in research laboratories have
been refined genetically by derivation from a single
oospore and maintained subsequently by propagation
of asexual inoculum (Holub and Beynon 1997).
Eur J Plant Pathol (2008) 122:91–109
95
Table 1 Two decades in the Linnaean genomics of Arabidopsis thaliana–oomycete natural history
Year Milestone
References
1988 Paul Williams rediscovers downy mildew (DM) and white rust (WR)
in European At, following an 80 year old trail.
1990 Natural variation of DM resistance in At is described as a model for
molecular genetic investigation.
1991 The DM isolate Emoy2 was borne from an oospore in a seedling of At
‘Columbia’. This isolate would be used to establish genetics in the
organism and provide the first reference genome of downy mildew
parasites in 2007.
1995 Mutation of NDR1 demonstrates that H.H. Flor’s notion of disease
resistance being conferred by single Rgenes can actually be a multigenic process and involve common links in the signalling of
defense against diverse pathogens. This established a precedent for
using oomycete and bacterial pathogens of At in comparative
laboratory experiments.
Systemic acquired resistance to bacterial disease and DM in At is
found to require salicylic acid.
Natural variation in WR resistance provides a complementary model
for molecular genetics of At–oomycete interactions.
1996 Mutation of EDS1 demonstrates that species level barriers (‘non-host’
resistance) to biotrophic parasites can be amenable to mutation and
genetic analysis. This gene and PAD4, which is also typically
required for DM resistance in At, were found to encode lipase-like
proteins.
1997 The first DM resistance gene RPP5 is cloned and found to encode a
member of the previously described TIRNBS-LRR class of
cytoplasmic receptor-like proteins.
Major R-gene clusters revealed on four chromosomes of At using a
powerful combination of recombinant inbred At populations and
DM isolates as physiological probes to map RPP loci.
1998 The bacterial resistance genes RPS4 and RPS5 were cloned and
demonstrated that non-host resistance in At can involve parasite
recognition mediated by classic (receptor-like) R-genes. This
established the use of a broadly virulent pathogen as a surrogate
vector for transient expression to test the ‘non-host’ recognition of
effector proteins. Interestingly, mutation of RPS5 can interfere with
recognition of some DM isolates.
R-like homologues provide a powerful class of molecular markers for
map-based new oomycete resistance genes in At and in crops such as
lettuce and potato.
RPP8 is cloned, and found to be a CC-NBS-LRR gene (similar to the
first bacterial resistance genes described in At) and subsequently
associated with viral resistance.
The multi-copy locus RPP1 contains several DM resistance genes
(TIR-NBS-LRR subclass) that differ in specificity.
DM resistance genes vary in how they confer defense via different
regulatory proteins.
2000 Whole genome sequence of At is announced as a public resource. The
‘Linnean genomics’ age of plant biology is soon launched with
ARABIDOPSIS 2010, an international community effort to ascribe
a function for all of the genes in At by 2010.
A single DM resistance gene (RPP7) can confer accumulative
(salicylic acid dependent and independent) defense responses.
Williams, PH (personal communication); Lindau 1901;
Gäumann 1918.
Koch and Slusarenko 1990.
Holub 2006.
Century et al. 1995, 1997.
Lawton et al. 1995.
Holub et al. 1995; Borhan et al. 2001.
Parker et al. 1996; Glazebrook et al. 1997; Falk et al.
1999; Jirage et al. 1999; Holub and Cooper 2004.
Parker et al. 1997.
Holub and Beynon 1997.
Warren et al. 1998; Gassmann et al. 1999; Holub 2007;
Rentel et al. 2008.
Aarts et al. 1998a, b; Botella et al. 1997; Speulman
et al. 1998.
McDowell et al. 1998; Cooley et al. 2000; Takahashi
et al. 2002.
Botella et al. 1998.
Aarts et al. 1998a, b; Eulgem et al. 2004.
AGI 2000; Holub 2007.
McDowell et al. 2000; Tör et al. 2002; Eulgem
et al. 2007.
96
Eur J Plant Pathol (2008) 122:91–109
Table 1 (continued)
Year Milestone
2001
2002
2004
2005
2006
2007
References
DM isolates collected from At appear to be phylogenetically distinct
from brassica isolates: referred to hence as subsp. arabidopsis
(HpA) or brassica (HpB).
RPP13 is cloned, encoding a protein homologous to RPP8 and
providing the most extreme benchmark for allelic diversification of a
receptor-like gene in At.
An At-Phytophthora model is described, providing Agrobacteriummediated transformation of an oomycete for host–parasite research
in At.
‘Gene-for-gene’ paradigm is established in the At-HpA pathosystem.
An outcross of HpA enables genetic evidence for five independent
At-recognizable effectors (ATR1, ATR4, ATR5, ATR8 and ATR13)
that correspond with different cloned DM resistance genes.
DM3 is cloned from lettuce, and found to encode a TIRNBS-LRR
protein similar to several RPP genes in At.
The DM parasite of crucifer species (previously Peronospora
parasitica) is renamed as Hyaloperonospora parasitica.
SGT1b and RAR1/PBS2 provide evidence for highly conserved
regulators (also found in monocots) and the likely involvement of
proteolysis in defense signalling.
A. thaliana-oomycete molecular ecology is launched.
The first At-recognized effector (ATR13) is cloned from HpA isolate
Maks9 and found to encode a small secreted protein that exhibits a
high degree of sequence variation amongst UK isolates of HpA.
An EDS1-independent WR resistance gene (RAC1) is cloned and
found to encode a TIR-NBS-LRR protein.
The broad spectrum late blight resistance gene Rb is cloned and found
to encode a CC-NBS-LRR protein.
Enhanced downy mildew resistance (DMR) mutants are described,
and launch the genetic analyses of induced accessibility for
oomycete parasites in At.
The first draft sequence of two oomycete genomes (Phytophthora
sojae and Ph. ramorum) is announced.
ATR1 is cloned from HpA Emoy2 and ignites the ‘Linnean genomics’ age
of oomycete effector biology by revealing a conserved RxLR motif also
found in ATR13, effector proteins from Phytophthora species, and
secreted proteins from the human malarial pathogen Plasmodium.
Draft genome sequence of HpA isolate Emoy2 is released by a
Phytophthora consortium.
Noco2 is an exception, which was derived and has
since been propagated from an original source of
asexual inoculum.
Characterising the extent of phenotypic variation
was an important step in establishing downy mildew
and white rust of A. thaliana as model pathosystems
for molecular genetics, following examples that
Williams had established for assessing these diseases
in vegetable brassica breeding (downy mildew exam-
Rehmany et al. 2000.
Bittner-Eddy et al. 2000; Rose et al. 2004.
Roetschi et al. 2001.
Gunn et al. 2002.
Shen et al. 2002.
Constantinescu and Fatehi 2002.
Austin et al. 2002; Muskett et al. 2002; Tör et al. 2002;
Tornero et al. 2002; Warren et al. 1999.
Damgaard and Jensen 2002.
Allen et al. 2004.
Borhan et al. 2004.
Song et al. 2004.
Van Damme et al. 2005; Holub 2006.
Tyler et al. 2006.
Rehmany et al. 2005; Holub 2007; Win et al. 2007.
http://pmgn.vbi.vt.edu/
ple in Fig. 3a; Holub et al. 1994, 1995). Incompatibility with HpA isolates is common among A.
thaliana accessions and much of the observed host
resistance is HpA-isolate specific and simply inherited
(Fig. 3b; Holub 2007). The naturally variable HpA
isolates described above were therefore readily identified and differentiated according to their respective
recognition by different combinations of downy
mildew resistance (RPP = resistance to Peronospora
Eur J Plant Pathol (2008) 122:91–109
97
Fig. 1 Arabidopsis thaliana and its natural associations with
parasitic oomycetes provide a rich and fascinating model for
molecular and evolutionary ecology. Arabidopsis thaliana is a
common host for two biotrophic oomycetes including Hyaloperonospora arabidopsis (HpA, causing downy mildew) and
Albugo candida subsp. ‘A’ (AcA, causing white blister rust);
and will also be exposed under field conditions to recurrent but
foiled attacks from closely related parasites (e.g., H. brassica
and A. candida subsp ‘B’) that proliferate on brassica crops and
other wild crucifers (e.g., Capsella bursa-pastoris). a The
relative degree of compatibility to different parasite subspecies
is compared in A. thaliana and B. oleracea; the estimates are
based on the percentage of susceptibility within diversity
collections of each host species (Holub et al. 1995; Holub,
unpublished). b Many naturally variable pathogen recognition
proteins (encoded by so-called R-genes) from just a few
standard accessions of A. thaliana are now known which
confer resistance to specific isolates of HpA (red labels), A.
candida (blue) or Pseudomonas syringae (black). All of the
molecularly characterised examples are members of a large
receptor-like ‘NB-LRR’ gene family (NB = nuceotide binding
site; LRR = leucine rich repeat domain; Holub 2001).
Preliminary analyses (Holub unpublished) indicate that the
known functional proteins occur at a frequency of <50% in a
UK diversity collection of 96 A. thaliana accessions, and many
are potentially quite rare. Estimates for downy mildew and
white rust resistance genes here have been based on cumbersome F2 allelism testing (populations derived from out-crossing
UK accessions to the standard accessions containing the known
R-genes), except where parentheses indicate cruder estimates
based only on using a single standard isolate to survey
resistance in the 96 accessions. These are overestimates;
however, they are sufficient to pose questions (right margin)
concerning the natural history of each variant R-protein, and
more theoretical consideration of the origins and fate of allelic
variation within a plant species. c For example (modified from
Holub 2001), attempts to illustrate how the very earliest means
of host defence (the first ‘source allele’), when an ancestral
host–parasite relationship began to co-evolve, may have been
replaced by a ‘new allele’ and presumably lost from the
historical DNA-based record. Several rounds of this ‘arms race’
may have occurred before a final solution arose in vascular
plants, with a highly adaptable capability of parasite recognition made possible by the accumulation in the gene pool of
allelic variation in multiple copies of highly mutable NB-LRR
proteins. These proteins enable individual plants, upon parasite
detection, to rapidly elicit inducible and highly conserved
(ancient) defence responses. It should be possible with
population-level experiments to investigate whether recycling
and ‘common versus rare’ variants of NB-LRR proteins
actually occur in the molecular ecology of A. thaliana–parasite
interactions
parasitica) genes in laboratory accessions of A.
thaliana (Holub and Beynon 1997). This HpA collection provided the ‘physiological probes’ for rapid,
iterative mapping of numerous RPP loci relative to the
molecular markers already available in recombinant
inbred populations of A. thaliana (Holub 1997).
Targets for molecular characterisation were easily
established (described below) on each of the five A.
thaliana chromosomes that represented the breadth of
phenotypic variation observed in inoculated cotyledons
98
Eur J Plant Pathol (2008) 122:91–109
Fig. 2 The natural diversity and genomic history in Arabidopsis thaliana from the British Isles provides a unique resource
for exploring molecular ecology of plants. a A large collection
of A. thaliana has been assembled for a multi-national
ARABIDOPSIS 2010 project which aims to generate a
database of genome-wide, high density DNA sequencing from
a global diversity collection of 1,152 accessions (Holub 2007).
This collection includes all of the UK accessions currently
available from public stock centres (Nordborg-Bergelson global
diversity collection, and donations from P.H. Williams and M.
Koorneeff) and a UK and Ireland diversity (UKID) collection
assembled by E.B. Holub. This combined material provides a
sample metapopulation of A. thaliana with a single accession
representing the local population at each of ca. 150 locations.
Extensive sampling of more than 100 established local
populations (>10 years old; ca. 1,200 accessions total) was
conducted in 2006, and this material was also included in the
first phase (low density genotyping) of the 2010 project. b
Satellite photographs of one heavily sampled region in
southeast England, where in 1988 P.H. Williams observed
downy mildew and white blister rust in A. thaliana and where
the molecular genetics of these pathosystems began 2 years
later in I.R Crute’s laboratory at the horticulture research
facility in East Malling, Kent (aerial view of buildings, lower
panel). c Epidemics have occurred most years in established A.
thaliana populations (>20 years old) that reside between
polytunnels. d An example of white rust occurring on rosette
(readily visible above as bright yellow tissue) and stem leaves
of an individual plant
(Fig. 3b). It is important to note that natural variation
exists in other characteristics of downy mildew or
white rust resistance including: polygenic but isolatespecific in seedlings; broad spectrum in seedlings
against the current HpA-collection; and adult leaf
resistance. Although these further examples are ecologically important, they have yet to be characterised at
the molecular level.
Natural variation in A. thaliana–AcA interactions is
comparatively sparse in providing clear examples of
genotype-specific resistance (Fig. 1a; Holub et al.
1995), so this closely related pathosystem was less
attractive for the opening phase of molecular genetics
research. As a consequence, this pathosystem remains
undeveloped as a potentially excellent model for
investigating such topics as species-level characteristics including host determinants of parasite speciation involving pathogen receptor-like genes (Fig. 1a;
Rehmany et al. 2000; Borhan et al. 2008), and
important aspects of compatible interactions including
induced accessibility (pathogen-activated defence
suppression) and source-sink dynamics affecting
carbohydrate metabolism (Chou et al. 2000; Cooper et
al. 2008; Holub and Cooper 2004; Tang et al. 1996).
Eur J Plant Pathol (2008) 122:91–109
99
Fig. 3 Molecular genetics has revealed tremendous diversity in
the ability of Arabidopsis thaliana to recognize genetic variants
of Hyaloperonospora arabidopsis (HpA, causing downy mildew) under laboratory conditions. Research began two decades
ago (see Table 1) with a, characterisation of phenotypic
variation in the incompatibility of cotyledons with respect to
the extent of host colonization and sporulation by the parasite,
and the relative degree of visible host response (summary text
above photographs). b These interaction phenotypes were
observed among several standard accessions of A. thaliana
(e.g., Columbia, Col-0; Landsberg erecta, Ler-0; and Niederzenz, Nd-0; labelled below photographs) following inoculations
with a diverse collection of HpA isolates (nine examples listed
in left hand column, below photos). One of the first HpA
isolates collected, Emoy2, is broadly avirulent and was
therefore chosen for an outcross to Maks9 to launch genetic
analyses in HpA (Holub 2006), and consequently, also provided
DNA for the first reference genome of downy mildews. The
host × parasite table (below photographs) summarizes genetic
analysis of downy mildew resistance in A. thaliana conferred
by RPP genes (recognition of Peronospora parasitica, the
previous name of HpA). Examples have been found on each of
the five chromosomes of A. thaliana, and most of them have
been molecularly characterised (RPP21 and RPP27 are exceptions). Five of them correspond with simply inherited avirulence
(shown in grey box) in HpA, suggesting that the ‘gene-for-gene’
hypothesis is often valid in this pathosystem (Table 1). Susceptibility to some isolates (e.g., Emoy2 and Hiks1) was only
observed in these experiments from transgressive segregation
within host populations derived by intercrossing Col-0 with
either Ler-0 or Nd-0
Only two UK isolates of AcA have thus far been named
including the type isolate Acem1 from East Malling,
Kent and Acks1 from Keswick, Cumbria (Borhan et al.
2004; Holub et al. 1995).
From a laboratory perspective, downy mildew and
white rust are most detrimental to wild-type A.
thaliana when infections are initiated at the seedling
stage in a susceptible host accession. Seedlings and
100
juvenile rosettes can readily succumb to downy mildew
or white rust, becoming chlorotic and stunted but plants
are still capable of reproducing (albeit at severely
reduced capacity). As plants get older, they are generally
more able to ‘outgrow’ downy mildew or white rust
infections with no obvious developmental effects,
although the parasites themselves are able to survive
endophytically (without symptoms) in older tissue of a
compatible host. This suggests that under natural
conditions, selection pressure by HpA and AcA will be
most acute during the establishment and competition
amongst early juvenile rosettes in wild populations of
A. thaliana, because debilitated plants may be less able
to compete for resources with other non-diseased
plants as well as being potentially more vulnerable to
attack by secondary pathogens and saprophytes and
more attractive to herbivores such as slugs and snails.
Using this rationale, the knowledge gained from
molecular genetic analyses of downy mildew and white
rust resistance in the cotyledon and seedling stage of A.
thaliana should be relevant to theoretical considerations of host–parasite co-evolution (Fig. 1c). However,
endophytic survival of AcA and HpA throughout the
life cycle of A. thaliana requires consideration in future
studies in molecular ecology aimed at resolving the
underlying selection drivers determining the observed
distribution, epidemiology and maintenance of genetic
variation in these symbiotic oomycetes. Similarly, host
determinants of HpA and AcA endophytism should also
be targeted for molecular investigation; for instance,
transgressive segregation for natural variation in A.
thaliana can generate recombinant plants which exhibit
hyper-susceptibility to downy mildew and white rust
(high levels of sporulation and severe stunting)
following inoculation of mature rosettes (Holub,
unpublished). Such recombinants can presumably arise
in natural populations but are presumably poor competitors when disease is prevalent and perhaps also
under seemingly disease-free conditions.
Two decades in the Linnaean genomics
of A. thaliana
Molecular milestones from A. thaliana-oomycete pathology have added appreciably to the Linnaean-like
effort to name and classify gene families (Table 1;
Holub 2001, 2006, 2007). For example, naturally
variable genes that control HpA isolate specific downy
Eur J Plant Pathol (2008) 122:91–109
mildew resistance were among the first pathogen
recognition genes (so-called R-genes) to be molecularly characterised from plants. These abundant genes in
monocots and dicots encode receptor-like proteins
characterised by a leucine-rich repeat (LRR) domain
that is typically highly mutable, and most known Rgenes also contain a nucleotide binding (NB) site. A
single accession of A. thaliana will contain more than
100 full length NB-LRR genes, many of which exhibit
unusually high levels of allelic variation amongst
accessions of the species collected from natural
populations (Borevitz et al. 2007; Clark et al. 2007;
Holub 2001). At least 10 such genes have been
identified as genes conferring either downy mildew
or white rust resistance and are distributed amongst
each of the five A. thaliana chromosomes; at least half
reside in major R-like gene clusters (Borhan et al.
2004, 2008; Holub 1997, 2001).
Laboratory researchers excelled at using artificial
variation generated randomly by irradiation and
chemical mutagenesis to reveal other key components
of induced defence (Table 1). These components (e.g.,
EDS1, NDR1, RAR1/PBS2 and SGT1b) represent
much smaller gene families more highly conserved
than R-genes. Such genes have been shown to be
essential for defence against a wide spectrum of
pathogens, as first demonstrated by the discovery of
NDR1, essential for resistance to plant pathogenic
bacteria and downy mildew. Some of these genes
have also been shown to represent essential and
commonly-occurring components of induced defence
across different families of vascular plants including
monocots (e.g., RAR1/PBS2 and SGT1b).
It is important to remember that all of these innate
defence genes, as with R-genes, have been identified
and defined functionally in an experimental context.
Each gene was discoverable because they were nonlethal and non-redundant (unique functional copy) in
the specific combination of host and parasite genotypes selected by investigators for mutagenesis
experiments. For instance, null mutation of sgt1b
exhibits full susceptibility to some HpA isolates (e.g.,
Cala2) in some genetic backgrounds of A. thaliana
including the most popular laboratory accession
Columbia (Tör et al. 2002). However, the same
mutant exhibits residual resistance to other isolates
of HpA and strong resistance to all bacterial isolates
tested thus far. Similarly, the effect of sgt1b mutation
can also be masked in other genetic backgrounds such
Eur J Plant Pathol (2008) 122:91–109
as Landsberg erecta in an RPP-dependent manner
(Tör et al. 2002). This effect may be due in some
cases to redundant expression of a second copy of the
gene (SGT1A) known to exist in Columbia A.
thaliana, although this hypothesis is difficult to test
decisively because double mutation of the gene pair
(sgt1a/ sgt1b) appears to be lethal (Azevedo et al.
2006; Holt et al. 2005).
In other words, the currently known R-genes and
other defence components were essentially ‘low
hanging fruit’ for discovery because, despite fortuitous examples like SGT1b, functional redundancy
and mutational lethality present major obstacles that
limit the scope of an approach to the dissection of
defence mechanisms in plants based purely on
molecular genetics. This is true even in the case of
excellent model organisms like A. thaliana. The
science has nevertheless progressed a long way
towards addressing Albert Ellingboe’s challenge to
identify the molecular ‘pieces of the (disease resistance) puzzle’ with genetics and mutational analyses
(Ellingboe 1976, 2001; Holub 2006). Now however,
complementary methods and techniques are required
to extend knowledge incrementally from the low
hanging fruit and fully reconstruct defence mechanisms in model systems (Glazebrook 2007; Kazan
and Schenk 2007; Shen and Schulze-Lefert 2007;
Staal and Dixelius 2007).
Nevertheless, for the purposes of this review, there
is already sufficient molecular understanding of
downy mildew resistance in A. thaliana to state what
now seem to be three features underlying innate
immunity in plants: (1) disease resistance typically
involves the activation of an inducible defence
process involving many other genes; (2) natural
genetic variation in disease resistance is most prevalent at the level of receptor-like proteins (containing a
highly mutable leucine-rich repeat domain), which
acts as a trigger for the induced defence; and (3)
exceptions to the above will arise from further
investigation as a consequence of genetic background,
plant tissue type and/or physiological age of tissue.
Linnaean genomics has also revolutionized our
understanding of plant pathogenic oomycetes as a
consequence of whole genome sequences that have
become available for several Phytophthora species
and HpA (Table 1). There is now a fervent interest in
revealing so-called effector proteins from parasitic
microorganisms that somehow affect biochemical
101
responses in a potential plant host (Grant et al.
2006; Kamoun 2007; Tör 2008; Win et al. 2007).
Microbial effectors that can be detected by matching
R-protein activity were previously referred to in plant
pathology literature as avirulence proteins or racespecific elicitors. Similarly, non-specific elicitors have
come in recent years to be referred to as PAMPs
(pathogen associated molecular patterns), an abbreviated term adopted from the research literature of
innate immunity in animals. Many ATR (A. thaliana
recognized) effectors were predicted in HpA following the genetic identification of numerous R-genes
conferring downy mildew resistance in A. thaliana
and classical genetic analysis of avirulence in Bremia
lactuca (Holub and Beynon 1997; Michelmore and
Wong 2008). However, confirmation of a ‘gene-forgene’ correspondence has only recently been confirmed for five cloned RPP genes, followed swiftly by
the molecular identification of the first two ATR
effectors (Table 1; Fig. 3b; reviewed by Holub 2007).
Downy mildew research in A. thaliana has benefited
enormously over nearly two decades from healthy
competition and collaboration amongst several research
groups. However, our current knowledge (summarized
in Table 1) would not have been achieved so quickly
and coincident with the precedents from molecular
investigations of bacterial resistance in A. thaliana
without the original catalytic fieldwork provided by P.
H. Williams in 1988. Two downy mildew resistance
genes (RPP1 and RPP5) would probably have been
discovered; however, HpA genetics and cloning of
HpA effectors would still only be a theoretical prospect
if Williams had not conducted his European collection
trip. Similarly, the involvement of major defence
components such as EDS1, NDR1 and SGT1b in
bacterial resistance would have been revealed but the
parallel with downy mildew resistance would likely
have come much later. Hence, Williams provided a
crucial link between the founders of experimental
botany and the modern laboratories of molecular plant
pathologists.
Establishing an experimental basis for molecular
ecology of plant–parasite interactions
Arabidopsis thaliana–oomycete symbioses could provide the basis for making the next crucial link,
between recent laboratory advances in molecular
102
biology of innate immunity in plants and genomicsbased field experiments of molecular ecology (Holub
2007). Seven key resources will be: (1) access to
microhabitats in the form of reasonably protected
field locations that have been inhabited naturally by
A. thaliana; (2) seed samples from these reference
habitats collected from a high proportion of individuals at each location; (3) ‘DNA finger-printing’ of
individuals derived from these initial seed lines
involving genome-extensive sequence information to
provide a base-line reference for future sampling; (4)
access to comparable locations uninhabited by A.
thaliana for hypothesis testing in future seed introduction experiments (including virgin sites which
could provide suitable conditions for A. thaliana,
and sites which are unsuitable for some unknown
selective factor despite the initial appearance of the
habitat); (5) satellite navigation (GPS) and photographic records enabling field biologists to accurately
re-visit the marked populations; (6) public participation that must include land owners of reference sites,
local and regional plant ecologists, and amateur
naturalists; and (7) online access for researchers and
educators to the public-sponsored data.
The best reference locations for field research will
have been well-established (>10 years old, but ideally
much older) natural populations of A. thaliana. In the
British Isles for instance, A. thaliana is predominantly
a winter annual (flowering May–June) throughout,
but it can occur as a summer–autumn annual
especially in wettest regions such as the Scottish
highlands and the Hebrides (or even well-watered
gardens with good slug control). The species relies
largely on animals for seed dispersal to establish new
populations, and human activity is particularly important so suitable locations for established populations will often be of man-made origin. Sites that
support enduring natural populations of A. thaliana
will include mural communities (described by Segal
(1969) as vertical, such as the head, vertical face and
foot of old stone or brick walls; and horizontal, such
as cracks in urban pavements), ballast of disused and
existing steam railway tracks, roadside verges, and
cultivated areas such as historic gardens (Fig. 4).
All such habitats can be found throughout the
British Isles, and even within regions such as the Lake
District National Park that became a protected
conservation area thanks to the investment and
leadership of Beatrix Potter (Holub 2007; Thompson
Eur J Plant Pathol (2008) 122:91–109
2007). It will be fascinating to see whether the
rich genomic history (DNA-based, whole genome)
that is currently being accumulated from A. thaliana
(Borevitz et al. 2007; Clark et al. 2007; Holub 2007)
will enable us to resolve whether this species is a
native in the British Isles or instead an archaeophyte
(i.e., pre-Medieval introduction by one or more
human migrations) like its close relative Capsella
bursa-pastoris (Preston et al. 2004).
Mural populations of A. thaliana will be particularly interesting for molecular ecology, especially
where A. thaliana can cycle continuously in consecutive years in essentially the same spot (Fig. 4a and
b). Human disturbance represents a major threat to the
survival of these self-maintaining mural populations.
The linear nature of old wall and railway habitats
provides pertinent opportunities to assess the accumulation and maintenance of genetic variation. It
should be possible to collect evidence for multiple
migration events or out-cross pollination events, as
well as for genetic differences that correlate with
contrasting features of microhabitat (e.g., heavy shade
vs. full sunlight along a wall extending several
kilometre; different exposure levels to atmospheric
pollutants), and for possibly predicting historical
movement of genetic variants along a wall. Although
A. thaliana is highly self-fertile, pollen dispersal and
outcrossing may be more important than expected for
movement and maintenance of natural variation
within established populations (Bakker et al. 2006),
especially on walls.
In a given region, the total available length of wall
habitats will far exceed the natural distribution of A.
thaliana, so with appropriate permission from property owners, there is excellent potential for experimental introductions such as reciprocal transplants of
seed from genetically distinct populations or ‘seeding’
of apparently non-diseased populations with oospores
of genetically defined HpA or AcA isolates. Defining
the suitable environmental conditions for establishment and extended survival of A. thaliana in these
natural habitats will, at the very least, be critical for
judging the relative merits of experiments that are
performed under controlled laboratory conditions, and
whether sites chosen for ‘common garden studies’
(Mitchell-Olds and Schmitt 2006) are representative
of habitat in a given geographic region.
In the British Isles, a good start has been made in
assembling the key resources outlined above. A
Eur J Plant Pathol (2008) 122:91–109
103
Arabidopsis thaliana habitats are typically of man-made
origin in the British Isles including: a in the cracks of
pavements; b on the head, vertical face and foot of old stone
or brick walls; c in the ballast of existing steam railway tracks,
d persisting in disused sections of railways; e in grassland
meadows and roadside verges; and f in cultivated settings (e.g.,
a flower bed in the Cambridge University Botanical garden).
The resident populations are typically well established in these
locations; for example, ‘X’ literally marks the spot in a where
three to five plants reside annually at this location on a street in
Stratford-on-Avon, and similarly in b for the position on top of
a drystone wall in the Lake District. White blister rust (Albugo
candida subsp. A) is the disease most commonly visible during
late spring in UK habitats (example shown in b, right panel);
however, downy mildew (Hyaloperonospora arabidopsis) can
also occur frequently during the juvenile rosette stage (October–March). Yellow arrowheads indicate the location of A.
thaliana plants in each of these habitats
Fig. 4
starter set of A. thaliana accessions has been available
from public stock centres, and this has been extended
with a metapopulation sample that was assembled
over 5 years between 1988 and 1993 (Fig. 2a). The
local population at each of the locations is represented
by a single accession (seed from one individual). In
2006, GPS marking was initiated of field sites that
were inhabited with established populations of A.
thaliana, and seed was collected from at least five
individuals per population (80% or more of individuals in each population; ca. 1,200 samples in total).
Many of the sites had been sampled in previous
forays, including wall populations in southeast England that P.H. Williams had visited in 1988. In most
cases, populations were found in the same site as in
previous forays indicating an age of at least 15–
20 years. Such walls are often more than 100 years
old. Many of the newly identified sites from 2006
were subsequently revisited in 2007 to confirm that
the populations were also recurrent. Unfortunately,
given the timing and logistics of handling seed
samples, it was not possible to thoroughly assess
disease and collect pathogen samples. The sole
purpose was to accumulate potential sites of scientific
interest and reference host material. Nonetheless, AcA
was observed in ca. 70% of the established populations in both the spring of 2006 and 2007 (e.g.,
Figs. 2d and 4b), whereas HpA was only found in a
few locations (<2%). These observations at least
identify sites for future host–parasite investigations.
For DNA fingerprinting, the complete collection of
A. thaliana from the British Isles shown in Fig. 2a has
been incorporated into a major international project
led by US scientists, including sampling from
104
established populations in 2006 (Holub 2007). This
project is aimed at providing laboratory researchers
with a global diversity collection of 1,152 accessions,
a large database of molecular markers (<10 kb
spacing), and linkage analysis tools as a research
capability for the mapping of genes that underlie
natural phenotypic variation in A. thaliana. The first
phase of the project, however, was to genotype more
than 5,000 accessions (mostly European and USA
samples) at 149 genome-wide loci containing SNP
(single nucleotide polymorphism) or indel (small
insertion or deletions) sequence variation. This relatively low-cost dataset provides a means for choosing
accessions with unique genotypes that represent
global diversity in an elite collection that will undergo
the further high-density genotyping. All of the British
accessions have been genotyped in the first phase, and
ca. 20% are likely to be advanced for inclusion in the
elite diversity collection. The low-density genotyping
should provide sufficient information for assessing
the frequency and distribution of genetic variation in
UK A. thaliana at different geographic scales and in
contrasting habitats, and for choosing prime diseased
and non-diseased sites for future field research.
Access to A. thaliana seed stocks and DNA
information will be possible via the North American
and European stock centres (www.biosci.ohio-state.edu/
pcmb/Facilities/abrc/abrchome.htm; www.arabidopsis.
info) and the central information website (www.
arabidopsis.org). The UK National Biodiversity Network (www.nbn.org.uk) provides an exciting precedent
for international efforts to release biodiversity information online, and will therefore provide a superb means
for releasing information about reference field sites for
A. thaliana in the British Isles.
Pump-priming molecular pathology in natural
populations of A. thaliana
Although the seven key resources described above are
at various stages of development, it is already
possible to envisage how the molecular ecology of
A. thaliana–parasite interactions will begin to unfold.
For instance, the conventional method of using
standard parasite isolates (diagnostic for different Rgenes) to assess wild accessions for compatibility
phenotypes indicates that most of the currently known
R-genes, conferring bacterial or oomycete resistance,
Eur J Plant Pathol (2008) 122:91–109
occur across the UK at a frequency well below 50%.
This may suggest that the resistance alleles are
transient, and are either newly-emergent alleles that
have yet to provide a selective advantage under
natural conditions, or are much older alleles that have
been persistently defeated by a virulent pathogen
across the species distribution (Fig. 1b). Alternatively,
they may be relatively old alleles that have been
recycled by undergoing fluctuations in frequency due
to changes in virulence of the pathogen population
(Fig. 1c; Holub 2001).
These hypotheses need testing by spatial and
temporal experiments in populations of A. thaliana
and should be extended to include other ecologically
relevant examples of natural variation (e.g., polygenic
and adult plant resistance). Seedling assays with HpA
and AcA isolates provide a simple and high-throughput means of generating phenotypic data (Fig. 5);
however the results obtained will lack precision for
genotyping purposes because uncharacterised R-protein/avirulent effector interactions may occur in the
sampled material which can confound interpretations
from the observed phenotypes. Additional data
generated from bacterial assays may eventually be
possible, using a broadly virulent isolate of Pseudomonas syringae as genetically engineered physiological probes to predictably deliver different avirulent
effectors from downy mildew and white rust parasites
(Holub 1997, 2007; Rentel et al. 2008).
The next step will be to generate phenotypic data
using the existing UK seed stocks and remaining elite
global diversity collection, to supplement the rich
DNA-based dataset. Once the prime reference locations have been identified, the major investigation can
commence with experiments designed to monitor
changes in known R-genes and oomycete effectors.
Low-cost genotyping and PCR-based assays that can
be used to detect specific alleles will be instrumental
as case studies in aiding progress, especially at fine
levels of spatial distribution where populations may
have, for example, extended along a kilometre or
more of wall and are exposed to variable physical and
biological conditions of microhabitat.
Concluding remarks
The natural history of A. thaliana–oomycete interactions was the topic of a reviews published a decade
Eur J Plant Pathol (2008) 122:91–109
105
Fig. 5 Arabidopsis thaliana from the grounds of a horticulture
research site in southeast England (located at East Malling
Research) provides one important example of a well-established
wild population (at least 25 years old) for investigating the
molecular ecology of downy mildew. The reference isolate of
Hyaloperonospora arabidopsis (HpA-Emoy2) was derived in
1991 from oospore-infested leaf tissue, collected from A. thaliana
plants that were growing naturally outside a polytunnel at this
location (Holub 2006; see Fig. 2c). Seed samples were subsequently collected in 1993, from 200 plants that were distributed
across the research site, and each seed line was increased by selfpollination to establish larger individual seed stocks of this sample
population for use in laboratory experiments. In a pilot experiment, 100 seed lines (arranged as illustrated by top row of
seedling photographs; divided in 10 subpopulations of 10 lines in
each) were inoculated as 7 day-old seedlings with different HpA
isolates including Emoy2, Emoc5 (also from the same East
Malling site), and Hiks1 (from Hampshire, ca. 250 km west of
East Malling). Interaction phenotypes (far right, vertical panel of
cotyledon photographs, along with brief description and colour
coding) were recorded for each combination of seed line and HpA
isolate. The pattern of colour coding summarizes the observed
phenotype from each combination (seed line × isolate) across the
host population. The red phenotype is specifically indicative of
downy mildew resistance mediated by RPP1 on chromosome 3
(recognizing both Emoy2 and Hiks1), and appears to occur at a
high frequency throughout this population. The only alternative
phenotype observed with Emoy2 was full susceptibility, whereas
two alternative resistance phenotypes were observed with Hiks1;
the RPP gene(s) conferring these examples of resistance have yet
to be determined. Interestingly, Emco5 inoculations revealed an
opposite pattern of susceptibility and resistance compared with
Emoy2. In this case, the GREEN or full immunity phenotype may
be indicative of RPP13 which is known to recognize Emco5 and
is closely linked to RPP1; however, this too has yet to be
determined). Nonetheless, these preliminary data indicate that two
of the most important RPP genes from previous laboratory
research most likely occur in this natural population of A. thaliana
as well as the matching avirulence effectors within the resident
HpA population
ago (Holub 1997; Holub and Beynon 1997). However, the emphasis then was on laboratory research and
the ‘hardcore’ molecular biology of innate immunity
in plants. Molecular ecology of plant–parasite inter-
actions at that time was an aspiration for a small
community of biologists. Times have clearly changed
with the complete genome sequence of the first A.
thaliana accession, rich DNA-sequence data from
106
another 20 A. thaliana accessions (Borevitz et al.
2007; Clark et al. 2007), and a dramatic fall in the
cost of DNA sequencing. The aspiration has progressed from plausibility to a very real and exciting
opportunity.
