Pesticidal Plants

Pesticidal Plants
From Smallholder Use

to Commercialisation
Edited by
Philip C. Stevenson, Steven R. Belmain and Murray B. Isman
Printed Edition of the Special Issue Published in Plants
www.mdpi.com/journal/plants
Pesticidal Plants
Pesticidal Plants
From Smallholder Use to Commercialisation
Special Issue Editors

Philip C. Stevenson
Steven R. Belmain
Murray B. Isman
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

Philip C. Stevenson Steven R. Belmain Murray B. Isman
University of Greenwich University of Greenwich University of British Columbia
UK UK Canada
Editorial Office
MDPI
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This is a reprint of articles from the Special Issue published online in the open access journal Plants
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Contents
About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface to ”Pesticidal Plants” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Sara Vitalini, Francesca Orlando, Valentina Vaglia, Stefano Bocchi and Marcello Iriti
Potential Role of Lolium multiflorum Lam. in the Management of Rice Weeds
Reprinted from: Plants 2020, 9, 324, doi:10.3390/plants9030324 . . . . . . . . . . . . . . . . . . . . 1
G. Mandela Fernández-Grandon, Steven J. Harte, Jaspher Ewany, Daniel Bray and

Philip C. Stevenson
Additive Effect of Botanical Insecticide and Entomopathogenic Fungi on Pest Mortality and the
Behavioral Response of Its Natural Enemy
Angela G. Mkindi, Yolice L. B. Tembo, Ernest R. Mbega, Amy K. Smith, Iain W. Farrell,
Patrick A. Ndakidemi, Philip C. Stevenson and Steven R. Belmain
Extracts of Common Pesticidal Plants Increase Plant Growth and Yield in Common Bean Plants
Marı́a J. Pascual-Villalobos, Manuel Cantó-Tejero, Pedro Guirao and Marı́a D. López

Fumigant Toxicity in Myzus persicae Sulzer (Hemiptera: Aphididae): Controlled Release of
(E)-anethole from Microspheres
Kelita Phambala, Yolice Tembo, Trust Kasambala, Vernon H. Kabambe, Philip C. Stevenson
and Steven R. Belmain
Bioactivity of Common Pesticidal Plants on Fall Armyworm Larvae (Spodoptera frugiperda)
Kateřina Kovařı́ková and Roman Pavela

United Forces of Botanical Oils: Efficacy of Neem and Karanja Oil against Colorado Potato
Beetle under Laboratory Conditions
Angela G. Mkindi, Yolice Tembo, Ernest R. Mbega, Beth Medvecky, Amy Kendal-Smith,
Iain W. Farrell, Patrick A. Ndakidemi, Steven R. Belmain and Philip C. Stevenson
Phytochemical Analysis of Tephrosia vogelii across East Africa Reveals Three Chemotypes that
Influence Its Use as a Pesticidal Plant
Liliana Ruiz-Vásquez, Matı́as Reina, Vı́ctor Fajardo, Matı́as López and

Azucena González-Coloma
Insect Antifeedant Components of Senecio fistulosus var. fistulosus—Hualtata
Naomi B. Rioba and Philip C. Stevenson

Opportunities and Scope for Botanical Extracts and Products for the Management of Fall
Armyworm (Spodoptera frugiperda) for Smallholders in Africa
v
Victor Jaoko, Clauvis Nji Tizi Taning, Simon Backx, Jackson Mulatya, Jan Van den Abeele,
Titus Magomere, Florence Olubayo, Sven Mangelinckx, Stefaan P.O. Werbrouck and
Guy Smagghe
The Phytochemical Composition of Melia volkensii and Its Potential for Insect Pest Management
Ricardo A. Rincón, Daniel Rodrı́guez and Ericsson Coy-Barrera

Botanicals Against Tetranychus urticae Koch Under Laboratory Conditions: A Survey of
Alternatives for Controlling Pest Mites
vi
About the Special Issue Editors
Philip C. Stevenson is a Professor of Plant Chemistry at the Natural Resources Institute in
the University of Greenwich (UK) where he leads Chemical Ecology research. He holds a dual
position as an NERC Merit researcher and Head of Biological Chemistry and In Vitro research at
the Royal Botanic Gardens, Kew (UK).
Phil’s research has focussed on the biological and ecological functions of plant chemicals and
understanding how these compounds can be used in support of sustainable agriculture. His work
includes research on pollen and nectar chemistry to determine their role in pollinator behaviour
and health and behavioural ecology, natural pest resistance in crops to identify breeding traits and
the optimisation of pesticidal plants (botanical insecticides) as benign and affordable alternatives to
synthetic insecticides. Phil’s research is or has been funded by UK Research and Innovation (BBSRC),
the European Union, Darwin Initiative, USDA and NSF (USA) and the Pewter Sowerby Foundation.
His research is published in over 100 international journal articles, including recent papers in Science,
Current Biology, Journal of Ecology, Ecological Monographs and Frontiers in Ecology and the Environment.
His work has been cited more than 5000 times. His international scientific role is represented through
positions on the Editorial Boards of journals including Subject Editor at the Bulletin of Entomological
Research, Regional Editor of Biopesticides International and the Editorial boards of Crop Protection,
Industrial Crops and Products, Plants (MDPI) and Plants, People, Planet. Phil also advises the UK’s
Department for Environment Food and Rural Affairs. He is a Fellow of the Royal Entomological
Society and Member of the British Ecological Society and International Society of Chemical Ecology.
Steven R. Belmain is a Professor of Ecology at the Natural Resources Institute in the University of
Greenwich (UK) where he leads the institute’s research on small mammals and wildlife management.
Steve’s research in the field of applied ecology has specialized in the sustainable management of
vertebrates and invertebrates as crop pests and disease vectors affecting people living in rural and
urban communities, improving the management of natural resources and advancing the livelihoods
of the poor. His work on the optimizing the use of pesticidal plants in sub-Saharan Africa stretches
back more than 20 years investigating their use in post-harvest insect pest management, their
application to a variety of field crops targeting a range of arthropod pests and their integration in to
agro-ecologically sustainable production systems. Further research interests include ecosystem
services, ecological pest management, agroecology and ecological engineering, entomology, chemical
ecology, eco-epidemiology of zoonoses, animal behaviour and small mammal ecology. As principal
investigator Steve has led more than 15 multidisciplinary research projects collaborating with
institutions in more than 50 countries across Europe, Africa and Asia with funding from the EU and
European governments, UN agencies, the World Bank and charitable foundations. He is Associate
Editor for the journal Wildlife Research, Fellow of the Royal Entomology Society, Fellow of the Higher
Education Academy, Coordinating member of the International Society of Zoological Sciences,
Committee member of the World Health Organization’s Global Leptospirosis Environmental Action
Network, Member of the British Ecology Society, and author of over 100 publications in peer-
reviewed journals, books and book chapters.
vii
Murray B. Isman was Professor of Entomology and Toxicology at the University of British Columbia,
Vancouver, and former Dean of the Faculty of Land and Food Systems. He performed extensive
research for over 35 years in the areas of insect toxicology and behavior, with particular emphasis
on the discovery and development of botanical insecticides. Others areas of research included
insect-plant chemical interactions, metabolism of plant toxins by insects, habituation to antifeedants
and insect memory, and non-target effects on pollinators and fish. He has authored over 200 refereed
publications (cited >20,000 times according to Google Scholar). Collaborative research with industry
has culminated in the commercialization of botanical insecticides partly developed in his laboratory
that are currently used in over a dozen countries, including the USA.
He is a former President of the International Society of Chemical Ecology, Phytochemical Society
of North America, and the Entomological Society of British Columbia. Murray has served on the
editorial boards of the Journal of Economic Entomology, Journal of Pest Science, Bulletin of Entomological
Research, Bulletin of Environmental Toxicology and Contamination, and the Journal of Chemical Ecology, and
is the founding Chief Specialty Editor (Pest Management) for Frontiers in Agronomy. He received the
Entomological Society of Canada’s Gold Medal for Excellence in Entomology, and is an elected Fellow
of the Entomological Society of America and the Royal Entomological Society (London).
viii
Preface to ”Pesticidal Plants”
Global perceptions about pesticides are changing as a consequence of their environmental
impacts, persistence, broad spectrum activities and non-target effects. As a result of this,
pesticide regulations are changing in some regions. For example, Europe has limited the
number of synthetic chemical products permitted for use in pest control. The increasing pressure on
synthetic products has reinvigorated efforts to seek alternative pest management options, including
new opportunities for plant-based solutions that are environmentally benign and tailored to different
farmers’ needs, from commercial to smallholder and subsistence farming.
This Special Issue captures some of the latest developments in research on pesticidal plants
and botanical pesticides from fundamental aspects, including the identification of bioactive plant
chemicals and the evaluation of their bioactivities against pests, mechanisms of activity, validation of
use in small-scale systems and commercial-scale pest management, including in horticulture and
disease vector control. Other work reports developments in the use of botanicals in rice weeds,
combination biopesticides and the growth-promoting properties of some plant extracts when used
to control pests. We also provide insight into how chemistry can vary dramatically and influence the
value and effectiveness of botanical insecticides in different locations. Three reviews assess wider
questions around the potential of plant-based pest management to address the global challenges of
new, invasive and established pests and previously underexploited pesticidal species of plant.
Philip C. Stevenson, Steven R. Belmain, Murray B. Isman