Much of the infrastructure is falling into place,
behind major advances in the molecular genetics of
downy mildew resistance in A. thaliana over the past
two decades, and the discovery of large numbers of
oomycete effectors providing raw material for another
complementary round of investigation in the downy
mildew parasites of A. thaliana and crops such as
lettuce (Michelmore and Wong 2008; Tör 2008; Win
et al. 2007). In parallel, the infrastructure for A.
thaliana field biology is beginning to emerge, which
has exciting potential for advancements in understanding the evolutionary context of a range of
developmental and physiological processes in plants.
The well-developed and timely geographic mosaic
theory of co-evolution provides the essential framework for constructive use of the resources described
in this review. Mathematical modelling that combines
ecological parameters (to explain spatial and temporal
changes in populations) with evolutionary genetics
(natural selection acting on multiple loci of interacting
species) represents an enormous challenge for this
exciting research field. However, a cross-disciplinary
‘systems biology’ approach is inevitable. Somehow,
all of the public investment in laboratory research of
A. thaliana will not make sense without further
practical applications in plant breeding, and equally,
without concerted contributions from a new generation of field botanists who are committed to aiding
conservation biology. I would hope that the likes of
A. DeBary and H.M. Ward would agree.
Acknowledgements The author is grateful to Robin Allaby,
Simon Bright, Ian Crute, Greg Gilbert, Cindy Morris and
Caroline Young for critical reading of the manuscript; to Joy
Bergelson for the opportunity to join the 2010 association
mapping project; and for generous support for related research
from the UK Biotechnology and Biological Sciences Research
Council and the Gatsby Charitable Trust.
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DOI 10.1007/s10658-008-9289-y
Comparative epidemiology of zoosporic plant pathogens
Mike J. Jeger & Marco Pautasso
Received: 4 October 2007 / Accepted: 4 February 2008
# KNPV 2008
Abstract Loss of zoospores has happened independently several times in different phylogenic lines and
has, it is claimed, no major phylogenetic significance.
But whether or not, how, and under which conditions
plant pathogens retain the ability to produce motile
asexual spores has fundamental importance from an
ecological and epidemiological perspective. Recent
molecular investigations of the early evolution of
fungi and oomycetes are shedding light on the issue
of zoospore loss in organisms able to cause plant
diseases. Zoospore loss may have accompanied the
development of new forms of dispersal adapted to the
terrestrial environment, or the simplification processes
which often follow the shift to parasitic or biotrophic
life-forms. In this review we consider hybridisation
events between Phytophthora species, long distance
dispersal of oomycetes, sporangia and zoospore
survival, direct and indirect infection processes and
newly observed sporulating structures. These aspects
are all relevant features for an understanding of the
epidemiology of zoosporic plant pathogens. Disease
management should not be based on the presumption
that the zoosporic stage is a weak link in the life
cycle. Oomycete plant pathogens show remarkable
M. J. Jeger (*) : M. Pautasso
Division of Biology, Imperial College London,
Silwood Park Campus,
Ascot SL5 7PY, UK
e-mail: m.jeger@ic.ac.uk
flexibility in their life cycles and ability to adapt to
changing environmental circumstances.
Keywords Fungal phylogeny . Landscape pathology .
Pathogen evolution . Plant epidemiology .
Zoosporangia
Introduction
Plant diseases are the outcome of the interaction of
plants with a variety of pathogenic organisms in a
disease-conducing environment. Many important
plant pathogens are zoosporic, i.e. with motile asexual
spores. Zoosporic plant pathogens cause significant
crop losses worldwide and are the object of a
substantial amount of epidemiological research. In
our use of the term ‘zoosporic plant pathogens’ we
include both zoosporic fungi and oomycetes. Although we wish to avoid becoming entangled in a
systematics debate, modern molecular phylogenetic
studies must be at the very heart of any attempt to
discuss the comparative epidemiology (Kranz 1980,
2003) of plant pathogens in relation to the evolutionary loss of zoospores, a feature present in both true
fungi and oomycetes. Zoospores, which are singlenucleated, formed in sporangia, and motile in aqueous
environments, are however a key feature in the life
cycle of many plant pathogens. They have been
thought to be a weak link, as zoospores have no cell
walls, which makes them particularly vulnerable and
112
transient (Lange and Olson 1983; Stanghellini 1997).
Here, we aim at a selective review of relevant
literature, focusing on a limited number of case
studies that we believe provide insights in the issue
of zoospore function and loss in plant pathogens. We
then move on to discuss the epidemiological and
ecological implications for sustainable plant disease
management. We emphasize plant pathogens within
the Straminipila1, mostly oomycetes, but refer also to
the true fungi with zoospores when appropriate.
Zoospores are common in oomycetes and less
common in the true fungi (Hardham et al. 1994;
Lebeda and Schwinn 1994; Judelson and Blanco
2005).
Phylogenetic and epidemiological significance
of zoospore loss
According to Dick (2002), “loss of the zoospore and
therefore flagellation is a feature of both the Peronosporales and Sclerosporales and has minor phylogenetic significance.” If the term ‘fungus’ is considered
to be an essentially physiological concept and not a
taxonomic one, then several independent phylogenetic lines of fungi have evolved (and lost) flagella (Dick
1997). We would not dismiss such a lack of
phylogenetic significance for zoospore loss, but argue
here that loss of flagellation and motility must have
considerable significance from an epidemiological
point of view. This follows from the differences in
dispersal potential, infection processes and survival
between pathogens with or without zoospores.
In spite of this, the ability to produce zoospores
varies amongst different groups of oosporic plant
pathogens. For example, it is usual in Albugo (e.g.
Whipps and Cooke 1978), variable in Plasmopara
(e.g. Kast and Stark-Urnau 1999), environmentallydependent in Phytophthora (e.g. Judelson and Blanco
2005), and lost in Hyaloperonospora (e.g. Slusarenko
and Schlaich 2003) and Peronosclerospora (e.g. Jeger
et al. 1998). Whenever organisms have evolved to
occupy niches in which their pre-existing complexity
1
Alternatively, Stramenopila: spelling of the taxon and of its
various derivatives urgently needs standardization. Analysis of
32 publications since 2000 breaks down into 20 using the
spelling above and 12 using the alternative. In some cases both
taxon spellings are given as keywords (Money et al. 2004;
Honda et al. 2007).
Eur J Plant Pathol (2008) 122:111–126
might have been superfluous, there has been the
potential for features not contributing to the fitness of
the species to be lost abruptly or over a period of
time.
Whether zoosporic loss happened during punctuated events or over longer periods of time can only be
the subject of speculation; given the paucity of the
fossil record for fungi and oomycetes alike, the
important point is that antagonistic interactions may
inherently lead towards simplification—once one
organism becomes dependent on another for its
sustenance it may discard features previously required
as a free-living organism. Parasitism, for instance, is
often accompanied by morphological simplification
involving, in the system we are interested here, the
evolution of sporangia originally water-dependent and
producing zoospores into sporangiophores producing
directly germinating conidia (Brasier and Hansen
1992).
There may be here a conceptual connection with
the argument that pathogens with higher genetic
diversity and thus evolutionary potential pose a
greater risk to plant populations, other things being
equal, as these pathogens will be more likely than
those with less genetic diversity to overcome the
defence apparatus of their host(s) (McDonald and
Linde 2002). Host specialization may on the one hand
lead to genetic impoverishment, as the pathogen no
longer needs the ability to infect various hosts, and
can thus discard the machinery upon which it relied to
successfully infect that host diversity; on the other
hand, host specialization may also lead to the creation
of new pathogen genetic diversity due to speciesspecific evolutionary arms races between host and
pathogen (Clay and Kover 1996).
For species of the genus Phytophthora, both
specialization to a single host and general aggressiveness towards a wide range of hosts are observed
(Brasier and Hansen 1992; Hardham 2007). For
example, P. cinnamomi affects several tree, shrub
and herbaceous species in the Jarrah forest of SouthWestern Australia (e.g. Shearer et al. 2007). A similar
wide range of potential and actual hosts is found with
P. ramorum (e.g. Rizzo et al. 2005). Conversely, P.
sojae (e.g. Tyler 2007), P. ilicis (Coyier 1981) and P.
porri (Smilde et al. 1995) are all examples of
Phytophthoras which are specialized to a single host
or to a taxonomically related group of hosts. This host
specialization implies a distinct co-evolution of attack
Eur J Plant Pathol (2008) 122:111–126
and defence in these pathosystems. Zoospore loss
seems not to be dependent on whether or not a certain
Phytophthora has undergone host specialization, but
rather on environmental conditions.
Increasing numbers of molecular studies are
elucidating the early evolution of various groups of
plant pathogens, including the true fungi (James et al.
2006a) and oomycetes (Göker et al. 2004; Tyler et al.
2006). Assembling the fungal tree of life (Bruns
2006) also provides insights on the issue of zoospore
loss in organisms able to cause plant diseases. The
ancestors of fungi are believed to have been simple
aquatic forms with flagellated spores (James et al.
2006a). Also the earliest fungi were aquatic and
lacked aerial spore dispersal. The traditional view is
then that a monophyletic core developed producing
zoospores (phylum Chytridiomycota, with the exception of Hyaloraphidium curvatum, where the presence
of flagella has never been reported; Ustinova et al.
2000). As opposed to that, loss of zoospores was
generally thought to have happened in the Zygomycota, with the exception of the single-flagellated
Olpidium (Lange and Olson 1976), which has now
been reclassified (James et al. 2006b). However,
recent molecular work based on a six-gene phylogeny
suggests that the Chytridiomycota are not monophyletic, and that at least four independent events of
zoospore loss can be traced back in the kingdom
Fungi (James et al. 2006a).
This surge of molecular activity is not just relevant
for the production of a more accurate phylogeny
(Tyler et al. 2006; Göker et al. 2007), but also for
applied epidemiology, as zoosporic fungi can act as
vectors of plant viruses (e.g. Teakle 1983; Adams
1991; Campbell 1996; Rochon et al. 2004), although
suspicions that oomycetes may be implicated in virus
transmission, e.g. Lagena radicicola and flame
chlorosis of cereals (Haber et al. 1991), have not
been confirmed. But before dealing with the ecological and epidemiological implications of zoospore loss
in oomycetes, we briefly discuss potential explanations for such an evolutionary development and some
case studies.
Explanations for the loss of zoospores
Loss of flagellated spores is believed to have been
concurrent with the development of new mechanisms
113
of spore production and dispersal (James et al.
2006a). When fungi moved on to the terrestrial
environment, some of them shed their ‘ancient
baggage’ which had made them successful in water,
and focused on new means of dispersal, more adapted
to the new life in periodically water-poor environments. For example, in the Peronosporales, Hyaloperonospora parasitica has no zoosporic stage in its life
cycle, and this has been related to its independence
from the aqueous environment (Slusarenko and
Schlaich 2003).
Alternatively, zoospore loss may have accompanied the development of parasitism and biotrophy. An
example is Peronospora, which is thought to derive
from a Phytophthora that lost the ability to produce
zoospores and became an obligate biotroph (Cooke et
al. 2000). There is a wide spectrum of angiosperm
hosts that is parasitised by the morphologically
‘advanced’ (i.e. lacking zoospores) genus Peronospora. For species-specific parasitic interactions, it
has been claimed that suppression and inhibition are
likely to be less important than attraction and growth
stimulation (Dick 2002).
There are many examples of zoosporic loss of
plant pathogens in relation to the presence or absence
of humidity in their typical environment. Prime case
studies are tropical graminaceous downy mildews of
sorghum and pearl millet (Jeger et al. 1998; Fig. 1).
On the one hand, Sclerospora graminicola produces
zoospores and affects pearl millet, which is generally
found in regions with higher temperatures and lower
rainfall than sorghum. Sorghum is affected by
Peronosclerospora sorghi, which does not produce
zoospores in spite of sorghum growing in regions of
higher humidity than those where pearl millet is
cultivated. Given that flagellated zoospores are
propagules for dispersal in the presence of humidity,
it is perhaps counter-intuitive that S. graminicola
should have kept zoospores whilst P. sorghi should
have lost them. Conversely, it can be argued that
zoospores are even more important in an arid
environment where water is available only rarely
and needs to be used efficiently.
There are recent examples where plant pathogens
have made a rapid transition to a new environment.
Turf grass rapid blight disease has recently emerged
as a terrestrial plant pathogen (Olsen 2007). It was
first observed in California in 1995 and was subsequently associated with high salinity irrigation in
114
Eur J Plant Pathol (2008) 122:111–126
Fig. 1 Sexual and asexual
phases of Sclerospora
graminicola a, c, f and
Peronosclerospora sorghi
b, d, e, g (from Jeger et al.
1998, with kind permission
of Blackwell)
water and golf courses. Preliminary diagnosis identified the pathogen as a species of the Labyrinthula
genus, which is associated with the marine environment. For example, L. zosterae causes marine grass
wasting disease (Olsen et al. 2003). The pathogen
(Fig. 2) was then aptly named as Labyrinthula
terrestis sp. nov. (Bigelow et al. 2005), as it is the
first observation of this type of organism (a straminipile; Leander and Porter 2001) on land plants. It is
Fig. 2 Vegetative cells of Labyrinthula terrestris illustrating
longitudinal cell division (photo, D. Bigelow, with kind
permission of American Phytopathological Society)
considered to have originated from a single infected
population and to share a recent common ancestor
with other labyrinthulids (Craven et al. 2005).
Labyrinthula terrestris builds digitate colonies in an
extracellular network produced by specialized organelles called bothrosomes and uses these networks to
move rapidly (Stowell et al. 2005). The disease has
spread onto golf courses in Arizona and nine other US
states; there has been a first report of a Labyrinthula
sp. on amenity turf grass in the UK (Entwistle et al.
2006). In many Labyrinthulid species there is an
absence of zoospore production, although biflagellated zoospores are clearly described (Amon and
Perkins 1968; Perkins 1973; Amon 1978). Perhaps
the formation of the extracellular network enables the
local but rapid movement of somatic cells analogous
to the swimming of zoospores.
Pythium species are root-infecting oomycetes
closely related to Phytophthoras (Brasier and Hansen
1992; Deacon and Donaldson 1993). They are
characterized by flexibility in their life cycle.
Oospores can either germinate directly or produce
cysts via sporangia and zoospores. Zoospores can also
be produced by sporangiophora on infected seedlings
(van West et al. 2003). Some species, e.g. P.
glomeratum from soil, are reported to produce no
sporangia or zoospores (Paul 2003) but as a rule
Pythium species do have the ability to undergo
Eur J Plant Pathol (2008) 122:111–126
zoosporogenesis (Walker and van West 2007). Other
species, such as P. helicoides, are reported to produce
only sporangia and zoospores in ebb-and-flow culture
systems (Kageyama et al. 2003; Fig. 3). Some related
oomycetes, e.g. Saprolegnia species, are able to
release a new secondary zoospore after encystment
of a primary zoospore. The secondary zoospore is the
better swimming spore (Walker and van West 2007).
Thus Pythium and related species such as Aphanomyces show remarkable flexibility in their life cycles
and the ability to respond and adapt to changing
environmental conditions.
Epidemiological and ecological implications
Zoospore loss has been reported widely in plant
pathogens, but it is important to relate this knowledge
to its potential epidemiological implications and to its
relevance for disease management (Jeger 2004; Madden
2006). We discuss here hybridisation events for
Phytophthoras, long distance dispersal for tobacco blue
mold, the relation of sporangia and zoospore release
with pathogen survival, infection processes (direct and
indirect germination), sporulating structures in PhytophFig. 3 Morphology and
germination mode of group
P of Pythium (scale bars =
20 μm). a Papillate sporangium, b zoospore formation
in a vesicle originating from
a sporangium, c hyphae
proliferating from the base
of the sporangium, d a
sporangium proliferating
from inside an old sporangial wall (from Kageyama et
al. 2003, with kind permission of Blackwell)
115
thora ramorum, integrating life cycles in P. syringae,
and epidemic modelling in P. infestans.
Hybridisation events
The advent of molecular phylogenetics has revealed
the potential for interspecific hybridisation of many
plant pathogens (Schardl and Craven 2003). Hybrids
may create devastating disease on both cultivated and
wild plants (Olsen and Stenlid 2002) and have the
potential to jump on new host species or to increase
their virulence on traditionally infected hosts. For
Phytophthora, the occurrence of multiple species in
the rhizosphere of individual nursery plants can
enhance the evolution and emergence of new tree
diseases (Brasier and Jung 2003). Natural hybrids of
P. nicotianae and P. cactorum have been observed in
glasshouse hydroponic systems (Bonants et al. 2000).
Similarly, there are reports of interspecific crosses
between Phytophthora sojae and P. vignae (May et al.
2003) and of nuclear hybrids from protoplasts of P.
parasitica and P. capsici followed by completion of
the parasexual cycle (Gu and Ko 2000). In vitro
fusion of zoospores of P. nicotianae and P. capsici has
been achieved (Érsek et al. 1995; English et al. 1999).
116
Eur J Plant Pathol (2008) 122:111–126
There has been much less work done with downy
mildews although genetic recombination through the
parasexual cycle has been demonstrated in Plasmopara halstedii (Spring and Zipper 2006).
The emergence and spread of the hybrid alder
Phytophthora is a good example of the potential of
hybridization events to create new pathosystems
(Brasier et al. 1995, 2004). Extensive field surveys
of riparian and plantation alder in Bavaria (Germany)
have revealed that symptoms were widespread on the
majority of river courses and one third of plantation
stands (Jung and Blaschke 2004; Fig. 4; see also
Gibbs et al. 1999 for Britain, and Streito et al. 2002
and Thoirain et al. 2007 for France). The source of
inoculum was traced back to young infected alder
plantations at sites that drain into the river system.
Rootstocks of alder plants might have been infected in
nurseries, possibly due to the presence of disease
propagules in irrigation water. The subsequent direct
spread of zoospores from infected plantations (during
seasonal flooding or waterlogged sites) to older and
naturally regenerating trees, as well as to river
catchments and riparian alders, can be seen as an
example of disease spread at the landscape level along
a physical network (Holdenrieder et al. 2004; Jeger et
al. 2007).
Long-distance dispersal
Long-distance dispersal of plant pathogens is a
fundamental process in the dynamic of plant epidemics, as it enables disease to jump from patch to patch
of susceptible hosts, overcoming efforts at containing
disease development with local control measures.
Long-distance spread of pathogens is helped by
man-made connectivity of previously separated continents creating what are known as ‘small-world’
networks, and is of concern given the lower disease
threshold of epidemics in such networks compared
with regular lattices (Pautasso and Jeger 2008).
Phytophthora infestans, the cause of potato late
blight, moves over long distances aerially by producing asexual sporangia which can infect plants by
germinating directly or by releasing zoospores (e.g.
Ristaino 2002). Natural long-distance spread of
sporangia of P. infestans is limited by exposure to
UVB radiation, the short infectious period of the
pathogen, and rapid mortality of the host plants
(Campbell 1999; Brown and Hovmøller 2002;
Fig. 4 Distribution in Bavaria of Phytophthora root and collar
rot of alders a along main rivers and streams and b in forest
alder stands (from Jung and Blaschke 2004, with kind
permission of Blackwell)
Zwankhuizen and Zadoks 2002), but disease expression may be facilitated by current and future climate
warming (Baker et al. 2005; Garrett et al. 2006;
Hannukkala et al. 2007; Jeger and Pautasso 2008).
Aylor (2003) assessed the critical gap width for
dispersal to be approximately 35–50 km. However,
P. infestans has been shown to spread rapidly and
over long distances due to movement of infected
tubers (Goodwin et al. 1998).
Eur J Plant Pathol (2008) 122:111–126
Long-distance dispersal of tobacco blue mold
(Peronospora tabacina) is another example of the
potential for plant pathogens to spread and act over
vast regions. Each year, blue mold advances in a wave
from the southern-most tobacco-growing regions to the
northern-most ones in the eastern USA (Aylor 1999).
This is consistent with the observed low rates of
genetic diversity in this pathogen throughout the USA
(Sukno et al. 2002). Calculated rates of advance range
from 9 to 18 km per day. Aylor (2003) estimated the
critical gap width for disease spread to be 102 km for
dispersal under full sun and 103 km under cloud cover,
depending on spore density. The effects on disease
spread of the mode of dispersal of inoculum, with
particular attention to Phytophthoras, was summarized
by Ristaino and Gumpertz (2000). In general, although
flagellated spores have epidemiological relevance, the
presence of absence of zoospores does not necessarily
have an impact on dispersal, particularly for foliar
pathogens.
Survival
The occurrence of full sun or cloud cover is an
important variable in plant epidemics, as it can affect
the survival of spores. Some chytrids have the ability
to survive periodic drying and high summer temperatures typical of cropping soils (Gleason et al. 2004).
There are many examples of the influence of
environmental conditions on oomycetes, both above
ground and below. Solar radiation is the dominant
factor determining survival of sporangia of Bremia
lactucae in California. Infection by sporangia that
have survived a day is only likely on cloudy days or
shaded leaves (Wu et al. 2000, 2005). However, there
is a lower ability of zoospores of P. infestans to
survive under the cool temperatures which favour
their development. Sporangia that do not form
zoospores under conditions favourable for formation
may be specially adapted for survival in the absence
of a host (Porter and Johnson 2004). Release of
zoospores from sporangia of Plasmopora viticola
occurred for at least seven days if free water was
available (Kast and Stark-Urnau 1999). Many sporangia of P. viticola do not survive during clear
daylight periods following their production. However,
with overcast conditions for 12–24 h, 50% still
released zoospores (Kennelly et al. 2007). The
formation of sporangia in P. viticola has been shown
117
to be photosensitive, with a prolonged period of dark
as a necessary condition (Rumbolz et al. 2002).
Assessment of survival abilities in soil, and hence
the influence of edaphic factors, depends on the
techniques used. Assays for detecting and quantifying
surviving P. capsici in soil differed in efficacy
according to propagule type: oospores, mycelial fragments, sporangia and zoospores. Zoospore inoculum
was detected at 10 propagules per gram (ppg) of soil,
whereas sporangia were detected at 1 ppg (Larkin et
al. 1995).
Infection processes
Host targeting is a fundamental strategy for zoosporic
plant pathogens to successfully infect their hosts
(Tyler 2002). This is true both in aquatic and
terrestrial environments. Zoospore chemotaxis was
observed in mangrove strains of Halophytophthora
vesicula (Leano et al. 1998). However, no evidence
for this phenomenon was obtained for Pythium
porphyrae parasitising the red alga Porphyra yezoensis (Uppalapati et al. 2001). For terrestrial pathosystems, it is known that host factors can influence the
development of Plasmopara viticola by (1) accelerating the release of zoospores from mature sporangia,
(2) coordinating the morphogenesis of the germ tube,
and (3) directing zoospores to stomata (Kiefer et al.
2002). Similar evidence for host-mediation of zoospore development was reported for Phytophthora
infestans infecting Solanum phureja (Oyarzun et al.
2004). However, Pythium oligandrum zoospores are
not attracted to hyphae of their fungal host, but if
encysted on hyphae show a significant germ-tube
emergence towards the host (Madsen et al. 1995).
Direct germination of conidia may be an advantage
in some cases. Conidia of Peronospora rubi germinate and infect most commonly through direct
penetration or enter through stomata (Williamson et
al. 1995). Conidia of Peronospora parasitica enter
through the stigma, ovary wall and establish in the
ovary enabling embryo infection and seed transmission (Jang and Safeeulla 1990). Direct germination
exists in Phytophthora drechsleri, where sporangia
are stimulated by microbial interaction in soil. With
indirect germination zoospore infectivity may be
suppressed (Hardy and Sivasithamparam 1991). A
study on the effect of the biocontrol bacterium
Burkholderia cepacia on Pythium aphanidermatum
118
indicated that although antibiosis was the main
mechanism involved in suppression there was some
contribution of competition for zoospore homing
compounds (Heungens and Parke 2000). This effect
was not apparent against Aphanomyces euteiches
zoospores.
Many studies have shown that temperature has an
important effect on zoospore infection. For example,
heat stress (40°C rather than 25 to 35°C) enhanced the
severity of root rot caused by Phytophthora cryptogea
on container-grown Chrisanthemum (MacDonald
1991). Also for P. cryptogea on Lycopersicon
esculentum, enhanced temperature (above 25°C) was
ineffective to counter established infection in summer-grown plants (Kennedy and Pegg 1990). Together with wetness duration, higher day temperature was
found to be associated with increasing incidence and
severity of P. cactorum on apple and pear fruits
(Grove and Boal 1991). However, citrus root colonization by P. citrophthora and P. parasitica was shown
to be restricted or limited above a certain temperature
threshold (27 and 33°C, respectively). A similar result
was obtained for early infection of Vitis vinifera by
Plasmopara viticola in Western Australia (Williams et
al. 2007). In general, the effect of temperature on
disease severity caused by zoosporic plant pathogens
will depend not only on the temperature preferences
of the pathogens, but also on the temperature
threshold at which they will tend to switch from
zoospore to sporangial infection (Judelson and Blanco
2005), and will be confounded by other factors such
as inoculum density and plant age (Raftoyannis and
Dick 2002).
Sporulation structures
In Phytophthora ramorum, the causal agent of sudden
oak death and ramorum dieback of many shrubby
species (Rizzo et al. 2005), sporangia and zoospores
are the elements driving the observed disease epidemic. Moralejo et al. (2006) observed structures
termed sporangiomata on susceptible woody species.
This is the first description of stromata produced by a
Phytophthora species, and may be a significant
environmental adaptation in P. ramorum. In particular, adaxial positioning suggests adaptation for rainsplash dispersal. Moreover, sub-epidermal positioning
of the stroma may in part protect from desiccation or
solar radiation and clustering of sporangia may
Eur J Plant Pathol (2008) 122:111–126
contribute to moisture retention. Oversummering
survival structures may provide a way to avoid the
challenge posed by the Mediterranean climate in the
current region of outbreak, as well as in other regions
with potentially susceptible hosts (Moralejo et al.
2006).
Integrating life cycles and predictive models
Oospore germination and zoospore infection in
Phytophthora syringae also pose a challenge to
understanding disease epidemiology and management. Phytophthora syringae persists as oospores in
fallen apple leaves. Oospores germinate by giving rise
to one or two sporangia and, when free water is
available, each sporangia produces 20 to 30 motile
spores. Undehised sporangia may germinate to create
a secondary sporangium which may produce zoospores or give rise to a tertiary sporangium, potentially an important adaptation providing flexibility in
response to variable environmental conditions. One
open question in this pathosystem is the long-term
viability of ungerminated zoospores. Harris and Xu
(2003) found that infection of fruit depended mainly
on sufficient rain being available to keep soil moist
for at least 2–3 days (oospore germination) and
wetness periods of at least 4 h (zoospore infection;
Fig. 5).
Typically mechanistic and/or forecasting models
should take account of zoospore behaviour, because
in many cases this factor seems to be essential in
understanding and predicting epidemic development.
Examples of various predictive models where zoospore activity could significantly improve forecasting
involve outbreaks of Phytophthora infestans (Johnson
et al. 1996; Aylor et al. 2001; Bourgeois et al. 2004;
Andrade-Piedra et al. 2005; Powell et al. 2005).
Disease management
Other than resistance breeding and sanitation, disease
management for zoosporic plant pathogens has relied
heavily on chemical control, and the emergence of
resistance has been observed repeatedly. Apparently,
the cost of fungicides used against Phytophthora
infestans on Solanum tuberosum accounts worldwide
for approximately 25% of the total sum spent on
fungicides on all crops (Erwin and Ribeiro 1996). In
Eur J Plant Pathol (2008) 122:111–126
Fig. 5 Observed and predicted percentage of a Phytophthora
syringae oospore activation, estimated as the percentage of
infected leaf discs, and b apple fruits infected by zoospores of
P. syringae, in relation to temperature and duration of wet
period; circle 10°C, square 12°C, triangle 14°C, inverted
triangle 16°C, diamond 18°C, hexagon 20°C; a solid line 10
and 12°C, dashed line 14 and 16°C; no models can be fitted to
data at 18 and 20°C; and b solid line 10, 12 and 14°C, dotted
line 16°C, dashed line 18 and 20°C (from Harris and Xu 2003,
with kind permission of Blackwell)
many cases the effects of oomycete fungicides have
been tracked through the various stages of zoospore
formation, release, motility, cyst formation, germination, and infection (e.g. Mitani et al. 2001; Reuveni
119
2003) and similarly for plant extracts (Rohner et al.
2004), secondary metabolites (Shimai et al. 2002) and
mineral supplementation (Xu and Morris 1998). In
relatively few studies has the relative effect of control
of sporangia/conidia and zoospores been directly
compared.
In a comprehensive study the response of Plasmopara halstedii to anti-oomycete fungicides varied
during ontogeny defined in terms of 13 developmental stages of the pathogen (Viranyi and Oros 1991). A
principal component analysis of responses formed
two main groupings with same separation of sporangial and zoosporic responses in one of the two groups.
Famoxadone used against P. infestans and Plasmopara halstedii inhibited zoospore release and caused
lysis of zoospores. Higher doses were required to
inhibit direct germination (Andrieu et al. 2001). In P.
infestans zoospore encystment and cyst germination
were highly sensitive to dimethomorph; direct sporangial germination less so (Stein and Kirk 2003).
Multi-drug resistant isolates of P. infestans significantly reduced sporulation and sporangial germination but not differentiation into zoospores (Ziogas et
al. 2006). Tomato treated with PGPR, and BABA for
induced systemic protection had reduced germination
of P. infestans sporangia and zoospores with a
marginally greater effect on sporangia (Yan et al.
2002; Fig. 6). Both direct and indirect germination of
sporangia of P. infestans were suppressed by a range
of calcium-modulating treatments, marginally greater
for indirect germination (Hill et al. 1998; Fig. 7).
From a biological control point of view, a different
line of work has built on the discovery that biosurfactants produced by the bacterium Pseudomonas
aeruginosa were an effective way to protect hydroponic plant specimens inoculated with four species of
Pythium and Phytophthora parasitica (Stanghellini
and Miller 1997). In order to achieve long-term
sustainability, strategies alternative to pesticides are
needed for the management of zoosporic plant
pathogens (Hoitink and Boehm 1999; Martin and
Loper 1999; Paulitz and Belanger 2001; Hong and
Moorman 2005). This research showed that rhamnolipids (Nitschke et al. 2005) produced by bacteria or
directly applied to plants are able to lyse the
membranes of zoospores (e.g. Kim et al. 2000; Maier
and Soberon-Chavez 2000; see also Tomlinson and
Faithfull 1979). Subsequent work showed that fluorescent pseudomonads colonizing the rhizosphere are
120
Eur J Plant Pathol (2008) 122:111–126
Sharma et al. 2007). Widespread adoption is dependent
on economic circumstances in different crop production
systems.
One often overlooked management strategy is the
effect of spatial and temporal mixtures of resistant and
susceptible species or varieties on diseases. Devoting
different fields to different crops and rotating crops
Fig. 6 Percent germination of a sporangia and b zoospores of
Phytophthora infestans on tomato leaves induced with plant
growth-promoting rhizobacteria (PGPR) strains SE34 and
89B61, β-amino butyric acid (BABA), and pathogen. Data
are means of two experiments (from Yan et al. 2002, with kind
permission of American Phytopathological Society)
able both to elicit systemic defence response in plants
and to affect the pathogenicity of zoosporic plant
pathogens (Haas and Defago 2005). The potential of
the approach has been confirmed empirically in various
pathosystems (e.g. Phytophthora capsici on Capsicum
annuum; Ristaino and Johnston 1999; Nielsen et al.
2006; Albugo occidentalis on Spinacia oleracea; Irish
et al. 2002; Pythium aphanidermatum on Cucumis
sativus; Folman et al. 2004; Phytophthora cryptogea on
Cicorium intybus; De Jonghe et al. 2005; Phytophthora
infestans on Solanum tuberosum; Lozoya-Saldana et al.
2006; Pythium aphanidermatum or Phytophthora spp.
on Lycopersicon esculentum; Calvo-Bado et al. 2006;
Fig. 7 Effect of [Ca2+] on sporangial germination by a hyphal
outgrowth (20°C) and b zoospore release (12°C). Data points
are means ± SE of three replicates, based on counts of 100
sporangia in each replicate (from Hill et al. 1998, with kind
permission of Kluwer)
Eur J Plant Pathol (2008) 122:111–126
from year to year is a traditional agricultural practice
which makes sense also as a control strategy for
zoosporic plant pathogens. Indeed, monocultures
grown year after year in the same soil are often
remarkably susceptible to disease, as exemplified by
potato late blight. A study of the effect of mixtures of
Solanum tuberosum varieties with differing levels
of susceptibility to P. infestans showed that mixtures
of an immune or near immune variety substantially
reduced disease on susceptible ones (Phillips et al.
2005). That host diversity can reduce potato blight
severity has been now shown repeatedly, although
with varying degrees (Garrett and Mundt 2000;
Garrett et al. 2001; Andrivon et al. 2003; Pilet et al.
2006). It is likely that the mechanisms underlying
these findings involve sporangial dispersal, as immune plants constitute a physical barrier and reduce
the overall density of susceptible individuals in a field
(Burdon and Chilvers 1982; Keesing et al. 2006; see
also Jactel and Brockerhoff 2007). At a landscape
level, a similar protective mechanism could be
implemented for sudden oak death. In this emerging
pathosystem, connectivity of woodland patches is
playing a key role in the spread of Phytophthora
ramorum and forests could be managed so as to
decrease connectivity of susceptible hosts (such as
bay laurel) by increasing the diversity of resistant
understory species (Condeso and Meentemeyer
2007). In tropical forests, Phytophthora and Pythium
species have been suggested as contributing to the
high tree diversity by producing density-dependent
mortality of seedlings close to parent trees (e.g.
Packer and Clay 2000; Hood et al. 2004; Pautasso et
al. 2005; Bell et al. 2006; Augspurger and Wilkinson
2007).
Conclusions
Loss of flagellated cells, zoospores, has occurred
independently in different phylogenetic lineages. No
single explanation is apparent for these evolutionary
losses. The case studies discussed in this review
suggest that it would be an oversimplification to view
lack of zoospores as progressing from free-living
aquatic to parasitic terrestrial organisms. Indeed,
oomycetes show remarkable flexibility (and redundancy) in ‘spore’ structure and function in relation to
their environment. Zoospores have perhaps mistaken-
121
ly been seen as the weak link in pathogen life cycles.
Evidence from disease management studies on the
best targets for control interventions is inconclusive
and needs further comparative analysis.
Acknowledgement This review is partly based on an invited
talk at the Downy Mildews Second International Symposium,
2–6 July 2007, Olomouc, Czech Republic. Many thanks to
Sandra Denman, Ottmar Holdenrieder, Geert Kessel, Alan
Slusarenko, Laetitia Willocquet, and two anonymous reviewers
for helpful comments and encouragement in approaching the
topic.
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DOI 10.1007/s10658-008-9291-4
Structure and variation in the wild-plant pathosystem:
Lactuca serriola–Bremia lactucae
Aleš Lebeda & Irena Petrželová & Zbyněk Maryška
Received: 18 August 2007 / Accepted: 14 February 2008
# KNPV 2008
Abstract Over the past decade, extensive research on
the wild-plant pathosystem, Lactuca serriola (prickly
lettuce)–Bremia lactucae (lettuce downy mildew), has
been conducted in the Czech Republic. Studies
focused on pathogen occurrence and distribution, host
range, variation in symptom expression and disease
severity, interactions of B. lactucae with another
fungal species (Golovinomyces cichoracearum) on
L. serriola, variation in resistance within natural
populations of L. serriola, the structure and dynamics
of virulence within populations of B. lactucae, sexual
reproduction of B. lactucae, and a comparison of
virulence structure and changes in B. lactucae
populations occurring in wild (L. serriola) and crop
(L. sativa) pathosystems. The incidence of B. lactucae
on naturally growing L. serriola and other Asteraceae
was recorded. Lactuca serriola was the most commonly occurring host species, followed by Sonchus
oleraceus. Over the duration of these studies, the
incidence of B. lactucae in L. serriola populations
varied between 45–87%. Disease incidence and
disease prevalence were partly related to the size,
density and different habitats of L. serriola populations. In addition to B. lactucae infection, infection by
A. Lebeda (*) : I. Petrželová : Z. Maryška
Faculty of Science, Department of Botany,
Palacký University in Olomouc,
Šlechtitelů 11, 783 71 Olomouc, Czech Republic
e-mail: ales.lebeda@upol.cz
the lettuce powdery mildew fungus (Golovinomyces
cichoracearum) was quite common, including coinfection. Variation in resistance to B. lactucae was
studied by using ten isolates (NL and BL races with
known virulence patterns) at a metapopulation level,
i.e. 250 L. serriola samples representing 16 populations from the Czech Republic (CZ). Our comparisons
revealed broad variation in host resistance among
host populations and also intrapopulation variability.