ix
plants
Article
Potential Role of Lolium multiflorum Lam. in the
Management of Rice Weeds
Sara Vitalini 1, *,† , Francesca Orlando 2,† , Valentina Vaglia 3 , Stefano Bocchi 3,‡ and
Marcello Iriti 1, *,‡
1 Department of Agricultural and Environmental Sciences, Università degli Studi di Milano, 20133 Milan, Italy
2 Department of Molecular and Translational Medicine (DMMT), Università degli Studi di Brescia,
25123 Brescia, Italy; francesca.orlando@unibs.it
3 Department of Environmental Science and Policy, Università degli Studi di Milano, 20133 Milan, Italy;
valentina.vaglia@unimi.it (V.V.); stefano.bocchi@unimi.it (S.B.)
* Correspondence: sara.vitalini@unimi.it (S.V.); marcello.iriti@unimi.it (M.I.)
† Those authors contributed equally to this work.
‡ Those authors contributed equally to this work.
Received: 21 February 2020; Accepted: 29 February 2020; Published: 4 March 2020
Abstract: The phytotoxic relationships between crops and weeds can cover a role in weed management,
reducing the use of chemical herbicides. Starting from the organic farmers’ experience, the study
aimed to define the inhibitory action of Lolium multiflorum Lam., used as a cover crop before rice
sowing, against Echinochloa oryzoides (Ard.) Fritsch, one of the main rice weeds. In vitro 7-day
assays were carried out in Petri dishes to compare the effect of different L. multiflorum Lam. parts,
in the form of aqueous extract or powder, on the seed germination and seedling growth of Oryza
sativa L. and E. oryzoides and to verify the hypothesis of a higher susceptibility of the weed. The
total polyphenolic content, as the potential source of allelochemicals, in the L. multiflorum parts was
measured. The results showed that both species suffer the phytotoxic action of L. multiflorum, but a
more marked effect against E. oryzoides was recorded. In according with the polyphenol quantities,
stem and inflorescence extracts showed the more significant species-specific inhibition. In all assays,
the weed showed a stronger reduction in the root length and seedling vigor index, and, in some cases,
also in the germination percentage and shoot length compared to rice.
Keywords: Italian ryegrass; barnyard grass; rice; cover crops; organic farming; weed control;
phytotoxic activity
1. Introduction
Weeds cause severe crop losses in rice production worldwide. The yield reductions in flooded
paddy fields are due to the presence of invasive aquatic and semi-aquatic species. Among them,
the species of the Echinochloa genus are the most common weeds in wetlands and water-saturated
conditions and are included in the list of the ten worst weeds in the world [1]. In particular, E. oryzoides
(Ard.) Fritsch, known as early watergrass, is a rice mimic with very similar emergence and flowering
times that allow it to achieve greater competitiveness than other Echinochloa species by influencing rice
in its early growth stages [2,3]. Therefore, any intervention able to control the incidence of this weed is
useful to give rice a competitive advantage.
Rice growers in temperate regions (Europe, US, Australia) are particularly attentive to weed
control. Within an established model of industrial agriculture, based on monoculture and high-input
systems, they face no option other than the application of synthetic herbicides because of the low
knowledge of alternative agronomic practices and plant-based solutions, as well as the unfeasibility of
hand-weeding due to the high labor costs [4]. However, the herbicide-based weed management has
Plants 2020, 9, 324; doi:10.3390/plants9030324 1 www.mdpi.com/journal/plants

Plants 2020, 9, 324
proved to be unable to solve the issues, leading to well documented resistance phenomena as in the
case of E. oryzoides [2,5,6].
In addition, the special attention of the European Union to the risks and hazards for humans,
animals and the environment associated with the use of chemical substances has led to the banning in
its member states of many herbicides, such as oxadiazon-based plant protection products, a compound
largely used in rice fields [7–9].
Given the above, there is the need to move toward new and more sustainable weed management
strategies [10,11].
In this context, the long-term experience of the organic farmers could be used to recognize
and set innovative and good practices, transferable somehow also to the conventional or integrated
systems [12,13]. They paid particular attention to solve the weed issue identified as the main cause of
yield variability and loss in the Mediterranean regions, which is the main challenge for organic rice
production [14,15].
Weed control in organic farming is carried out through crop rotation and the use of cover crops,
smother crops and green mulching, which are important for regulating the weed seed population in
the soil and the plant population in the field [16,17]. In this regard, several factors influence the weed
growth such as competition for space, water and nutrients, changes in temperature and shade as well
as toxic microbial products, soil pH and release of allelochemicals [18]. Especially allelopathy, defined
as the release of compounds from the living or dead tissues of a plant species with strong phytotoxic
effect towards another one, is a phenomenon which deserves attention in the study of alternative pest
management options, thanks to its potential action in weed suppression [19,20]. It is known that some
species or varieties, used as cover crop or crop in rotation, produce relevant amounts of allelochemicals
significantly affecting the weed germination and growth [21].
Allelopathic compounds could be used to control weeds in both organic and conventional
agriculture: in the first case, through the direct cultivation of allelopathic species, respecting the
ban on the use of any products for herbicide purposes and with the principle of low-external-input
farming [22]; while, in the second case, through the marketing and use of plant-based herbicides able
to support and integrate weed management, reducing the need for synthetic herbicides.
Particularly for rice, a participatory research carried out by Orlando and co-workers [23] with a
small group of organic farmers in North Italy identified a strategy based on the cultivation of Lolium
multiflorum Lam. as the most promising practice for weed control. In the study area (Po Valley, between
Piedmont and Lombardy regions), characterized by a typical Mediterranean climate, 94% of national
rice production is concentrated. The farming systems are mainly based on continuous flooding and a
wide use of pesticides and herbicides that caused the highest groundwater and surface water pollution
in the country [24]. L. multiflorum, known as Italian ryegrass, is used by rice growers during the
winter season as a cover crop. Then, in May, the rice is sown directly among the standing plants of
L. multiflorum and, subsequently, its biomass is mowed, chopped or rolled, producing green mulch. The
farmer’s empirical knowledge suggested to the researchers the existence of an allelopathic suppressive
action of L. multiflorum versus Echinochloa spp., with a chemical inhibition of weed germination and
growth, beyond the well-known competitive effect of green mulching.
Accordingly, the present study was aimed to verify the inhibitory activity of different organs of
L. multiflorum against E. oryzoides. Two in vitro bioassays were carried out in order to evaluate the
possible release of phytotoxic chemicals from the cover crop separately from other factors occurring
simultaneously in the field able to influence the weed growth and from the complex dynamics of the
soil seed bank. The impact of the L. multiflorum biomass aqueous extracts and its powder was assessed
versus the germination and seedling growth of both E. oryzoides and O. sativa to highlight a potential
species-specific action of L. multiflorum.
2
Plants 2020, 9, 324
2. Results
2.1. Stem Effects