In the CZ populations, 45 resistance phenotypes
were recorded, the most frequent were race-specific
reaction patterns. Structural and temporal changes in
virulence variation of B. lactucae populations on L.
serriola were studied during 1998–2005. Altogether,
313 isolates of B. lactucae originating from the Czech
Republic were examined for the presence of 32
virulence factors (v-factors), and 93 different virulence phenotypes (v-phenotypes) were recorded. A
study of v-factor frequency showed that common vfactors in B. lactucae populations match some of the
race-specific resistance genes/factors (Dm genes or Rfactors) originating from L. serriola. The highest
frequency was recorded by v-factors v7, v11, v15–17,
and v24–30. In contrast, v-factors (e.g. v1–4, 6, and
10) matching Dm genes originating from L. sativa
were very rare. This demonstrates the close adaptation
of B. lactucae virulence to the host (L. serriola)
genetic background. Temporal changes in virulence
frequencies over the period were recorded. In many vfactors (v11, v14, v16, and v25–28), fluctuations were
128
observed, some (v14 and v17) shifting to higher
frequencies, and others (v5/8 and v23) decreasing.
The occurrence of mating types was studied (1997–
1999) in a set of 59 B. lactucae isolates. Both
compatibility types (B1 and B2) were recorded;
however the majority of the isolates (96%) were type
B2. A comparative study of B. lactucae virulence
variation between the wild (L. serriola) and crop (L.
sativa) pathosystems showed major differences. Migration and gene flow between both pathosystems and
the potential danger of wild B. lactucae populations
for cultivated lettuce are discussed. This paper
summarizes comprehensive and unique research on
an oomycete pathogen (B. lactucae) that is shared
between a crop (lettuce, L. sativa) and its close wild
relative (prickly lettuce, L. serriola). The data
demonstrate clear evidence about race-specific interactions, variation and changes in virulence, and
coevolutionary relationships in the wild pathosystem
L. serriola–B. lactucae. Conclusions contribute to the
broadening and better understanding of gene-for-gene
systems in natural host–pathogen populations and
their relationships to crop pathosystems.
Keywords Disease incidence . Disease prevalence .
Gene flow . Gene-for-gene . Host range .
Intra- and inter-population variability .
Lettuce downy mildew . Lettuce powdery mildew .
Metapopulation . Migration .
Natural plant communities . Prickly lettuce .
Race-specific resistance . Virulence structure .
Wild- and crop-pathosystems
Introduction
Plant pathogens play a substantial role in the structure,
dynamics, and evolution of natural plant communities.
They may cause increased mortality, reduced fitness of
individual plants, or dramatic shifts in the structure or
composition of plant populations and communities.
However, they may also help to maintain plant species
diversity, and enhance the genetic diversity and structure of host populations (Gilbert 2002). The first
detailed studies focusing on wild-plant pathosystem
structure and function were published in the 1980s
(Burdon 1987; Dinoor and Eshed 1984), and research
on host–pathogen interactions in natural communities
is a rapidly growing area of investigation in plant
Eur J Plant Pathol (2008) 122:127–146
pathology (Burdon et al. 2006). Most of these studies
have focused on interactions between host plants and
plant parasitic fungi. Investigations of interactions
between host plants and oomycetes are still very rare
(Lebeda 2002; Lebeda and Schwinn 1994), with one of
the most extensively studied of such pathosystems
being Lactuca spp.–Bremia lactucae (Lebeda et al.
2002, 2007c).
Bremia lactucae (lettuce downy mildew) is an
oomycete pathogen of cultivated lettuce (Lactuca
sativa) and many other species of Asteraceae (Lebeda
et al. 2002) that is distributed worldwide. The most
common wild host species of this pathogen is Lactuca
serriola (prickly lettuce), and it can also be frequently
found on Sonchus species (Lebeda et al. 2002;
Lebeda and Petrželová 2004a; Lebeda and Syrovátko
1988; Petrželová and Lebeda 2004b). However, it is
well documented that B. lactucae is highly hostspecific and mostly limited to a single plant genus
(Crute and Dixon 1981; Lebeda and Syrovátko 1988).
Thus, except for certain Lactuca species, weedy
growing Asteraceae cannot serve as a source of
inoculum for cultivated lettuce (Lebeda and Syrovátko
1988) and vice versa.
The interaction between L. sativa and L. serriola
and B. lactucae generally conforms to a gene-for-gene
relationship (Crute 1992a, b), in which resistance is
determined by dominant Dm resistance genes (or Rfactors) in the hosts, matched by dominant avirulence
factors in the pathogens (Hammond-Kosack and
Jones 1997). Detailed analyses of the genetics of
these host–parasite interactions (Crute and Johnson
1976; Farrara et al. 1987) made it possible to interpret
the variability of virulence in B. lactucae individuals
and populations in terms of virulence factors
(v-factors) and virulence phenotypes (v-phenotypes;
Lebeda 1981, 1982).
Several different mechanisms of resistance to B.
lactucae have been identified in cultivated and wild
lettuce (Lebeda et al. 2001a). Most of the resistance is
considered to be race-specific (Lebeda et al. 2002,
2007b). This type of resistance has a big disadvantage
as it does not provide durable protection against
lettuce downy mildew and the introduction of new
resistant cultivars is often followed by the appearance
of new virulent pathogen races (Lebeda and Schwinn
1994; Lebeda and Zinkernagel 2003a). During the last
few decades, lettuce resistance breeding has focused
on searching for and utilizing novel sources of
Eur J Plant Pathol (2008) 122:127–146
resistance to B. lactucae from wild Lactuca species
(Lebeda et al. 2002, 2007b). However, these new
resistances could be quickly overcome by B. lactucae
isolates from wild pathosystems (Lebeda 2002;
Lebeda et al. 2002).
During the last few decades, studies of host
resistance, variation and distribution of B. lactucae
virulence phenotypes have focused on the population
level and only on cultivated lettuce (L. sativa; e.g.
Crute 1987; Lebeda and Zinkernagel 2003a). So far,
there have been no studies of interactions between
Lactuca spp.–B. lactucae in natural populations,
especially from the viewpoint of host resistance,
pathogen virulence, and their temporal and spatial
dynamics (Lebeda 2002). In the Czech Republic,
studies of the wild L. serriola–B. lactucae pathosystem
were initiated at the beginning of the 1980s (Lebeda
1984, 1986; Lebeda and Boukema 1991; Lebeda and
Syrovátko 1988). However, more detailed research
focusing on the structure, spatial and temporal changes
in this pathosystem, including interactions with the
crop (L. sativa) pathosystem and coevolutionary
studies, began only recently (Lebeda 2002; Lebeda
et al. 2002; Lebeda and Petrželová 2004a, b; Petrželová
and Lebeda 2003, 2004a, b, c).
In populations of B. lactucae, sexual reproduction
has an important role in genetic recombination
(Michelmore 1981) and is considered to be the major
source of virulence variation (Crute 1992b; Lebeda and
Schwinn 1994). Bremia lactucae is predominantly
heterothallic, and two sexual compatibility types
(mating types), designated B1 and B2, have been
described (Michelmore 1981). Lebeda and Schwinn
(1994) documented sexual reproduction in populations
of B. lactucae occurring on lettuce (L. sativa), but
studies focussing on pathogen isolates from L. serriola
have been more limited (Lebeda and Blok 1990). These
reports documented both mating types of B. lactucae.
The aim of this paper is to describe and analyze
patterns of variation in interactions between naturally
growing L. serriola populations and B. lactucae. This
report includes both previously published and new
data (collected between 1998 and 2006) about the
structure and dynamics of this pathosystem from the
viewpoint of host range, disease distribution and
severity, and the occurrence of various types of
symptoms, variation and spatial distribution of host
resistance and pathogen virulence, temporal dynamics
and microevolutionary shifts in B. lactucae popula-
129
tions on naturally growing populations of L. serriola.
Of particular interest are the interactions between the
wild- and crop (Lactuca sativa) pathosystems. Coincidence of B. lactucae with Golovinomyces cichoracearum and its potential for competitive interactions
is also considered.
Host range of B. lactucae in natural populations
of Asteraceae plants
The natural incidence of B. lactucae on wild Asteraceae
species was surveyed in two main areas of the Czech
Republic (Fig. 1) during the period 1999–2006, with
the main focus on populations of weedy L. serriola
populations and associated Asteraceae plants. Field
surveys usually took place between May and early
September. Whenever possible, locations were visited
repeatedly during the growing season. During the
course of this study, B. lactucae was recorded on eight
Asteraceae species (Table 1). It is evident that, in the
Czech Republic, the most common host species of B.
lactucae are L. serriola and Sonchus oleraceus;
however sparse occurrence was also observed on
Arctium tomentosum, Carduus crispus, Cirsium
arvense, Lapsana communis, Sonchus arvensis and
Sonchus asper (Table 1).
Bremia lactucae is an obligate biotrophic pathogen
with a broad host range within the Asteraceae. On the
lettuce crop (L. sativa), B. lactucae has a worldwide
distribution (e.g. Achar 1996; Crute 1987; Datnoff et
al. 1994; Lebeda 1979; Lebeda and Zinkernagel
2003a; Marlatt 1974; Sharaf et al. 2007; Trimboli
and Crute 1983). It has also been recorded on more
than 200 other Asteraceae species from about 40
genera of the tribes Lactuceae, Cynareae and Arctotideae (Crute and Dixon 1981; Koike and Ochoa
2007; Lebeda et al. 2002). However, information
about the natural distribution and patterns of variation
of B. lactucae populations on wild composites is very
rare (Lebeda and Syrovátko 1988). Recently, B.
lactucae was noted as a common disease on L.
serriola in The Netherlands (Hooftman et al. 2007);
however, only sporadic occurrence has been recorded
in other European countries, such as Austria, France,
Germany and Switzerland (Lebeda et al. 2001b), and
Slovenia and Sweden (Doležalová et al. 2001).
Detailed data on the distribution of B. lactucae within
Europe are lacking. Only in the Czech Republic has
130
Eur J Plant Pathol (2008) 122:127–146
Fig. 1 Areas and locations
(by dots) in the Czech
Republic surveyed for the
natural distribution of Bremia lactucae in the period
1998–2006
the natural occurrence of B. lactucae been studied
more intensively during the last decade, with L.
serriola and Sonchus species (especially S. oleraceus)
found as its most common hosts (Lebeda et al. 2007a;
Petrželová and Lebeda 2004b).
However, despite its broad host range, B. lactucae
was found to be highly host-specific. Cross-inoculation
laboratory experiments showed that pathogen popula-
Table 1 Distribution of Bremia lactucae in populations of
Lactuca serriola and other Asteraceae plants occurring in the
same plant associations (1999–2006)
Species
Arctium lappa
Arctium tomentosum
Carduus acanthoides
Carduus crispus
Cirsium arvense
Cirsium canum
Cirsium oleraceum
Cirsium vulgare
Lactuca serriola
Lapsana communis
Sonchus arvensis
Sonchus asper
Sonchus oleraceus
Taraxacum spp.
Total
Number of
populations
Proportion (%)
of infected
populations
Observed Infected
within evaluated
Asteraceae species
3
82
22
49
144
6
7
18
768
9
14
7
64
50
1,243
0
2
0
1
3
0
0
0
563
1
4
3
36
0
613
0
0.33
0
0.16
0.49
0
0
0
91.84
0.16
0.65
0.49
5.88
0
100
tions occurring on wild Asteraceae mostly cannot
serve as an inoculum source for cultivated lettuce, and
inter-specific transmission was demonstrated only
within the genera Lactuca and Sonchus (Lebeda and
Syrovátko 1988). In another wild plant pathosystem,
where the pathogen is the smut fungus Microbotryum
violaceum, Carlsson-Granér (2006) recently showed
that in a spatially fragmented metapopulation, the
pathogen can alter its host species, which can
increase disease spread. For cultivated lettuce,
populations of B. lactucae on weedy-growing L.
serriola plants, represent a very important danger to
the race-specific resistance genes that originated
from L. serriola and were introduced to lettuce
(Lebeda 1984, 1989; Lebeda et al. 2002, 2007b).
Our previous (Petrželová and Lebeda 2004b) and
recent data (Table 1) documented the relatively high
frequency of B. lactucae on Sonchus species.
Recently, Vieira and Barreto (2006) suggested the
possibility of lettuce (L. sativa) infection with B.
lactucae originating from Sonchus spp. However,
there is still no direct evidence for natural transmission of B. lactucae between the genera Lactuca and
Sonchus, or vice versa.
Variation in phenotypic expression of B. lactucae
infection on naturally growing L. serriola plants
Large variation in phenotypic expression of B.
lactucae infection on L. serriola plants was observed.
During the disease survey, in addition to epidemiolog-
Eur J Plant Pathol (2008) 122:127–146
ical data (disease incidence, prevalence and severity) for
each evaluated L. serriola population, the types of
disease symptoms on leaves were also recorded.
Generally, B. lactucae is described as a pathogen
causing light green, yellow or (on older leaves) necrotic
lesions visible on the upper surface of lettuce leaves.
Those lesions are often surrounded by larger leaf veins,
and under optimal conditions are covered with sporulation on the lower leaf surface. But it is known that
there may be variation in lesion types and sporulation
on infected lettuce leaves (Crute and Dixon 1981).
Disease symptoms on L. serriola may be divided
into several groups according to the basic character of
response, shape, abundance of lesions and intensity of
sporulation (Fig. 2a,b). The first category of symp-
Fig. 2 Variation in expression of symptoms of B.
lactucae on naturally
infected leaves of L. serriola; a Symptoms without
any visible leaf necrosis; a1
both sides of the same leaf
(a–e yellowing lesions well
localized by main veins),
a→e increasing percentage
of leaf area with strongly
sporulating lesions (on abaxial side) of B. lactucae; f
and g frequent, small and
localized lesions with unusual leaf discolouration,
covered on abaxial side of
the leaf by sporulating B.
lactucae; a2 abaxial side of
the leaf (h–k: different
examples of diffuse occurrence of sporulating lesions
of B. lactucae); b Symptoms of B. lactucae infection are connected with the
leaf-tissue (abaxial side) necrosis (a→d: an example of
increasing percentage of
necrotic lesions with reduced sporulation of
B. lactucae)
131
toms includes responses without any visible leaf
necrosis (Fig. 2a). These may be characterized either
by small, discrete chlorotic spots surrounded by veins
that are variable in abundance on leaves according to
the progress of infection, or by minute spots dispersed
over the leaves with only a few conidiosporangiophores growing from each spot. Sometimes, dispersed
sporulation over large parts or even the whole leaves
with no obvious borders may be observed. However,
there may be discrete chlorotic spots involving larger
parts of leaves, with profuse sporulation on the lower
leaf surface when conditions are suitable for asexual
reproduction.
The second main type of macroscopic response is
connected with leaf-tissue necrosis (Fig. 2b). Both
132
small necrotic spots or larger ones were observed.
Both may be characterized by limited to profuse
pathogen sporulation.
Symptoms of B. lactucae infection on L. serriola
showed considerable variability in macroscopic
disease expression. In contrast, on other evaluated
wild-host composites, little or no variation was
found; only typical lesions surrounded by bigger
veins and covered with the sporulating pathogen
were observed. The main reason for this difference
may be the broad genetic diversity of L. serriola
populations, characterized by the occurrence of a
large number of race-specific resistance genes and/or
factors (Kuang et al. 2006; Lebeda et al. 2002;
Lebeda and Petrželová 2004b, 2007; Table 6 and
Fig. 4), which may differ in their phenotypic
expression. Despite the high variation in symptom
expression found among evaluated L. serriola populations, our field observations showed that infected
plants within individual populations generally displayed similar disease symptoms. However, no
experimental data are available to compare whether
different symptoms observed on naturally infected
plants are directly linked to the presence of specific
R-factors in L. serriola, or if it is a more complex
phenomenon which involves interactions among
host plant and pathogen populations and the environment (Cooke et al. 2006; Drenth 2004; Frantzen
2000; Zadoks and Schein 1979). These interactions
could be very variable, substantially influenced by
environmental factors, such as ambient temperature
(Judelson and Michelmore 1992).
Natural distribution of B. lactucae and disease
prevalence in populations of L. serriola
Disease incidence, seasonal and temporal dynamics of
B. lactucae in natural populations of L. serriola
Two parameters were used to assess the distribution of
B. lactucae in natural populations of L. serriola.
Disease incidence was expressed as the percentage of
occurrence of B. lactucae on surveyed sites and on
populations of L. serriola. Disease prevalence was
assessed in each host population by using a visual 0–3
scale (Lebeda 2002; Petrželová and Lebeda 2004b).
Bremia lactucae was found frequently (ranging from
ca 60 to 85%) in populations of L. serriola at surveyed
Eur J Plant Pathol (2008) 122:127–146
localities (Fig. 3). Disease was recorded at all
developmental stages of the host plants. Bremia
lactucae can be found throughout the growing season
(April to September, in some extreme cases even up to
October or November), as long as weather conditions
are suitable for host plant growth. However, the
highest disease incidence was recorded from June to
August.
Disease prevalence in populations of L. serriola
Disease prevalence in infected L. serriola populations
was surveyed, and some seasonal fluctuations within
individual years were recorded. In some populations
visited repeatedly during the growing season, it was
possible to document the progress of infection;
however, in other populations, no changes were
observed. Also, the proportion of populations with
different levels of disease prevalence fluctuated
slightly among individual years. However, it appears
that, under natural conditions, disease prevalence of
B. lactucae infection mostly does not reach higher
levels (Table 2).
Prevalence of B. lactucae in different types of habitats
and populations of L. serriola
Possible influences of habitat type (Table 3) as well as
of the size and density (Table 4) of host populations
on the incidence and disease prevalence of B.
lactucae were also considered (Petrželová and Lebeda
2004b). From these perspectives, there were some
significant differences in the disease prevalence,
which were most pronounced in urban areas (Table 3)
with frequent occurrence of solitary host plants or
small groups of plants, also in agricultural areas, or in
habitats with moist substrates (Tables 3 and 4).
Interactions of B. lactucae with G. cichoracearum
on L. serriola
The natural incidence of other fungal pathogens on L.
serriola plants was also recorded. Only Golovinomyces
cichoracearum, the causal agent of powdery mildew in
Asteraceae (Braun 1995) was found to be of particular
importance for incidence and prevalence of B. lactucae
in the host populations. We focused on the coincidence of both pathogens with results summarized
in Table 5. It is evident that both pathogens are widely
Eur J Plant Pathol (2008) 122:127–146
1
Frequency of L. serriola populations
Fig. 3 Fluctuation of Bremia lactucae infection in
natural populations of Lactuca serriola. Figure shows
among-year fluctuations in
proportion of healthy (noninfected) and B. lactucae
infected populations of L.
serriola within all populations surveyed in a given
year
133
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
1998
1999
2000
2001
2002
2003
2004
2005
2006
Year
healthy
Table 2 Temporal variation in Bremia lactucae prevalence in
natural populations of Lactuca serriola
Year
1998
1999
2000
2001
2002
2003
2004
2005
2006
Mean
P
Number of evaluated Disease prevalence/%
L. serriola
of populations
populations
0
1
2
36
77
136
132
53
59
101
100
109
41.7*
22.1
38.2
12.9*
39.6*
55.9*
23.8
16.0*
22.9
30.3
<0.000
41.7*
63.6*
38.2*
59.1*
35.8*
35.6*
66.3*
55.0
57.8*
50.3
<0.002
3
8.3
8.3
10.4
3.9
17.7
5.9
20.4*
7.6
18.9
5.7
3.4*
5.1
9.9
0.0
21.0*
8.0
16.5
2.8
11.1
5.3
<0.000 <0.200
Table shows among-year variation in proportion of L. serriola
populations differing in disease prevalence (proportion of
individuals within a population diseased with Bremia lactucae).
For simplification we used visual 0–3 scale for expressing
different levels of disease prevalence (Lebeda 2002; Petrželová
and Lebeda 2004b): 0 no symptoms of B. lactucae infection in
a surveyed L. serriola population; 1 low disease prevalence; 2
medium disease prevalence; 3 high disease prevalence
Mean=average of disease prevalence for the whole period
(1998–2006)
P level for observed vs. expected (average) chi-square test
Methodology for recording of field observations used over the
study period was continuously supplemented with some new
parameters (Petrželová and Lebeda 2004b). As a result, all data
are not complete for all years in Tables 2, 3 and 4.
*P<0.05 for differences between two percentages (marked
value of prevalence and mean value/under line/) used software:
StatSoft, Inc. (2001). STATISTICA Cz, Version 6. Www.
StatSoft.Cz
infected
distributed on L. serriola under natural conditions;
however, B. lactucae is more frequent. Nevertheless,
the percentage of populations where both pathogens
were recorded together was rather high (Table 5).
Some differences were found in the incidence and
prevalence of these pathogens during the growing
season and among individual years (Table 5).
Generally, the dynamics of a pathogen on a weed is
completely different than in a crop. In a dynamic
weed population, pathogen epidemiology is likely to
be intimately related to the ecology of its host,
influencing the host’s abundance, spatial distribution,
and genetic diversity. Temporal variation in these
factors affects the ability of the pathogen to reproduce
and spread (Cousens and Croft 2000). Other important factors for the incidence of a pathogen in weed
populations include general climatic conditions and
specific microclimatic conditions at individual sites.
The first pre-condition of pathogen incidence is the
presence of its host. Results summarized in this paper
show that B. lactucae occurs in a patchy fashion
wherever appropriate hosts grow and conditions are
suitable for pathogenesis. Populations of B. lactucae
and L. serriola are a good example of a host–
pathogen metapopulation structure (McDermott and
McDonald 1993; Thrall et al. 2001), where individual
fragmented populations are linked together by the
transport of spores and gene-flow.
Long-term research has been focused on the
structure and dynamics of B. lactucae populations
on L. serriola (Lebeda 2002). Within the genus
Lactuca, L. serriola (prickly lettuce) is the most
frequent weed species in Europe, especially within the
134
Eur J Plant Pathol (2008) 122:127–146
Table 3 Comparison of disease prevalence in populations of Lactuca serriola occurring in different types of habitats (1998–2006)
Habitata
Total No. of
observed populations
Along transport corridors (ditches, roadsides)
Agricultural areas (fields, field margins, field roads)
Ruderal areas, dust-heaps, debris, building sites, piles of soil
Urban areas (pavements, lawns, parking sites)
Uncultivated areas, fallows
Moist with biological material (dunghills, compost-heaps)
Mean
P
385
215
114
61
86
22
Disease prevalenceb/% of populations
0
1
2
3
26.0
15.3*
22.8
59.0*
23.2
9.1*
25.9
<0.000
55.5
60.0
55.3
34.5
51.2
59.1
52.6
<0.135
12.5
19.1
17.5
4.9*
19.8
22.7
16.1
<0.025
6.0
5.6
4.4
1.6
5.8
9.1
5.4
<0.358
StatSoft, Inc. 2001. STATISTICA Cz, Version 6. Www.StatSoft.Cz
a
Categorization according Petrželová and Lebeda (2004b)
b
Categorization same as used in Table 2
P level for observed vs. expected (average) Chi-square test
*P<0.05 for differences between two percentages (marked value of prevalence and mean value/under line/)
last two decades as it has undergone a big population
explosion connected to human activity (Hooftman et
al. 2006; Lebeda et al. 2001b). It is a pioneer plant
which colonizes disturbed areas (Feráková 1977;
Lebeda et al. 2004) in various ruderal habitats, often
along transport corridors (Lebeda et al. 2001b). It
produces many achenes which can disperse over long
distances, often generating large populations of
hundreds or thousands of plants (Petrželová and
Lebeda 2004b; Weaver and Downs 2003). However,
our field observations showed that these habitats
typically undergo a succession, where L. serriola is
replaced by another species. For this reason, the
natural distribution of B. lactucae on L. serriola
Table 4 Comparison of disease prevalence in populations of Lactuca serriola differing in the size and density (1998–2006)
Population sizea,
c
Individual plantsd
Group of several dispersed plantse
Compact group of more plantsf
Extensive and dense growthg
Large areas with L. serriola (Lactuca fields)h
Mean
P
Total No. of
observed populations
89
211
364
136
69
Disease prevalenceb/% of populations
0
1
2
3
34.8*
29.4
22.8
15.4
11.6*
22.8
<0.003
51.7
53.1
56.9
52.9
60.9
55.1
<0.904
5.6*
12.3
15.9
22.1*
18.8
14.9
<0.029
7.9
5.2
4.4
9.6
8.7
7.2
<0.588
P level for observed vs. expected (average) Chi-square test, * P<0.05 for differences between two percentages (marked value of
prevalence and mean value /under line/; StatSoft, Inc. 2001. STATISTICA Cz, Version 6. Www.StatSoft.Cz)
a
Categorization according to Petrželová and Lebeda (2004b)
b
Categorization same as used in Table 2
c
Approximate number of plants
d
<5
e
5–10
f
11–50
g
51–100
h
>100
Eur J Plant Pathol (2008) 122:127–146
135
Table 5 Temporal variation in the incidence/co-incidence of
Bremia lactucae and Golovinomyces cichoracearum in populations of Lactuca serriola
Year
1998
1999
2000
2001
2002
2003
2004
2005
2006
In totala
Total
number
of evaluated
populations
36
77
136
132
53
59
101
100
109
750
Incidence of B. lactucae
and G. cichoracearum (%)
Single infection
B. lactucae
G. cichoracearum
Coincidence
of both
pathogens
22.2
37.7
29.4
48.5
n.d.
32.2
49.5
32.0
45.0
38.8
22.2
19.5
16.2
5.3
n.d.
23.7
13.9
9.0
11.0
13.5
36.1
40.3
32.4
38.6
n.d.
11.9
26.7
52.0
32.1
34.7
n.d. not determined during the main season
a
L. serriola populations (total number of evaluated populations,
year 2002 excluded); % of incidence/co-incidence for the
period 1998–2006
cannot be assessed only from the viewpoint of
individual populations, but also at a larger geographical scale, i.e. metapopulation size.
In comparison to crops, weed populations mostly
occur as mixtures of genotypes, and pathogen
incidence is thus strongly dependent on the availability of susceptible host plants (Cousens and Croft
2000). Host genetic diversity has considerable influence on the occurrence of a pathogen and its
variation, especially in pathosystems operating on a
gene-for-gene basis (Burdon 1997). Thus, despite its
persistence in the area during the studied period, it
was not possible to predict the incidence of B.
lactucae in particular sites due to the unpredictable
dynamics of the host populations. Further, we
observed a large discrepancy between disease incidence and disease prevalence (in the present study,
expressed as the degree of infection in evaluated
populations, Table 2). Although L. serriola populations with B. lactucae infection were widely distributed, the prevalence of infection in most populations
was very low (Table 2). In natural populations such
negative relationships between disease incidence and
disease prevalence may be caused by higher levels of
connectivity within the fragmented host metapopula-
tion (Carlssson-Granér and Thrall 2002). Indeed,
though L. serriola is distributed in a patchy fashion,
it is very common in many plant associations.
The distribution of B. lactucae and its interaction
with L. serriola must be also considered from the
viewpoint of interactions with the physical environment. Differences in disease incidence during the main
season of B. lactucae development were related to
some extent with local temperature and rainfall in July
and August. Bremia lactucae has a narrow optimal
range of conditions for growth (Crute and Dixon
1981), preferring cool temperatures and relatively high
humidity. Weather variables are considered crucial in
the infection and epidemiology of Bremia lactucae on
cultivated lettuce (Scherm and van Bruggen 1994; Su
et al. 2004). A recent study (Mieslerová et al. 2007)
demonstrated that in extremely dry and hot summer
months (as was the case in 2003), more than 50% of
populations were free of infection symptoms. In
contrast, in the cool and wet Augusts of 2005 and
2006, a high frequency of infected L. serriola
populations was observed (Mieslerová et al. 2007).
The age of host plants can also influence disease
severity (Petrželová and Lebeda 2004b). From our
empirical data, it is evident that plants initially infected
at the early stages of development (leaf rosette
formation or bolting) expressed higher degrees of
infection under optimal conditions than plants infected
as adults. This agrees with experimental data reported
by Crute and Dickinson (1976). The success of a
pathogen in a host population may also be influenced
by its interactions and possible competition with other
pathogens attacking the same host plants (Lindow
2006). Our recent research has focused on the coincidence of B. lactucae and Golovinomyces cichoracearum (Mieslerová et al. 2007). Golovinomyces
cichoracearum, in comparison with B. lactucae, has a
shorter period of incidence during the growing season,
and its epidemics start later (mostly in June or July),
when B. lactucae is already widespread among L.
serriola plants (Lebeda 2002; Petrželová and Lebeda
2004b). However, where it did occur, G. cichoracearum was able to develop heavy and extensive
infections within a few (3–4) weeks (Petrželová and
Lebeda 2004b). The peak of incidence of both
pathogens was approximately the same, i.e. August.
Both pathogens can co-occur within the same population of L. serriola and either of them may dominate,
depending strongly on different environmental optima
136
for their growth and development, with humidity being
the most pronounced parameter (Mieslerová et al.
2007). In relationship to disease severity and interactions of both pathogens, the phenomenon of induced
resistance must also be seriously considered in natural
plant pathosystems (Newton and Pons-Kühnemann
2007).
Host–pathogen interactions play an important role
in plant populations and may have some impact on
plant fitness and demography (Thrall and Burdon
2003, 2004). However, our knowledge of these
ecological parameters in the L. serriola–B. lactucae
interaction is still very limited. From our data, it is
evident that there are differences in the expression of
disease symptoms and disease intensity within and
among individual plants inside populations and
between populations, leading to some reduction in
leaf surface (Fig. 2) and the number of leaves;
however, we never observed the infection of reproductive (floral) parts of L. serriola plants. Recently,
the first data about this phenomenon showed that the
impact of inheriting Bremia resistance on reproductive plant fitness is small (Hooftman et al. 2007).
Nevertheless, it was observed that infected individuals
had fewer leaves at the beginning of the bolting phase
than did non-infected plants, and for individual seed
weight there was a significant interaction between
mainlines and Bremia infection. However, the total
seed weight per head (capitulum) was not altered by
Bremia infection, and it was concluded that Bremia
infection did not affect these fitness components in
any consistent manner (Hooftman et al. 2007).
Eur J Plant Pathol (2008) 122:127–146
and BL24) with known virulence patterns (van
Ettekoven and van der Arend 1999) and were
characterized by means of resistance phenotypes.
Some basic results are given in Table 6 and Fig. 4.
A substantial proportion of the Czech L. serriola
populations showed high levels of susceptibility to the
B. lactucae isolates used (Table 6). Overall, 30% of
the host individuals studied were completely susceptible, and this phenotype was widely distributed
among studied populations (in 12 of 16 populations).
A completely resistant phenotype was recorded in
five populations, represented by 24 (9.6%) samples
(Fig. 4). While most plants expressed intermediate
levels of race-specific resistance, only four populations showed relatively high levels of resistance. Most
plants were susceptible to at least one to three races of
B. lactucae (among them very often BL21 and BL24).
Despite their high levels of susceptibility, Czech
populations of L. serriola were variable in terms of
their resistance to ten races of B. lactucae. Presence of
race-specific resistance was very common. Both interand intra-population variation of resistance were
found. In total, 45 different resistance phenotypes
were recognized in the studied populations of L.
serriola. However, 80% of the individual plants
evaluated were represented by only eight resistance
phenotypes; the remaining phenotypes were generally
rare. When compared to other European populations
of L. serriola, Czech populations most closely resemTable 6 Variation of resistance within European metapopulations of Lactuca serriola (Lebeda and Petrželová 2004b;
Lebeda et al. 2007a)
Populations
Number of L. serriola populations
High levela of
Variation of resistance in populations of L. serriola
Research was focused on the determination of
resistance variation within and among Czech populations of L. serriola as well as on the evaluation of
variation within other European populations of prickly lettuce (Lebeda and Petrželová 2004b, 2007).
Samples of L. serriola were collected in 2001 within
the framework of the EU project ‘Gene-Mine’ (for
details see Lebeda et al. 2007a). In total, 250
individual plants from 16 Czech populations of L.
serriola were screened following previously described methods (Lebeda and Zinkernagel 2003b)
for resistance against 10 races of B. lactucae (NL1,
NL5, NL12, NL14, NL15, NL16, BL17, BL18, BL21
Total
Resistance Susceptibility RaceIntraspecific population
response variation
in racespecificity
CZ
D
NL
UK
Totally
a
4
0
0
0
4
9
6
2
0
17
0
1
6
10
17
3
9
0
0
12
16
16
8
10
50
Relative variation in mean resistance of populations evaluated
as a relative proportion of samples with prevalence of
susceptibility, resistance and race-specificity to the used set of
B. lactucae races
Eur J Plant Pathol (2008) 122:127–146
137
100
L. serriola individuals (%)
90
80
70
60
50
40
30
20
10
0
Resistance
Susceptibility
Race-specificity
Fig. 4 Percentage of Lactuca serriola individuals (in total n=
250), with different reaction patterns to ten races of Bremia
lactucae, within the sampled Czech host populations (n=16; each
population represented by ca 16 individual plants, from each
plant tested ca 30 achenes/seedlings/). Three basic categories of
reaction patterns were distinguished: Resistance L. serriola
individual plants were resistant to all ten races of B. lactucae
used; Susceptibility L. serriola individual plants were susceptible
to all ten races of B. lactucae used; Race-specificity differential
reaction patterns to races of B. lactucae used was recorded
bled resistance structures observed in their German
counterparts (Table 6). However, while German populations expressed greater levels of intra-population
variation, no population was completely resistant
(Table 6) despite the widespread occurrence of resistant
individuals among the populations. Lactuca serriola
populations in the Netherlands also expressed a high
level of intra-population variation; however, their
responses showed higher levels of race-specificity
(Table 6). Unlike the Czech, German and Dutch
populations, disease responses of populations from the
United Kingdom were much more uniform (Table 6).
During the last few decades, lettuce resistance
breeding has focused on the identification and
incorporation of novel sources of resistance to B.
lactucae from wild Lactuca spp., especially from L.
serriola, L. saligna and L. virosa (e.g. Beharav et al.
2006; Bonnier et al. 1992; Jeuken and Lindhout 2002;
Lebeda et al. 2002, 2007b; Lebeda and Zinkernagel
2003b; Maisonneuve 2003). Mapping the distribution
patterns of resistance and virulence can help us
understand co-evolutionary dynamics in plant pathosystems (e.g. Carlsson-Granér 2006; Carlsson-Granér
and Thrall 2002; Delmotte et al. 1999; Laine 2006;
Thrall and Burdon 2003) and select appropriate
resistance sources and crop-breeding strategies (Lebeda
et al. 2002, 2007b). However, there is only limited
information about the distribution of resistance to B.
lactucae in natural populations of its hosts (Lebeda and
Petrželová 2004b, 2007). To date, most such studies
have been based on evaluation of genebank germplasm
samples (Lebeda et al. 2007b) which may not represent
the structure of natural host populations. Only in
Europe there has been extensive field collections aimed
towards obtaining large population samples of L.
serriola (Doležalová et al. 2001; Křístková and Lebeda
1999; Lebeda et al. 2001b, 2007a).
The interaction between L. serriola and B. lactucae
in Europe is an exciting model for such studies
(Lebeda et al. 2001b). First, L. serriola is a highly
invasive species and recently, due to the increasing
ruderalization of the environment, it has become quite
a common weed in both agricultural and natural plant
ecosystems (Hooftman et al. 2006; Lebeda et al.
2001b, 2004). Furthermore, it is closely related to the
cultivated lettuce, considered to be its progenitor
(Lebeda et al. 2001b, 2007b). It is also used very
extensively as a source of resistance against B. lactucae
in lettuce breeding, and many of its race-specific
resistance genes have been introduced into commercial
lettuce cultivars (Lebeda et al. 2002, 2007b). From
these perspectives, it is probably the only plant
pathosystem where we can precisely study the structure, dynamics and interactions between the wild- and
crop-pathosystems (Lebeda 2002; Lebeda et al. 2007c)
at both the individual and population levels.