The obtained data showed a significant impact of the L. multiflorum stem aqueous extract on all
the considered indices, except for the mean germination time (MGT), in both target species, but with a
more evident effect against E. oryzoides than O. sativa, (p-values ≤ 0.05 for the interaction “species × L.
multiflorum stem extract treatment”) (Table 1).
Table 1. Germination indices measured for E. oryzoides and O. sativa under the effect of different
concentrations of L. multiflorum stem extract.
Stem Extract
Germination Root Length Shoot Length
Species Concentration MGT SVI
(%) (mm) (mm)
(%)
0 100.0 ± 0.0 a 5.0 ± 0.0 56.2 ± 14.5 a 34.0 ± 3.9 a 9008 ± 1764 a
1 83.2 ± 13.6 ab 5.8 ± 0.4 64.0 ± 4.0 a 28.0 ± 4.0 ab 6613 ± 657 b
E. oryzoides
10 76.8 ± 17.4 ab 5.6 ± 0.5 62.0 ± 4.0 a 26.7 ± 0.6 b 5323 ± 227 b
20 50.0 ± 35.6 bc 5.8 ± 0.5 58.0 ± 0.0 a 26.0 ± 0.0 b 2787 ± 0 c
50 22.0 ± 22.8 c 5.0 ± 0.0 9.7 ± 6.7 b 22.0 ± 2.6 b 1198 ± 647 c
100 20.0 ± 12.2 c 5.5 ± 0.6 4.0 ± 2.2 b 14.5 ± 1.0 c 482 ± 21 c
F 13.756 2.892 36.382 22.701 43.232
p-value 0.000 * 0.052 0.000 * 0.000 * 0.000 *
0 96.8 ± 4.6 a 5.0 ± 0.0 43.0± 13.5 a 19.8 ± 2.6 a 6072 ± 1525 a
1 96.6 ± 3.5 a 5.0 ± 0.0 49.0 ± 1.0 a 19.0 ± 2.0 a 6364 ± 276 a
10 96.6 ± 3.5 a 5.0 ± 0.0 52.0 ± 3.0 a 18.3 ± 5.5 a 6805 ± 1009 a
O. sativa
20 96.6 ± 3.5 a 5.0 ± 0.7 47.0 ± 2.0 a 15.7 ± 1.5 ab 6042 ± 149 a
50 79.0 ± 12.1 a 5.0 ± 0.0 20.2 ± 10.8 b 11.0 ± 1.6 bc 2564 ± 1235 b
100 61.6 ± 21.1 b 5.2 ± 0.4 8.6 ± 2.1 b 8.6 ± 1.1 c 1003 ± 403 b
F 9.922 0.286 19.413 15.045 24.718
p-value 0.000 * 0.916 0.000 * 0.000 * 0.000 *
Interaction species × treatment
F 6.709 1.396 2.999 2.513 8.130
p-value 0.000 * 0.252 0.025 * 0.05 0.000 *
Values are mean ± standard deviation, asterisk and different letters indicate statistically significant differences at
p-value ≤ 0.05 among treatments in each species. F-value and p-value of the ANOVA test. MGT, mean germination
time; SVI, seedling vigor index.
In particular, the extract, from 20% to 100% concentration, significantly reduced the E. oryzoides
germination percentage (by 50%–80%), while O. sativa germination was affected only by 100% extract
concentration with a 36.4% decrease compared to the control. Moreover, stem extract was able to inhibit
E. oryzoides root and shoot elongation (up to 93% and 57%, respectively) by significantly lowering the
seedling vigour index (SVI) values for all used concentrations (p-value = 0.000). Otherwise, only the
treatments with 50% and 100% extract concentrations were effective on O. sativa whose SVI, root and
shoot length were reduced by 58%–83%, 53%–80% and 44%–57%, respectively.
Bioassay carried out with stem powder provided less evident effects (Table 2).
3
Plants 2020, 9, 324
quantity of L. multiflorum powdered stems.
Powdered Stem Germination Root Length Shoot Length

Species MGT SVI
Guantity (g/dm2 ) (%) (mm) (mm)
0.00 100.0 ± 0.0 5.0 ± 0.0 a 92.7 ± 3.6 a 36.5 ± 1.5 12925 ± 371 a
E. oryzoides 0.4 92.5 ± 9.6 5.3 ± 0.1 ab 43.1 ± 24.5 b 37.6 ± 3.7 7444 ± 2530 b
0.8 95.0 ± 5.0 5.4± 0.3 b 38.4 ± 11.4 b 35.3 ± 2.9 6973 ± 1180 b
F 1.964 7.226 20.427 0.780 23.126
p-value 0.186 0.011 * 0.000 * 0.482 0.000 *
0.00 90.0 ± 10.0 5.0 ± 0.1 64.7 ± 3.9 27.6 ± 1.9 8297 ± 804
O. sativa 0.4 90.0 ± 7.1 5.2 ± 0.1 55.8 ± 19.6 23.8 ± 4.1 7214 ± 2381
0.8 88.8 ± 4.5 5.1 ± 0.1 54.3 ± 11.9 21.4 ± 5.4 6632 ± 1338
F 0.118 2.000 0.878 2.923 1.321
p-value 0.890 0.178 0.441 0.092 0.303
F 0.762 3.384 6.715 1.588 5.573
p-value 0.478 0.052 0.005 * 0.227 0.011 *
No significant results were detected for O. sativa in relation to the measured indices. Similarly,
stems were not able to affect germination percentage and shoot growth of E. oryzoides. On the other
hand, at 0.4 and 0.8 g/dm2 , the treatment increased its MGT by 8% and decreased the root length up to
59% by significantly influencing SVI, reduced by 42% and 46%, respectively. In this case, the interaction
“species × L. multiflorum stem powder treatment” was significant (p-values ≤ 0.05) only for root length
and SVI.
In general, the results obtained for the L. mutiflorum stems showed a higher susceptibility of the
weed than the crop in their responses to the increasing concentrations (Tables 1 and 2).
2.2. Inflorescence Effects

Similarly to the stems, L. multiflorum inflorescence extract affected the seed development of both
studied species showing a greater inhibitory action on E. oryzoides compared to O. sativa, particularly
on the three seedling growth parameters, namely SVI, root and shoot length (p-values < 0.05 for the
interaction “species × L. multiflorum inflorescence extract treatment”) (Table 3). Otherwise, there is no
preferential effect by the extract in reducing the germination of the one of the two species.
Their germination percentage was remarkably lowered by 50% and 100% extract concentrations
(p-values = 0.000). In the first case, the germinated seeds of O. sativa and E. oryzoides were 38% and 18%,
respectively, while 100% extract concentration was able to completely inhibit them (0% germination).
In addition, the 50% extract concentration decreased root length of both species by 83% and 92% than
controls, as well as their shoot length (−33% and −70%) by significantly reducing the corresponding SVI
values (−86% and −95%). Notably, the E. oryzoides shoot elongation was also affected by inflorescence
20% extract concentration (−26%).
Like the extract, also L. multiflorum powdered inflorescences placed in direct contact with O. sativa
and E. oryzoides seeds significantly affected all measured indices, except for MGT (Table 4).
4
Plants 2020, 9, 324
concentrations of L. multiflorum inflorescence extract.
Inflorescence Mean Shoot

Germination Root Length Seedling Vigor
Species Extract Germination Length
(%) (mm) Index
Concentration (%) Time (mm)
0 100.0 ± 0.0 a 5.0 ± 0.0 66.2 ± 14.5 a 34.0 ± 3.9 a 9008 ± 1764 a
1 83.2 ± 7.5 a 5.6 ± 0.5 76.0 ± 1.0 a 33.3 ± 2.0 a 7733 ± 264 a
10 90.2 ± 5.6 a 5.8 ± 0.4 68.0 ± 9.0 a 29.3 ± 2.5 ab 8405 ± 978 a
E. oryzoides
20 94.8 ± 3.0 a 5.6 ± 0.5 62.0 ± 4.0 a 25.0 ± 0.6 b 9255 ± 772 a
50 18.0 ± 21.7 b 5.3 ± 0.6 4.3 ± 0.6 b 10.3 ± 2.1 c 407 ± 276 b
100 0.0 ± 0.0 c n.d. n.d. n.d. n.d.
F 100.627 2.179 27.971 39.963 32.699
p-value 0.000 * 0.113 0.000 * 0.000 * 0.000 *
0 96.8 ± 4.6 a 5.0 ± 0.0 43.0± 13.5 a 19.8 ± 2.6 a 6072 ± 1525 a
1 96.8 ± 5.6 a 4.6 ± 0.5 44.3 ± 1.5 a 17.7 ± 0.6 a 5794 ± 508 a
10 95.0 ± 5.4 a 4.6 ± 0.5 40.3 ± 3.5 a 18.3 ± 1.5 a 4825 ± 222 a
O. sativa
20 88.4 ± 2.6 a 5.0 ± 0.7 48.7 ± 1.5 a 15.2 ± 4.1 ab 5806 ± 327 a
50 37.0 ± 20.1 b 5.2 ± 0.4 7.2 ± 3.4 b 13.3 ± 2.5 b 825 ± 554 b
100 0.0 ± 0.0 c n.d. n.d. n.d. n.d.
F 101.674 1.385 21.825 3.155 27.790
p-value 0.000 * 0.275 0.000 * 0.048* 0.000 *
F 1.39 2.764 4.45 17.641 4.656
p-value 0.265 0.051 0.007 * 0.000 * 0.006 *
quantity of L. multiflorum powdered inflorescences.
Powdered
Inflorescence MGT SVI
(%) (mm) (mm)
Quantity (g/dm2 )
0.00 100.0 ± 0.0 a 5.0 ± 0.1 90.6 ± 5.9 a 36.2 ± 1.7 a 12688 ± 647 a
E. oryzoides 0.4 56.4 ± 35.1 b 5.2 ± 0.2 9.8 ± 9.9 b 28.8 ± 3.7 b 2649 ± 627 b
0.8 22.0 ± 31.9 b 5.6 ± 0.8 2.9 ± 0.8 b 14.9 ± 5.0 c 920 ± 57 c
F 10.165 2.121 175.721 33.891 436.013
p-value 0.003 * 0.182 0.000 * 0.000 * 0.000 *
0.00 92.0 ± 8.4 a 5.0 ± 0.0 63.2 ± 5.1 a 25.4 ± 1.3 a 8122 ± 355 a
O. sativa 0.4 72.5 ± 10.9 b 5.1 ± 0.1 12.6 ± 8.3 b 18.8 ± 6.4 b 2401 ± 1489 b
0.8 30.0 ± 12.2 c 5.0 ± 0.0 3.5 ± 1.1 c 11.3 ± 1.4 c 466 ± 223 c
F 44.506 2.889 160.545 16.963 99.309
p-value 0.000 * 0.095 0.000 * 0.000 * 0.000 *
F 2.292 3.357 15.724 2.014 21.465
p-value 0.127 0.055 0.000 * 0.160 0.000 *
The germination percentage decreased by 21%–67% and 44%–78%, respectively; the root length by
80%–94% and 89%–97%, shoot length by 26%–56% and 21%–59%, SVI by 70%–94% and 79%–93%, due
to both used quantities (0.4 and 0.8 g/dm2 ). The species showed a similar response to the treatments
both as regards the germination percentage and the shoot length (p-values > 0.05). Accordingly, the
interaction “species × L. multiflorum inflorescence powder treatment” was significant only for root
5
Plants 2020, 9, 324
length and SVI (p-values = 0.000) confirming the tendency towards greater susceptibility of E. oryzoides
shown by the previous results.
2.3. Root Effects