138
Our recent research shows that Czech populations
of L. serriola generally display a low level of
resistance to B. lactucae, which correlates well with
the high frequency of disease occurrence recorded
during field observations (Petrželová and Lebeda
2004b; Table 2 and Fig. 3). However, the frequent
occurrence of differential reaction patterns to the B.
lactucae races tested (originating only from L. sativa)
indicates that all the resistance recorded in these L.
serriola populations is race-specific (Lebeda and
Petrželová 2004b, 2007; Fig. 4), supporting previously reported results (Lebeda et al. 2002; Lebeda and
Petrželová 2001). Both inter- and intra-population
variation in race-specificity was recorded in the Czech
metapopulation of L. serriola. When the spatial
distribution of individual resistance phenotypes was
assessed, they were randomly distributed over the
study area with no obvious aggregation of populations with more resistant and/or susceptible phenotypes. One of the probable explanations for recorded
resistance patterns over the Czech metapopulation of
L. serriola is that host migration events among locally
adapted populations play an important role in shaping
resistance structure.
A completely different situation was found within
other European metapopulations of L. serriola (Lebeda
et al. 2007a). Although the same resistance phenotypes could be found in different countries, other
European populations generally have completely
different resistance patterns with an increasing
frequency of race-specificity towards the Atlantic
coast. Among-population variation in resistance is
largely affected by the level of patchiness and
connectivity of populations within the larger metapopulation (Carlsson-Granér and Thrall 2002). In the
case of data summarized in this paper, this may be an
explanation for greater differentiation, both among and
within L. serriola populations observed in continental
Europe, while the decrease in variation was most
pronounced for populations in the United Kingdom,
where only one form of the host, L. serriola f.
integrifolia, is prevalent (Lebeda et al. 2004). These
populations are spatially isolated from host populations
in continental Europe, both by distance per se, but also
by their island location (Lebeda and Petrželová 2005).
In a previous study with a limited number of plant
samples, it was concluded that populations of L.
serriola in Britain were commonly homogeneous for
the B. lactucae resistance phenotype. There was no
Eur J Plant Pathol (2008) 122:127–146
evidence for extensive resistance gene ‘pyramiding’ or
population heterogeneity as defence strategies against
B. lactucae in natural populations of L. serriola (Crute
1990). However, some variation in the level of field
resistance was recorded (Crute 1990) which agrees
with observed levels of field resistance in wild Lactuca
spp. germplasm (Lebeda 1990).
In natural pathosystems, disease dynamics is the
most important factor driving the diversity and
distribution of host-resistance genotypes (Laine
2006). In the crop pathosystem, L. sativa–B. lactucae,
geographic differences in virulence among pathogen
populations from various countries and growing areas
are relatively well known (e.g. Crute 1987; Lebeda
and Zinkernagel 2003a) and the same should be
expected in the wild pathosystem, L. serriola–B.
lactucae (Lebeda 2002; Lebeda and Petrželová
2004a; Petrželová and Lebeda 2004c). However, for
L. serriola we still lack sufficient data about relevant
pathogen populations in much of Europe except for
the Czech Republic, hindering our understanding of
coevolutionary trends within this pathosystem. Thus,
we can only suppose that, at the larger spatial scale,
different L. serriola populations may have been
exposed to differential selection pressures by B.
lactucae and as a result evolved different patterns of
resistance.
Temporal changes in variation of virulence
in populations of B. lactucae occurring on L.
serriola
Samples of B. lactucae from naturally infected wild
populations of L. serriola and of cultivated lettuce (L.
sativa) were collected and used for the virulence
screening. Tests were carried out according to
methods described previously (Lebeda 2002; Lebeda
and Zinkernagel 2003b). Altogether, 313 isolates of
B. lactucae from L. serriola were collected during the
period of searching (23 in 1998, 31 in 1999, 78 in
2000, 43 in 2001, 8 in 2002, 19 in 2003, 51 in 2004
and 60 in 2005).
Virulence of isolates was examined by screening
on a standard differential set (van Ettekoven and van
der Arend 1999). More detailed characterization was
made on 56 L. sativa and L. serriola genotypes
(Lebeda and Zinkernagel 2003b) with well characterized patterns of race-specific resistance (Dm-genes or
Eur J Plant Pathol (2008) 122:127–146
139
R-factors). By use of both differential sets, 32
virulence factors (v-factors) were determined; however four of them (v32, v33, v41 and v42) are not
included in the set of data (see Fig. 5).
Variation in virulence among natural populations
of B. lactucae in the Czech Republic was studied
from 1998 to 2005 and was analyzed at both
individual and population levels. Results related to
the analysis of virulence at the level of individuals
have been partly (for the period 1997–2000) previously published (Lebeda and Petrželová 2004a;
Petrželová and Lebeda 2004c). Variation in virulence
and its temporal changes were quantified by relative
frequencies of virulence factors (v-factors) in the
tested isolates (Lebeda 1981, 1982; Fig. 5). With only
a few exceptions (v18, v32, v33, v37, v41 and v42),
most of the examined v-factors were recorded in
populations of B. lactucae on L. serriola during the
whole study period. Nevertheless, there were substantial differences in frequencies recorded for individual
v-factors (Fig. 5). Medium to high frequencies were
recorded for v-factors v5/8, v7, v11, v14–17 and v23–
30. In contrast, factors v1–4, v6, v10, v12, v13, v35,
v36 and v38 were detected in extremely low
frequencies (Fig. 5). Frequencies of v-factors were
not uneven with values varying by year. Fluctuations
were most pronounced for factors v5/8, v11, v14,
v16, v23 and v25–28, with v5/8 and v23 showing a
rapid decrease within natural populations of B.
lactucae (Petrželová and Lebeda 2004c). On the other
hand, factors v14 and v17 increased.
Virulence patterns on the differential set recorded
for individual isolates were described as virulence
phenotypes (v-phenotypes). The complexity of
recorded B. lactucae v-phenotypes varied broadly,
with 6 to 33 Lactuca spp. differentials being infected
by the tested isolates. The distribution of isolates
based on the number of virulent responses showed a
normal (Gaussian) distribution curve each year, with
average virulence ranging from 9 to 15 virulent
responses. In total, 93 different v-phenotypes were
identified among 313 isolates tested in the period
1998 to 2005 (Table 7). Mean variation of vphenotypes differed among years, ranging from
24.4% in year 2000 to 87.5% in year 2002. However,
the basic spatial-distribution pattern of individual vphenotypes was very similar among years. Each year,
there was one or a few v-phenotypes prevailing in
examined populations, and broad variation of other
unique ones (Lebeda et al. 2007c). Some pathogen
populations were also tested for the existence of intrapopulation variation. Different v-phenotypes were
recorded even among pathogen isolates collected
from L. serriola plants growing in close proximity
in one host population (Lebeda and Petrželová
2004a).
A primary main focus for our long-term research
on Lactuca spp.–B. lactucae pathosystems has been
the determination of virulence variation within B.
lactucae populations, documenting its temporal and
spatial dynamics, and comparing pathogen populations in crop (L. sativa) and wild (L. serriola)
1
0,9
0,8
Frequency
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
1
2
3
4 5/8 6
7 10 11 12 13 14 15 16 17 18 23 24 25 26 27 28 29 30 35 36 37 38
v-factor
Fig. 5 Frequency of v-factors recorded in the sampled set of Bremia lactucae isolates (n=313) collected in the Czech pathogen
populations on Lactuca serriola during the period 1998–2005
140
Eur J Plant Pathol (2008) 122:127–146
Table 7 Variation of virulence phenotypes in populations of
Bremia lactucae on Lactuca serriola in the period 1998–2005
Year
1998
1999
2000
2001
2002
2003
2004
2005
Total
No. of tested No. of determined Variation among
isolates
v-phenotypes
studied isolates (%)
23
31
78
43
8
19
51
60
313
12
13
19
29
7
11
16
34
93
52.2
41.9
24.4
67.4
87.5
57.9
31.4
56.7
29.7
pathosystems. At the population level, virulence can
be thought of as the average ability of a pathogen
population to overcome the diversity of resistance
genes present in the corresponding host population
(Thrall and Burdon 2003). From this viewpoint, B.
lactucae populations occurring naturally on L. serriola showed highly complex patterns of virulence in
relation to the L. serriola and/or L. sativa differentials
with resistance derived from L. serriola (Lebeda
2002; Lebeda and Petrželová 2004a; Petrželová and
Lebeda 2004c). Such complexity of pathogen isolates
may have its origin in response to the heterogeneity of
the host populations (Bevan et al. 1993), and it is
supposed that, in gene-for-gene based pathosystems,
broadly virulent isolates of pathogen are more likely
to occur in highly diverse and resistant host populations (Thrall and Burdon 2003). Many papers have
highlighted the importance of parallel studies of the
structure and dynamics of the host and pathogen
populations (e.g. Burdon and Jarosz 1991; CarlssonGranér and Thrall 2002; Delmotte et al. 1999; Laine
2006; Thrall and Burdon 2000, 2003), which can
bring new insights into the behaviour of the two
interacting species.
Recently, we compared the resistance of Czech
populations of L. serriola (Fig. 4) and the virulence of
B. lactucae populations occurring within the same
area. While B. lactucae isolates originating from
naturally infected L. serriola generally showed high
compatibility to L. serriola they were typically
incompatible with L. sativa differentials, with the
exception of those L. sativa genotypes carrying racespecific resistance genes derived from L. serriola
(Lebeda 1989, 2002; Lebeda and Petrželová 2004a).
Interestingly, when samples from L. serriola populations were screened for resistance to races of B.
lactucae with v-phenotypes generally able to overcome
resistance in L. sativa they typically showed relatively
low levels of race-specific resistance (Lebeda and
Petrželová 2004b). This raises the question of why B.
lactucae isolates with relevant virulence are not more
common in natural populations of B. lactucae. Only a
few such isolates were found in one year (1998) in a
region where lettuce is frequently cultivated (Lebeda
2002). If we suppose that populations of B. lactucae on
L. sativa and L. serriola are fully inter-connected,
isolates with such v-phenotypes should have a selection advantage in L. serriola populations and would
appear more frequently. However, it was not true in the
populations we studied, and perhaps it occurs only
under certain conditions. A logical explanation is that
B. lactucae populations on L. sativa and L. serriola are
highly isolated by their host specificity, and therefore
co-evolution in the wild and crop pathosystems is
operating independently.
From this viewpoint, L. serriola appears to be a
good source of resistance genes for cultivated lettuce
(Lebeda et al. 2007b), and cross-inoculation experiments with isolates from L. sativa may reveal new
sources of resistance (Beharav et al. 2006; Lebeda
and Zinkernagel 2003b). However, the occasional
occurrence of isolates with combined v-phenotype
structure recorded both in the wild (Lebeda and
Petrželová 2004a; Petrželová and Lebeda 2004c) and
crop pathosystems (Lebeda et al. 2007c) indicates that
genotype and gene flow between both pathosystems
is possible, which may increase variation in both
pathogen populations, especially when both host
species are grown in close proximity (Lebeda 2002;
Lebeda and Petrželová 2004a). Furthermore, in
lettuce cultivars with resistance derived from L.
serriola (Lebeda et al. 2002, 2007b), the probability
of an ‘escape of virulence’ from natural pathogen
populations is much higher (Lebeda 1984), and it may
increase when encountering wild and crop populations of pathogen that undergo sexual recombination
(Lebeda and Blok 1990).
Despite the complexity of responses, broad variation in virulence of B. lactucae to L. serriola differentials was found, as seen in the variable distribution
of v-factors among populations. From our data, it is
evident that virulence structure in B. lactucae pop-
Eur J Plant Pathol (2008) 122:127–146
ulations occurring on L. serriola is very dynamic,
undergoing both qualitative and quantitative shifts
(Lebeda and Petrželová 2004a; Petrželová and Lebeda
2004c). In gene-for-gene systems, it is supposed that
the genetic structure of host and pathogen populations
follow each other in a dynamic interaction (Burdon
1997; Burdon et al. 1996), so a long-term decrease in
particular v-factors (e.g. v5/8 or v23) in B. lactucae
populations may be evidence of co-evolution taking
place. However, increases in the frequency of other vfactors (e.g. v14 or v17) were less marked than the
decreases, and may just be considered as evidence for
year-to-year fluctuations over a longer time period.
On the level of individuals, no obvious changes in
mean virulence were found during the study period,
but there was a large variation in recorded virulence
patterns (v-phenotypes) and their distribution over the
pathogen metapopulation. Furthermore, great differences in the prevalence of individual v-phenotypes
were observed, and many rare ones were recorded just
once during our investigations. Similar population
structures with uneven distribution of v-phenotypes,
where just a few predominated have also been
reported for other pathogens of wild plants (Bevan
et al. 1993; Burdon and Jarosz 1991). It seems that
populations of B. lactucae tend, on one hand, to higher
diversity, but on the other hand to a higher prevalence
of particular v-phenotypes. These v-phenotypes may
have a selective advantage at a given time and place,
which may be largely influenced by fluctuations in
local variation in host resistance and the environmental
conditions contributing to the establishment of infection in natural plant populations.
Comparison of B. lactucae virulence variation
between wild (L. serriola) and crop (L. sativa)
pathosystems
In comparison to B. lactucae isolates originating from
L. serriola, isolates originating from cultivated lettuce
generally displayed a highly complex response to L.
sativa differentials and expressed a completely different virulence structure. A comparison of the frequencies of the most important v-factors (from the
viewpoint of resistance breeding, see e.g. Lebeda et
al. 2007b) in both pathosystems during the study
period is illustrated in Fig. 6. Numerous v-factors
were detected in both pathosystems; however, their
141
frequencies differed considerably. Many of the compared v-factors were more common in the crop
pathosystem (e.g. v1–4, v6, v10, v12, v13, v36,
v38). More or less equal frequencies were recorded
for factors v7, v11 and v16, while differences
between factors v5/8, v14 and v15 were more distinct
(Fig. 6). In this case, it is very interesting that all
complementary race-specific resistance genes to these
v-factors originate from L. serriola (Lebeda et al.
2002). Factor v18 was recorded only on L. sativa, and
factor v17 was found only on L. serriola (Fig. 6).
Finally, factor v37 was not found in either pathosystem. In the crop pathosystem temporal shifts in
frequencies of some v-factors were also recorded
(Lebeda et al. 2007c).
Isolates with combined virulence (for L. sativa and
L. serriola) structure were recorded only occasionally.
Differences were also recorded in the dynamics of
occurrence of v-phenotypes in both pathosystems.
Most of the v-phenotypes found on L. sativa were
unique and did not appear repeatedly in other
pathogen populations or in subsequent years (Lebeda
et al. 2007c; Petrželová and Lebeda 2004a).
Large differences in virulence were recorded
between populations of Bremia lactucae occurring
on L. sativa and L. serriola. Populations in crop and
wild Lactuca–B. lactucae pathosystems have different
structures of v-factors; individual v-factors occur in
different frequencies and differences were recorded in
their spatial and temporal population structure and
dynamics. These findings show that individual B.
lactucae populations substantially differ in their
specificity to the host species and virulence to the
host genotypes, respectively. However, we observed
some unexpected overlaps in virulence structure
between B. lactucae populations on L. sativa and L.
serriola (Fig. 6).
Temporal shifts in virulence were recorded in both
pathosystems. In the crop pathosystem, the changes
are largely influenced by increasing usage of cultivars
with newly introduced race-specific resistance genes
(Lebeda and Zinkernagel 2003a). Characterization of
the evolutionary forces driving the wild pathosystem,
L. serriola–B. lactucae, will require more detailed
studies of resistance patterns and their changes in L.
serriola populations (Lebeda et al. 2001b, 2007c).
The virulence data for both pathosystems are very
unique and comparable results are not available for
any other crop and wild pathosystems.
142
Eur J Plant Pathol (2008) 122:127–146
1
0,9
0,8
Frequency
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
1
2
3
4
5/8
6
7
10
11
12
13
14
15
16
17
18
36
37
38
v-factor
L. sativa
L. serriola
Fig. 6 Comparison of frequency of the most important vfactors recorded in the sampled set of Bremia lactucae isolates
collected in the Czech pathogen populations on Lactuca sativa
(n=93 isolates) and Lactuca serriola (n=313 isolates) during
the period 1998–2005. v1, v2, v3, v4, v10, v12, v13, v14—vfactors matching Dm genes or R-factors in cvs of L. sativa. v7,
v15, v16, v17, v23, v24, v25, v26, v27, v28, v29, v30—vfactors matching Dm genes or R-factors in L. serriola. v5/8, v6,
v11, v18, v38—v-factors matching Dm genes or R-factors in
cvs of L. sativa derived from L. serriola. v36, v37—v-factors
matching Dm genes or R-factors in cvs of L. sativa derived
from L. saligna. v35—v-factor matching Dm gene or R-factor
in cvs of L. sativa derived from L. virosa. Relative frequencies
of individual v-factors were expressed as the ratios between the
number of isolates with given v-factor and the total number of
isolates investigated for the presence of considered v-factor
Sexual reproduction and occurrence of mating
types in B. lactucae populations on L. serriola
and L. serriola are not completely compatible when
pairing together to produce oospores under laboratory
conditions (Petrželová and Lebeda 2003). This is
additional evidence for at least some isolation of both
pathosystems (L. sativa versus L. serriola–B. lactucae).
We may assume that the possible danger of the natural
formation of new virulent races of B. lactucae by the
crossing of pathogen isolates from crop and wild
lettuce is quite low; this is also supported by the results
from virulence analyses (Fig. 6).
The occurrence of mating types was studied in a set of
59 B. lactucae isolates originating from 33 naturally
infected and wild populations of L. serriola in the
Czech Republic, including two isolates from Germany
and France. The isolates were collected in the period
1997–1999. Both compatibility types were recorded;
however, the majority of the isolates (96%) were
determined as type B2, supporting the observation that
sexual reproduction of B. lactucae on naturally
growing L. serriola plants is rare (Petrželová and
Lebeda 2003).
As was stated before, sexual recombination is
considered to be important for generating considerable genetic variation in virulence in populations of B.
lactucae on L. sativa (Crute 1992b; Lebeda and
Schwinn 1994). However, our previous (Lebeda and
Blok 1990) and recent results indicate that its
importance for the pathogen populations on L.
serriola is questionable (Petrželová and Lebeda
2003). From the practical viewpoint it is interesting
that isolates of B. lactucae originating from L. sativa
Conclusions and future developments
From the results summarized in this paper, it is
evident that the wild pathosystem, L. serriola–B.
lactucae, is very complex, variable and dynamic. The
frequency of pathogen incidence in host populations
is very high, but disease prevalence is rather low.
Other host plant species have no substantial influence
on B. lactucae epidemiology. Pathogen incidence is
most strongly influenced by ecological factors, including host habitat, density of host populations, and
climatic and microclimatic conditions. Host plants are
Eur J Plant Pathol (2008) 122:127–146
also frequently infected by powdery mildew (Golovinomyces cichoracearum); the frequency of co-infection
by both pathogens is about 35%. We expect some
competition for leaf niche, and the phenomenon of
induced resistance may play a role in co-infection, but
we lack clear experimental evidence to document this
phenomenon.
Research showed that race-specific resistance is the
dominant pattern in populations of L. serriola. A
broad spectrum of resistance phenotypes (altogether
45) was detected in host populations occurring in the
Czech Republic. In some populations, individuals
with completely resistant or completely susceptible
reactions were detected. Intra-population variability
was rather common. Comparison of host populations
from continental Europe with those from the UK
showed substantial differences. The island populations were much more homogeneous with regard to
variation in resistance, and reactions were always
race-specific in inoculation studies.
From the viewpoint of virulence, the pathogen
population is enormously variable, and most of the
known v-factors were detected. However, there are
substantial differences in the frequency of individual
v-factors. In the pathogen population, v-factors that
match R-factors originating from L. serriola prevail,
but v-factors matching race-specific Dm genes from
cultivated lettuce are very rare. At the individual
level, we recorded many v-phenotypes, but only a few
were common in B. lactucae populations. Comparative studies of the virulence structure of pathogen
populations in wild and crop pathosystems clearly
demonstrated completely different compositions of vfactors and v-phenotypes. It seems that there is no
direct epidemiological linkage between both pathosystems. However, some unexpected overlaps in
virulence structure were recorded, and these differences may be evidence for the existence of potential
migration or gene flow between the pathosystems.
This phenomenon, although rare, may be quite
important and could influence the stability of resistance derived from L. serriola in cultivated lettuce.
The data obtained about this pathosystem demonstrate that B. lactucae belongs to a group of pathogens
with high evolutionary potential (sensu McDonald
and Linde 2002), and the wild host population is
extremely variable from the viewpoint of resistance.
The evolutionary forces operating in and between
both pathosystems are not well known. Future
143
research on these pathosystems can contribute to our
understanding of this exciting area of plant pathology
and oomycete population biology. We propose that
these pathosystems are quite suitable as model systems
for the study of structure and variability from a spatial
and temporal viewpoint, and that interactions between
the wild and crop pathosystems are especially relevant
for crop improvement and agricultural production.
Acknowledgements Critical reading and valuable remarks by
Dr. M. P. Widrlechner (USDA-ARS, Iowa State University,
Plant Introduction Station, Ames, Iowa, USA) and Dr. P. H.
Thrall (CSIRO Plant Industry, Canberra, Australia) are gratefully acknowledged. We are grateful to Dr. Milena Kršková for
help with statistical analyses of the data. This research was
supported by grants MSM 6198959215 and QH71229 (NAZV)
and by the National Programme of Genepool Conservation of
Microorganisms and Small Animals of Economic Importance
of the Czech Republic.
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Eur J Plant Pathol (2008) 122:147–155
DOI 10.1007/s10658-008-9346-6
Development of detection systems for the sporangia
of Peronospora destructor
Roy Kennedy & Alison J. Wakeham
Received: 6 November 2007 / Accepted: 9 June 2008
# KNPV 2008
Abstract A monoclonal antibody that recognises
components of the wall of sporangia of Peronospora
destructor was raised. Tests using spores of higher
fungi and other species of mildew demonstrated the
specificity of the monoclonal. The antibody was used
to develop lateral flow devices for sporangia of
P. destructor. A competitive lateral flow format was
developed which could detect onion downy mildew
sporangia. Five-microliter gold anti-mouse IgM solution pre-mixed with 10 μl of P. destructor monoclonal
antibody (EMA 242) proved the optimal concentration for detection of sporangia of P. destructor when
applied to sample pads of lateral flow devices. Limits
of approximately 500 sporangia of P. destructor could
be detected by the absence of a test line on the lateral
flow device within test samples. Using a scanning
densitometer improved the sensitivity of detection.
Further development and validation of the test is
required if it is to be used for risk assessments of
onion downy mildew in the field.
Keywords Onion downy mildew .
Monoclonal antibody . Peronospora destructor .
Lateral flow assay . Detection . PTA ELISA .
Immunofluorescence
R. Kennedy (*) : A. J. Wakeham
Warwick HRI, University of Warwick, Wellesbourne,
Warwick, Warwickshire CV35 9EF, UK
e-mail: Roy.Kennedy@warwick.ac.uk
Introduction
Foliar diseases of onion crops (onion downy mildew
and Botrytis leaf blight) can cause heavy yield losses
in bulb and salad onion crops. Onion downy mildew
(Peronospora destructor) is the most serious disease
in bulb and salad onions in the UK (Gilles et al. 2004;
Clarkson et al. 2000). Actual yield losses in bulb
onions of 60 to 75% have been recorded (Cook 1932;
Cruickshank 1958). These losses mainly result from
severe infections in bulb onion crops causing early
defoliation, reduced bulb sizes and poor storage
quality of bulbs (Rondomanski 1967). In salad
onions, yield losses can be as high as 100% with
whole crops being discarded, as downy mildew
symptoms on the plant make them unmarketable.
Fungicidal control of onion downy mildew is difficult
and fungicides are only effective if they are applied
before or immediately after disease first appears in the
crop (Kennedy 1998). The environmental requirements for infection and sporulation by P. destructor
have been reported (Yarwood 1937, 1943; Hildebrand
and Sutton 1982). Mathematical models describing
climatic effects on sporulation and infection have
been described (Gilles et al. 2004; Battilani et al.
1996; Jesperson and Sutton 1987). However, despite
the rapid development of onion downy mildew and
the requirements for reductions in fungicide usage by
consumers, the practical use of these systems in risk
assessment has been limited to date.
148
New approaches in forecasting diseases of onion
crops based on estimation of spore numbers in air
samples have been reported (Carisse et al. 2005;
Berger 1970). Detection and quantification of airborne spore numbers can be used to predict disease
accurately before it is visible in the crop. Peaks of
airborne spores are always detected prior to crops
becoming infected. It has been reported that one or
two peaks in sporangial concentration in the air of
the potato blight pathogen Phytophthora infestans
preceded the first observed symptoms of the disease
in the field (Bugiani et al. 1998; Phillon 2003). In
these studies the information on spore number had to
be collected manually using a microscope which was
slow and time consuming. Tests which can be
conducted in the field are necessary if information
on air-borne inoculum concentration is to be of more
practical value. However, there are few reported
systems for detecting and differentiating airborne
spores (Wakeham et al. 2004). Molecular techniques
exist for detection of spores using Hirst types spore
samplers (Williams et al. 2001). Day et al. (2002)
reported the development of cell flow cytometric
differentiation of air-borne sporangia of P. infestans
using ‘in field’ systems. Both techniques required
either laboratory processing of results or the development and use of sensitive equipment not fully
demonstrated under field conditions. The development and use of detection systems for estimating airborne spore numbers would be a further development
in risk assessment for onion downy mildew. This
study reports on the development of an immunomonitoring system for conidia of P. destructor.
Materials and methods
Production of P. destructor immunogen for antibody
production
The isolate (PD HL00) of P. destructor used in the
study was as reported by Gilles et al. (2004). Leaf
surface wax material of ten onion sets (Allium cepa
cv. White Lisbon) was removed by gentle agitation
with sheep’s wool (found to remove leaf wax without
leaf damage) prior to inoculation with P. destructor.
Twenty-five 20-μl droplets of P. destructor (1×104
conidia ml−1 H2O) were applied to each sheep’s wooltreated leaf. To induce infection, inoculated plants
Eur J Plant Pathol (2008) 122:147–155
were incubated in high humidity for three days after
which plants were removed and placed in a temperaturecontrolled glasshouse (18°C) for a further 2 weeks.
Inoculated plants were returned to a high humidity
environment for a period of 48 h to induce sporulation
by P. destructor on infected leaves.
Collection of P. destructor spores from leaf surfaces
A hand-held Burkard surface cyclone sampler (Burkard
Manufacturing Co., Rickmansworth, Herts, UK) was
used to collect sporangia of P. destructor from the
surface of the infected leaf material. The 2 ml
Eppendorf containing the collected spores was removed and 1 ml of chilled sterile distilled water
(SDW) was added. The collected P. destructor
sporangia were suspended in water and 0.5 ml volume
of chilled SDW was added. The sporangial suspension
was filtered through a stainless steel membrane
(47 μm pore size) to remove any large contaminating
material. The liquid phase was collected and bacterial
and other small leaf contaminants removed by
filtering using a polyester membrane (10 μm pore
size). The filtrate was collected and resuspended in
1 ml phosphate buffered saline solution, pH 7.0
(PBS). Bright field microscopy was used to determine
the presence of P. destructor sporangia which were
adjusted to a concentration of 3.5×104 conidia ml−1.
Immunization of mice with P. destructor sporangia
The spore suspension was agitated, using a Gallenkamp
spinmix, continuously for a period of 5 min after 3 h
at 0–4°C. A microfuge (MSE Microcentaur) was used
at 13 rpm for 5 min to separate particulate spore
material from the soluble spore fraction of the
sample. The soluble fraction of the sample was
retained and concentrated at first by freeze-drying
(Modulyo 4 k, Edwards) and then rehydrating to a
final volume of 100 μl PBS. Two Balb C female mice
(coded 7996, 7997) were immunised (by intraperitoneal injection) each with 50 μl of the concentrated
soluble P. destructor sporangial preparation mixed
with an equal volume of Titermax adjuvant. All
further immunisations were as described above. Tail
bleeds were taken seven days after the second
immunisation procedure and a PTA-ELISA (described
below) was carried out to determine whether the mice
had produced an immune response to P. destructor.
Eur J Plant Pathol (2008) 122:147–155
The mice received a final pre-fusion boost of the
P. destructor soluble sporangial immunogen mixed
with adjuvant (100 μl). The spleen of mouse 7996
was removed 4 days later and the fusion was carried
out according to those methods reported by Dewey
(1992). Hybrids were fed on days 3, 6 and 10 and cell
culture supernatants screened by PTA ELISA and
immunofluorescence 14 days after cell fusion for the
presence of antibodies which recognised sporangial
components of P. destructor.
Monoclonal antibody screening
Plate trapped antigen ELISA (PTA ELISA)
One hundred μl of P. destructor soluble sporangial
washings in PBS were aliquoted in to each of 96-well
Polysorp microtitre well strips (Nunc, Roskilde,
Denmark; Cat. No.469957). The strips were incubated
overnight in an enclosed chamber at 18°C. Unbound
material was then removed and the microtitre wells
were washed once with 200 μl PBS. The microtitre
wells were blocked with 200 μl of 1% Casein buffer
(1% (w/v) casein PBS) and incubated at 37°C for
45 min. Residual blocking buffer was removed and
wells were washed four times for 1 min each with
200 μl PBS, 0.05 % Tween 20 and 0.1 % Casein
(PBSTw C). Each well received 100 μl of fusion
hybridoma tissue culture supernatant mixed with
PBSTw C. Following incubation in a Wellwarm
shaker incubator (30°C) for a period of 45 min as
above, wells were washed three times for 1 min each
with 200 μl PBSTw. A DAKO duet amplification
system was used according to manufacturer’s instructions (DAKO Ltd, Cambridge, UK) to amplify the
signal generated by bound tissue culture supernatant
antibodies. Wells were washed as described above
and 100 μl of 3,3′,5,5′-tetramethylbenzidene substrate
(Sigma, UK) was added to each well. The reaction
was stopped by adding 25 μl of a 20% 1 M H2S04
solution to each well. Absorbance at 450 nm was
determined with a Biohit BP800 ELISA plate reader
(Alpha Laboratories, Hampshire, UK).
Immunofluorescence
Twenty microliters of a 1×103 spores ml−1 P. destructor
conidial spore suspension was aliquoted to individual
multiwell glass slides (Cel-Line/Series Scientific Corp,
149
USA; Cat No. 10-3404). Following air drying any
unbound spore material was removed with a PBSTwC
wash. Material remaining bound to the multiwell glass
slides was incubated with 20 μl of hybridoma tissue
culture supernatant antibodies (TCS) mixed with
PBSTwC for a period of 30 min at room temperature.
A counterstain of Evans blue and Eriochrome black
was incorporated within the TCS antibody suspension
to quench P. destructor spore autoflourescence
(Kennedy et al. 1999). Each multiwell received a
wash as described above and following air drying was
incubated with anti-mouse antibodies which had been
conjugated to fluorescein isothiocyanate dye. A
counter-stain was included to ensure quenching of
sporangial autoflourescence. Incubation was carried
out at room temperature in darkness to prevent photobleaching of the conjugated antibody. The processed
multiwells received a final wash of PBSTwC and after
air drying were mounted and viewed by episcopic
fluorescence microscopy for the presence of antibody/
fluorescein-tagged sporangia of P. destructor. Hybridoma antibody tissue culture supernatants, identified as
positive to P. destructor sporangial material using
either PTA ELISA and/or IF, were selected and twice
cloned to monoclonal Ab status.
Selection of specific P. destructor monoclonals
To determine specificity, the selected P. destructor
monoclonal cell lines were determined by PTA-ELISA
and IF against a range of fungal species. Tests were
carried out on spores and mycelium taken from pure
cultures of Bremia lactucae, Peronospora parasitica,
Paecilomyces variotii, Botrytis cinerea, B. squamosa,
Stemphyllium sp., Aureobasidium pullulans, Phoma
betae, Ascochyta rabei, Fusarium culmorum, Penicillium roquefortii, Pyrenophora teres and sporangia of
P. destructor. Stains used in these tests are as
designated in Kennedy et al. (2000). With the
exception of P. destructor, P. parastica, B. lactucae
and Ascochyta (all of which were grown directly on
plant material) the fungal species used in the reactivity
tests were grown on a synthetic medium covered with
a sterile Supor membrane filter prior to inoculation.
Fourteen days after inoculation (of cultures grown
on agar) 5 ml of PBS (pH 7.5) solution was applied to
the culture surface. Surface washings were taken by
gently stroking the culture surface with a glass
spreader. All collected spore concentrations were
150
Eur J Plant Pathol (2008) 122:147–155
Fig. 1 Lateral flow crosssection (5 mm strip)
Membrane
Conjugate pad
Sample pad
Absorbent Pad
Laminate backing card
adjusted to a final concentration of 1×105 spores ml−1
PBS. The spore solutions were individually aliquoted
into each microtitre well (100 μl per well) of a
Polysorp microtitre strip. The wells were covered and
incubated overnight at 4°C. Unbound material was
removed and the microtitre wells were washed once
with 200 μl PBS. An ELISA was carried out as
previously described.
Development of a competitive lateral flow assay
format for the detection of conidia of P. destructor
A competitive lateral flow format (Fig. 1), comprising
a Millipore 135 HiFlow™ cellulose ester membrane
direct cast onto 2 ml Mylar backing (Millipore Corp,
USA.), an absorbent pad (Schleicer and Schuell,
Germany) and a sample pad (Millipore Corp., USA)
was constructed for the detection of P. destructor
sporangia. Control lines of an anti-mouse serum were
sprayed directly onto the membrane surface using a
flat bed air jet dispenser (Biodot Ltd, West Sussex,
UK). A collected soluble fraction of a P. destructor
sporangial sample, prepared as described earlier, was
adjusted to a protein concentration of 500 μg ml−1 ,
250 μg ml−1 and 125 μg ml−1 in PBS and applied as a
test line again using a flat bed air jet dispenser.
Membranes were air-dried at 35°C for a period of 4 h.
The test and control line-labelled lateral flows were
cut in to 5 mm strips and each strip housed within a
plastic case (Schleicer & Schuell, Germany).
cell line (coded EMA 242) before drop application to
a 5 mm sample pad and air-drying. Variable concentrations of gold conjugated antibody EMA 242 were
applied to different sample pads to investigate the
antibody conjugate concentration which gave optional
test line formation on the lateral flow device (lfd).
Sample conjugate Ab pads were attached to each lfd
strip as shown in Fig. 1. A test antigen of a 60 μl
sporangial suspension (3×103 P. destructor sporangia) was then applied to the sample pad of an lfd strip.
The competitive lateral flow devices (clfd) were
viewed 5 min post-sample application. For each test,
a spore-free suspension was applied to a clfd as a
negative control. The variable antibody dilutions of
MAb used in these tests are shown in Table 1.
Visual detection threshold of a competitive lateral
flow device employing two membrane types
for P. destructor sporangia
Studies were carried out using a clfd format for the
detection of known concentrations of P. destructor
spores. Two different membrane types were examined:
a Millipore 135 HiFlow™ cellulose ester membrane
direct cast on to 2 ml Mylar backing and a Millipore
Table 1 Antibody dilutions in the samples pad and
corresponding P. destructor test line concentration
P. destructor MAb type
and dilution factor EMA 242
P. destructor protein
concentrations μg ml−1
at test line
Antibody conjugation
A British Biocell gold anti-mouse IgM solution was
pre-mixed (conjugated) with a selected hybridoma
1 in 160
1 in 320
1 in 640
500
500
500
250
250
250
125
125
125
Eur J Plant Pathol (2008) 122:147–155
Semi-quantitative tests with lateral flow format
using Millipore 135 HiFlow™ cellulose ester membrane
A 5 μl British Biocell gold anti-mouse IgM solution
was pre-mixed with 10 μl EMA 242 and then applied
drop-wise to lateral flow sample pads at a test volume
of 15 μl, each dried as previously described. This was
chosen as the optimal concentration for detecting
approximately 500 sporangia of P. destructor. Sporangia of P. destructor in sample buffer were applied
drop-wise (70 μl) to the sample pads of the preprepared lateral flows. Sporangial concentrations
ranged from 240 to 960 sporangia applied. The lateral
flow devices were viewed 20 min post-sample
application for the formation of a test and control
line and test line optical density values were generated using a BioDot lateral flow reader (BioDot,
Chichester). A negative control of lateral flow
running buffer alone (0 downy mildew conidia) was
also included within these tests.