Unlike stems and inflorescences, L. multiflorum root extract was not able to affect, at any used
concentration, both MGT and germination percentage in the studied species. Furthermore, only
50% and 100% extract concentrations showed cases of significant impact on other considered indices
(Table 5).
concentrations of L. multiflorum root extract.
Root Extract
Species Concentration MGT SVI
(%) (mm) (mm)
(%)
0 100.0 ± 0.0 5.0 ± 0.0 66.2 ± 14.5 a 34.0 ± 3.9 a 9008 ± 1764 a
1 93.2 ± 7.5 5.6 ± 0.5 68.3 ± 3.5 a 32.0 ± 1.0 a 8740 ± 120 a
10 91.6 ± 2.6 5.6 ± 0.5 63.7 ± 1.5 a 32.3 ± 0.6 a 8787 ± 123 a
E. oryzoides
20 93.2 ± 4.6 5.4 ± 0.5 67.0 ± 7.0 a 32.0 ± 2.0 a 9557 ± 537 a
50 98.0 ± 4.5 5.2 ± 0.4 46.8 ± 4.0 b 32.2 ± 4.1 a 7734 ± 1040 a
100 92.0 ± 8.4 5.4 ± 0.5 21.8 ± 2.9 c 24.8 ± 2.7 b 4321 ± 820 b
F 1.870 1.171 22.461 5.315 14.805
p-value 0.140 0.352 0.000 * 0.004 * 0.000 *
0 96.8 ± 4.6 5.0 ± 0.0 43.0± 13.5 a 19.8 ± 2.6 a 6072 ± 1525 a
1 100.0 ± 0.0 5.0 ± 0.0 43.0 ± 4.0 a 20.7 ± 3.5 ab 6323 ± 752 a
10 95.0 ± 5.4 4.6 ± 0.5 48.7 ± 1.5 a 25.3 ± 1.5 a 6920 ± 724 a
O. sativa
20 94.8 ± 3.0 4.8 ± 0.4 47.6 ± 3.5 a 25.0 ± 2.0 a 6760 ± 579 a
50 96.2 ± 3.3 5.0 ± 0.0 57.6 ± 6.0 a 26.4 ± 4.5 a 7784 ± 1184 a
100 92.2 ± 6.1 5.0 ± 0.0 23.0 ± 6.5 b 14.4 ± 4.4 b 3304 ± 696 b
F 1.867 1.680 10.479 7.595 10.370
p-value 0.138 0.178 0.000 * 0.001 * 0.000 *
F 2.371 1.543 6.227 2.357 2.761
p-value 0.59 0.201 0.000 * 0.06 0.033 *
At 100% extract concentration, a reduction by 27% was observed in the O. sativa and E. oryzoides
shoot length (p-values < 0.05) and by about 50% for their SVI values (p-values = 0.000). Roots decreased
by 47% and 61% (p-values = 0.000), respectively.
Lastly, the significant interaction “species × L. multiflorum root extract treatment” with respect to
root length and SVI (p-values < 0.05), thanks also to the effect of the 50% extract concentration on the
roots of E. oryzoides (−17%), showed a greater inhibition of the growth of weed seedlings compared to
that of rice.
The results of L. multiflorum root powder bioassay supported previous data on E. oryzoides
showing that both used quantities (0.4 and 0.8 g/dm2 ) significantly influenced only its root elongation
(decrease between 36% and 38% compared to the control) and SVI (decrease between 29% and 30%).
Contrastingly, the powdered roots showed no effect against O. sativa, for which all the values of
the measured indices were comparable to those of controls (Table 6). On the basis of these results,
the interaction “species × L. multiflorum root powder treatment” was significant, showing a greater
reduction in E. oryzoides root length and SVI.
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quantity of L. multiflorum powdered roots.
Powdered Root Germination Root Length Shoot Length

Species MGT SVI
Quantity (g/dm2 ) (%) (mm) (mm)
0.00 100.0 ± 0.0 5.0 ± 0.1 90.6 ± 5.9 a 36.2 ± 1.7 12688 ± 647 a
E. oryzoides 0.4 94.0 ± 5.5 5.0 ± 0.1 57.9 ± 9.7 b 37.3 ± 1.4 8984 ± 1389 b
0.8 94.0 ± 5.5 5.1 ± 0.2 56.2 ± 21.9 b 39.2 ± 3.9 8872 ± 1965 b
F 3.000 1.471 9.275 1.704 11.394
p-value 0.088 0.268 0.004 * 0.223 0.002 *
0.00 92.5 ± 8.3 5.0 ± 0.0 63.2 ± 5.1 25.4 ± 1.3 8166 ± 346
O. sativa 0.4 92.0 ± 8.4 5.0 ± 0.1 62.7 ± 12.6 31.8 ± 4.5 8639 ± 1427
0.8 90.0 ± 7.1 5.1 ± 0.1 52.7 ± 8.4 29.5 ± 5.3 7478 ± 1697
F 0.139 1.600 2.063 3.205 1.015
p-value 0.872 0.242 0.170 0.077 0.391
F 0.467 0.286 4.843 1.724 6300
p-value 0.632 0.754 0.017 * 0.200 0.006 *
2.4. Seed Effects

The phytotoxic activity of L. multiflorum seeds was also assessed. Their aqueous extract impacted
similarly on both target species that achieved growth values comparable to those of their controls for
all considered indices (p-values > 0.05) (Table 7). The germination percentage of treated E. oryzoides
and O. sativa was greater than 90%. MGT was the same as for untreated seeds while the root and shoot
development showed insignificant differences as well as SVI values (p-values > 0.05).
Table 7. Germination indices measured for E. oryzoides and O. sativa under the effect of L. multiflorum
seed extract.

Species Seed Extract MGT SVI
(%) (mm) (mm)
0% 96.7 ± 5.2 5.7 ± 0.4 42.0 ± 6.5 23.5 ± 3.0 6323 ± 785
E. oryzoides
100% 91.7 ± 20.4 5.7 ± 0.1 50.8 ± 13.7 24.5 ± 7.2 6703 ± 1866
F 0.338 0.037 2.034 0.086 0.212
p-value 0.574 0.850 0.184 0.775 0.655
0% 90.0 ± 25.3 4.7 ± 0.1 51.4 ± 5.1 23.3 ± 5.1 5432 ± 2640
O. sativa
100% 98.0 ± 12.1 4.7 ± 0.1 50.1 ± 3.6 21.8 ± 2.7 6040 ± 1308
F 1.356 0.448 0.271 0.425 0.256
p-value 0.271 0.519 0.614 0.529 0.624
F 1.640 0.146 2.301 0.387 0.024
p-value 0.215 0.706 0.145 0.541 0.878
Values are mean ± standard deviation. F-value and p-value of the ANOVA test. MGT, mean germination time; SVI,
seedling vigor index.
2.5. Polyphenol Content in L. multiflorum Extracts