Results
1.2
EMA 242
1
EMA243
0.8
0.6
0.4
0.2
0
Bremia lactucae
Peronospora parasitica
Peronospora destructor
Stemphyllium
Aureobasidium pullulans
Paecilomyces variotii
B. squamosa
B. cinerea
Pyrenophora teres
Penicillium roq.
Fusarium culmorum
Eleven hybridoma cell lines were identified (using
PTA ELISA) as producing antibodies which recognised components associated with the sporangial
material of P. destructor. A preliminary screen against
a range of plant fungal pathogens identified three
Only eight of the eleven cell lines were identified as
producing antibodies which recognised components
directly associated with the conidia of P. destructor
when visualised by immunofluorescence. Of these,
six were excluded following preliminary reactivity
studies (data not shown). Those selected for further
testing by IF were EMA 242 and 243 (Table 2). EMA
240 did not react with material directly associated
with P. destructor; however, an area of diffuse
speckling was noted surrounding the spore. In
immunofluorescence studies both EMA 242 and 243
reacted with the spore wall of P. destructor and
retained a high level of specificity when tested against
other fungal species. This indicated a high degree of
similarity between these cell lines.
Ascochyta rabei
Plate trapped antigen ELISA (PTA ELISA)
Immunofluorescence
Phoma betae
Monoclonal antibody screening
tissue culture supernatants for expansion to monoclonal status. These were selected, cloned to monoclonal
antibody status and coded EMA 240, 242, and 243.
Monoclonal antibody cell line EMA 240 was not used
in reactivity tests as it was observed to react with other
downy mildew species when tested by ELISA (data
not shown). Monoclonal antibodies EMA 242 and 243
reacted to their homologous antigen (P. destructor),
and demonstrated a high level of specificity when
tested against other fungal species (Fig. 2).
PTA ELISA absorbance value (450 nm)
240 HiFlow™ cellulose ester cast membrane. The lfd
devices were prepared as described above and a test
line of 250 μg ml−1 P. destructor soluble antigen in
PBS was applied. The membranes were air-dried at
35°C, cut into 5 mm strips and each strip housed
within a plastic case as previously described.
A known sporangial concentration of P. destructor
sporangia (60 μl) was mixed with EMA 242 gold
conjugate (5 μl) to produce a final antibody dilution
of either 1:150, 1:400 or 1:600. The mixture was
applied to the sample pad of each clfd and results
viewed 5 min post-sample application. For each
membrane type a ‘spore free suspension’ was mixed
with MAb EMA 242 gold conjugate to act as a
negative control.
151
Fungal species tested
Fig. 2 Reactivity of monoclonal antibodies EMA 242 and 243
to a range of airborne fungal species as tested by PTA ELISA
(each value represents the mean of two replications, SD 0.0892)
152
Eur J Plant Pathol (2008) 122:147–155
Table 2 Reactivity of monoclonal antibodies EMA 242 and 243 to a range of airborne fungal species as tested by
immunofluorescence
Fungal species tested
Phoma betae
Aschochyta rabei
Fusarium culmorum
Penicillium roqueforti
Botrytis cinerea
Botrytis squamosa
Paecilomyces variottii
Aureobasidium pullulans
Stemphyllium Peronospora
Peronospora destructor
Peronospora parasitica
Bremia lactucae
Isolate Code
WPhbl
cbs765.01
C2751
C2709
Cbs121.39
Cbs105.23
C2745
C1718
Wsa1
WPd05
WHpBo717
WB103
EMA 242
EMA 243
Mycelium
Spores
Mycelium
Spores
☒
Not
☒
Not
☒
☒
Not
Not
☒
Not
Not
Not
☒
☒
Not Tested
☒
☒
☒
☒
☒
☒
☑
☒
☒
☒
Not
☒
Not
☒
☒
Not
Not
☒
Not
Not
Not
☒
☒
Not Tested
☒
☒
☒
☒
☒
☒
☑
☒
☒
tested
tested
tested
tested
tested
tested
tested
tested
tested
tested
tested
tested
tested
tested
☒ No fluorescence observed
☑ fluorescence observed denoting reactivity
cbs The Centraalbureau voor Schimmelcultures
W Warwick HRI Culture collection, c Rothamsted culture collection
Assessment of competitive lateral flow assay format
for the detection of P. destructor
At a test line application of 500 μg ml−l spore protein
deposition, test line inhibition (i.e. no test line
development) was observed when a P. destructor
spore sample was mixed with gold conjugated EMA
242 at a dilution >1:160. For all negative control
samples (i.e. no P. destructor spores present), control
and test line development was observed for each
competitive lateral flow device. Using an Ab dilution
of 1:640 gave no test or control lines for either sporepositive or spore-negative samples.
At a test line concentration of 250 μg ml−l protein
(spore) deposition, strong test and control line
development was observed at detection antibody
(Ab) dilutions of 1:160 and 1:320 when a spore-free
suspension was applied. Testing a positive sample of
P. destructor and the detection antibody at a dilution
of 1:160 gave rise to a barely visible test line, but a
strong control line. At an antibody dilution of 1:320,
test line depletion was complete. At a test line
concentration of 125 μg ml−l , testing a P. destructor
spore-free suspension, gave test and control line
development when a detection antibody dilution of
1:160 was used. Using a positive P. destructor spore
sample gave rise to a clear control line but no test line
development (a positive test for the competitive
lateral flow format). At all other antibody dilutions,
control lines were barely visible and no test line
development was noted for any of the samples tested.
Visual detection threshold of a competitive lateral
flow device employing two membrane types
for P. destructor sporangia
Using a Millipore HiFlow™ 135 Membrane and
antibody EMA 242 gold conjugate at a dilution of
1:150, test line formation was observed for all spore
samples tested. This denoted that the detection
sensitivity of the test was poor and unable to detect
2,000 sporangia of P. destructor. However, by
diluting the activity of the antibody conjugate to
1:400, test sensitivity was improved (Table 3a,
Fig. 3a). At an antibody conjugate dilution of 1:600,
the test became void with no test line formation for
any of the samples tested.
Using a Millipore HiFlow™ 240 membrane
competitive lateral flow device and EMA 242
conjugated to gold spheres at a dilution of 1:150, test
line formation was again observed for all spore
samples tested. As previously noted, by diluting the
activity of the antibody to 1:400, the sensitivity of the
test was improved (Table 3b). For this membrane, an
Eur J Plant Pathol (2008) 122:147–155
153
Table 3 Detection of sporangia of P. destructor with varying antibody dilutions using (a) Millipore HiFlow™ Membrane 135 (b)
Millipore HiFlow™ Membrane 240
(a)
No P. destructor spores in sample
EMA 242 Ab dilution
1 in 150
1 in 400
1 in 600
(b)
EMA 242 Ab dilution
1 in 150
1 in 400
1 in 600
0
✓
✓
✘
62
✓
✓
✘
125
✓
✓
✘
250
✓
✓
✘
500
✓
✘/✓
✘
1000
✓
✘/✓
✘
2000
✓
✘/✓
✘
0
✓
✓
✓
62
✓
✓
✓
125
✓
✓
✓
250
✓
✓
✓
500
✓
✓
✘
1000
✓
✘
✘
2000
✓
✘
✘
✓ Clear test line (P. destructor not detected in sample
✘ no test line development (P. destructor presence detected by clfd)
✘/✓ weak test line development
antibody dilution of 1:600 was required to achieve a
detection assay where sporangia in excess of 250
could be detected (Fig. 3b, Table 3b).
Semiair-quantitative lateral flow prototype tests
with onion downy mildew
The results of using increasing amounts of P. destructor
sporangia on the lateral flow device are shown in
Table 4. When a negative sample (0 sporangia) was
applied to a lateral flow device, strong test line
development was observed. As spore concentrations
increased, the test line colour formation decreased.
When a P. destructor sporangial concentration of 960
was applied to a lateral flow device, no test line
development was observed. Using a Bio-dot lateral
flow reader, an optical density value of 2.2 was
observed with control suspensions (0 sporangia).
However, when 960 sporangia of P. destructor was
added to the device, the optical density of the line
decreased to 0.3.
Discussion
In this study detection tests for P. destructor sporangia
were developed although these have not yet been used
in the field. Detection of the presence of P. destructor
sporangia could be important in onion downy mildew
control regimes. Control of plant pathogens could be
improved if inoculum could be detected quickly in the
field directly by the grower. Airborne inoculum plays
a vital role in the development of epidemics caused
by Botrytis leaf blight on onion crops (Carisse et al.
2003, 2005). In this work, a linear relationship was
found between number of lesions on plants and airborne Botrytis conidial concentrations. Airborne
conidial concentrations of 25 to 35 conidia m−3 of
air were associated with 2.5 lesions per leaf. When
detection of Botrytis inoculum was used as a control
criterion under field conditions, it led to a reduction in
fungicide usage of 75 and 56% in 2002 and 2003. A
similar relationship between spore number and disease intensity has been reported for Cercospora apii
on celery (Berger 1969). In both these studies,
microscopes were used to determine spore numbers
from air samples.
One of the objectives of the work reported in this
paper was to construct rapid tests for sporangia of
P. destructor. If rapid tests were suitable for use in the
field they could be used potentially to detect
sporangia of P. destructor using air samplers as a
means of forecasting the onset of disease development. To date no strains of onion downy mildew have
been reported in the UK. However it is likely that the
antibodies used to construct lateral flow tests developed in this study would react equally with all
populations of P. destructor found in the field. By
using techniques outlined in this paper, early detection of P. destructor in samples could be made
possible. The lateral flow device would, however,
need to be tested with portable air samplers in the
154
Eur J Plant Pathol (2008) 122:147–155
(a)
low levels, especially in large cropping areas. Additionally, the symptomology of the disease on young
plants is poorly understood and observed. By detecting the presence of P. destructor sporangia, it would
be possible to determine action thresholds for onion
crops at different stages in their development. The
lateral flow device, if used to detect sporangia of
P. destructor in the field, would require validation in
different onion-producing areas.
Using advanced monitoring techniques, the optimal
criteria for applying fungicide applications to an onion
crop could be investigated. Disease development
might also be detected in the absence of visible
symptoms. This is a critical point in considerations
of disease control, since if early applications of
fungicide can be targeted to when P. destructor
sporangia are present, improved control could be
achieved. Rapid diagnostic tests, similar to those
reported in this study, exist for identifying Phytophthora
spp. (Lane et al. 2007). However the test kits reported
reacted to a range of Phytophthora spp. which are
commonly found in soils. These lateral flow devices
and those reported by Thornton et al. (2004) were used
to identify infected plant tissues in soil samples.
However, in studies reported in this paper, lateral flow
devices reacted selectively to P. destructor sporangia
(no infected plant material present). If the device were
to be used in conjunction with air samples, it would
pose fewer problems in comparison to using lateral
flow devices for detecting infected soil or plant tissues.
Detecting P. destructor sporangia would be particularly useful early in the season as a method of
preventing disease transfer between over-wintered
salad onion crops and bulb onions grown as sets or
as seeded crops. The use of weekly estimates of
inoculum in air samples has also been reported
(Kennedy and Wakeham 2006) for other diseases,
notably Pyrenopeziza brassicae (light leaf spot of
horticultural and arable brassicas). Tests which can be
Limit of detection
(b)
Fig. 3 Development of a competitive lateral flow for onion
downy mildew conidia a competitive lateral flow test employing detection antibody concentration of 1:400, b competitive
lateral flow test employing detection antibody concentration of
1:600
field to determine the optimal trapping format for
P. destructor sporangia. Trapping formats for sporangia would also need to be integrated with numbers
of sporangia found above infected crops in the
absence of onion downy mildew symptoms on plants.
However, by integrating the device with the output
from a scanning densitometer, the sensitivity of the
device can be improved to enable it to detect the
presence of sporangia of P. destructor at lower
concentrations. Scanning densitometers are becoming
more portable and their use might enable low
numbers of P. destructor sporangia to be detected
using a lateral flow device. Symptoms of onion
downy mildew within crops are difficult to detect at
Table 4 Optical density values of the test line at varying
P. destructor sporangial numbers
Onion downy mildew
sporangial number
0
240
480
960
Optical density
value
2.2
1.8
0.7
0.3
Eur J Plant Pathol (2008) 122:147–155
conducted in the field are necessary if information on
air-borne inoculum concentration is to be of more
practical value. Results of the trials reported in this
paper demonstrate the development of lateral flow
devices that can detect plant pathogenic inoculum.
Potential exists for linking these estimates of
P. destructor inoculum to mathematical models describing the environmental factors which affect onion
downy mildew sporulation (Gilles et al. 2004). Use of
this approach might improve the efficiency of both the
inoculum detection system and disease forecasts. However this would require investigation in future work.
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Eur J Plant Pathol (2008) 122:157–167
DOI 10.1007/s10658-008-9290-5
Fungicide modes of action and resistance in downy mildews
Ulrich Gisi & Helge Sierotzki
Received: 24 September 2007 / Accepted: 11 February 2008 / Published online: 9 April 2008
# KNPV 2008
Abstract Among oomycetes, Plasmopara viticola on
grape and Phytophthora infestans on potato are
agronomically the most important pathogens requiring
control measures to avoid crop losses. Several chemical classes of fungicides are available with different
properties in systemicity, specificity, duration of
activity and risk of resistance. The major site-specific
fungicides are the Quinone outside inhibitors (QoIs;
e.g. azoxystrobin), phenylamides (e.g. mefenoxam),
carboxylic acid amides (CAAs; e.g. dimethomorph,
mandipropamid) and cyano-acetamide oximes (cymoxanil). In addition, multi-site fungicides such as mancozeb, folpet, chlorothalonil and copper formulations
are important for disease control especially in mixtures
or in alternation with site-specific fungicides. QoIs
inhibit mitochondrial respiration, phenylamides the
polymerization of r-RNA, whereas the mode of action
of the other two site-specific classes is unknown but not
multi-site. The use of site-specific fungicides has in
many cases selected for resistant pathogen populations.
QoIs are known to follow maternal, largely monogenic
inheritance of resistance; they bear a high resistance risk
for many but not all oomycetes. For phenylamides,
U. Gisi (*) : H. Sierotzki
Research Biology, Syngenta Crop Protection,
4332 Stein, Switzerland
e-mail: ulrich.gisi@syngenta.com
U. Gisi
Institute of Botany, University of Basel,
4056 Basel, Switzerland
inheritance of resistance is based on nuclear, probably
monogenic mechanisms involving one or two semidominant genes; resistance risk is high for all oomycetes. The molecular mechanism of resistance to QoIs is
mostly based on the G143A mutation in the cytochrome
b gene; for phenylamides it is largely unknown.
Resistance risk for CAA fungicides is considered as
low to moderate depending on the pathogen species.
Resistance to CAAs is controlled by two nuclear,
recessive genes; the molecular mechanism is unknown.
For QoIs and CAAs, resistance in field populations of
P. viticola may gradually decline when applications are
stopped.
Keywords CAA fungicides . Cytochrome b gene .
G143A substitution . Inheritance of resistance .
Monogenic resistance . Phenylamide fungicides .
Phytophthora infestans . Plasmopara viticola .
QoI fungicides . Recessive resistance .
Segregation of resistance
Introduction
In spite of cultural practices and breeding for resistant
cultivars, downy mildews are among the most
devastating plant diseases. To avoid yield losses,
disease control is required mainly by using chemical
products. The economic importance of single downy
mildew diseases (within Peronosporales and Sclerosporales) can be ranked according to the size of crop
158
area treated with chemicals. In 2006, the sales value of
the global fungicide market for the control of diseases
caused by oomycetes was about US$ 1.2 billion. By
far the biggest segment of downy mildews worldwide
is represented by Plasmopara viticola on grape
(54%), followed by Pseudoperonospora cubensis on
cucurbits (12%), Bremia lactucae on lettuce (8%),
Peronospora spp. on leek and onion (6%), on tobacco
(4%), on field crops such as peas, brassicas and sugar
beet (each 3%) and on soybeans and corn (each 2%),
Pseudoperonospora humuli on hops and Plasmopara
halstedii on sunflower (each 1%), and the systemic
pathogens Peronosclerospora and Sclerophthora spp.
in corn (1%) (Syngenta internal data). Not included in
this list are pathogens of the Pythiales, especially
Phytophthora spp., of which Phytophthora infestans
on potato and tomato is the most predominant
segment (about the same as the P. viticola segment).
Phytophthora infestans is included in this review,
because most fungicides controlling downy mildews
are also active against Phytophthora spp., and the
modes of action and mechanisms of resistance have
often been studied in Phytopthora spp., which are
easier to handle under laboratory and glasshouse
conditions than the biotrophic downy mildews.
Chemical control is the most effective measure
currently used to protect crops from downy mildews.
Surprisingly, the rather ‘old’ multi-site fungicides
including dithiocarbamates (e.g. mancozeb), phthalimides (folpet), chloronitriles (chlorothalonil) and copper
formulations account still for about 50% of the downy
mildew fungicide market. Among the single-site fungicides, four chemical classes dominate the market: the
Quinone outside inhibitors (QoIs; ‘strobilurins’, mainly
azoxystrobin, famoxadone, fenamidone), the phenylamides (PAs, mainly mefenoxam), the carboxylic acid
amides (CAAs; mainly dimethomorph, iprovalicarb,
benthiavalicarb, mandipropamid) and the cyanoacetamid-oximes (cymoxanil). Smaller market shares
are taken by phosphonates (mainly fosetyl-Al), dinitroanilines (fluazinam), carbamates (propamocarb) and
plant defence inducers such as the benzothiadiazoles
(BTH, acibenzolar-S-methyl/Bion).
For many decades, multi-site contact fungicides
were the only compounds available for the control of
downy mildews. Within the last 30 years, chemical
control of downy mildews has undergone dramatic
changes with the detection and introduction of singlesite fungicides such as cymoxanil (1976), fosetyl-Al
Eur J Plant Pathol (2008) 122:157–167
(1977), phenylamides (1977–1983), propamocarb
(1978), CAAs (1992–2005) and QoIs (1996–2000;
Gisi 2002). Generally, single site-fungicides act
against a very specific step in the metabolism of
pathogens and have only few side effects on other
processes or non-target organisms. Most single-site
fungicides penetrate into the leaf and are protected
against wash-off by rain; some are also systemic and
move into untreated parts of the plant. In contrast to
multi-site fungicides, most single-site inhibitors bear a
high intrinsic risk of causing the evolution of resistant
pathogen sub-populations. This development is a
common phenomenon in agricultural practice and is
based on the selection of resistant individuals by the
use of fungicides. However, a robust disease control
programme will result also in a successful resistance
management, because the probability of resistant
survivors is smaller if the initial inoculum density is
low. Therefore, all agronomic measures reducing
disease pressure will also contribute to a reduced
fungicide resistance risk. Effective resistance management and successful disease control are supported by the
use of effective fungicide rates, alternation and mixtures
of fungicides, appropriate spray intervals and an early
onset of applications in the disease cycle.
QoI fungicides
QoI fungicides are inhibitors of mitochondrial respiration; they inhibit the electron transport at cytochrome b
(complex III) by binding to the Qo site, the ubiquinol
oxidizing pocket, which is located at the positive, outerside of mitochondrial membranes. In the Qo pocket, the
amino acid glutamic acid (Glu) at position 272 of the ef
protein loop is responsible for binding to an oxygen
moiety in the toxophore of the fungicide molecule (O–
H–N bridge; Gisi et al. 2002). The cytochrome b (cyt b)
gene is the molecular target for QoI fungicides; it is
located in the mitochondrial genome. Long before the
introduction of agricultural QoI fungicides, resistance
to QoI molecules (e.g. myxothiazol) was described as
being based on several mutations in the cyt b gene in a
range of genera such as yeast (Saccharomyces),
bacteria (Rhodobacter), protozoa (Paramecium), sea
urchin, algae (Chlamydomonas) and mice (Di Rago
et al. 1989; Geier et al. 1992; Degli-Esposti et al. 1993;
Brasseur et al. 1996). However, it was not known
which mutation would appear in plant pathogens.
Eur J Plant Pathol (2008) 122:157–167
Frequency ( % ) of
isolates in population
a
100
2000 (N = 81)
80
2001 (N = 33)
2002 (N = 728)
60
2003 (N = 90)
40
2004 (N = 247)
2005 (N = 178)
20
2006 (N = 40)
0
<0.1
0.1-1
1-10
10-100
Frequency of A143 allele
(resistance) in bulk sample
b
Frequency ( % ) of
isolates in population
Sierotzki et al. (2000a, b) detected the G143A
substitution (exchange of glycine by alanine at
position 143) for the first time in QoI-resistant isolates
of Blumeria (Erysiphe) graminis f.sp. tritici and
Mycosphaerella fijiensis. This substitution is based
on a single nucleotide polymorphism in the triplet at
position 143 from GGT to GCT in the cyt b gene. It
was described in the following years in resistant
isolates of many important plant pathogen species
such as P. viticola, P. cubensis, Venturia inaequalis
and Mycosphaerella graminicola (Heaney et al. 2000;
Steinfeld et al. 2002; Gisi et al. 2002). It is associated
with high levels of resistance (high resistance factors
RF) or ‘complete’ resistance which leads to a
complete loss of disease control if QoIs are used as
solo products. A second mutation, F129L (exchange
of phenylalanine by leucine at position 129) was
discovered in resistant isolates of a few pathogen
species such as P. viticola (Sierotzki et al. 2005),
Pythium aphanidermatum (Gisi et al. 2002) and
Pyrenophora teres (Sierotzki et al. 2007), resulting
in a ‘partial’, less pronounced resistance leading to
reduced disease control. However, in P. infestans,
Bremia lactucae, Peronospora spp. and in all rust
genera (e.g. Puccinia, Uromyces, Phakopsora, Hemileia), no resistant isolates (and no mutations) were
detected until now. For rusts, the lack of resistance
(based on G143A) has been elucidated recently: an
intron is present in cyt b between positions 143 and
144 which has to be spliced for correct transcription
and translation. The splice site recognition is based on
a GGT triplet (De La Salle et al. 1982). If mutated
from GGT to GCT, splicing will not occur resulting in
a non-functional cytochrome b which is lethal (Grasso
et al. 2006).
QoI resistance in P. viticola populations in Europe
was first detected in 2000 (Heaney et al. 2000) and
evolved quickly with a rapid increase of resistant
isolates reaching a frequency in 2003 of 70% to 80%
in France and about 30% in northwest Spain (Galicia;
Fig. 1a,b). In the following years, the frequency of
resistance remained more or less stable in the two
countries (except for France with a further increase to
about 90% in 2006). In the north of Italy and in
Switzerland, frequencies reached high levels in
certain areas, whereas in Portugal, Germany and
Austria, they are still low (Sierotzki et al. 2008).
Since the collected leaves always represented a bulk
population, the measured frequency of resistance
159
100
80
2001 (N = 7)
2002 (N = 37)
60
2003 (N = 36)
40
2004 (N = 40)
2005 (N = 31)
20
2006 (N = 32)
0
<0.1
0.1-1
1-10
10-100
Frequency of A143 allele
(resistance) in bulk isolates
Fig. 1 Frequency of bulk isolates carrying the A143 allele
(resistance to QoIs) in Plasmopara viticola populations
collected in 2000 to 2006 in France (a) and in Spain (b; after
Sierotzki et al. 2008)
(A143 allele in Q-PCR tests) is representative for
the entire population at a specific vineyard. However,
if single sporangiophore isolates are picked from
these samples, they are always either completely
sensitive (100% G143 allele) or completely resistant
(100% A143 allele); heteroplasmic stages were never
detected.
At a specific trial site in Brazil (Holambra), QoI
treatments were carried out during several years until
November 2000, when they were stopped for three years
(2001–2003), re-started again in 2004, stopped in
2005 and started again in 2006. A decline of
resistance was observed when QoI applications were
stopped and a rapid increase when QoIs were used
again (Fig. 2a,b; Sierotzki et al. 2008). This fluctuation
of resistance resulting from the use strategy of QoIs
might be based on a reduced fitness of QoI-resistant
isolates, as it was described also by Heaney et al.
(2000) and Genet et al. (2006). Similar declines of
160
Eur J Plant Pathol (2008) 122:157–167
a
QoI used
QoI used
no QoI
no QoI
80
60
40
b
0
0
Nov 05
0
Mar 05
0
Mar 04
Nov 01
Mar 01
Nov 00
0
Nov 03
20
Nov 02
A143 in population
Frequency (%) of
100
A143 in population
Frequency (%) of
100
80
60
40
AZ solo
20
AZ + Mancozeb
0
12.12. 18.12. 25.12. 02.01. 08.01. 15.01. 23.01.
Date in 2006/07
six applications (one per week)
Fig. 2 Change in frequency of QoI-resistant isolates (carrying
the A143 allele) in Plasmopara viticola populations as a result
of different QoI usages at a trial site location (Holambra) in
Brazil. a season-long applications (six treatments),stop and restart of azoxystrobin applications between 2000 and 2005; b six
applications (one per week) of azoxystrobin (AZ) solo or in
mixture with mancozeb in the 2006/2007 season (after Sierotzki
et al. 2008)
resistance after QoI treatments were stopped have also
been observed in oospore populations of P. viticola in
some vineyards in Italy (Toffolatti et al. 2006) and in
P. cubensis populations in glasshouses in Japan (Ishii
2003 personal communication).
In order to investigate the segregation pattern of
QoI resistance, a sensitive P2 and a resistant P1
mating type single sporangiophore isolate (Scherer
and Gisi 2006) were crossed by co-inoculating a 1:1
sporangial mixture onto grape leaves. After 14 days
of incubation, plenty of oospores were produced in
the leaves which were further incubated in dry
conditions in the dark for another 8 weeks. Then,
the rotted leaves with oospores were ground to
powder, mixed with perlite and moistened with water
for inducing oospore germination (Gisi et al. 2007b)
as originally described for P. infestans (Rubin and
Cohen 2006). Young grape leaves were incubated on
top of the oospore/perlite mixture for 1 to 3 weeks
until first sporangiophores appeared which were
picked and propagated for producing F1 progeny
isolates. Based on the mitochondrial origin of QoI
resistance, a maternal inheritance of resistance (0: 1 or
1: 0, depending whether resistance is in the male or
female parent) was expected. Surprisingly, a segregation of resistance r:s=8:23 (or ~1:3) was observed; all
resistant offspring carried the A143, the sensitive
offspring the G143 allele (Fig. 3; Blum and Gisi
2008). Possible reasons for the unexpected segregation might be mitochondrial leakage, irregularities in
the mating process or involvement of a recessive
nuclear gene with epistatic regulation of the mitochondrial gene. The first possibility can be ruled out
because no heteroplasmic stages were detected with
Q-PCR; the second hypothesis is based on ‘femaleness’ of isolates (both parents may have the potential
for oospore production) and was described for
P. infestans (Judelson 1997). Thus, many basic
features of the biology of P. viticola are still not well
understood. Indeed, if inheritance of QoI resistance
does not follow a 0:1 (in a single cross) or 1:1 pattern
(in populations, assuming P1: P2 ratio is about 1:1),
evolution of resistance is not easy to predict, not even
under ‘controlled’ conditions. The G143A mutation
was obviously very rare in unselected populations of
P. viticola prior to the use of QoI fungicides, but was
quickly selected through the continuous use of these
fungicides. It is an open question as to whether the
G143A mutation (A143 allele) might be lost when
QoI applications are stopped, as quickly as it
appeared through selection. In addition, P. viticola
is a pathogen with a high rate of sexual recombination and high genetic diversity resulting in many
different genotypes every season (Scherer and Gisi
2006) but with a low migration rate resulting in local
epidemics. Thus, resistance evolution might be
driven mainly by ‘local’ processes (micro-climate,
fungicide use strategies, disease pressure on different
varieties).
161
100
sensitive F0 (F10.17, P2):: EC 50 = 0.85 mg l-1
resistant F0 (F02.3, P1): EC 50 > 100 mg l-1
10
1
35
r F0 (P1)
20
29
34
11
12
5
10
4
8
27
30
19
3
18
s F0 (P2)
1
21
2
7
9
15
32
25
14
13
17
39
33
31
36
0.1
16
Fig. 3 Sensitivity to
azoxystrobin (AZ; EC
50 mg l−1) of F1 progeny
isolates derived from a cross
between an AZ- sensitive
and AZ-resistant parent
(F0, black columns) in
Plasmopara viticola (after
Blum and Gisi 2008)
Sensitivity, EC50 (mg AZ l-1)
Eur J Plant Pathol (2008) 122:157–167
F1 progeny (n = 31)
Phenylamide fungicides
Phenylamide fungicides such as mefenoxam (metalaxylM), metalaxyl and benalaxyl inhibit ribosomal RNA
synthesis, specifically RNA polymerization (polymerase complex I; Davidse 1995). The molecular target
gene is unknown and no sequence data and no
mutations are available, although phenylamides have
been in use for more than 25 years. Resistance to
phenylamides developed rather quickly after their
introduction in many pathogen species of the oomycetes such as P. viticola, P. cubensis, Peronospora
tabacina, B. lactucae and P. infestans (Table 1).
Although resistance is widely spread nowadays, the
frequency in populations rarely reaches 100%; it
fluctuates not only from year to year but also within
the season. In French vineyards, resistance in
P. viticola populations in 1987 to 1998 varied from
15 to 75% (Gisi 2002). In 2004, the proportion of
sensitive, intermediate and resistant isolates was 35%,
45%, and 20%, respectively (samples from France;
Gisi et al. 2007b) and 15%, 40% and 45%, respectively,
in 2006 (samples from France, Italy, Spain, Germany;
Fig. 4a). Resistance increased during the season
(Fig. 4a) and was higher in mefenoxam-treated than
untreated fields (Fig. 4b). The decrease of sensitive and
the increase of intermediate and resistant isolates
during the season was observed earlier over a period
of four consecutive years (Fig. 4c) and seems to follow
a seasonal pattern every year. Similar observations
were also made for P. infestans populations (Gisi
and Cohen 1996). Whether sexual recombination,
Table 1 Published cases of resistance to phenylamides in field populations of important pathogens in Peronosporales (first
publication, in chronological order)
Pathogen species
Host plant
References
Pseudoperonospora cubensis
Phytophthora infestans
Plasmopara viticola
Peronospora tabacina
Phytophthora cinnamomi
Phytophthora parasitica
Bremia lactucae
Pythium spp.
Plasmopara halstedii
Phytophthora erythroseptica
Peronospora viciae
Pythium spp.
Peronospora destructor
cucurbits
potato
grape
tobacco
avocado
tobacco
lettuce
turf, carrot
sunflower
potato
pea
ornamentals
onion
Reuveni et al. 1980
Davidse et al. 1981
Staub and Sozzi 1981
Bruck et al. 1982
Darvas and Becker 1984
Shew 1985
Crute 1987
Sanders and Soika 1988; White et al. 1988
Albourie et al. 1998
Lambert and Salas 1994
Falloon et al. 2000
Moorman and Kim 2004
Wright 2004
162
b 100
100
90
80
EC50, mg MFX l-1
Frequency (%) of isolates
in population
a
70
60
50
40
30
20
c
10
1
resistant
intermediate
0.1
0.01
sensitive
10
0
0.001
early
resistant
Frequency (%) of isolates
in population
Fig. 4 Sensitivity to
mefenoxam (sensitive,
intermediate, resistant;
proportion of isolates in (a)
and (c), EC 50 in (b)) of
Plasmopara viticola isolates
collected in France, Italy,
Spain and Germany in 2006
(N=49) early, mid and late
in the season (a) and from
mefenoxam-untreated or
treated fields (b), and in
France (Armagnac area) in
four consecutive years
(1997 to 2000, represented
as the average of the
four years) early (N=132),
mid (N=85) and late (N=
110) in the season
Eur J Plant Pathol (2008) 122:157–167
mi d
l ate
intermediate
sensitive
MFX untreated
MFX treated
N = 32
N = 17
Number of tested isolates
100
90
80
70
60
50
40
30
20
10
0
early
resistant
mi d
l ate
intermediate
pathogenic fitness and/or over-seasoning capacity of
resistant isolates play a major role for these dynamics
is still to be elucidated.
Inheritance of phenylamide resistance in the F1
progeny was studied in crosses of P. viticola (Fig. 5a)
and P. infestans (Fig. 5b) by co-inoculation of
mefenoxam-sensitive and resistant parents according
to the method described for QoI fungicides (Fig. 3).
The segregation pattern of phenylamide resistance in
P. infestans corresponded to the expected Mendelian
mechanisms for inheritance based on one semidominant gene: all F1 progeny isolates were intermediates (Fig. 5b, Knapova et al. 2002). However, in
P. viticola the segregation pattern was somewhat
unexpected (Fig. 5a) with a proportion of r:i:s=
21:8:2 (Blum and Gisi 2008). In this experiment, the
sensitive parent may be considered as ‘truly sensitive’
or as intermediate (depending on categorization of
EC50 value in the bioassay). The segregation of
resistance in the F1, based on one semi-dominant
gene in the resistant parent should result either in an
entirely intermediate progeny (if the other parent is
considered as sensitive) or in r:i=1:1 (if the other
sensitive
parent is considered as intermediate). The observed
segregation pattern does not fit to either of the
expected segregation. If resistance is based on one
dominant gene, the expected segregation would differ
even more from the observed pattern. Since for
P. viticola, no other data on resistance segregation in
the F1 are available in the literature, it remains unclear
whether the genetic background of mefenoxam
resistance is different in P. viticola compared to
P. infestans, or whether sexual recombination and
oospore production follows some yet unknown
modifications in P. viticola as was speculated to occur
for the inheritance of QoI resistance (see above).
Similarly, a distorted segregation pattern was also
described in P. infestans when oospores were produced
in planta as compared to in agar cultures (Van der Lee
et al. 2004; Rubin and Cohen 2006). In addition, the
segregation pattern of phenylamide resistance in
P. viticola was described for F2 progeny by Gisi et al.
(2007b): a proportion of s:i:r=1:2.7:2 was observed
which was considered to be based on one semidominant gene affected by minor genes as described
for P. infestans (Judelson and Roberts 1999). In
Eur J Plant Pathol (2008) 122:157–167
a
100
sensitiveF0
F0(F10.17,
(F10/17a,P2):
P2):EC
EC 50
50 = 0.15
sensitive
0.15 mg/L
mg/L
mg
l-1
resistantF0
F0(F02.3,
(F02.3, P1):
P1): EC
EC 50
50 >
> 100
mg
resistant
100 mg
m l-1
10
1
0.1
0.01
F1 progeny (n = 31)
32
r F0 (P1)
19
32
14
30
27
18
17
8
16
3
1
7
5
13
11
10
33
35
12
29
31
15
20
2
21
25
39
36
4
s F0(P2)
0.001
9
Sensitivity, EC 50 (mg MFX l-1)
Fig. 5 Sensitivity to
mefenoxam (MFX; EC
50 mg l−1) of F1 progeny
isolates derived from a cross
between an MFX-sensitive
and MFX-resistant parent
(F0, black columns) of a
Plasmopara viticola (after
Blum and Gisi 2008) and of
b Phytophthora infestans
(after Knapova et al. 2002)
163
100
sensitive parent: EC 50 = 0.0015 mg/L
mg l-1
resistant parent: EC 50 = 85 mg
mg/L
l-1
open bars are selfs
10
1
0.1
0.01
summary, the risk of resistance for phenylamide
fungicides is considered as high, the evolution as fast
with a certain stabilization effect over time, and a reappearance of sensitivity in unselected populations
after sexual recombination.
CAA fungicides
The biochemical mode of action of CAA fungicides
(including dimethomorph, flumorph, iprovalicarb,
benthiavalicarb, mandipropamid) is still speculative;
potential targets are phospholipid biosynthesis (Griffiths
et al. 2003) and cell wall deposition (Jende et al. 2002;
Cohen and Gisi 2007; Gisi et al. 2007a). Although
CAAs may interfere with cell membranes, it is
doubtful whether the observed effects on phosphocholinetransferase, the last step in the Kennedy pathway of
lecithin biosynthesis (Griffiths et al. 2003) can be
considered as primary effects caused by CAA fungicides. Similarly, the observed changes in cell wall
architecture and deposition during germination of
cystospores (Jende et al. 2002) may be a secondary
effect, because some of the key enzymes of cell wall
F1 progeny (n=35)
44
7
r parent
5
3
4
1
15
13
50
11
17
18
53
27
10
16
46
26
47
56
6
14
30
8
24
23
19
22
25
43
41
45
21
54
28
0.001
s parent
Sensitivity, EC 50 (mg MFX l-1)
b
biosynthesis such as glucanases and synthases of β-1,3
glucans and cellulose may not be inhibited directly
(Mehl and Buchenauer 2002; Gisi et al. 2007a). Most
likely, the target site for CAA fungicides may be
membrane-bound at the interface between plasmalemma
and cell wall (Syngenta internal data). So far, the target
gene(s) have not been identified and no mutations
conferring resistance are known, although CAAresistant field isolates of P. viticola are available.