Figure 1 shows the polyphenol content in the aqueous extracts of the different investigated L.
multiflorum parts measured using the Folin-Ciocalteau reagent.
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Figure 1. Total polyphenols detected in the aqueous extracts of the various L. multiflorum organs.
The highest content, equal to 590 mg GAE/100 g plant part, was identified in the inflorescences
extract. Gradually lower quantities were found in stems (390 mg GAE/ 100 g), roots (190 mg GAE/
100 g) and seeds (120 mg GAE/100 g), in accordance with the decreasing phytotoxic activity recorded
for the various L. multiflorum parts against E. oryzoides and O. sativa.
3. Discussion
Different studies reported the allelopathic activity of some Lolium species including
L. multiflorum [25–28]. Usually, it is treated more as a weed capable of undermining the crop
rather than as a crop cultivated with a function in the weed control and, therefore, in the crop protection.
For example, Lehoczky and co-workers [26] described the inhibitory effects of aqueous extract obtained
from L. multiflorum shoots on some of the main grown crops such as Hordeum vulgare L., Triticum
aestivum L. and Zea mays L. However, other authors investigated the impact of decaying residues from
L. multiforum used as a cover crop on O. sativa seedling development obtaining opposite results due to
both inhibitory and stimulating effects [29–31]. To the best of our knowledge, very few data refer to
the effectiveness of L. multiflorum against the weed growth [32]. Moreover, the relationship between L.
multiflorum and E. oryzoides was never investigated.
In this context, our results are particularly interesting and can be at the basis of the weed
management strategies adopted by organic farmers who cultivated L. multiflorum before rice.
L. multiflorum showed a preferential action with impacts significantly different and more severe on
E. oryzoides rather than on O. sativa. Both L. multiflorum treatments, i.e., aqueous extracts and powder,
obtained from all the investigated organs—inflorescences, stems and roots—showed a significant
inhibitory effect on the weed. In particular, the stem and inflorescence aqueous extracts and the
inflorescence powder significantly affected both seed germination and seedling growth, while the root
aqueous extract and the stem and root powder reduced the root length. The root development of
E. oryzoides showed always a greater reduction than those of O. sativa. In addition, species-specific
phytotoxic effects were evident for the inflorescence and stem extracts, also regarding the shoot
development and seed germination.
Additionally, O. sativa suffered the inhibitory effect of the aqueous extracts from L. multiflorum.
In particular, inflorescence and stem extracts were able to reduce both the seed germination and the
seedling growth (i.e., root and shoot elongation), while the root extracts affected only the seedling
growth. On the other hand, the powder treatments showed minor activity and only those obtained
from inflorescence had significant effect, inhibiting the seed germination and the seedling growth.
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Therefore, the data showed the existence of a phytotoxic activity by L. multiflorum, instead of its
stimulating effects on rice, and, in general, a more marked action of the inflorescence, followed by
stems and roots.
Finally, the seed aqueous extract was unable to affect E. oryzoides neither O. sativa. In both bioassays,
all their growth parameters reached high values, similar to those of controls. The ineffectiveness of
L. multiflorum seeds in influencing the development of other seeds could be attributed to the fact that
the phytotoxic substances present in the cover crop are synthesized in a subsequent growth stage of
the plant.
The preferential impact of L. multiflorum on the root development confirmed previous data
documenting that the phytotoxic effect most observed in vegetative structures occurs on the root
system [20]. Nevertheless, some studies on the relationships between species showed the different
impact of the aqueous biomass extract on the measured variables. For example, Hoffman et al. [33]
reported significant inhibition of root and shoot growth, without effect on germination, while
Turk et al. [34] documented the decrease of germination and no reduction of the hypocotyl as well
as Han et al. [35] recorded the inhibition of germination and root development but no effect on the
shoots. On the other hand, the activity of phytotoxic compounds and their effects such as reduction in
seed germination and seedling growth are caused by a variety of specific interactions and cannot been
explained by just a single mode of action [28].
Lastly, polyphenols are a heterogeneous group of substances produced by the secondary
metabolism of plants, where, in relation to chemical diversity, they play different roles. They
can be simple low molecular weight compounds or complex structures conjugated with sugar
moieties useful to plants for their structure, pigmentation, pollination, defense from predators and
pathogens. Furthermore, their action as allelochemicals is known and investigated [36–39]. The different
phytotoxicity of the investigated L. multiflorum organs could be partially related to the decreasing
polyphenol values detected starting from the inflorescences.
Some allelopathic compounds were previously isolated from the aqueous leachates of decaying
L. multiflorum residues. In particular, benzenepropanoic acid has proven to be effective in inhibiting the
root and shoot growth of rice seedlings [29]. Other compounds such as caffeic acid, p-coumaric acid,
ferulic acid and hydrocinamic acid were identified in the water fraction obtained from the fermentation
of L. multiflorum shoots and roots. These phenolic acids seem to be responsible for the ability of the
extract to reduce the shoot and root elongation in different rice cultivars [31]. Furthermore, the same
type of extract was also able to affect the growth of two wheat cultivars [32].
In conclusion, the data obtained from our in vitro tests substantially confirmed the farmer empirical
observations regarding the use of L. multiflorum as a cover crop, namely the corresponding reduction
of E. oryzoides incidence, and explained the O. sativa poor density observed in certain fields under
the same practice. In their opinion, L. multiflorum appears to negatively affect both the weed and rice
development in the early growth stages, but O. sativa is less influenced than E. oryzoides, and on this
thin difference it is possible to play for giving the crop a competitive advantage over the weed.
The farmers’ empirical knowledge comes from their direct long-time experiences in managing
complex agro-ecosystems or are drawn from the rural tradition, thus including and safeguarding a
stock of precious knowledge often fragmented or lost. It could be not a coincidence that in the past
century, when the local farms combined the rice production with livestock, the cultivation of forage
species such as L. multiflorum in rotation with rice was a common practice. Hence, the study results
validate the usefulness of the farmers’ contribution in participatory research as a valuable guide for
scientific inquiry and as a support for innovations in sustainable agriculture.
Moreover, L. multiflorum could be considered the starting point to formulate new plant-based and
eco-friendly herbicides, functional to reduce the use of more dangerous synthetic compounds and the
consequent environmental pressure due to agronomic practices in the rice area.
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4. Materials and Methods
4.1. Plant Material

Seeds of O. sativa (cv. Rosa Marchetti), E. oryzoides and L. multiflorum were obtained from ‘Terre di
Lomellina’ organic farm located in the northern Italy (GPS coordinates: 45◦ 10 28.329 N 8◦ 35 44.198 E).
They were stored at 4 ◦ C until use after surface sterilization with 1% sodium hypochlorite by shaking
for 7 min and repeatedly rinsing with distilled water.
In the same farm, fresh plants of L. multiflorum were also collected. Their inflorescences, stems
and roots were separately air-dried at room temperature (25 ◦ C) in the shade and preserved in paper
bags until extraction.
4.2. Aqueous Extract Bioassay

The aqueous extract of each powdered part—inflorescences, stems and roots—of L. multiflorum was
prepared mixing a suitable amount with distilled water (1:10, w/v) and shaking it at room temperature
for 24 h. Afterwards, the mixture was filtered through gauzes to remove residues and centrifuged at
4500 rpm for 30 min. The obtained extracts were used as such (100%) and diluted with distilled water
to give final concentrations of 1%, 10%, 20% and 50%.
Otherwise, in order to simulate the leaching from seeds, 60 unsterilized seeds were placed in
30 mL of distilled water on an orbital shaker, at room temperature, for 24 h. Subsequently, the obtained
extract was filtered before use.
Ten sterilized seeds of E. oryzoides and O. sativa were sown into each Petri dish (90 mm diameter)
containing 2 filter papers and 4 mL of each extract or its dilution were added. The same volume
of distilled water was used as a control (0%). Petri dishes prepared in a vertical laminar flow hood
and sealed with parafilm were kept in a growth chamber (25 ◦ C/16 h light and 18 ◦ C/8 h dark) for
seven days.
Concerning the inflorescence, stem and root extracts, five Petri dishes were realized for each
combination of “species × L. multiflorum treatment”, according with the following randomized block
design: two species (E. oryzoides and O. sativa) × six levels of concentration (100%, 50%, 20%, 10%,
1%, 0%) × three L. multiflorum organs (inflorescences, stems and roots) × five replicates. A similar
experimental design with five repetitions was followed for the seed extract, considering only two
levels of concentration (100% and 0%) and one L. multiflorum organ (seed).
4.3. Plant Part Powder Bioassay

Different quantities (0.4 and 0.8 g/dm2 ) of each powdered part—inflorescences, stems and roots—of
L. multiflorum were spread on two filter papers in Petri dishes (90 mm diameter). Afterwards, ten
sterilized seeds of E. oryzoides and O. sativa, respectively, were placed and soaked with 5 mL of distilled
water. The same volume of distilled water was used in the control samples (0 g/dm2 of powder).
Petri dishes prepared in a vertical laminar flow hood and sealed with parafilm were kept in a growth
chamber (25 ◦ C/16 h light and 18 ◦ C/8 h dark) for seven days. Five Petri dishes were realized for each
combination of “species × L. multiflorum treatment” according with the following randomized block
design: two species (E. oryzoides and O. sativa) × three levels of quantity (0.5 g, 0.25 g, 0 g) × three
L. multiflorum organs (inflorescences, stems and roots) × five replicates.
4.4. Seedling Growth Parameter and Germination Indices

The number of germinated seeds in each Petri dish was recorded daily. At the seventh day, the
length of their radicles and shoots was measured on graph paper under a stereomicroscope. The
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collected data were used to calculate the germination percentage, SVI [40] and MGT [41], respectively,
by the following equations:
Germinated Seed Number

Germination Percentage = × 100 (1)
Seed Total Number
SVI = (Mean Root Length + Mean Shoot Length) × Germination Percentage (2)

D × Germinated Seed Number
MGT = , (3)
Germinated Seed Number
where D is the number of days from the beginning of germination.
4.5. Determination of Polyphenolic Content

The total polyphenolic content of the aqueous extracts was determined colorimetrically by the
Folin-Ciocalteau method described by Scalbert et al. [42] with slight modifications. Briefly, 0.5 mL of
each extract was added to 2.5 mL of 10% Folin-Ciocalteau reagent, previously diluted with distilled
water. After 3 min, 2 mL of 7.5% sodium carbonate solution was added. The mixture was incubated
in the dark for 1 h at room temperature and its absorbance was measured at 765 nm using a UV-vis
spectrophotometer (Jenway 7205). A calibration curve was prepared with gallic acid standard solution
at various concentrations (10 to 100 mg/L). The results were expressed as mg gallic acid equivalent
(GAE)/100 g dry plant part. All the measurements were taken in triplicate and the mean values
were calculated.
4.6. Statistical Analysis

The data were analyzed, with the support of IBM SPSS software, through the analysis of variance
carried out separately for each bioassay (i.e., extract and powder bioassays) and L. multiflorum organs
(i.e., inflorescences, stems, roots, seeds). The germination indices (i.e., germination percentage, SVI,
MGT, root length, shoot length) measured for the two species (i.e., E. oryzicola and O. sativa) under
different treatments were taken into account as dependent variables.
The one-way ANOVA and the Turkey’s-b post hoc test were performed in order to establish
the significant effect (at α ≤ 0.05) of the treatments with L. multiflorum (i.e., the different levels of
concentration or quantity in extract and powder bioassay, respectively), on the species, and describe
the homogenous subsets.
Moreover, the two-way ANOVA was performed, considering as factors the treatments with
L. multiflorum and the species, in order to highlight the significant interaction (at α ≤ 0.05) between
“species × L. multiflorum treatments”, and then highlighting the species-specific effects of the treatments
and the different behaviors or susceptibility between the rice crop and the weed.
Author Contributions: Co-first authors, S.V. and F.O; co-last authors, S.B. and M.I. Conceptualization, S.B., M.I.
and S.V.; methodology, F.O., M.I., V.V. and S.V.; validation, M.I. and S.V.; formal analysis, F.O. and V.V.; investigation,
V.V. and S.V.; resources, S.B. and M.I.; data curation, F.O. and S.V.; writing—original draft preparation, F.O. and
S.V.; writing—review and editing, F.O., M.I. and S.V.; visualization, F.O., V.V. and S.V.; supervision, M.I. and S.V.;
project administration, M.I. and S.V.; funding acquisition, S.B. and M.I. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: This research was supported by “Risobiosystems Project” (Italian Ministry Mipaaf funds, for
research and innovation in the organic rice sector), and by EcorNaturaSì s.p.a.. We gratefully thank Dr. Stefano
Gomarasca for his help in plant identification and seed treatment; all the members of the multi-actor community
“RisoBioVero”, the farmers network “Noi Amici della Terra” and the farm “Terre di Lomellina di Rosalia Caimo
Duc” for the efforts in promoting the networking, knowledge exchange and dissemination of best practices; the
farm “Una Garlanda” as the early pioneer of the practice.
Conflicts of Interest: The authors declare no conflict of interest.
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Kawada, K.; Katsura, K.; Oikawa, Y.; et al. Involvement of carnosic acid in the phytotoxicity of
Rosmarinus officinalis leaves. Toxins 2018, 10, 498. [CrossRef]
37. Scavo, A.; Rialb, C.; Molinillob, J.M.G.; Varelab, R.M.; Mauromicalea, G.; Maciasb, F.A. The extraction
procedure improves the allelopathic activity of cardoon (Cynara cardunculus var. altilis) leaf allelochemicals.
Ind. Crops Prod. 2019, 128, 479–487. [CrossRef]
38. Fiorentino, A.; Della Greca, M.; D’Abrosca, B.; Oriano, P.; Golino, A.; Izzo, A.; Monaco, P. Lignans, neolignans
and sesquilignans from Cestrum parqui l’Her. Biochem. Syst. Ecol. 2007, 35, 392–396. [CrossRef]
39. Della Greca, M.; Fiorentino, A.; Monaco, P.; Previtera, L.; Zarrelli, A. Effusides I-V: 9, 10-dihydrophenanthrene
glucosides from Juncus effusus. Phytochemistry 1995, 40, 533–535. [CrossRef]
40. Abdul-Baki, A.A.; Anderson, J.D. Vigor determination in soybean seed by multiple criteria. Crop Sci. 1973,
13, 630–633. [CrossRef]
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
13
plants
Article
Additive Effect of Botanical Insecticide and
Entomopathogenic Fungi on Pest Mortality and the
Behavioral Response of Its Natural Enemy
G. Mandela Fernández-Grandon 1, *, Steven J. Harte 1 , Jaspher Ewany 1 , Daniel Bray 1 and
Philip C. Stevenson 1,2
1 Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB,
UK; s.j.harte@gre.ac.uk (S.J.H.); EwanyJaspher@yahoo.com (J.E.); d.bray@gre.ac.uk (D.B.);
p.c.stevenson@gre.ac.uk (P.C.S.)
2 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
* Correspondence: m.fernandez-grandon@gre.ac.uk; Tel.: +44-0-1634-883057
Received: 25 December 2019; Accepted: 29 January 2020; Published: 1 February 2020
Abstract: Sustainable agricultural intensification employs alternatives to synthetic insecticides for

pest management, but these are not always a direct replacement. Botanical insecticides, for example,
have rapid knockdown but are highly labile and while biological pesticides are more persistent, they
are slow acting. To mitigate these shortcomings, we combined the entomopathogenic fungus (EPF)
Metarhizium anisopliae with pyrethrum and evaluated their efficacy against the bean aphid, Aphis fabae.
To ascertain higher trophic effects, we presented these treatments to the parasitoid, Aphidius colemani,
on an aphid infested plant in a Y-tube olfactometer and measured their preferences. Aphid mortality
was significantly higher than controls when exposed to EPF or pyrethrum but was greater still
when exposed to a combination of both treatments, indicating an additive effect. This highlights the
potential for applications of pyrethrum at lower doses, or the use of less refined products with lower
production costs to achieve control. While parasitoids were deterred by aphid infested plants treated
with EPF, no preference was observed with the combination pesticide, which provides insight into the
importance that both application technique and timing may play in the success of this new technology.
These results indicate the potential for biorational pesticides that combine botanicals with EPF.
Keywords: biopesticide; organic pesticide; Y-tube olfactometer; pyrethrum; parasitoid;

entomopathogenic fungi; leaf disc assay; insect behavior; survival analysis
1. Introduction
Pesticidal plant extracts are an important component of sustainable integrated pest management
(IPM) and can offer an effective alternative to synthetic pesticides for management of pests, especially
for smallholders [1]. Pesticidal plants typically have lower impact on higher trophic levels, including
natural enemies of pests, so are better suited to sustainable production [2,3] and are locally available at
a lower cost than synthetic chemicals [4]. The most important commercial botanical pesticides include
pyrethrum and neem products [5]. Pyrethrum is a natural insecticide extracted from the flowers of
Chrysanthemum cinerariaefolium and Chrsanthemum cineum [6,7] which has been used for controlling
field, household, and storage pests, and parasites in livestock and humans [8–10]. A combination
of awareness of the negative impacts of synthetic pesticides and increased pressure from regulatory
authorities on permitted chemicals has seen the global demand for biopesticides grow over the past
decade at an estimated 15% per annum [11–13], compared to 3% per annum for synthetic pesticides [11].
Natural pyrethrum contains six entomotoxic compounds: cinerin I and II, pyrethrin I and II
and jasmolin I and II [14]. Pyrethrins enter the insect body via ingestion or contact, penetrate the