In spite of an intensive monitoring programme, no
resistant isolates have been detected in P. infestans
populations (Cohen et al. 2007; FRAC CAA working
group reports, www.frac.info), although CAA fungicides (dimethomorph) have been used commercially
for more than 10 years. Also, enforced selection
experiments and mutagenesis did not yield isolates in
P. infestans with stable resistance to CAAs (Bagirova
et al. 2001; Stein and Kirk 2004; Yuan et al. 2006;
Cohen et al. 2007; Rubin et al. 2008). Therefore,
resistance risk for CAA fungicides in P. infestans can
be considered as low. The entire genus Pythium is
insensitive to CAA fungicides; therefore, there are no
resistance issues for CAAs in this genus. However,
CAA-resistant isolates have been detected in P.
164
Eur J Plant Pathol (2008) 122:157–167
applications were stopped. At locations with low
proportions of CAA resistance at the beginning of the
season, six applications (full recommended rates)
caused a more rapid increase of resistance when the
product was used solo as compared to when used in
mixture (with Folpet). Interestingly, the proportion of
resistance in the populations at the Stein and Estillac
sites was lower at the beginning of the season than at
the end of the previous season (Fig. 6). These
observations suggest that CAA-resistant isolates of
P. viticola may be less fit in the absence of selection
pressure than sensitive isolates. The decline of
resistance in the absence of CAA treatments is an
additional element supporting the classification of
resistance to be moderate for CAAs in P. viticola (as
compared to high for QoI and phenylamide fungicides;
www.frac.info).
viticola populations for several years in some regions
of France and Germany (Gisi et al. 2007b), but no
serious product failures were reported. Mean resistance factors are often >300, and resistant isolates are
stable when transferred onto untreated grape leaves
(Gisi et al. 2007b). Also, in P. cubensis, resistant
isolates have recently been detected in a few trial site
locations, one each in South Korea, Israel and USA
(FRAC CAA working group reports, www.frac.info)
and in China (Zhu et al. 2007). When a CAAsensitive and CAA-resistant single sporangiophore
isolate of P. viticola were crossed (method see above),
resistance segregated in the F1 in a 0:1 (entire F1
progeny sensitive) and in the F2 in a 1:9 pattern,
suggesting that two recessive nuclear genes are
involved in CAA resistance (Gisi et al. 2007b). Based
on this segregation pattern, resistance risk for
CAA fungicides in P. viticola was estimated to be
moderate.
The evolution of resistance to CAA fungicides in
P. viticola populations was followed over several
years at four different trial sites (Les Barges and Stein
in Switzerland, Marsillargues and Estillac in France)
in response to different spray programmes (treatments
stopped or applications as solo product or in mixture
with multi-site fungicides; Fig. 6). At locations with
fully resistant initial populations (resulting from
season-long selection with recommended rates during
several years), resistance clearly declined (in some
cases to zero) within 2 years after mandipropamid
The multi-site inhibitors such as copper formulations,
dithiocarbamates like mancozeb, phthalimides like
folpet and chloroisophthalonitriles like chlorothalonil
are non-systemic, preventive fungicides forming a
protectant barrier at the surface of the plant against
pathogens, and inhibit pathogen development prior to
penetration into the tissue. They interact mostly
unspecifically with many biochemical steps in the
pathogen metabolism, such as the formation of
Marsillargues
no treatment
solo
80
60
40
20
0
early
late
2003
late
2004
late
2005
% resistant isolates
100
100
% resistant isolates
% resistant isolates
Les Barges
100
mixture
100
solo
60
40
20
0
early
2003
mixture
60
40
20
early late
2003
early late
2004
late
2004
late
2005
Estillac
80
0
no treatment
80
Stein
% resistant isolates
Fig. 6 Evolution of
resistance to CAA
fungicides at four trial sites
during the season and over
several years as a result
of season-long application
of mandipropamid (six to
eight applications) as a solo
product or in mixtures with
multi-site fungicides and
after treatments were
stopped
Other fungicide classes against oomycetes
early late
2005
mixture
solo
80
60
40
20
0
early
late
2003
early
late
2004
early
late
2005
Eur J Plant Pathol (2008) 122:157–167
complexes with enzymes possessing sulphydrylgroups (Gisi 2002). As a consequence, the enzymes
are inactivated leading to a general disruption of
metabolism and cell integrity. Based on the multi-site
mode of action, resistance to such inhibitors has never
developed and is unlikely to evolve. Multi-site fungicides are important elements in spray programmes
(about 50% of the total oomycete fungicide market)
either as stand alone products or in mixtures with
single-site fungicides to improve their activity and to
delay resistance evolution.
Based on its short persistence, the systemic cyanoacetamide oxime fungicide cymoxanil is used against
oomycetes always in mixtures with multi-site fungicides. The biochemical mode of action of cymoxanil
is unknown. Reduced sensitive (or resistant) isolates
have been reported in field populations of P. viticola
(but not of P. infestans) in several vineyards of Italy
and France (Gullino et al. 1997; Genet and Vincent
1999). Depending on the proportion of resistance in
populations, the curative activity of cymoxanil can be
significantly reduced. The dinitroaniline fungicide
fluazinam is an uncoupler of phosphorylation from
electron transport by disrupting the proton gradient;
as a consequence, ATP production is blocked (Gisi
2002). Resistance in field populations of oomycetes
has never been reported. The carbamate fungicide
propamocarb is reported to affect the permeability of
cell membranes; as a consequence, leakage of cell
components has been observed, but the precise
biochemical mode of action is not well understood.
Field isolates resistant to propamocarb have been
detected in Pythium species (Moorman and Kim
2004). Within the chemical class of phosphonates,
fosetyl-Al and its breakdown product phosphorous
acid (H3PO3) are readily taken up by plant tissue and
translocated systemically within the phloem (symplastically). Fosetyl-Al may have an indirect effect
against downy mildews by stimulating the plant
defence reactions; but also a direct antifungal activity
has been reported (change in phosphorylated sugar
content and cell wall composition; Gisi 2002).
Nevertheless, the primary site of action is not known,
and resistant isolates in field populations have never
been detected. For ‘true’ plant defence inducers such
as acibenzolar-S-methyl (Bion), it is generally assumed that pathogens cannot develop resistance very
easily, because several mechanisms would have to be
overcome (Gisi 2002).
165
Conclusions
Resistance evolution depends on the specific action of
the fungicide (biochemical and molecular mechanisms), the agronomic usage of the fungicide and
the pathogen biology. These parameters have been
used by FRAC for resistance risk assessment for
all chemical classes (‘FRAC grid’ in Monograph
1, FRAC classification lists, www.frac.info). In
P. viticola, fungicide resistance has emerged quite
quickly (within a few years) after product introduction
for almost every chemical class of single-site fungicides (phenylamides, cymoxanil, QoIs, CAAs),
whereas in P. infestans it emerged only for phenylamides (Kuck and Russell 2006). Therefore, resistance
risk assessment has to be carried out carefully and for
each single pair of fungicide and pathogen species. In
order to delay resistance evolution and ensure robust
disease control for as long as possible, all available
chemical classes of fungicides such as QoIs, phenylamides, CAAs, cyanoacetamide-oximes, phosphonates
and multi-site fungicides should be integrated in
a spray programme, either in sequence and/or in
mixtures. Fungicide applications should start before
or at the onset of the epidemics; the recommended rates
and spray intervals have to be strictly followed and
adapted to the local disease and weather conditions.
Acknowledgements We acknowledge the excellent experimental contributions of Maya Waldner, Regula Frey, Dominique Edel,
Noemy Kraus and Mathias Blum.
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Eur J Plant Pathol (2008) 122:169–183
DOI 10.1007/s10658-008-9327-9
Activity of carboxylic acid amide (CAA) fungicides against
Bremia lactucae
Yigal Cohen & Avia (Evgenia) Rubin & Dror Gotlieb
Received: 31 August 2007 / Accepted: 5 May 2008
# KNPV 2008
Abstract Four carboxylic acid amide (CAA) fungicides, mandipropamid (MPD), dimethomorph (DMM),
benthiavalicarb (BENT) and iprovalicarb (IPRO) were
examined for their effects on various developmental
stages of Bremia lactucae, the causal agent of downy
mildew in lettuce, in vitro and in planta. Spore
germination in vitro or on leaf surfaces was inhibited
by all CAA fungicides (technical or formulated). MPD
was more effective in suppressing germination than
DMM or BENT, whereas IPRO was least effective.
CAA induced no disruption of F-actin microfilament
organisation in germinating spores of B. lactucae. CAA
applied to germinating spores in vitro prevented further
extension of the germ tubes. When applied to germinated spores on the leaf surface they prevented
penetration. Preventive application of CAA to intact
plants inhibited infection. MPD was more effective in
suppressing infection than DMM or BENT, whereas
IPRO was least effective. Curative application was
effective at ≤3 h post-inoculation (hpi) but not at
≥18 hpi. CAA (except IPRO) applied to upper leaf
surfaces inhibited spore germination on the lower
surface and hence reduced infection. CAA suppressed
sporulation of B. lactucae on floating leaf discs and
when sprayed onto infected plants two days before onset
: D. &Gotlieb
Rubin
Y. Cohen
Cohen (*)
(*): &A.A.(.(E.)
Rubin
D. Gotlieb
The Mina and Everard Goodman Faculty of Life Sciences,
Bar-Ilan University,
Ramat Gan 52900, Israel
e-mail: ycohen@mail.biu.ac.il
of sporulation. BENT and DMM were more effective in
suppressing sporulation than MPD or IPRO. Epidemics
of downy mildew in shade-house grown lettuce were
suppressed by CAA. A single spray applied to five-leaf
plants before transplanting controlled the disease for
50 days. The results suggest that CAA are effective
inhibitors of spore germination and therefore should be
used as preventive agents against downy mildew of
lettuce caused by B. lactucae.
Keywords Benthiavalicarb . Cinnamic acid amides .
Dimethmorph . Disease control . Downy mildew .
Iprovalicarb . Lettuce . Mandelic acid amides .
Mandipropamid . Oomycete . Valinamid carbamates
Introduction
Mandipropamid (MPD) is a new mandelic acid amide
fungicide (Lamberth et al. 2006) which together with
dimethomorph (DMM) and flumorph (cinnamic acid
amides), iprovalicarb (IPRO) and benthiavalicarb
(BENT; valinamid carbamates), belongs to the carboxyl acid amide (CAA) fungicides (Anon. 2006).
CAA fungicides are effective against oomycete foliar
plant pathogens.
Field studies indicate that MPD is highly effective
against late blight in potato and tomato, downy mildew
in grapes and several downy mildews in vegetable
crops (Harp et al. 2006; Huggenberger et al. 2005;
170
Huggenberger and Kuhn 2006). A recent report (Harp
et al. 2007) has shown effective control of downy
mildew in lettuce with Revus (a.i. MPD). The
fungicide was shown (Hermann et al. 2005) to quickly
bind to the wax layer of the leaf surface thus providing
strong rain-fastness and long-lasting efficacy. A small
amount of MPD is taken up by the leaf tissue,
providing curative and translaminar activities against
disease. All four CAA fungicides belong to one crossresistance group, as field isolates of Plasmopara
viticola exhibit resistance to all (Gisi et al. 2006).
A recent study (Cohen and Gisi 2007) provided a
comprehensive analysis of the effects of three CAA
fungicides, MPD, DMM and IPRO, on all stages in
the asexual life-cycle of Phytophthora infestans. The
most sensitive stage to CAA was shown to be
germination of cystospores and sporangia (direct
germination) and the most active CAA was MPD.
Nano-molar doses of MPD were sufficient to block
spore germination in vitro and in vivo. Mycelium
growth and sporulation were less sensitive to CAA.
Of the three CAA fungicides tested, MPD was most
effective in suppressing late blight epidemics in
shade-house grown potatoes (Cohen and Gisi 2007).
Benthiavalicarb-isopropyl, another CAA fungicide,
was not studied in the previous research (Cohen and
Gisi 2007). It was reported (Miyake et al. 2003) to be
strongly inhibitory to germination of sporangia and
cystospores, mycelial growth and sporulation of
various oomycetes. The compound has not only strong
preventive activity, but also curative and penetrant
activity, with excellent residual effects and rainfastness.
Several attempts were made to reveal the mode of
action of CAA. Morphological studies (Albert et al.
1988, 1991; Bagirova et al. 2001; Cohen et al. 1995;
Cohen and Gisi 1996; Dereviagina et al. 1999;
Hermann et al. 2005; Huggenberger et al. 2005; Jende
et al. 1999, 2001; Matheron and Porchas 2000; Miyake
et al. 2003; Reuveni 2003; Stenzel et al. 1998)
indicated that DMM, IPRO and BENT, as well as the
experimental CAA XR-539 (Young et al. 2005),
inhibit cell wall deposition/assembly in cystospores of
P. infestans. Biochemical studies (Griffiths et al. 2003)
with the mandelamide SX 623509 in mycelium of P.
infestans suggested alterations in phospholipid biosynthesis, with an inhibition of phosphatidylcholine
(lecithin) biosynthesis as a main target. Unpublished
data (Cohen and Gisi) indeed indicate that lecithin
(phosphatidylcholine) compromised the inhibitory ac-
Eur J Plant Pathol (2008) 122:169–183
tivity of CAA on cystospore germination. Recently,
Zhu et al. (2007) compared a flumorph-sensitive and a
flumorph-resistant Phytophthora melonis and suggested that the primary site of action of flumorph is
the disruption of F-actin organisation.
The aim of the present study was to examine the
effects of CAA on the development of Bremia
lactucae, the causal agent of downy mildew in lettuce,
in vitro and in planta. Here, four CAA fungicides
were tested, including benthiavalicarb (BENT) which
was not tested with P. infestans.
Materials and methods
Fungicides
Four CAA (carboxylic acid amide) fungicides were
used: mandipropamid (MPD; Syngenta, mw=412),
dimethomorph (DMM; BASF, mw=266), iprovalicarb (IPRO; Bayer, mw=320) and benthiavalicarb
(BENT; Kumiai Chemicals, mw=339). Technical
grade fungicides were dissolved in DMSO (10 mg
ml−1) and diluted in double distilled water (DDW) to
the desired concentrations. Formulated fungicides
used were: mandipropamid 250SC; dimethomorph
(Forum) 50WP and iprovalicarb 50WG. Benthiavalicarb (10SC) in Agsolex-8 (N-octylpyrrolidone) was a
gift from Makhteshim, Beer Sheba, Israel. All concentrations are represented in units of active ingredient (a.i.).
Pathogen
All studies, unless stated otherwise used isolate ISR60 of B. lactucae Regel (Sharaf et al. 2007) carrying
13 virulence factors (0, 1, 2, 3, 4, 5/8, 6, 7, 10, 11, 13,
15, 16, 17) obtained from A. Beharav, The Institute of
Evolution, Haifa University, Israel. Some studies used
isolates BL-18, BL-21, BL-24 and BL-25 (a gift from
A. Lebeda, Olomouc University, Czech Republic).
Isolates were maintained by repeated inoculation of
lettuce cotyledons in Petri dishes in a growth chamber
(15°C, 12 h light/day).
Plants
The susceptible lettuce (Lactuca sativa) cv. Noga (cup
type; Hazera Genetics, Mivhor, Israel) was used. For
Eur J Plant Pathol (2008) 122:169–183
growth chamber studies, plants were grown from
seeds in 175 ml pots containing 40 g peat/vermiculite
mixture (1/1, v/v) to give 20 plants per pot. Plants
grown in the greenhouse (18–32°C) were used 1 week
after sowing, when two cotyledon leaves had developed. In other experiments, plants were grown either
in Speedling (Hishtil, Petah-Tiqwa, Israel) trays
(25 ml cells), one plant per cell and used when they
had five to six true leaves, or in 0.5-l pots and used at
the five to six true leaf stage.
Application of compounds
Compounds were diluted in water to a series of
concentrations and applied to lettuce plants by spraying
onto the upper leaf surfaces to initial run-off. Depending
on the experiment, a compound was applied either
before or after inoculation. In other experiments,
compounds were applied as 10 μl droplets to detached
cotyledon leaves, true leaves, or leaf discs. In the field,
compounds were applied by spraying to initial run-off
with the aid of a backpack sprayer.
171
recorded per depression with the aid of a dissecting
microscope at ×160.
F-Actin distribution
Spores were mixed (1:1) with water (as control),
0.01 mg l−1 MPD or 0.1 mg l−1 DMM and applied to
depressions in glass slides (20 μl per depression, n=9).
Slides were kept in moist Petri dishes at 13°C for 18 h
in the dark to allow for spore germination. Spores were
then fixed for 10 min with formaldehyde (3.7%, 10 μl
per depression), collected from the depressions,
washed twice, and treated for 10 min with Alexa
Fluor® 488 phalloidin (Invitrogen, Molecular Probes,
Eugene, OR, USA). F-Actin distribution was visualised with a confocal microscope (Karl Zeiss, LSM-510
META), and images captured with a Zeiss AxioCam
camera and analysed using Zeiss AxioVision software.
Germination of spores on leaves
Spores of B. lactucae were collected from freshly
sporulating lettuce leaves into ice-cold DDW, their
density adjusted to 1×104 spores per milliliter and then
sprayed onto the upper leaf surfaces of the test plants
to initial run-off with the aid of a glass atomiser. Plants
were thereafter placed in a dew chamber (18°C, in the
dark) for 20 h and then transferred to a growth
chamber at 20°C (12 h light/day, 100 μE m−2 s−1).
At 5 days post-inoculation (dpi) plants were placed in
Perspex boxes (100% relative humidity) for two days
to induce sporulation of the pathogen.
Tests were performed with either cotyledon leaves or the
first formed true leaf detached from 7- or 10 day-old
plants, respectively. Cotyledons or leaves (n=4) were
placed on moistened filter paper in 9 cm Petri dishes
and each inoculated on the upper surface with a 20 μl
droplet of spore suspension (containing 500 spores)
mixed 1:1 with CAA. Dishes were incubated at 13–15°
C for 20 h in the dark. A 10 μl droplet of 0.02%
calcofluor (Polyscience Inc., Warrington, PA, USA)
was added to each cotyledon and leaf, and germination
was visualised with the aid of an UV epifluorescence
microscope (Olympus AX70) equipped with an excitation filter of 390–420 nm and an emission filter of
425–450 nm. Bremia lactucae structures fluoresced
blue; 100 spores were recorded per sample.
Disease assessment
Temporary exposure of spores to CAA
The number of sporulating plants was determined at
the cotyledon stage with the aid of a magnifying lens
(×10) at 7 dpi, unless stated otherwise.
The effect of temporary exposure of spores to CAA
was assessed by incubating spores in 0, 0.5 or 1 mg l−1
CAA on ice for 1 h, then washing with water and
allowing them to either germinate for 6 h at 13°C in
the dark in vitro or to infect lettuce leaves.
Inoculation
Germination of spores in vitro
Spores were mixed (1:1) with CAA and applied to
depressions in glass slides (20 μl per depression,
n=3). Slides were kept in moist Petri dishes at 13°C
for 20 h in the dark. Germination of 100 spores was
Pathogen development in planta
To detect pathogen structures inside the tissue,
leaves were cleared by boiling in 90% ethanol for
172
MPD
DMM
BENT
IPRO
60
40
20
0
0 0.0005 0.005 0.05
CAA, mg l
c
30
0.5
5
% Germination
MPD
DMM
BENT
IPRO
15
10
5
0
0 0.0005 0.005 0.05 0.5
100
50
0
5
200
MPD
DMM
BENT
IPRO
150
100
50
0
0 0.0005 0.005 0.05
f
MPD
DMM
BENT
IPRO
40
20
0.5
5
-1
CAA, mg l
60
BL-24
350
300
250
MPD
DMM
BENT
IPRO
200
150
100
50
0
g
100
0.5
-1
CA A, m g l
h
BL-25
350
80
MPD
DMM
BENT
IPRO
60
40
Y
20
100
BL-25
250
MPD
DMM
BENT
IPRO
200
150
100
50
0
5
0 0.0005 0.005 0.05
CAA, mg l
j
ISR-60
80
MPD
DMM
BENT
IPRO
60
40
20
Germ-tube length, µm
CA A, m g l
-1
0.5
5
-1
300
0
0 0.0005 0.005 0.05
0.5
0 0.0005 0.005 0.05
5
Germ-tube length, µm
CA A, m g l
% Germination
0.5
BL-21
250
5
BL-24
0 0.0005 0.005 0.05
i
0 0.0005 0.005 0.05
-1
0
% Germination
150
d
20
80
MPD
DMM
BENT
IPRO
200
-1
25
e
250
CAA, mg l
BL-21
CA A, m g l
BL-18
300
-1
Germ-tube length, µm
% Germination
80
Germ-tube length, µm
b
BL-18
100
Germ-tube length, µm
a
% Germination
Fig. 1 a–j Percentage spore
germination (means and
standard deviation of the
mean, n=100; germ-tube
length, >5 μm) and germtube length of four European (BL-18 to BL-25) and
one Israeli (ISR-60) isolates
of B. lactucae in vitro in the
presence of technical CAA
compounds (MPD mandipropamid, DMM dimethomorph, IPRO iprovalicarb,
BENT benthiavalicarb).
k Epifluorescence micrographs of calcofluor-stained
B. lactucae spore germination in vitro in the presence
of 0.0005 mg l−1 of four
technical CAA compounds.
Scale bar=100 μm. l Germination of B. lactucae spores
in the presence of water and
CAA: Only very small
germ-tubes are produced in
the latter (arrows). Scale
bar=20 μm
Eur J Plant Pathol (2008) 122:169–183
0.5
5
-1
ISR-60
200
160
MPD
DMM
BENT
IPRO
120
80
40
0
0
0 0.0005 0.005 0.05
CAA, mg l
0.5
-1
20 min, and then placed in 0.05% aniline blue in
70 mM potassium phosphate buffer (pH 8.9) at
4°C for 24 h. Stained leaves were placed on glass
slides, a drop of 0.02% calcofluor applied to the
5
0 0.0005 0.005 0.05
CAA, mg l
0.5
5
-1
surface and then examined by epifluorescence
microscopy, as above (Cohen et al. 1989, 1990).
Spores and germ tubes on the leaf surface fluoresced
blue; β-1–3 glucans in walls of intercellular hyphae
Eur J Plant Pathol (2008) 122:169–183
173
Fig. 1 Continued
and callose deposited around haustorial necks
fluoresced yellow.
Sporulation on leaves, plants and leaf discs
Leaves of plants with one true leaf were detached at
7 dpi and floated on technical CAA in 5 cm Petri
dishes at 18°C for 48 h (12 h light/day). Intensity of
sporulation was estimated with the aid of a magnifying lens (×10), and spore counts for leaves floating on
100 mg l−1 were made using a cytometer. Plants at the
cotyledon stage were sprayed with technical CAA at
4 dpi, the number of sporulating plants counted at
7 dpi and spore counts measured at 11 dpi. Leaf discs
(1 cm diam) from infected 10-leaf plants at 7 dpi were
floated on 1 ml of either water or formulated CAA at
20°C for 20 h in the dark in six-well titer plates, and
the number of sporophores per disc was determined
by fluorescence microscopy after staining with calcofluor. Similar discs were incubated at 18°C with 12 h
174
Eur J Plant Pathol (2008) 122:169–183
trays in the greenhouse, and transplanted when they
had five true leaves into polystyrene containers
(1.2× 0.6× 0.2 m) filled with peat and vermiculite
(1:1, v/v), to give 10 plants per container. Containers
were located in shade-houses in the field at Bar-Ilan
University Farm. Shade-houses were covered with
white plastic nets (50 holes per square inch, mesh)
to avoid aphid and viral infections. In 2006, plants
were sprayed twice with three concentrations of
formulated MPD, with the first spray at 4 weeks
after planting, when plants had reached the 10–12
leaf stage. The second spray was applied after a
further 8 days. In 2007, plants were sprayed once
with 500 mg l−1 of each of three formulated CAA
fungicides, before transplanting into the shadehouse. BENT was not tested outdoors because no
formulated solo product was available. In both
experiments plants were inoculated with a spore
suspension of B. lactucae (1 × 103 ml−1) in the
evening on the same day as the treatment. After
inoculation, plants were covered with plastic sheets
light per day for 2 days, and the number of spores
produced assessed as above.
Translaminar efficacy
Leaves formed fifth from the stem base were
detached from 10-leaf plants, sprayed on upper leaf
surface with 100 mg l−1 of four technical CAA
fungicides, and placed on wet filter paper in 20×20×
3 cm plates. After 3 h, leaves were inverted and
inoculated on their lower surface with spores of B.
lactucae, and spore germination determined at 20 hpi
as before. The number of sporulating lesions was
counted at 10 dpi.
Shade-house experiments
Two experiments were conducted during 2006–
2007 with whole plants of L. sativa cv. Noga to
evaluate the efficacy of CAA in controlling B.
lactucae. Plants were raised from seeds in Speedling
% Germination
100
80
IPRO
60
DMM
40
MPD
20
0
0
0.0005 0.005
0.05
0.5
Formulated CAA, mg l
5
-1
Germ-tube lenght, µ m
b
a
200
150
IPRO
DMM
100
MPD
50
0
0
0.0005 0.005
0.05
0.5
Formulated CAA, mg l
5
-1
c
Fig. 2 Percent spore germination (means and standard deviation of the mean, n=100) (a) and germ-tube length (b) of B.
lactucae isolate ISR-60 on leaf surfaces treated with three
formulated CAA compounds (see Fig. 1 for abbreviations).
c Epifluorescence micrographs of calcofluor-stained B. lactucae
spore germination on leaf surfaces treated with water (control)
and technical MPD. Scale bar=50 μm
Eur J Plant Pathol (2008) 122:169–183
175
Fig. 3 Laser scanning confocal micrographs of phalloidin-stained B. lactucae spore germination in vitro showing no effect of CAA
on F-actin organization in the germinating spores (arrow small germ-tube). Scale bar=20 μm
for the night to ensure infection. In the first experiments, CAA was applied with a hand sprayer, or a
manual backpack sprayer, at a rate of about 20–
30 ml per plant. Disease severity was recorded at 42
or 50 dpi by counting the number of downy mildew
lesions developing on 10 plants in a container.
Fig. 4 Epifluorescence micrographs of calcofuorstained germinating spores
of B. lactucae after adding
0.005 mg l-1 MPD 3 h after
start of germination in vitro.
a Germ-tube at 3 h after
start of germination (time of
treatment). Control (b) and
treated (c) germ-tubes at
48 h after start of germination. Scale bar=20 μm
Results
Spore germination
Four European and one Israeli isolate of B. lactucae
were tested for sensitivity to technical CAA fungi-
176
Eur J Plant Pathol (2008) 122:169–183
cides in vitro. CAA strongly suppressed spore
germination, but enabled spores to produce very
small germ-tubes (Fig. 1l). True germination was
considered to have occurred only when a spore
produced a germ-tube of ≥5 μm (spore diam
∼20 μm).
Figure 1a–f shows that the European isolates BL18, BL-21 and BL-24 were totally inhibited by
0.0005 mg l−1 (lowest concentration tested) of MPD,
DMM and BENT, and by 5 mg l−1 of IPRO.
Strangely, low concentrations of IPRO stimulated
germination of BL-24 (Fig. 1e). BL-25 (Fig. 1g, h)
was similarly sensitive to MPD and IPRO, but
required ×100 and ×1,000 more BENT and DMM,
respectively, to be suppressed fully. The Israeli isolate
ISR-60 (Fig. 1i, j) was more sensitive to IPRO, and
controlled fully with 0.5 mg l−1, compared with the
European isolates. It was highly sensitive to MPD and
BENT (totally inhibited with 0.0005 mg l−1) and
×100 less sensitive to DMM.
Figure 1k shows the in vitro germination of B.
lactucae spores in water and in four technical CAA
fungicides at 0.0005 mg l−1 (for numerical data see
Fig. 1a–j). At inhibitory concentrations, all fungicides
allowed the formation of very short germ-tubes (about
one fifth of the spore size, Fig. 1l) in about 20% of the
spore population.
Figure 2 shows the germination of B. lactucae
spores in the presence of three formulated CAA
fungicides (0.0005–5 mg l–1) on plant leaf surfaces.
At 0.0005 mg l−1, MPD (1.2 nM) was significantly
more effective than DMM or IPRO, causing 90%
inhibition. Formulated IPRO in planta was much
more effective than technical IPRO in vitro (compare
with Fig. 1). All fungicides strongly affected not only
the number of germinating spores (Fig. 2a, c) but also
Fig. 5 Epifluorescence micrographs of germinating spores of
B. lactucae treated with technical CAA (0.005–5 mg l−1; see
Fig. 1 for abbreviations) and water at 3 h post-inoculation.
Penetration of B. lactucae into lettuce leaf tissue occurs in
water-treated leaf tissue but not in CAA-treated tissue. Arrow
indicates point of penetration (yellow fluorescence). Aniline
blue staining followed by calcofluor staining; scale bar=20 μm
Eur J Plant Pathol (2008) 122:169–183
b
Leaf discs , CAA mixed with sporangia
Preventive, cotyledons
100
100
80
MPD
60
DMM
40
IPRO
20
% Sporulating plants
% Successful infections
a
177
80
MPD
60
DMM
BENT
40
IPRO
20
0
0
0
0.0005 0.005 0.05
0.5
5
0
50
0.1
1
10
100
Technical CAA, mg l
d
Preventive, cotyledons
Curative, cotyledons
100
100
80
60
MPD
DMM
40
IPRO
20
0
% Sporulating plants
% Sporulating plants
c
0.001
-1
-1
Formulated CAA, mg l
80
MPD
60
DMM
40
IPRO
20
0
0
0.1
0. 39
1. 56
6. 25
25
100
-1
Formulated CAA, mg l
0
62.5
125
250
500
1000
-1
Formulated CAA, mg l
e
Fig. 6 Downy mildew development in lettuce plants treated
with CAA (see Fig. 1 for abbreviations). a CAA mixed with
spores of B. lactucae before inoculation (means and standard
deviation of the mean, n=40). b, c CAA applied preventively
4 h before inoculation (n=80). d CAA applied curatively 1 dpi
(n=80). Disease symptoms recorded at 7 dpi. e The appearance
of the treated and inoculated plants shown in c at 21 dpi
178
Microscopic observations (Fig. 4) made in vitro (13°C,
darkness) showed that CAA (technical, 0.005–50 mg
l−1) added to spores at 3 h after the start of germination
inhibited further extension of germ-tubes. At 3 h, when
CAA was added, 50% of the spores produced a germtube of 40 μm (±standard error of 10 μm); at 48 h
(45 h after adding CAA) mean germ-tube length in
control, water-treated spores was 250±50 μm. In
contrast, spores treated with ≥0.005 mg l−1 of technical
MPD, DMM or BENT showed no extension of the
germ-tubes. Spores treated with IPRO at 0.005, 0.05,
0.5, 5 and 50 mg l−1 produced germ-tubes of 150±30,
100±20, 80±20, 60±10, and 40±10 μm, respectively.
Spores incubated in water showed 80–90% germination
(germ-tube=30–50 μm). Spores exposed to 0.5 or 1 mg
l−1 MPD or DMM for 1 h on ice failed to germinate or
infect leaves when the CAA was removed by washing
with water. In contrast, spores exposed to 0.5 or 1 mg
l−1 BENT or IPRO for 1 h germinated in vitro and
produced lesions when inoculated onto detached leaves
equivalent to control spores.
Effect on infection
Spores were mixed with formulated CAA and
inoculated onto lettuce leaf discs laid lower surface
uppermost on moist filter paper in 9 cm Petri dishes.
a
Lesions, % of control
Effect of CAA on germ-tube growth
Temporary exposure
0h
100
80
MPD
60
DMM
40
BENT
IPRO
20
0
0
0.005
b
5
50
3h
100
80
MPD
60
DMM
40
BENT
IPRO
20
0
0
0.005
0.05
0.5
5
50
-1
CAA, mg l
Lesions, % of control
Spores were allowed to germinate on lettuce leaf surfaces
at 13°C, and CAA added at 3 hpi when 50% of the
spores had germinated with a germ-tube of 40±10 μm.
Leaves were incubated for an additional 17 h at 13°C to
allow penetration of the host. At 20 hpi, epifluorescence
microscopy showed that penetration occurred in watertreated control leaves (Fig. 5, arrow) but not in leaves
treated with CAA at 0.005–50 mg l−1, except IPRO
which stopped penetration at ≥5 mg l−1 (Fig. 5).
0.5
-1
c
Effect of CAA on penetration
0.05
CAA, mg l
Lesions, % of control
the size of the germ-tubes (Fig. 2b, c). BENT was not
available as a formulated solo product; a formulation
(10% a.i.) was therefore prepared in Agsolex-8 and used
for testing germination in vitro. Results confirm that
BENT at 0.005 mg l−1 (lowest concentration tested)
allowed 11% germination (88% inhibition relative to
water control). The germinated spores produced a
germ-tube of 11 μm compared to 170 μm in the water
control.
Phalloidin staining indicated no disruption by
CAA in B. lactucae spores (Fig. 3). Spores germinating in water (18 h, 13°C, darkness) produced a germtube of about 80 μm. The F-actin (red) was
concentrated in the distal, growing part of the germtube, moving with the cytoplasm towards the tip. In
CAA-treated spores, most produced no germ-tube,
while some produced a germ-tube of about 3–5 μm.
In non-germinating spores the red stain remained
within the spore. In those that produced a small germtube, the red stain representing the F-actin microfilaments moved into the germ-tube (Fig. 3).
Eur J Plant Pathol (2008) 122:169–183
18 h
100
80
MPD
60
DMM
40
BENT
IPRO
20
0
0
0.005
0.05
0.5
5
50
-1
CAA, mg l
Fig. 7 Control of downy mildew lesion development in lettuce
by CAA (see Fig. 1 for abbreviations) applied in a at 0 hpi, b at
3 hpi, and c at 18 hpi
Eur J Plant Pathol (2008) 122:169–183
179
The number of successful infections (sporulating
lesions) at 10 dpi is shown in Fig. 6a. MPD was
most effective while IPRO was least effective. The
inhibitory concentrations of the three CAA fungicides
resemble those required to inhibit spore germination
in vitro (Fig. 1). Microscopic observations made at
1 dpi revealed that the failure to infect (for example in
the presence of MPD) was a consequence of inhibited
b
Floated leaf
5
Float leaf, 100 mg l-1
25
4
MPD
3
DMM
BENT
2
IPRO
1
0
0
0.01
0.1
1
10
100
Spores per leaf, x1000
Sporulation intensity
a
spore germination. In control inoculated plants,
primary and secondary vesicles were seen in epidermal cells from which hyphae were emerging.
Preventive application of four technical or three
formulated CAA fungicides to 7 day-old lettuce
plants efficiently protected against downy mildew.
Plants were sprayed with CAA at various concentrations and inoculated 4 h later. The proportion of
20
15
10
5
0
-1
Technical CAA, mg l
c
WATER
d
Cotyledon stage plants, 7dpi 100 mg l -1
MPD
DMM
BENT
IPRO
Cotyledon stage plant, 11 dpi 100 mg l -1
Spores per plant, x1000
% Sporulating plants
100
80
60
40
20
0
WATER
Spores per 24 mm disc, x1000
e
MPD
DMM
BENT
50
40
30
20
10
0
WATER
IPRO
MPD
DMM
BENT
IPRO
Floating leaf discs
60
50
40
MPD
DMM
30
BENT
20
IPRO
10
0
0
1
10
100
-1
Technical CAA, mg l
Fig. 8 Inhibition of sporulation of B. lactucae by CAA (see
Fig. 1 for abbreviations), shown as sporulation intensity (visual
assessment, means and standard deviation of the mean, n=20)
(a), spores per leaf (n=10) (b), percent sporulating plants
(n=20) (c), spores per plant (n=20) (d) and spores per leaf disc
(n=10) (e). a and b First-formed leaves floating on CAA. c and
d Whole plants at cotyledon stage, 7 and 11 dpi respectively,
sprayed with CAA. e Leaf discs (24 mm diam) floating on
CAA
180
Eur J Plant Pathol (2008) 122:169–183
45 hpi did not affect symptom production relative to
untreated inoculated controls.
plants showing sporulation of B. lactucae at 8 dpi is
shown in Fig. 6b and c, and the appearance of the
plants treated with formulated CAA at 21 dpi is
shown in Fig. 6e. The fungicides differed in their
efficacy in the order of MPD > DMM > BENT >
IPRO.