Plants 2020, 9, 173
epidermis and are distributed throughout the body in the haemolymph [7]. The insecticide disrupts the
insect’s peripheral and central nervous systems by causing repetitive discharges of nerves, resulting in
paralysis [15]. Pyrethrins have a rapid “knockdown” effect preceding insect death [7,14] and insects
usually die in a few minutes or hours following exposure to a fatal dose [8].
Pyrethrins influenced the development of some of the most widely used synthetic insecticides—the
pyrethroids, including cypermethrin, permethrin, deltamethrin, fenvalerate and bifenthrin. The drive
towards different synthetic pyrethroids was to increase their stability in the environment, providing
effective control for longer. This has proven successful as pyrethroids are routinely used as agricultural
insecticides with high adoption rates internationally [8].
The active components in pyrethrum are highly labile in ultraviolet (UV) light, non-persistent,
and are less toxic to humans and the environment [9,16–18]. Although the lack of persistence has
previously limited use of natural pyrethrum as an agricultural insecticide [8,15] it is less disruptive to
IPM programs that include beneficial insects than conventional insecticides.
One of the emerging technologies as part of IPM is the use of entomopathogenic fungi (EPF) [19],
which can be used to control a wide range of agricultural pests [20]. They have no negative effects
on human health [21]. Entomopathogenic fungi are specific pathogens of insects that can infect their
hosts through the external cuticle [20]. Rapid penetration and infection of a susceptible host occurs at
high humidity [20], but spores can remain viable on the cuticle during unfavorable conditions and
penetrate when humidity rises, even if only for a short time [22].
Upon successful penetration, the fungi develop, colonize the insect’s internal organs and the
insect eventually dies. It is after insect death that hyphae emerge, followed by spore formation and
production of numerous conidia on the cadaver [20]. EPF, such as Metarhizium anisopliae (Metschnikoff)
used in this study, can only complete their lifecycles and increase populations by producing numerous
conidia after the death of infected hosts [23] which will disperse, infecting more insects and continue
the propagation cycle.
There are successful synergies and compatibilities of EPF with different plant-based pesticides
for improved pest control. For instance, the combination of sub-lethal doses of the EPF Beauvaria
bassiana and neem extract increased mortality against whitefly, Bemisia tabaci, nymphs when neem
insecticide was drenched with simultaneous application of B. bassiana on tomatoes plants [24,25].
Beauvaria bassiana (isolate PL63) was compatible with botanical extracts from Trichilia catigua leaves and
effectively controlled insect pests in Brazil [26]. Another study by Shoukat et al. [27], revealed that both
M. anisopliae and B. bassiana showed a synergistic effect when mixed with neem extract, Azadirachta
indica, and increased mortality against 3rd instars of Culex pipiens in the field. These examples of the
compatibility of EPF with pesticidal plant extracts suggests potential in combining EPF and pyrethrum
to improve efficacy through the rapid knockdown of pyrethrum and the persistent control offered by
EPF to control agricultural pests and combat pest resistance.
A benefit that may be realized through a combination biopesticide over synthetic pesticides
could be reduced impact on beneficial insects in the environment through decreased exposure and
greater specificity. EPF has been reported to be pathogenic to beneficial insects, including parasitoids.
B. bassiana was found to infect and kill adult Aphidius colemani with infection rates ranging from 46.3%
to 60% [28] and between 57.6 to 66% [29] under greenhouse environments. Shipp et al. [29] did not
recommend the use of adult A. colemani together with B. bassiana for pest control in greenhouses.
The exposure of A. colemani to different EPF strains such as M. anisopliae is still to be explored.
However, time of application of EPF has been manipulated to reduce parasitoid mortality and
affect the use of EPF together with parasitoids in pest control. For instance, parasitoid Aphidius
matricariae and B. bassiana (strain EUT116) were effective against the aphid, Myzus persicae when the
fungus was applied 96 hours after the release of parasitoids [30]. Beauvaria bassiana (strain PL63) and
the aphid parasitoid, Diaeretiella rapae were recommended against M. persicae [31]. Another study by
Mohammed and Hatcher [32], revealed that introducing EPF Lecanicillium muscarium six days after
releasing A. colemani was effective against M. persicae in a greenhouse environment. Based on these
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results, usage of selected EPF isolates and applying EPF after parasitism can reduce detrimental effects
on parasitoids. However, to inform the effective use of EPF with the beneficial insects, it is important
that we understand their interaction with the fungi.
In this study, we assessed the efficacy of pyrethrum, Metarhizium anisopliae EPF and the combination
of both on mortality of the aphid pest, Aphis fabae, and how the aphid parasitoid, Aphidius colemani,
responded to the odors associated with these compounds. The components were selected for this
proof of principle because of the ready availability of pyrethrum in low-income areas and the known
efficacy of this M. anisopliae strain against this highly problematic aphid pest. To gauge the potential
in establishment of EPF on the aphid population, we also recorded incidence of visible fungal
establishment with hyphae seen emerging from the cuticle and the number of offspring produced
by the aphids following exposure. We found that both pyrethrum and the EPF led to a significant
increase of mortality on A. fabae which was further enhanced when they were presented in combination.
Visible fungal growth occurred more rapidly on aphids treated with the combination biopesticide,
indicating establishment in population could be accelerated through the multimodal action. It was
also shown that its associated parasitoid, A. colemani, preferentially selects plants that do not contain
EPF when foraging using odor, however, the preference exhibited is absent in the combined treatment.
2. Results
2.1. Aphid Mortality Assay
2.1.1. Survival
No significant interaction was found between the application rate of EPF 0 CFU mL−1 (carrier oil
only), 1 × 106 CFU mL−1 or 1 × 108 CFU mL−1 and pyrethrum presence (10 ppm pyrethrins) or absence
(0 ppm) on aphid survival (χ2 = 0.70, df = 2, p = 0.70). However, significant independent effects of both
pyrethrum (χ2 = 6.56, df = 1, p = 0.01) and EPF concentration (χ2 = 16.8, df = 2, p = 0.001) were found
on aphid survival (Figure 1). Addition of pyrethrum led to a 40.5 h reduction in predicted mean aphid
survival at 0 CFU mL−1 EPF (from 80.1 h to 39.6 h). At 1 × 106 CFU mL−1 survival was reduced by 29.2 h
through addition of pyrethrum (from 67.3 h to 35.3 h), and by 10 h at 1 × 108 CFU mL−1 (from 19.7 h
to 9.7 h). Survival was reduced significantly through addition of EPF compared to No EPF (χ2 = 6.9,
df = 1, p = 0.009), and from 1 × 106 CFU mL−1 to 1 × 108 CFU mL−1 EPF (χ2 = 9.9, df = 1, p = 0.002).
Figure 1. Cont.
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Figure 1. Survival of individual A. fabae exposed to M. anisopliae alone (solid line) or in combination
with pyrethrum (dotted line). Entomopathogenic fungus (EPF) was tested at 0 colony forming units
(CFU) mL−1 (carrier oil only, top graph), 1 × 106 CFU mL−1 (middle graph) and 1 × 108 CFU mL−1 .
2.1.2. Hyphal Growth on Insect Surface

Overall, aphids (n = 40) which had not been treated with EPF (0 CFU mL−1 ) showed no hyphal
growth up to 192 h after treatment. Aphids not treated with EPF were therefore excluded from
further analysis of time until visible fungal growth. Increasing concentration of treatment from
1 × 106 CFU mL−1 to 1 × 108 CFU mL−1 significantly reduced time until hyphal growth (χ2 = 10.74,
df = 1, p = 0.001). Addition of pyrethrum at 10 ppm pyrethrins also significantly reduced time until
hyphae formation was observed (χ2 = 10.74, df = 1, p < 0.001). However, no interaction was found
between EPF level and pyrethrum treatment on time until hyphal growth was observed (χ2 = 2.37,
df = 1, p = 0.12). Addition of pyrethrum reduced predicted mean time until growth was seen by 84 h at
1 × 106 CFU mL−1 (from 226 h to 142 h) and 63 h at 1 × 108 CFU mL−1 (from 170 h to 107 h) (Figure 2).
Figure 2. Predicted time (± upper and lower quantiles) until hyphal growth was observed on individual
A. fabae exposed to M. anisopliae alone or in combination with pyrethrum. CFU: colony forming units.
2.1.3. Number of Offspring

Due to a very low offspring count in the first block of replicates, only aphids tested in the second
block of treatments were used in the analysis. In this block, a significant interaction was found between
EPF level (0 CFU mL−1 (carrier oil only), 1 × 106 CFU mL−1 or 1 × 108 CFU mL−1 ) and pyrethrum
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presence (10 ppm pyrethrins) or absence (0 ppm) on total number of offspring produced by each aphid
(χ2 = 7.01, df = 2, p = 0.03). At 0 CFU mL−1 and 1 × 106 CFU mL−1 , addition of pyrethrum resulted in
significantly fewer offspring produced (Tukey’s test, p < 0.05, Figure 3). However, no significant effect
of pyrethrum was found on number of offspring produced by aphids exposed to 1 × 108 CFU mL−1
of EPF.
Figure 3. Predicted mean number (± 95% confidence intervals) of offspring produced by individual
A. fabae exposed to M. anisopliae alone (white bars) or in combination with pyrethrum (10 ppm pyrethrins;
grey bars). EPF was tested at 0 colony forming units (CFU) mL−1 (carrier oil only, left), 1 × 106 CFU mL−1
(middle) and 1 × 108 CFU mL−1 (right).
2.2. Parasitoid Dual-Choice Assays

When presented with the choice of aphid-infested or uninfested plant material through a
dual-choice assay, more female parasitoids chose the aphid-infested material (exact binomial test,
n = 50, p = 0.0066) (Figure 4). This served as a positive control to confirm that parasitoids would
orientate towards A. fabae in the absence of visual cues. All other experimental treatments used only
aphid-infested bean plants. When presented with a choice between two aphid-infected plants, one of
which had been treated with pyrethrum, no significant difference was found between proportion of
parasitoids choosing an untreated plant, and a plant which had been treated with pyrethrum (exact
binomial test, n = 50, p = 0.322). However, significantly fewer parasitoids chose the EPF-treated
compared to the untreated plant (exact binomial test, n = 50, p < 0.001). When the pyrethrum was
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presented alongside the EPF and compared to an untreated plant, the parasitoid displayed no preference
(exact binomial test, n = 50, p = 0.203) (Figure 4). For all the replicates there was only one non-responder
recorded for failing to make a choice in the allotted time (treatment containing pyrethrum + EPF), this
individual was excluded from the analysis.
Figure 4. Parasitoid, A. colemani, responses to treatments in a Y-tube olfactometer. N = 50 for

each treatment, with the number of individuals choosing each side indicated on the relevant bar.
Where ‘Infested’ refers to aphid colonized plant, Py is treatment with pyrethrum and EPF is treatment
with entomopathogenic fungi, Metarhizium anisopliae (ICIPE 62) *** Indicates p < 0.01, ns = not significant.
3. Discussion
Our working hypotheses for this study were:
1. Efficacy of pyrethrum and EPF would be enhanced when presented in combination.