Effect on sporulation
BENT was most suppressive to sporulation: at 10 mg
l−1 it caused 82% inhibition, while the other fungicides showed 0–17% inhibition at this concentration
(Fig. 8a). At 100 mg l−1, BENT was most suppressive
and MPD least suppressive to sporulation (Fig. 8b).
For whole plants at the cotyledon stage, both the
number of sporulating plants (Fig. 8c) and the number
of spores produced per plant (Fig. 8d) were reduced
strongly by DMM and BENT, and moderately by
MPD. In contrast, IPRO had no effect. The number of
spores produced on leaf discs at 7 dpi and treated with
technical CAA is shown in Fig. 8e. BENT was most
inhibitory to sporulation, reducing the number of
spores by 85% at 10 mg l−1 compared to 48–55%
with the other fungicides. Other experiments revealed
that BENT formulated with Agsolex-8 prevented
sporophore emergence from stoma. BENT at 6.25 or
25 mg l−1 reduced the number of sporophores by 75%
and 100%, respectively, relative to water controls
(=130 sporophores per disc). The other fungicides had
no effect on sporophore formation.
Post-infection effects
Formulated CAA fungicides exhibited reduced control efficacy when applied at 1 dpi, when penetration
of the pathogen into the leaf had already taken place
(Fig. 6d). Much higher concentrations were required
for inhibition of disease compared to preventive
application suggesting that mycelium growth in planta
is less sensitive to CAA than spore germination.
Post-infection efficacy was also tested with technical CAA in detached leaves. Leaves were inoculated and treated with the compounds at 0, 3, 18 or
45 hpi, and lesion production was assessed at 8 dpi
(Fig. 7). The efficacy of the fungicides was strongly
dependent on the time of their application postinoculation. When applied at 0 h after inoculation
(Fig. 7a) they were highly efficient (except IPRO) in
inhibiting disease, probably due to their strong
inhibitory effect on spore germination. When applied
at 3 hpi efficacy decreased and the order of efficacy
was BENT ≥ DMM > MPD > IPRO (Fig. 7b). At
18 hpi (Fig. 7c), MPD and IPRO lost efficacy
whereas BENT and DMM showed full suppression
of the disease at 5 and 50 mg l−1, respectively. This
suggests that BENT and DMM might affect pathogen
development after penetration. Application of CAA at
Data on germination of spores and sporulating lesions
on detached leaves inoculated on the undersurface
following treatment with CAA on the upper surface
are given in Fig. 9a and b. Percentage inhibition of
b
100
20
80
16
Lesions per leaf
% Germinating spores
a
Translaminar efficacy
60
40
20
12
8
4
0
0
WATER
MPD
DMM
BENT
IPRO
Fig. 9 Germination of B. lactucae spores on lower leaf surface
(means and standard deviation of the mean, n=100) (a), and
lesion development (n=10) (b), in detached leaves treated with
WATER
MPD
DMM
BENT
IPRO
CAA (see Fig. 1 for abbreviations) on upper leaf surface and
inoculated on lower leaf surface
Eur J Plant Pathol (2008) 122:169–183
a
2006. 2 sprays, 27 d
120
Lesions per plant
spore germination at 20 hpi was 94, 54, 58 and 0
(Fig. 9a), and percentage inhibition of lesion at 10 dpi
formation was 73, 90, 79 and 0 for MPD, DMM,
BENT and IPRO, respectively (Fig. 9b). The data
show that while MPD was superior to the other
fungicides in translocation to the opposite surface of
the leaf in assessments of spore germination, DMM
was superior in inhibiting lesion formation. This
suggests that DMM might have post-germination
effects on B. lactucae.
181
100
80
60
40
20
0
0
Shade-house experiments
125
250
500
-1
MPD, mg l
b
2006. 2 sprays, 42 d
1200
Lesions per plant
Data from the experiments to assess the efficiency of
CAA for controlling epidemics in shade-house grown
plants are presented in Fig. 10. In 2006, all three
concentrations were effective similarly, providing
about 95% and 90% protection at 27 and 42 days
after the second (last) spray, respectively (Fig. 10a
and b). In 2007, a single spray of CAA provided
about 90% protection at 50 days after treatment
(Fig. 10c).
Discussion
1000
800
600
400
200
0
0
125
250
500
-1
MPD, mg l
c
Lesions per container (10 plants)
Carboxylic acid amide (CAA) fungicides are shown
here to control effectively (with intrinsic differences)
downy mildew in lettuce in growth chamber and
shade-house experiments. The toxicity of four CAA
compounds towards B. lactucae has been compared
quantitatively in vitro and in planta, by exposing the
pathogen to CAA at various stages of its life-cycle:
spore germination, germ-tube extension, penetration,
colonisation, sporophore formation, spore production
and epidemics in the field.
It appeared that spore germination was the stage in
the life-cycle most sensitive to CAA. Germination in
vitro and on leaf surfaces was inhibited with nanomolar concentrations of MPD (0.0005 μg ml−1) and
micromolar concentrations of DMM and BENT
(0.005 μg ml−1) or IPRO (0.5 μg ml−1). Similar
results were reported recently for germination in vitro
of sporangia and cystospores of P. infestans (Cohen
and Gisi 2007). Unlike cystospores or sporangia of P.
infestans which produce no germ-tubes in CAA
(Cohen and Gisi 2007), spores of B. lactucae produce
a small germ-tube (3–5 μm) in the presence of CAA.
The formation of these germ-tubes suggests that the
2007. 1 spray, 50 d.
350
300
250
200
150
100
50
0
Control
MPD
DMM
IPRO
Fig. 10 Downy mildew development in shade-house grown
lettuce plants treated with 125, 250 or 500 mg l−1 MPD
(formulated) at 27 (a) and 42 (b) days after the second (last)
spray. c Downy mildew development in shade-house grown
lettuce plants treated with three CAA compounds (formulated;
see Fig. 1) applied once at a dose of 500 mg l−1. Lesions per ten
plants in a box recorded at 50 days after spraying. n=10 boxes
per dose treatment per fungicide
182
polarity of the spores of B. lactucae, required for
germ-tube formation, is not disturbed by CAA.
Phalloidin is a specific stain for F-actin microfilaments and was applied recently by Zhu et al. (2007)
to flumorph-treated cystospores and mycelium of P.
melonis in vitro. They concluded that flumorph
disrupts microfilament organisation. In the present
study, no such disruption in F-actin organization was
seen in spores of B. lactucae treated with MPD or
DMM. More work is needed to elucidate the mode of
action of CAA and the target site in oomycetes.
When germ-tubes were allowed to be formed
during 3 h in water (reaching ∼40 μm long), CAA
stopped their further extension in vitro. Equivalent
experiments on leaf surfaces showed that CAA
prevented them from producing appressoria and
penetrating the host. Experiments also determined
that post-penetration stages of B. lactucae infection
are less sensitive to CAA and require much higher
doses to be controlled. As disease control by CAA is
a consequence of inhibition of spore germination and
germ-tube extension, only preventive application
might reduce disease development effectively.
CAA compounds applied to upper lettuce leaf
surfaces were shown here to inhibit spore germination
of B. lactucae on the lower leaf surface. This suggests
that CAA travels across the leaf (translaminar movement) to reach the lower surface of the opposite,
untreated side of the leaf. CAA compounds greatly
differ in their translaminar movement, with MPD being
superior to others. Similar results were obtained for
MPD compared to DMM and IPRO in potato and
tomato (Cohen and Gisi 2007). Studies with C14-MPD
showed that within 1.5 h after application, 4.9% and
1.3% of the applied compound reached the mesophyll
of potato and grape leaves, respectively (Hermann
et al. 2005). Three days after application, 54.9%,
36.4% and 8.7% of the applied radioactive MPD were
on the potato leaf surface (water wash), bound on the
surface and in wax (organic solvent wash), and in the
leaf extract, respectively. Seven days after application,
49% of the C14 MPD applied to potato leaves could be
recovered from leaf surface and wax (Hermann et al.
2005). This prolonged binding of MPD to the leaf
cuticle, its good rain-fastness (Hermann et al. 2005)
and its pronounced translaminar mobility may ensure
its prolonged availability to lower leaf surfaces. No
data, however, are available for how much C14-MPD is
present on the opposite, lower, surface of the leaf.
Eur J Plant Pathol (2008) 122:169–183
Miyake et al. (2003) reported that BENT has not only
strong preventive activity, but also curative and
penetrant activity, with excellent residual effects and
rainfastness.
The biotrophic nature of B. lactucae does not allow
testing the effects of CAA on mycelium growth or
sporulation in vitro. Therefore, the interpretation of in
planta experiments should consider also the incorporation of the CAA compounds into the leaf tissue.
Indeed, application of CAA to detached leaves at 18 h
post-inoculation, when primary and secondary vesicles
have already developed in the epidermal cells, did not
prevent the pathogen from completing its life-cycle by
producing new spores. Nonetheless, two compounds,
BENT and DMM, did partially inhibit sporulation, and
BENT also suppressed sporophore development.
CAA fungicides were found to be highly effective
in controlling downy mildew in lettuce in the shadehouse experiments. Unfortunately, doses applied were
too high to distinguish differences in efficacy among
the compounds. Previous data with late blight control
in potato (Cohen and Gisi 2007) showed superior
activity of MPD over DMM, and of DMM over
IPRO. More studies are required to evaluate the
performance of CAA, including BENT, in the field.
In conclusion, CAA fungicides were found here to
be extremely potent inhibitors of spore germination of
B. lactucae, as they were in P. infestans (Cohen and
Gisi 2007). CAA inhibited germ-tube extension when
applied to germinating spores. The compounds had
poor curative efficacy when applied at 1 dpi against
B. lactucae, as was determined previously for P.
infestans (Cohen and Gisi 2007). All the compounds,
except IPRO, showed translaminar movement across
lettuce leaves, with MPD superior to DMM and
BENT. When applied to mature lesions, BENT was
superior to the other compounds in suppressing
sporulation of B. lactucae. CAA fungicides, therefore,
should be used preventively to achieve the best
control of downy mildew in lettuce.
Acknowledgement We are grateful to Dr. Alex Perelman of
Bar-Ilan University for his assistance with confocal microscopy.
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DOI 10.1007/s10658-008-9285-2
Beta-aminobutyric acid-induced resistance in grapevine
against downy mildew: involvement of pterostilbene
Ana R. Slaughter & Mollah Md. Hamiduzzaman &
Katia Gindro & Jean-Marc Neuhaus &
Brigitte Mauch-Mani
Received: 29 August 2007 / Accepted: 31 January 2008
# KNPV 2008
Abstract BABA, a non-protein amino acid, was used
to induce resistance in grapevine against downy
mildew. BABA-induced resistance was observed in
the susceptible cv. Chasselas as well as in the resistant
cv. Solaris. Following BABA treatment, sporulation
of Plasmopara viticola was strongly reduced and the
accumulation of stilbenes increased with time following
infection. Induction of trans-piceide, trans-resveratrol
and, more importantly, of trans-ɛ- and trans-δ-viniferin
and trans-pterostilbene was observed in BABA-primed
Chasselas. On the other hand, induction of transresveratrol, trans δ-viniferin and trans-pterostilbene
was observed in BABA-primed Solaris. The accumuA. R. Slaughter : M. M. Hamiduzzaman : J.-M. Neuhaus :
B. Mauch-Mani (*)
Laboratory of Molecular and Cellular Biology,
University of Neuchâtel,
Rue Emile-Argand 11, Case Postale 158,
CH-2009 Neuchâtel, Switzerland
e-mail: Brigitte.mauch@unine.ch
Present address:
M. M. Hamiduzzaman
Department of Plant Physiology and Botany Department,
Stockholm University,
Lilla Frescativagen 5,
Stockholm 106 91, Sweden
K. Gindro
Swiss Federal Research Station for Plant
Production of Changins-Wädenswil ACW,
Case Postale 1060,
CH-12 Nyon, Switzerland
lation of stilbenes in BABA-primed Solaris was much
higher than that found in BABA-primed Chasselas.
Furthermore, BABA-treatment of Solaris led to a rapid
increase in transcript levels of three genes involved in
the phenylpropanoid pathway: phenylalanine ammonia
lyase, cinnamate-4-hydroxylase and stilbene synthase.
BABA-primed Chasselas showed increased transcript
levels for cinnamate-4-hydroxylase and stilbene synthase. Here we show that pre-treatment of a susceptible
grapevine cultivar with BABA prior to infection with
P. viticola primed the accumulation of specific phytoalexins that are undetectable in non-BABA-primed
plants. As a result, the susceptible cultivar became
more resistant to downy mildew.
Keywords Phytoalexin . Plasmopara viticola .
Priming . Stilbenes . Vitis vinifera
Abbreviations
BABA
beta aminobutyric acid
BABA-IR BABA-induced resistance
Introduction
Downy mildew caused by Plasmopara viticola is one
of the most serious diseases in vineyards worldwide.
Both susceptible and resistant cultivars can be
colonised by P. viticola zoospores, but in resistant
ones, the development of the parasite is rapidly
186
inhibited. The most cultivated grape cultivars (Vitis
vinifera) are susceptible to P. viticola and the control of
downy mildew requires regular fungicide applications.
The application of copper-containing fungicides to
control downy mildew causes accumulation of this
heavy metal in soil and groundwater resulting in
toxic effects to the environment. Unfortunately, the
replacement of such copper-based fungicides by
synthetic fungicides with specific modes of action has
promoted the development of resistant isolates of
P. viticola (Matasci et al. 2008). For these reasons,
alternative strategies are needed.
In grapevine, the most frequently observed and
best-characterised defence reactions upon fungal
infection are the deposition of lignin (Dai et al.
1995) and other phenolic compounds, increased
peroxidase activity, accumulation of stilbene phytoalexins and the synthesis of pathogenesis-related
(PR) proteins (Derckel et al. 1999). These defence
mechanisms also seem to be present in susceptible
cultivars, but in general, they are not activated or are
delayed during the infection process. Because grapevine
is an agriculturally and economically important crop
plant, the defence mechanisms of that plant against
pathogenic microorganisms, including phytoalexin
production, have attracted considerable attention.
Phytoalexins from the Vitaceae have been the subject
of numerous studies, because these compounds are
thought to have implications in both phytopathology
and human health (Jeandet et al. 2002).
Phytoalexins are low molecular weight antimicrobial
secondary metabolites (Kuc 1995). They have been
shown to possess biological activity against a wide
range of pathogens and can be considered as markers
for plant disease resistance. Although phytoalexins
display an enormous chemical diversity, phytoalexins
from the Vitaceae seem to constitute a restricted group
of molecules belonging to the stilbene family, the
skeleton of which is based on the trans-resveratrol
structure (3,5,4′-tryhydroxystilbene) (Fig. 1a) (Jeandet
et al. 2002). Other compounds considered as oligomers
of resveratrol and termed viniferins have also been
found in grapevine as a result of infection or other
stresses. Resveratrol is also glycosylated as piceide (5,4′dihydroxystilbene-3-O-β-glucopyranoside) (Fig. 1b)
and pterostilbene (3,5-dimethoxy-4′-hydroxystilbene)
(Fig. 1c) is a dimethylated resveratrol derivative (Jeandet
et al. 2002). Recently, it was shown that an isomer of
ɛ-viniferin (Fig. 1d), δ-viniferin (Fig. 1e), is one of the
Eur J Plant Pathol (2008) 122:185–195
Fig. 1 Chemical structures of stilbene phytoalexins: a resveratrol, b piceide, c pterostilbene, d ɛ-viniferin and e δ-viniferin
major stilbenes produced from resveratrol oxidation in
grapevine leaves infected by P. viticola (Pezet et al.
2003). Pezet et al. (2004b) tested the toxicity of these
stilbenes against zoospores of P. viticola and found that
δ-viniferin and pterostilbene were the most toxic
stilbenes.
The phenylpropanoid pathway is an important
pathway in secondary plant metabolism, yielding a
variety of phenolics with structural and defencerelated functions. These phenolic compounds include
lignins, phenolic acids, flavonoids and stilbenes. In
addition, enzymes such as phenylalanine ammonia
lyase (PAL; EC 4.3.1.5), cinnamate-4-hydroxylase
(C4H; EC 1.14.13.11) and 4-coumarate:coenzyme A
ligase (4CL, EC 6.2.1.12) are considered to be crucial
to phenylpropanoid metabolism. A number of reports
have shown that phenylpropanoid derivatives are
capable of protecting plants against various biotic
(infection by viruses, bacteria, fungi) and abiotic (low
and high temperatures, UV-B light, wounding)
stresses (Sgarbi et al. 2003; Solecka and Kacperska
2003). Stilbene synthase (STS) (EC 2.3.1.95) catalyses
the last step of the phenylpropanoid biosynthesis
pathway leading to the formation of stilbene phytoalexins. Expression of STS genes is often induced in
response to biotic and abiotic stresses (Jeandet et al.
2002).
Eur J Plant Pathol (2008) 122:185–195
In recent years, much attention has been focused
on the activation of a plant’s own defence system,
known as induced resistance (Sticher et al. 1997). The
non-protein amino acid β-aminobutyric acid has
previously been shown to induce resistance against
many different oomycetes and to be effective in
inducing resistance against various downy mildews
(reviewed in Jakab et al 2001; Cohen 2002). It has
been speculated that BABA deteriorates penetrated
host cells, blocking the translocation of nutrients into
the haustoria, therefore prohibiting further mycelial
growth and sporangial production (Steiner and
Schönbeck 1997). Experiments with 14C-labelled
BABA showed that it was not metabolised in tomato
(Cohen and Gisi 1994) or in Arabidopsis (Jakab et al.
2001), ruling out the involvement of a BABA
metabolite acting as an antimicrobial compound in
the plant. BABA-mediated resistance is therefore
most likely to be based on the activation of host
resistance mechanisms. Recently, in grapevine, it has
been shown that callose deposition as well as defence
mechanisms depending on the phenylpropanoid and
the jasmonic acid (JA) pathways all contributed to
BABA-IR (Hamiduzzaman et al. 2005).
In order to obtain a clearer picture of BABA-IR in
grapevine against downy mildew, we looked at the
involvement of stilbene phytoalexins and the expression
of genes encoding enzymes involved in the phenylpropanoid pathway. Different staining techniques were
used to visualise the biochemical changes that occurred
at the cellular level between BABA and water-treated
resistant and susceptible cultivars. After treatment of
plants with BABA and water, we performed quantitative
HPLC analysis of stilbenes and investigated the level of
expression of the genes involved in the biosynthesis of
stilbenes (PAL, C4H and STS) by quantitative real-time
PCR.
Materials and methods
Plant material
Cuttings of V. vinifera cvs Chasselas and Solaris, the
latter resulting from a cross of Merzling x GM 6493,
carried out by the Weinbauinstitut in Freiburg,
Germany, are sensitive and resistant to P. viticola,
respectively. They were obtained from the grape
collection at the Swiss Federal Research Station for
187
Plant Production in Changins, Switzerland and were
grown in glasshouses. The cuttings were maintained
in growth chambers (16 h light at 22°C, 8 h dark at
18°C and 60% relative humidity) until they had five
fully developed leaves. They were then used for
subsequent treatments and artificial inoculations.
Treatment and inoculation of plants
Cuttings from both cultivars were either soil drenched
with an aqueous solution of BABA (1 mM) or with
water (Hamiduzzaman et al. 2005) 2 days prior to
inoculation with P. viticola. For the inoculum, leaves
infected with P. viticola were harvested and sporangia
were collected by vacuum aspiration, as described by
Gindro et al. (2003). The abaxial leaf surfaces were
inoculated by spraying an aqueous suspension of
sporangia (5×104 sporangia ml−1). The plants were
then covered with transparent plastic bags and placed
in growth chambers under the conditions described
above. Samples were collected from both BABA- and
water-treated leaves 0, 3, 7, 24, 48 and 72 h after
inoculation and either used immediately for further
analysis or frozen in liquid nitrogen and stored at
−80°C. The experiments were carried out in duplicate.
Microscopic examination
Mycelia, sporangiophores and sporangia in inoculated
leaves were stained with lactophenol-trypan blue
(Keogh et al. 1980) and examined with bright field
microscopy. Leaf material was placed on a glass slide
in water, covered with a coverslip and examined with
an epifluorescence microscope with an UV excitation
filter (BP 340–380 nm, LP 425 nm). Blue fluorescence
was used as an indicator of the presence of resveratrol
(Dai et al. 1995). Flavonoid accumulation was
visualized by using Wilson’s reagent (Dai et al.
1995); leaves were immersed for 15 min, mounted
in glycerol (75%) and examined under UV light with
an epifluorescence microscope (BP 340–380 nm, LP
425 nm). Flavonoids fluoresced yellow and gallic
acid derivatives (GAD) fluoresced blue.
Preparation of samples and HPLC analysis
At 0, 3, 7, 24, 48 and 72 h post-infection (hpi), three
pieces of leaves were excised from inoculated leaves.
Three replicates were made for each time point, each
188
cultivar and each treatment. Leaf samples were
weighed and placed in a microfuge tube and 50 μl
of MeOH were added. The tubes were placed in a
thermo-regulated shaker at 60°C for 10 min and then
placed on ice for 5 min. The methanolic extracts
(30 μl) were analysed for stilbenes as described by
Pezet et al. (2003).
Preparation of cDNAs
Total RNA was isolated from frozen leaf tissue using
a modified CTAB extraction and lithium chloride
precipitation method according to Iandolino et al.
(2004). The quantity of total RNA was determined
with a Nano-Drop ND-1000 spectrophotometer
(NanoDrop Technologies, Wilmingon, DE, USA); in
addition the quality of RNA was verified by the
absorbance ratios (A260/A280) of 1.8 to 2.0. For
quantitative real-time PCR analysis, RNA was treated
with Turbo-DNase I (Ambion) according to the
manufacturer’s instructions. For cDNA synthesis, 1 μg
of RNA was reverse-transcribed using oligo(dT)18
and Superscript III reverse transcriptase (Invitrogen
Life Technologies) following the instructions of the
manufacturer.
Real-time PCR for expression analysis of C4H, PAL
and STS
Expression analysis of the genes VvC4H, VvPAL and
VvSTS was done by real-time PCR, using the SYBER
green method on an iCycler (Bio-Rad) real-time
cycler. Each PCR reaction (20 μl) contained
0.25 mM of each primer, cDNA and 1x ABsolute
QPCR SYBR Green mix (ABgene). The thermal
cycling conditions were 95°C for 15 min followed by
95°C for 30 s, 56°C for 30 s, and 72°C for 35 s for 40
cycles, followed by a melt cycle from 60°C to 95°C.
The primers used were as follows: VvC4H-F (5′AGTCCAAGTCACCGAGCCTGAT-3′) and VvC4HR (5′-TAGCAAGCCACCATGCGTTCAC-3′) for
VvC4H (gene fragment obtained from a suppressive
subtractive hybridisation SSH library constructed
from P. viticola-infected grapevine), VvPAL-F (5′TTGGTGCCACTTCACATAGGAG-3′) and VvPALR (5′-AATCTGATGCCGGAGTAGCCTT-3′) for
VvPAL, and VvStSy-F (5′-CTCGAACCATCCGTCA
GAAGAG-3′) and VvStSy-R (5′-CCTACGATTA
Eur J Plant Pathol (2008) 122:185–195
CAGCTGCAGACC-3′) for VvSTS. With all cDNAs
used, the above primer sets gave single PCR products,
which were verified by determining the melt curves
for the products at the end of each run and by analysis
of the products using gel electrophoresis. The
efficiency of the primers was tested in preliminary
experiments with serial dilutions of cDNA samples
and maintained an E value of between 0.97 and 0.98.
The expression of the three genes was normalised
relative to Elongation Factor 1-α (VvEF1-α) using the
primers VvEF1-F (5′-GAACGTTGCTGTGAAG
GATCTC-3′) and VvEF1-R (5′-CGCCTGTCAACCT
TGGTCATGA-3′). All samples were measured in
triplicate, every run included the VvEF1-α control
for each sample, and experiments were repeated
twice. The Gene-X software (Bio-Rad) was used to
calculate the mean normalised expression of the genes
(Vandesompele et al. 2002).
Results
Microscopic observation of the biochemical changes
in infected grapevine cuttings treated
with or without BABA
Successful infection with P. viticola in susceptible
Chasselas becomes apparent about 6 days after
infection as a white, downy growth, mostly on the
lower side of the leaves where P. viticola emerged
from the stomata. Sporulation of P. viticola was
strongly reduced in BABA-primed cuttings (Fig. 2b)
compared to water-treated control plants (Fig. 2a) and
the infection sites on BABA-primed cuttings were
surrounded by necrotic groups of cells (Fig. 2b).
Extensive hyphal growth and spread were observed
within 3 days inside the leaf tissue of water-treated
cuttings, whereas hyphal growth and spread of
P. viticola was reduced in BABA-primed cuttings
(data not shown). The biochemical changes at the
cellular level were analysed at different time points
(1–5 dpi) in BABA-and water-treated plants by using
different staining techniques. Infected, non-BABAprimed plants displayed only the red fluorescence of
chlorophyll in both plants stained with Wilson’s
Reagent (Fig. 2c) or unstained plants (Fig. 2e) when
visualised under UV light. However, infected, BABA-
Eur J Plant Pathol (2008) 122:185–195
189
Fig. 2 Development of
P. viticola and accumulation
of stilbene compounds in
grapevine (Chasselas).
Plants were soil drenched
with water (a, c, e) or 1 mM
BABA (b, d, f) 2 days prior
to challenge inoculation
with sporangia of P. viticola
(5×104 sporangia ml−1). a
White sporulation of
P. viticola on water-treated
cutting (6 dpi); b development of necrosis surrounding infection sites in
BABA-primed cutting
(6 dpi). Infected leaves were
stained with Wilson’s
reagent and analysed by
epifluorescence microscopy
(c and d) or analysed by
autofluorescence without
prior staining (e and f).
Water-treated controls show
red chlorophyll fluorescence
(c and e). Yellow fluorescence indicates flavonoid
accumulation (d) and blue
auto-fluorescence indicates
presence of resveratrol in
the tissues. Bars=50 μm
primed plants displayed a yellow fluorescence when
stained with Wilson’s Reagent (Fig. 2d) indicating the
possible presence of flavonoids. The blue autofluorescence of infected, BABA-primed plants on the other
hand suggests an accumulation of resveratrol in the
tissues (Fig. 2f).
Accumulation of stilbene phytoalexins in P. viticola
infected grapevine cuttings treated with and without
BABA
The trans-form of five major stilbene phytoalexins:
ɛ-viniferin, δ-viniferin, piceide, resveratrol and pter-
190
ostilbene were quantified. Trans-piceide was the only
phytoalexin detected in both BABA- and watertreated Chasselas and Solaris for the six investigated
time points (Fig. 3a). There was a significant increase
in trans-piceide in BABA-primed Chasselas cuttings
compared to the water-treated cuttings with the
highest concentration occurring at 72 h pi. In contrast,
trans-piceide increased significantly in BABA-primed
Solaris cuttings compared to water-treated cuttings
with the highest concentration occurring at 24 h pi.
With the BABA concentrations used for our experiments no direct induction of phytoalexins was
observed and accumulation occurred only in infected
or primed/infected plants. During the 72 h analysis,
trans-resveratrol was quantitatively the most abundant
stilbene produced in BABA-primed Chasselas and
Solaris (Fig. 3b). In both BABA-primed Chasselas
and Solaris there was a marked increase in transresveratrol starting at 24 h pi and accumulating
quantitatively to about 150 and 191 μmol mg FW−1
for Chasselas and Solaris, respectively, at 72 h pi.
Very slight amounts of trans-resveratrol were detected
in water-treated Chasselas at 72 h pi (8 μmol mg FW−1),
whereas in water-treated Solaris, trans-resveratrol was
detected at 24 h pi with a significant increase at 48 h (5
and 97 μmol mg FW−1, respectively). Trans-ɛ-viniferin
was only detected after 24 h pi and was not detected in
water-treated Chasselas (Fig. 3c). For BABA-primed
Chasselas, production started at 24 h pi, reaching a
plateau at 48 h pi (28 μmol mg FW−1). The highest
concentration of ɛ-viniferin was found in water-treated
Solaris at 48 h pi (97 μmol mg FW−1) and then
decreased to 56 μmol mg FW−1 at 72 h. The
production of ɛ-viniferin increased steadily in BABAprimed Solaris starting at 24 h until 72 h where it
reached the same amount as in water-treated Solaris at
48 h pi. Trans-δ-viniferin accumulated transiently
starting at 24 h pi and peaking at 72 h only for
Fig. 3 Quantitative analysis of the accumulation of several
stilbenes in leaf samples of a susceptible and resistant cvs
Chasselas and Solaris, respectively, over time after inoculation
with P. viticola. Chasselas cuttings were treated with water
(grey bars) or BABA (white bars) and Solaris cuttings were
also treated with water (striped bars) or BABA (black bars)
2 days prior to inoculation. The analysed stilbenes were transpiceide, trans-resveratrol, trans-ɛ-viniferin, trans-δ-viniferin
and pterostilbene. Values presented are means±standard error
of the mean. The experiment was repeated twice with similar
results
Eur J Plant Pathol (2008) 122:185–195
Eur J Plant Pathol (2008) 122:185–195
water-treated Solaris and BABA-primed Chasselas and
Solaris (Fig. 3d). The highest concentration was found
in BABA-primed Solaris at 100 μmol mg FW−1,
whereas that for BABA-primed Chasselas and watertreated Solaris the concentration of δ-viniferin was the
same at 49 μmol mg FW−1 at 72 h pi. Pterostilbene
was detected at relatively lower levels compared to the
quantity of the other phytoalexins (Fig. 3e). It was only
detected in BABA-primed cuttings, starting at 24 h pi
for BABA-primed Chasselas and 12 h later for BABAprimed Solaris. Pterostilbene accumulated transiently
for both BABA-primed cuttings peaking at 72 h pi with
the same concentrations of 31 μmol mg FW−1 for both
BABA-primed samples.
Response of grapevine genes involved
in the phenylpropanoid biosynthesis pathway
to P. viticola infection after treatment
with and without BABA
The expression pattern of three genes involved in the
phenylpropanoid biosynthesis pathway was analysed
using quantitative real-time PCR: the first gene encodes
phenylalanine ammonia lyase (PAL), the first enzyme
of the pathway, the second gene encodes cinnamate-4hydroxylase (C4H), the enzyme which catalyses the
conversion of cinnamate into 4-hydroxy-cinnamate, a
key reaction of the phenylpropanoid pathway, and the
third gene encodes stilbene synthase (STS), the enzyme
responsible for the synthesis of stilbenes such as
piceides and resveratrol.
In infected, BABA-primed Solaris plants, the
expression of PAL increased rapidly and transiently.
There was no significant transcript accumulation of
PAL in infected, BABA-primed Chasselas and in
infected water-treated Chasselas and Solaris. The
induction by BABA in infected Solaris peaked at 7 h
with a maximum intensity 15-fold higher, than in both
water-treated and BABA-primed infected Chasselas.
Cinnamate-4-hydroxylase (C4H) was rapidly and
transiently induced peaking at 7 h for both BABAprimed infected plants and for water-treated infected
Chasselas. There was no significant change in expression of C4H in water-treated infected Solaris. In both
BABA-primed infected plants STS was transiently
induced, reaching maximum levels at 7 h (5-fold and
21-fold induction for Solaris and Chasselas, respectively). The level of expression of STS decreased for
the first 24 h and increased again between 24 and
191
48 h pi. For both water-treated cultivars there was no
significant accumulation of STS transcript levels for
STS.
Discussion
The resistance inducer BABA has been shown to
work mainly through priming of defence responses by
sensitising the plants to respond faster and more
adequately to the exposure to a given stress situation
(Jakab et al. 2001; Conrath et al. 2002; Prime-A-Plant
Group et al. 2006). Previous work in our group has
shown that BABA induced resistance against
P. viticola in both the susceptible Chasselas and the
resistant Solaris cultivars of grapevine. This observed
resistance in BABA-primed Chasselas plants as well
as the basal resistance of Solaris depended to a large
extent on the deposition of callose, which was
positively correlated with BABA- and jasmonic
acid-induced resistance (Hamiduzzaman et al. 2005).
Here, we looked at the involvement of phytoalexins
and investigated three genes involved in the phenylpropanoid biosynthesis pathway in BABA-IR in
grapevine against downy mildew.
Phytoalexins have long been accepted as being
important in the defence mechanisms of plants against
phytopathogenic microorganisms. Previous work has
demonstrated that two biochemical processes are
indicative of downy mildew resistance in grapevines.
One is the synthesis of callose in stomata (Gindro
et al. 2003) and the second is the synthesis of
resveratrol and its subsequent oxidation to ɛ- and δviniferins (Langcake 1981; Pezet et al. 2003, 2004a).
The obvious priming for accumulation of different
flavonoids in BABA-primed, infected grapevine plants
compared to untreated water controls (Fig. 2d and f)
observed at the microscopic level led us to further
investigate the involvement of phytoalexins in BABAIR at the biochemical and molecular level.
Not all stilbenes are equally toxic to P. viticola
zoospores. Resveratrol is not a toxic compound as a
consequence of its hydrophilic character (Dercks and
Creasy 1989). According to Pezet et al. (2004a),
piceide has never shown any toxic activity against
P. viticola zoospores, even at concentrations greater
than 1,000 μM. In addition, resveratrol was found to
be glycosylated to form piceide in susceptible
cultivars (Gindro et al. 2003). In our experiment,
192
BABA-treatment led to an induction of trans-piceide
accumulation in the susceptible Chasselas cultivar to
levels higher than found in the resistant Solaris
cultivar. BABA treatment did not seem to have any
effect on piceide accumulation in Solaris (Fig. 3a). On
the other hand BABA-treatment significantly induced
the accumulation of trans-resveratrol in both susceptible and resistant cultivars. After 48 h pi the amount
of trans-resveratrol in the BABA-primed susceptible
Chasselas was equal to that found in the non-treated
resistant Solaris and after 72 h pi the application of
BABA led to an induction of trans-resveratrol in both
cultivars to a comparable amount (Fig. 3b). In
susceptible cultivars, resveratrol is synthesised in
large amounts, but it is rapidly glycosylated into the
non-toxic compound piceide, which could explain the
high concentration of trans-piceide in BABA-primed
Chasselas due to the high concentration of transresveratrol. This was not the case in non-treated
Chasselas plants.
The oxidation products of resveratrol, the viniferins,
are active against P. viticola; δ-viniferins is five times
more toxic than ɛ-viniferin (Pezet et al. 2004b). Both
ɛ- and δ-viniferin have been found in low concentrations in susceptible cultivars (Pezet et al. 2004a). We
also found both viniferins in the susceptible Chasselas
but only in BABA-primed plants at 48 h pi (Fig. 3c
and d). Treatment of the resistant cv. Solaris with
BABA did not seem to have a significant effect on the
accumulation of ɛ- and δ-viniferins. It is important to
note that an increase in resveratrol synthesis occurred
at 48 h pi for BABA-primed Chasselas providing an
important pool for the synthesis of viniferins that also
occurred at 48 h pi.
Pterostilbene is as toxic for P. viticola as δviniferin, but it is usually absent or its concentration
is too low to contribute to resistance mechanisms
(Pezet et al. 2004a). We found no detectable amounts
of pterostilbene in both water-treated Chasselas and
Solaris; however, BABA-treatment induced a significant increase in pterostilbene in both infected
cultivars even though the amounts were lower
compared to the other stilbenes analysed. In BABAprimed Chasselas the level of pterostilbene at 72 h pi
was comparable to that of BABA-primed Solaris
(Fig. 3e).
Pezet et al. (2004a) concluded that there were two
different types of reactions that could be observed
between susceptible and resistant cultivars. In suscep-
Eur J Plant Pathol (2008) 122:185–195
tible cultivars, resveratrol is synthesised in large
amounts after infection, but it is rapidly glycosylated
into piceide, which is a non-toxic compound, whereas
in resistant cultivars resveratrol is also synthesised
in large amounts but it is rapidly oxidised into the
more toxic viniferins. From our experiments BABAtreatment of the susceptible Chasselas cultivar made
it respond more like resistant Solaris and more
importantly BABA-treatment was able to prime the
production of pterostilbene in both cultivars which,
along with δ-viniferin, have been shown to be the
most toxic stilbenes affecting zoospore mobility and
disease development of P. viticola (Pezet et al.
2004b). BABA-treatment was also able to prime
δ-viniferin accumulation in Chasselas to levels comparable to non-treated Solaris at 72 h pi but not as
high as in BABA-primed Solaris (Fig. 3d).
The grapevine stilbenes are flavonoid-type phytoalexins and are formed via the phenylpropanoid
pathway, and synthesis of these stilbenes only occurs
if PAL genes and other genes encoding downstream
enzymes are induced (Jeandet et al. 2002). In our
experiments, only BABA-treatment of Solaris was
able to prime the expression of PAL in the first 7 h pi.
There was no induction in PAL expression in BABAprimed Chasselas or in water-treated Chasselas and
Solaris (Fig. 4a). On the other hand BABA-treatment
of Solaris also lead to the priming of the expression of
C4H in the first 7 h pi, whereas there was no
significant induction in C4H expression in BABAprimed compared to water-treated Chasselas for the
first 7 h pi. However, 24 h pi a significant decrease in
C4H expression was observed in the water-treated
Chasselas whereas in BABA-primed Chasselas the
expression level of C4H was comparable to levels
found in BABA-primed Solaris (Fig. 4b). In tobacco,
Blount et al. (2000) showed that PAL activity and
levels of phenylpropanoid compounds were reduced
in leaves and stems of plants in which C4H activity
had been genetically down-regulated. However, C4H
activity was not reduced in plants in which PAL
activity had been down-regulated by gene silencing
(Blount et al. 2000). These authors hypothesised that
flux into the phenylpropanoid pathway is controlled,
at least in part, via feedback regulation of PAL sensed
through the production of cinnamic acid. Furthermore,
when reduced below a threshold of 20% to 25% of
wild-type activity, PAL becomes a rate-limiting step
for lignin biosynthesis in tobacco (Bate et al. 1994;
Eur J Plant Pathol (2008) 122:185–195
Fig. 4 Transcript accumulation of genes involved in the
phenylpropanoid biosynthesis pathway. Leaf samples from the
susceptible cv. Chasselas were treated with water (open
triangles) or BABA (closed diamonds) 2 days prior to
inoculation with P. viticola. The same water-treatment (open
circles) or BABA-treatment (closed squares) was done for the
resistant cv. Solaris. The three genes analysed by quantitative
real-time PCR were: phenylalanine ammonia lyase (PAL),
cinnamate-4-hydroxylase (C4H) and stilbene synthase (STS).
Expression levels of the three genes were normalised relative to
elongation factor 1-α. Values presented are means±standard
error of the mean. The experiment was repeated twice with
similar results
Howles et al. 1996). Reduced C4H activity is also
correlated with reduced levels of lignin and phenolics
(Sewalt et al. 1997). Even though BABA-treatment
did not prime the expression of PAL in Chasselas, it
primed the expression of C4H, which was maintained
193
at comparable levels to those in BABA-primed Solaris
thereby allowing an increase in lignin biosynthesis for
BABA-primed Solaris and Chasselas but not for the
water-treated Solaris and Chasselas.
Stilbene synthase is a further key branch-point
enzyme in the phenylpropanoid pathway leading to
the production of resveratrol. The expression of STS
is often induced in response to biotic and abiotic
stresses. We found that at the transcriptional level,
BABA-treatment of both resistant and susceptible
cultivars induced an accumulation of STS expression
in two peaks: one at 7 h pi and the other after 24 h pi
(Fig. 4c). There was no priming of STS expression in
either water-treated cultivars. Similar results were
previously obtained with grapevine cell suspensions
elicited with cell walls of Botrytis cinerea (Liswidowati
et al. 1991) as well as with cell walls of Phytophthora
cambivora (Wiese et al. 1994). The induction of STS
expression found in the BABA-primed Chasselas and
Solaris cultivars corresponded to an accumulation of
resveratrol whereas a low STS gene expression in the
water-treated cultivars corresponded to low levels of
resveratrol (Figs. 3b and 4c).
Grapevine plants have various possibilities to
defend themselves against an attack by P. viticola.
As shown above, the phenylpropanoid pathway
leading to the accumulation of phytoalexins as well
as lignification has a major influence on the expression of resistance. However, the plants have also the
possibility to mount other early defenses such as
callose deposition at the stomatal entry points (Gindro
et al. 2003; Hamiduzzaman et al. 2005). Since the
addition of the PAL inhibitor AIP does not significantly compromise BABA-IR in Solaris (Hamiduzzaman
et al. 2005), it seems likely that the contribution of
callose deposition can compensate for the lack of
phenylpropanoid pathway products. The exact contribution of each of these defense mechanisms to final
resistance is, however, not yet clear.
Our results point to a prominent role for phytoalexins as a component of BABA-IR. Of special
interest is the fact that BABA treatment is able to
prime the plants to accumulate a specific phytoalexin,
pterostilbene, that is otherwise not present in the
plants. Interestingly, pterostilbene is extremely effective
in interfering with zoospore mobility and general hyphal
development and its specific priming might help to
develop better methods of protection of grapes against
downy mildew and possibly other diseases.
194
Acknowledgements This project was funded by the National
Centre of Competence in Research (NCCR) Plant Survival, a
research programme of the Swiss National Science Foundation.
We thank Mr. J. Taillens (Agroscope-RAC, Changins, Nyon)
for the grapevine cutting production and Dr. S. Godard
(Agroscope-RAC, Changins, Nyon) for the valuable help with
the measurements of phytoalexins.
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Eur J Plant Pathol (2008) 122:197–206
DOI 10.1007/s10658-008-9334-x
Effects of garlic (Allium sativum) juice containing allicin
on Phytophthora infestans and downy mildew of cucumber
caused by Pseudoperonospora cubensis
Daniela Portz & Eckhard Koch & Alan J. Slusarenko
Received: 29 January 2008 / Accepted: 8 May 2008
# KNPV 2008
Abstract The volatile antimicrobial substance allicin
(diallylthiosulphinate) is produced in garlic when the
tissues are damaged and the substrate allicin (Sallyl-L-cysteine sulphoxide) mixes with the enzyme
alliin-lyase (E.C.4.4.1.4). Allicin undergoes thioldisulphide exchange reactions with free thiol groups
in proteins and it is thought that this is the basis of its
antimicrobial action. At 50 μg ml-1, allicin in garlic
juice inhibited the germination of sporangia and cysts
and subsequent germ tube growth by Phytophthora
infestans both in vitro and in vivo on the leaf surface.
Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic
juice containing allicin over the range tested (55–
110 μg ml−1) with an effectiveness ranging from
approximately 45–100%. Similarly, in growth room
experiments at concentrations from 50–1,000 μg ml−1,
allicin in garlic juice reduced the severity of cucumber
D. Portz : A. J. Slusarenko (*)
Department of Plant Physiology,
RWTH Aachen University,
52056 Aachen, Germany
e-mail: Alan.slusarenko@bio3.rwth-aachen.de
E. Koch
Federal Research Centre for Cultivated Crops (JKI),
Institute for Biological Control,
Heinrichstr. 243,
64287 Darmstadt, Germany
downy mildew caused by Pseudoperonospora cubensis
by approximately 50–100%. These results suggest a
potential for developing preparations from garlic for
use in specialised aspects of organic farming, e.g. for
reducing pathogen inoculum potential and perhaps
for use under glass in horticulture.
Keywords Natural fungicides . Tomato leaf blight .
Plant antibiotic . Antimicrobial . Phytoanticipin
Introduction
Downy mildews and diseases caused by oomycetes in
general are among the most destructive and economically important agricultural problems world-wide.
According to Gisi (2002) almost 17% of the world
fungicides market in 1996 was for agents used in
downy mildew control. Effective control by planting
resistant varieties is in many cases not possible and
disease management problems have been compounded
by the emergence of fungicide-resistant/tolerant variants of several oomycete pathogens (Gisi 2002; Urban
and Lebeda 2006, 2007; Urban et al. 2007). Furthermore, the increasing public demand for organicallygrown produce, and the intended phasing out by the
EU of the use of copper-containing formulations, has
precipitated an urgent need for alternative control
methods. In this regard resistance-inducing treatments
and substances conditioning systemic acquired resis-
198
Eur J Plant Pathol (2008) 122:197–206
tance (SAR) are considered an alternative (MauchMani 2002; Körösi et al. 2007) and there is increased
interest in developing treatment strategies based on
natural plant defence products (KonstantinidouDoltsinis and Schmitt 1998; Konstantinidou-Doltsinis
et al. 2006; Slusarenko et al. 2008).
We have reported previously that the natural
antimicrobial substance allicin, which is a volatile
phytoanticipin produced in garlic (Allium sativum)
upon wounding, is active against a broad range of
phytopathogenic organisms in vitro and in planta
(Curtis et al. 2004) and indeed there are several
reports of garlic preparations containing allicin being
used to treat plant disease (e.g. Ark and Thompson
1959; Russell and Mussa 1977). Allicin (diallylthiosulphinate) is produced in garlic when the substrate
alliin (S-allyl-L-cysteine sulphoxide) mixes with the
enzyme alliinase (alliin-lyase, E.C.4.4.1.4; see diagram below). The antimicrobial
2
O
H
S
C
O
alliinase
H2O
NH2
COOH
alliin
S
+ 2pyruvate + 2NH 3
S
allicin
activity of garlic juice had long been known and
Cavallito and Bailey (1944) showed that this activity
was due to allicin, which they reported to be as active
against test bacteria as penicillin. Allicin crosses the
cell membrane easily and undergoes thiol-disulphide
exchange reactions with free thiol groups in proteins
(see diagram below). It is thought that these properties
are the basis of its antimicrobial action (Miron et al.
2000; Rabinikov et al. 1998). Allicin thus has several
O
2R
SH +
thiol
S
S
allicin
2R
S
S
+ H2O
mixed disulphide
targets in the cell and this makes it difficult for
organisms to develop resistance to it.
The use of natural products in plant protection,
either directly or as starting points for targeted
enhancement of desirable qualities by industry, has
been reviewed recently (Slusarenko et al. 2008) and
the current paper presents results using garlic juice
containing allicin to combat diseases caused by the
important plant pathogenic oomycetes Phytophthora
infestans and Pseudoperonospoa cubensis. The effect
of allicin in garlic juice was tested on the germination
rate and subsequent germ tube growth of sporangia
and cysts of P. infestans in vitro and in vivo on the
surface of tomato leaves. The effectivity of allicin in
garlic juice was tested in reducing leaf infection of
tomato seedlings by P. infestans, and cucumber
seedlings by Pseudoperonospora cubensis was also
tested under growth room conditions.
Materials and methods
Cucumber/Pseudoperonospora cubensis
Plant cultivation
Plants were cultivated in 8×8 cm plastic pots filled with
a 1:2 parts mixture of sand: commercial potting substrate
(Fruhstorfer® Erde Typ T; Industrie-Erdenwerk Archut,
Lauterbach). Twelve seeds of Cucumis sativus cv.
Chinesische Schlange were sown in each pot. The pots
were watered carefully and kept in a growth room at
20°C (cycle of 16/8 h light/dark). After 1 week the
seedlings were transplanted to fresh pots (one plant per
pot).
Inoculation
P. cubensis was maintained on plants grown as
outlined above. Fresh inoculum was prepared from
plants 10 days after previously being inoculated with
P. cubensis. Plants were incubated overnight in a
moist chamber to encourage sporulation and sporangia were harvested by washing the lower leaf surface
with water. The resulting suspension was adjusted to
5×103 sporangia ml−1 using a haemocytometer.
Plants were harvested approximately 3 weeks after
transplanting when the second true leaf was expanded. The upper, non-expanded leaves were excised and
the first and second leaves sprayed with the treatment
solution on both sides using a chromatography
sprayer. Control plants were sprayed with water or
with 0.2% Cuprozin Flüssig ™ (460.6 g l−1 copper
hydroxide) (Spiess-Urania, Hamburg). After 24 h the
first and second leaves were inoculated using a
chromatography sprayer on both sides with a suspension of P. cubensis sporangia (5×103 ml−1). The pots
were then incubated overnight at 15°C in a moist
chamber and the following day returned to the growth
room. Disease was rated 2 weeks after inoculation by
Eur J Plant Pathol (2008) 122:197–206
estimating the percentage of the affected leaf area. The
effectivity of the treatment was calculated according to
Abbott (1925):
% Effectivity ¼
affected leaf area ðcontrolÞ affected leaf area ðtreatmentÞ
100
affected leaf area ðcontrolÞ
199
allowed to dry (approx. 2 h) before being sprayinoculated. As a soil drench a single application of
5 ml of the appropriate dilution of garlic juice was
applied per 7×7 cm pot containing a single plant.
Five to seven intact tomato seedlings were inoculated
per experiment and each experiment was repeated at
least three times. A representative set of results for
each experiment is shown.
Tomato/Phytophthora infestans
Preparation of garlic juice
Plant cultivation
Tomato seeds (cv. Hoffmanns Rentita®, Schmitz &
Laux GmbH, Hilden, Germany) were sown in
seedling trays for germination in moist potting
compost covered with fine moistened sand and
incubated at 22°C in a light/dark cycle of 16/8 h.
After germination, 1 week-old seedlings were transferred to individual 7×7 cm pots and grown on for a
further 2 weeks.
Inoculation
The P. infestans isolate used in this work was kindly
donated by Bayer CropScience AG, Monheim. The
virulence of the isolate was ensured by regular
passaging through potato tuber discs. Phytophthora
infestans was cultivated under sterile conditions on
tomato juice agar (TJA) at 18°C in the dark (TJA=3 g
CaCO3, 12 g PDB (Difco™), 20 g agar (AppliChem
GmbH), 200 ml tomato juice (Fa. Krings Fruchtsaft
GmbH, Mönchengladbach) made up to a volume of
1 l and autoclaved at 121°C for 15 min). Phytophthora infestans inoculum was prepared by washing
the surface of 8 day-old Petri plate cultures with
cold (10°C), sterile deionized water and sieving
through a plastic kitchen sieve. Sporangia were
adjusted to a concentration of 4–5×104 ml−1 with a
haemocytometer. Zoospores were released from sporangia after approximately 2 h at 10–12°C. After
spray-inoculation, plants were placed in a seedling
tray and covered with a transparent plastic lid in the
growth chamber at 20°C with a light/dark cycle of
16/8 h.
Treatment with garlic juice
Unless otherwise stated, 3 week-old tomato plants
were sprayed with diluted garlic juice and the leaves
Garlic bulbs were purchased from the supermarket
and stored at 4°C in the dark until required. Axillary
buds from the composite garlic bulb were peeled,
weighed and a domestic juicer (Turmix Fabr. Nr.
1068, Turmix AG, 8645 Jona, Switzerland) was used
to extract the juice. The juice was poured into a sterile
50 ml Falcon tube and centrifuged at 5,000 rpm
(3,000×g) for 10 min in order to separate the majority
of the pulp from the liquid (Megafuge 1.0R, Heraeus
Instruments, Osterode, Germany). Floating debris was
removed from the top of the liquid with a spatula and
discarded. Filtering under pressure separated the
remaining pulp from the pure extract (Diaphragm
Vacuum Pump, Vacuubrand GmbH + Co., Wertheim,
Germany). The filtrate was transferred into a second
sterile 50 ml Falcon tube and sealed. The average
yield was approximately 1 ml of extract from 3 g
fresh weight of garlic tissue and typically contained
approximately 5 mg ml−1 allicin (determined by
HPLC). The garlic extract was used either immediately after appropriate dilution or stored undiluted at
10°C for a maximum of 2 weeks. Dilutions were
carried out with de-ionized water. Appropriate
amounts of stock solution to give the required end
dilution in Petri plates were incorporated into agar
medium kept just molten at 45°C. Plates were poured
immediately after adding and mixing the stock.
Determination of allicin by HPLC
The method used was based on that of Krest and
Keusgen (2002). Garlic juice was diluted 1:10 with
HPLC-grade water and 1.5 ml of a 0.05 mg ml−1
solution (in methanol) of butyl-4-hydroxybenzoate
(internal standard). To protect the column, this
mixture was first filtered through a polyethersulfonmembrane (0.2 μm pore size, Steriflip, Millipore)
before 20 μl were injected into the HPLC (Kontron
200
system with diode array detector, Kontron Instruments GmbH, Neufahrn). Using the HPLC software
Geminyx (version 1.91) a mixed gradient elution
(solvent A, 30% (v/v) HPLC grade methanol adjusted
to pH 2.0 with 85% (v/v) orthophosphoric acid;
solvent B, 100% HPLC grade methanol) was performed. Spectra were recorded between 200–600 nm
during elution with detection at 254 nm for the
chromatogram.
Effect of garlic juice on P. infestans sporangium
and cyst germination in vitro
Droplets (20 μl) of inoculum suspension, prepared as
described above and containing sporangia and zoospores, were pipetted onto the surface of 1% agar
containing 50 μg ml −1 allicin. Control plates
contained no allicin. Plates were sealed with Micropore™-tape and incubated in a plastic container with
moistened tissue paper at 18°C in the dark for 4 h.
Germination rate and germ tube length were measured
using a microscope (Leica DM R) at 50- to 200-fold
magnification. At least 50 sporangia or encysted
zoospores were scored for germination per plate and
photographed using a JVC digital camera (KY-F75U)
and Discus software (Version 32, Hilgers Co.,
Königswinter, Germany). Germ tube lengths of at
least 15 germinated sporangia or cysts were measured
per plate.
Effect of garlic juice on P. infestans sporangium
and cyst germination in vivo on tomato leaves
After spraying 3-week-old tomato plants with diluted
garlic juice containing 50 μg ml−1 allicin and
allowing them to dry, leaves were excised and placed
in plastic boxes (12×12 cm) on moistened tissue
paper. Droplets (20 μl) of sporangial or cyst suspensions were then pipetted onto the leaves and the lids
placed on the boxes for incubation for 4 h in the dark
at 20°C. The leaf lamina under the droplets was then
excised and stained with acid fuchsin (modified after
Carmichael 1955). Excised leaf segments were fixed
and decolourised for 48 h at 60°C in aqueous chloral
hydrate (2.5 g ml−1). Leaf segments were then stained
for 1–2 h in 0.01% acid fuchsin-lactophenol solution
and de-stained in 50% (v/v) glycerol before viewing
using a confocal laser-scanning microscope (Leica
TCS SP, using Leica software TCS NT) at 630- to
Eur J Plant Pathol (2008) 122:197–206
1000-fold magnification (excitation 543 nm; emission
filter 575–640 nm, 63× PL APO w, and 100× PL
FLUOTAR oil objective lenses).
Statistical treatments
Raw data were first tested for normal distribution and
variance homogeneity using Sigmastat® 3.1 (SYSTAT
software 2004) to a limit of P≤0.05. If the data
showed normal distribution and variance homogeneity they were subjected to parametric statistic tests to
show significant differences (t-test or one-way
ANOVA) to a probability of P≤0.05. Non-normal
data were analysed with either the Mann–Whitney
Rank Sum Test for two groups or the Kruskal–Wallis
ANOVA on Ranks for more than two groups. If these
treatments pointed to a significant difference between
groups, a post hoc test (Dunn’s or Tukey’s) was used
to determine which groups differed significantly at the
P≤0.05 level.
Results
Pseudoperonospora cubensis/Cucumis sativus
pathosystem
Cucumber plants were sprayed with either dilutions of
garlic juice, water (untreated controls) or Cuprozin™,
and spray-inoculated the next day with a suspension
of sporangia of P. cubensis (Fig. 1A). Two weeks
after inoculation infected leaf areas were estimated
(Fig. 1B, Table 1). Dilutions of garlic juice over a
wide range of allicin concentrations (50–1,000 μg
ml−1) led to a reduction in disease severity which
compared favourably with the degree of disease
control achieved with a copper-containing commercial fungicide (Cuprozin™).
Phytophthora infestans/Lycopersicon esculentum
pathosystem
Effects of garlic juice on P. infestans germination
and growth in vitro
The effect of garlic juice on P. infestans in vitro was
assessed by investigating the effects on sporangial
and cyst germination and on germ tube growth. Garlic
juice (50 μg ml−1 allicin) caused a clear reduction in
Eur J Plant Pathol (2008) 122:197–206
201
100
Water
50 µg ml–1 allicin
Germination [%]
80
60
a
a
40
b
b
20
0
Sporangia
Cysts
Fig. 2 Influence of garlic juice in agar (50 μg ml−1 allicin) on
the germination of sporangia and encysted zoospores of P.
infestans (in vitro). Means of nine replicate Petri plates of
sporangia and cyst preparations. Columns which differ significantly from one another are marked with a different letter (ttest, P≤0.05)
Effects of garlic juice on P. infestans germination
and growth in vivo
Fig. 1 Leaf of cucumber showing A, the spray inoculation
procedure and B, symptoms 14 days after inoculation with P.
cubensis (5×103 sporangia ml−1)
the germination of encysted zoospores and of sporangia under conditions where they germinate directly
with a germ tube (i.e. behave like conidia) (Fig. 2).
Hyphal growth from germinated sporangia or cysts
was also reduced by the presence of garlic juice in the
medium (50 μg ml−1 allicin) (Fig. 3).
The behaviour of sporangia and cysts on the tomato leaf
surface after treatment with garlic juice is shown in
Fig. 4. It can be seen that the inhibitory in vitro effects
of garlic juice are mirrored in the in vivo behaviour of
sporangia and cysts on the tomato leaf surface.
Effects of garlic juice on disease severity in tomato
leaves inoculated with P. infestans
To assess whether the inhibitory effects of garlic juice
on P. infestans observed in vitro and in vivo on the
leaf surface translated into an effect on disease
development, a systematic investigation on tomato
Table 1 Effect on disease severity of spraying garlic juice containing allicin at the concentrations shown onto leaves of 40-day-old
cucumber plants 24 h prior to spray inoculation with 5×103 conidia ml−1 of P. cubensis
Treatment
Water control
Allicin 1000 μg ml−1
Allicin 500 μg ml−1
Allicin 200 μg ml−1
Allicin 100 μg ml−1
Allicin 50 μg ml−1
Cuprozin™ (0.2%)c
Average effectivitya (%)
Infected leaf area (%) ±SD
Experiment 1
Experiment 2
Experiment 3
73.3±17.1
N.T.b
N.T.
3.7
19.0
8.2
N.T.
33.8±8.9
0.2
1.0
2.8
N.T. ±
N.T. ±
20.0±9.8
81.8±9.9
0.4
1.0
2.0
N.T.
N.T.
20.0±11.9
Plants (four per experiment, eight leaves in total) were scored 2 weeks after inoculation.
a
According to Abbott (1925), see “Materials and methods” section
b
N.T. Not tested
c
Equivalent to 0.92 g Cu(OH)2 l−1
>99
96–98
84–94
55
52
41–76
202
A
Average germtube length [µm]
Fig. 3 Influence of garlic
juice on germ tube growth
from germinating sporangia
and encysted zoospores of
P. infestans (in vitro). A
Means of ∼75 measurements (sporangia) and ∼135
measurements (cysts). Columns which differ significantly from one another are
marked with a different letter (Mann–Whitney Test,
P≤0.05). B Untreated sporangia. C Untreated cysts. D
Sporangia on agar incorporating garlic juice to give a
final concentration of 50 μg
ml−1 allicin. E Cysts on agar
incorporating garlic juice to
give a final concentration of
50 μg ml−1 allicin. Bar=
50 μm
Eur J Plant Pathol (2008) 122:197–206
800
a
Water
50 µg ml–1 allicin
700
600
500
400
300
200
a
b
100
b
0
leaf infections was carried out. Firstly, potential
phytotoxic effects of garlic juice on leaves were
monitored. As shown in Table 2, spraying tomato
leaves of 3-week-old plants with dilutions of garlic
juice containing 200–800 μg ml−1 allicin led to leaf
damage in category 2 (<2.5% of the leaf area showing
chlorosis or necrosis), the least severe, and only at the
highest concentration tested.
The effect on disease development of spraying
tomato leaves with a single application of garlic juice
containing a range of allicin concentrations 2 h before
inoculation with P. infestans is shown in Fig. 5 (for a
photograph showing the appearance of control and
allicin-treated leaves see Fig. 6 in Slusarenko et al.
Sporangia
Cysts
B
D
C
E
2008). In the experiment shown in Fig. 5, control
tomato plants had lesions covering 77% of the leaf
area 4 days after inoculation (dai). As can be seen,
spraying with garlic juice very effectively reduced
disease development, with a 1:50 dilution (110 μg
ml−1 allicin) suppressing lesion development completely (Fig. 5).
The effectivity of a single pre-inoculation spray
with garlic juice containing a low concentration of
allicin (60 μg ml−1), which did not completely
suppress disease development, decreased with time
but was still apparent 10 dai (data not shown). Thus,
in plants treated with 60 μg ml−1 allicin the affected
leaf area increased from 16% at 4 days to 37% at 10
Eur J Plant Pathol (2008) 122:197–206
203
Fig. 4 Influence of garlic
juice on germ tube growth
from germinating sporangia
and encysted zoospores of
P. infestans on the tomato
leaf surface (in vivo) shown
after acid fuchsin staining
under a confocal laser scanning microscope excitation,
543 nm, emission, 575–
640 nm; Scale bars = 50 µm
(A & B), 25 µm (C & D). A
Untreated sporangia showing germination and healthy
germ tube growth. B Sporangia on a leaf sprayed
with garlic juice (50 μg
ml−1 allicin) approximately
2 h prior to inoculation,
germinated at a lower rate
and has formed abnormal
germ tubes with reduced
growth. C Untreated cyst
showing normal germ tube
growth. D Ungerminated
cysts on a leaf sprayed with
garlic juice (50 μg ml−1
allicin)
dai. In the untreated controls, however, the infected
leaf area was 60% after 4 days and increased to 63%
by 10 dai.
The effect of various garlic juice application times
in relation to the time of inoculation with P. infestans
was investigated and it was found that the nearer to
the inoculation time that allicin was sprayed, the more
effective a given dosage was in suppressing disease
development (Fig. 6). In contrast, spraying leaves
with garlic juice 24 h after inoculation had little
effect. Direct spraying onto leaves was also compared
with a single application as a soil drench. As can be
seen in Fig. 7, allicin was more effective when
sprayed on the leaves than when applied to the soil.
Discussion
Curtis et al. (2004) previously reported that dilutions
of garlic juice containing allicin were effective in
reducing the production of conidiophores and
Table 2 Phytotoxicity scores for individually potted 3-weekold tomato seedlings sprayed to run-off with dilutions of garlic
juice containing various concentrations of allicin
Concentration of allicin in garlic juice
(μg ml−1)
0
200
400
800
Phytotoxicity
categorya
1
1
1
2
a
Phytotoxicity categories (Gorog née Privitzer et al. 1988): 1=
no damage, 2=<2.5% leaf area damaged (showing chlorosis or
necrosis), 3=<5% leaf area damaged. The scale progresses to
9=100% leaf damage. Scores<2 are considered acceptable in
screens of potential candidates for plant protection substances.
Plants were allowed to dry, and then pots were placed under
plastic hoods for 4 days before the hoods were removed. Plants
were incubated in a growth chamber (22°C, cycles of 18 h light
6 h dark) and scored 6 days after spraying.
204
Eur J Plant Pathol (2008) 122:197–206
120
bc
Effectivity [%]
Effectivity [%]
100
80
60
ab
40
20
b
100
c
bc
80
b
ab
a
a
60
40
20
a
0
Control
1:100
1:85
1:65
1:50
dilution
dilution
dilution
dilution
55 µg ml–1 65 µg ml–1 85 µg ml–1 110 µg ml–1
allicin
allicin
allicin
allicin
Fig. 5 Dose-dependency of disease control by allicin in garlic
juice in the P. infestans/tomato leaf pathosystem. Three-weekold plants (cv. Hoffmans Rentita) were sprayed with garlic
juice, the leaves allowed to dry (approx. 2 h) and then sprayinoculated with 4–5×104 sporangia ml−1. The effectivity of
treatment (Abbot 1925) is shown at 4 dai. Columns which
differ significantly from one another are marked with a different
letter (Dunn’s Test, P≤0.05)
oospores in downy mildew of Arabidopsis caused by
Hyaloperonospora parasitica. In the present study
these observations are extended to show that macroscopic disease symptoms of cucumber downy mildew
can be markedly reduced by spraying the leaves with
garlic juice containing a range of allicin concentrations 24 h prior to inoculation. The disease reduction
compared very favourably with a commercial copperfungicide treatment and suggests that development of
garlic products for at least small-scale application
such as in glasshouse situations might be feasible and
100
b
Effectivity [%]
80
60
a
40
a
20
a
0
48 h before
inoculation
24 h before
inoculation
2 h before
inoculation
24 h after
inoculation
Fig. 6 Influence of time between treatment with garlic juice
(70 μg ml−1 allicin) and time of inoculation on effectivity in the
P. infestans/tomato pathosystem at 4 dai. Columns which differ
significantly from one another are marked with a different letter
(Dunn’s Test, P≤0.05)
0
250 µg ml–1 100 µg ml–1 150 µg ml–1 100 µg ml–1 50 µg ml–1
allicin
allicin
allicin
allicin
allicin
drench
drench
spray
spray
spray
Fig. 7 Comparison of the effectivity of garlic juice containing
allicin as a soil drench or a foliar spray in the P. infestans/
tomato pathosystem at 4 dai. Columns which differ significantly from one another are marked with a different letter (Tukey’s
Test, P≤0.05)
desirable as an alternative to standard treatments
(Fig. 1, Table 1). Resistance of P. cubensis against
conventional fungicide treatments is increasing (Urban and Lebeda 2006, 2007; Urban et al. 2007) and
because allicin appears to have a multi-site mode of
action (Portz et al. 2005; Slusarenko et al. 2008) it
will presumably be difficult for pathogens to mutate
to resistance against it, thus conferring a strong
advantage on allicin-based disease treatments.
The inhibitory effect of allicin on the vegetative
mycelial growth of P. infestans and the reduction of
potato tuber colonization by allicin in the gas phase
have been reported previously (Curtis et al. 2004).
Now, the inhibitory effects of garlic juice containing
allicin on the germination of sporangia and encysted
zoospores and subsequent reduction in germ tube
growth, both in vitro and on the tomato leaf surface
(Figs. 2, 3, 4) are reported. These effects presumably
contribute to the reduction in infection seen in
inoculated tomato seedlings (Fig. 5). The tomato
leaf/P. infestans pathosystem was used in preference
to potato/P. infestans because it is easier to work with
in the laboratory. Nevertheless, since it appears that
the effect of allicin is directly against the pathogen,
rather than via an induced resistance mechanism
(Curtis et al. 2004), it seems likely that a similar
degree of control might be expected in the potato/P.
infestans pathosystem, particularly in view of the
effects of allicin in reducing tuber colonisation at least
under controlled conditions (Curtis et al. 2004). The
effectivity of garlic juice in reducing disease in
tomato leaves was very high and approached 100%
Eur J Plant Pathol (2008) 122:197–206
at an allicin concentration of 110 μg ml−1 (Fig. 5). In
fungicide screening, substances are usually only
considered for further development if they do not
cause leaf damage above category 2 (<2.5% leaf area
affected) on a scale of 1–9 (Gorog née Privitzer et al.
1988) (see Table 2). Garlic juice was assessed at
various dilutions for phytotoxicity, and disease control was achieved at allicin concentrations well below
those where phytotoxicity was observed (Table 2,
Fig. 5). Thus, allicin in garlic juice would not be
excluded in a conventional screening programme
based on this criterion.
The effectivity of the allicin treatment in reducing
disease on tomato seedlings is more pronounced in
the early stages after treatment. If allicin is working
mainly via a reduction of successful infections by
killing a certain proportion of the spores and
subsequently by suppressing germ tube growth from
surviving propagules, then a time-lag in disease
development would be expected until inoculum levels
had reached those present before the sanitation
treatment. However, the dynamics of disease development in fungicide-treated plants are difficult to
model and disease development often deviates from
the ideal mathematical description (Jeger 1987). In
control plants not treated with allicin, the disease level
4 dai was already high and this increased only
marginally in subsequent days. In the allicin-treated
plants the affected leaf area increased from 16% at
4 days to 37% by 10 dai. Thus, even a single
treatment with allicin at a dose (60 μg ml−1) below
that necessary to completely eradicate disease
(∼110 μg ml−1, see Fig. 5) is already effective at
reducing the rate of disease progress over a substantial time period.
The data presented in Fig. 6 show the effectivity of
a single allicin treatment in relation to the time of
inoculation and support a low-persistence, contactfungicide type of effect for allicin. Thus, the effectivity of the treatment increases with decreasing time
before inoculation (from 48 to 24 h), is maximal when
inoculation takes place approximately 2 h after
treatment with garlic juice, and is least effective at
later times after inoculation (e.g. 24 h) when the
pathogen has already penetrated the leaf and is
perhaps less easily accessed by allicin. In this regard
the kinetics of allicin behaviour on the leaf surface,
and its uptake by the leaf, are aspects which need
further investigation.
205
In downy mildew of Arabidopsis it was shown that
treatment of the plant with garlic juice did not lead to
the accumulation of SAR markers (Curtis et al. 2004)
and the authors suggested that garlic juice was
exerting its antimicrobial effect directly on the
pathogen rather than via inducing SAR in the plant.
The data presented in Fig. 6 for tomato support this
conclusion and extend it to a further pathosystem.
Interestingly, in the tomato/P. infestans pathosystem, applying garlic juice as a soil drench was also
effective at reducing disease levels, although a better
degree of control was achieved with lower concentrations of allicin as a direct spray on the leaves
(Fig. 7). As stated earlier, allicin appears to act
directly against the pathogen and it is unclear whether
the disease reduction after applying garlic juice as a
soil drench is due to the action of allicin against the
pathogen via the gas phase, or whether allicin is also
taken up via the roots and transported systemically
within the plant. Allicin is readily membrane-permeable (Miron et al. 2000; Rabinikov et al. 1998;
Slusarenko et al. 2008) and could therefore enter the
symplast in the roots, but whether it is transported
within the plant is unknown at present. In this regard,
it is perhaps important to mention that it is difficult to
quantify allicin in the gas phase because the temperature of the injection port in the GC is too high and
leads to modifications producing other polysulphides
(Block 1992).
The potential for allicin in garlic juice to be used as
an effective control agent against diseases caused by
oomycetes is clear, although there is scope for
optimisation of treatment regimes, and field testing
is certainly necessary. Very clearly, transfer from the
laboratory to the field/glasshouse is a stumbling block
which many otherwise promising compounds fail to
negotiate successfully (Slusarenko et al. 2008). This
may also prove true for allicin in garlic juice. Also, it
will be necessary to carry out organoleptic assessment
of harvested plant parts to ensure the absence of
undesireable flavour notes in any development of
garlic products for plant protection. Neither garlic
juice nor allicin are named presently as plant
protection substances specifically permitted for organic farming in the EU (Directive 2092/91). However, it is not likely that these substances, which are a
common foodstuff or a component thereof, have
properties that would not allow them to be added to
the list in the future. Furthermore, chemical modifi-
206
cation of allicin, which has an activity comparable to
several conventional antibiotics (Cavallito and Bailey
1944; Curtis et al. 2004; Slusarenko et al. 2008), to
enhance its desirable properties and reduce its
undesirable ones, might even lead to a new multitarget plant protection compound useable in conventional agriculture and horticulture.
Acknowledgements RWTH Aachen University provided a
student assistantship (D.P.) and financial support. Technical
assistance by Ulrike Noll (Aachen) and Monika Eitzen-Ritter
(Darmstadt) is gratefully acknowledged. Ales Lebeda and
Nikolaus Schlaich are thanked for critical reading of the
manuscript.
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