2. The biopesticides would affect parasitoid plant/host preference.
Through the evaluation of aphid mortality, hyphae formation and offspring production after
exposure to the biopesticides, we found that efficacy was enhanced when the components were
presented in combination. This additive effect of combination was observed through reduced survival,
more rapid formation of hyphae and reduced fecundity. Increased mortality was recorded as EPF
concentration increased, though from a practical perspective, the level of mortality achieved with lower
dosage may be sufficient in pest control programs and may even be preferable if it permits low-level
persistence of the host for biocontrol agents. One of the difficulties in controlling aphid populations
lies in their high rate of fecundity. The significant reduction in fecundity noted with exposure to the
combination treatment could be critical in more effective control as it may curb the exponential rate of
population growth. However, in this study only ten replicates were evaluated for offspring production
in each treatment. This was due to only four offspring being produced in the initial block of replicates.
It is not clear why this number was this low. To evaluate this further we suggest the experiment be
repeated and assessment to include the intrinsic rate of increase and other population metrics to gain
greater insight into how this is likely to affect population dynamics.
The increase in mortality may be due to the bimodal effects of the combined treatment as the
immediate attack on the nervous system provided by the pyrethrum leave individuals more susceptible
to infection from the EPF. This concurs with previous studies on pests that have shown additive
or synergistic effects when EPF and pesticidal plant products are presented in combination [33–35].
In addition to the increased mortality with a combined biopesticide application, it was notable that
the time lag before hyphae were observed was shortened in the lower dosage application (226 h to
142 h). This difference of 84 h could have a considerable impact on the viability of such a product and
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especially when considered over multiple generations of the pest. One of the most widely recognized
drawbacks to EPF application is the slow-acting nature of the product [35–38]. Although it can offer
sustained control, this relies on its establishment within the population through propagation via the
host. A guiding principle behind the exploration of a combined biopesticide is for a product that
overcomes the short-lived nature of pyrethrum and the slow-acting nature of the EPF. An accelerated
rate of establishment, as indicated by this study, could be a critical advantage to such a product.
However, we recognize that this is a laboratory evaluation of the interaction under controlled conditions
and with limited replication. The real impact of this would need to be established through longer
running trials which assess the formation of conidia from the host and population suppression over
multiple generations in a more field-realistic situation.
The results of the mortality experiments show strong support for the development of combined
biopesticides as a new tool for IPM. In addition to a direct increase in pest mortality, there are
indications that propagation of the EPF may be occurring more rapidly and fecundity of the pest is
being suppressed. Furthermore, it is important to note the pyrethrin dosages applied in the trials
(10 ppm) were a fifth of that recommended for effective pest control in the field. This was used to
allow any potential synergy or additive effect of combination to be recorded as it was identified in
preliminary trials that mortality was too high at 50 ppm to determine these effects. The mortality
observed at this low dosage of pyrethrins when presented in combination with the EPF has greater
practical and commercial appeal for this technology. Refinement of pyrethrum remains a relatively
expensive process and one limited by the technology available in an area. The use of less refined
material could lead to lower cost production, a reduction of impact on non-target species and greater
potential for formulation in and for lower income countries. We also identify that there are various
shortcomings to the use of these biopesticides which a combination approach will need to overcome
and will assess through field trials.
Evidence from the Y-tube olfactometer behavioral assays supported our second hypothesis that
the behavior of the parasitoid would be affected by volatiles from treated materials. The control
demonstrated that the parasitoid was able to detect A. fabae feeding on the bean plants and would
move towards them, behaviors indicative of foraging. When EPF was applied as the only variable
present, wasps showed a preference for the plant-aphid treatment without the EPF. The avoidance
of EPF by natural enemies has previously been observed in studies looking at ladybirds, Coccinella
septempunctata, and anthocorids, Anthocoris nemorum [39,40] respectively. The behavior possibly confers
fitness benefits as the wasp may reduce its exposure to the fungal pathogen. However, studies on
parasitoid Cephalonomia tarsalis showed no avoidance behavior in response to the EPF B. bassiana [41].
The avoidance behavior of parasitoids to EPF may be species dependent, which highlights the
importance of study on commercially relevant organisms. It is interesting to note that EPF avoidance
observed in this study was absent in the presence of pyrethrum which could indicate either an
interaction between the components or the perception of the EPF-aphid-plant treatment.
Future research should consider other aspects of the behavior of beneficial insects and elucidate
the mechanisms behind this observed preference for non-EPF treated material. Experiments could be
performed to disentangle the interaction between plants, aphids and EPF to identify whether individual
components or the interaction is responsible for the deterrence that was observed. Next steps in this
direction would be to evaluate the direct impact of relevant EPF strains to the parasitoids, the odors
involved in potential repellency from EPF and how these behaviors affect success in field settings.
Our findings align with what has been found previously that plant-based insecticides can be
complemented by addition of EPF. The full potential of such a technology is still to be explored and
different formulations should be investigated using different EPF and botanical components. It is
also important that these experiments are taken out of the laboratory and into the field to assess
their efficacy in highly variable field conditions. In future testing we also suggest that the impact on
beneficial insects in the environment is considered as a high priority and should extend to include
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pollinators as well as natural enemies. Their susceptibility to the combined formulations should be
assessed and the findings should inform the future use of this technology.
4.1. Insect Rearing

Aphids, Aphis fabae, were obtained from colonies at Harper Adams University. The population
was maintained on potted broad bean plants at temperatures of 27 ◦ C, on a 12 h, L:D cycle. Fresh plant
material was introduced into the colony if alatae were seen to form with old material removed following
24 h.
Parasitoid species, Aphidius colemani, were used in laboratory bioassays. The parasitoids were
obtained from Bioline Agro-Sciences Ltd, UK from a different rearing background to that used in
experiments. It has previously been found that parasitoids 2 to 5 days old display greater fecundity
and higher rates of parasitism [42–44]. For these experiments female A. colemani 3 to 5 days old were
used to increase the likelihood of host seeking behavior.
4.2. Entomopathogenic Fungi

The commercially available strain of Metarhizium anisopliae isolate, ICIPE 62, in an oil emulsion
was obtained from Real IPM Kenya (Madaraka, Thika, Kenya) and used for the experimental work.
Dilutions were conducted as necessary to obtain the concentration of colony forming units (CFU)
required for trials.
4.3. Preperation of Pyrethrum

Semi-refined pyrethrum product was supplied by Botanical Extracts EPZ Ltd (Twiga Crescent,
Export Processing Zone, Athi River, Kenya) for use in the experimental work. The product was analyzed
to ensure the correct dosage of active ingredient was used in experimental work. Pyrethrins were
analyzed using an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, United States)
consisting of modular quaternary pump, degasser, auto-sampler, column oven and photodiode array
detector. Separation of entomotoxic pyrethrum constituents was achieved on a Waters X-Select
T3 column (250 × 4.6 mm, 3.5 μm) and a guard column with the same characteristics, all kept at
25 ◦ C. The chromatographic conditions were: flow rate 1 mL min−1 , sample injection volume of
10 μL and mobile phases; A (100% H2 O), B (100% MeOH) and mobile phase C (1% formic acid).
27/68/5 (A/B/C) which was held for 2 mins, raised to 5/90/5 over 22 min (24 min total), followed by
wash and re-equilibration steps. The photodiode array detection was conducted by scanning between
200 and 600 nm.
Individual compounds in each sample were identified by comparing their retention times and
UV–Vis spectra with those of a standard pyrethrum sample purchased from Sigma (Gillingham, Dorset,
UK). Quantitative determination of the target compounds in the extracts was performed using external
calibration curves in the concentration range of 0.05 to 1 mg mL−1 (detection at 225 nm).
Total w/w% of the active compounds was 17%, comprised of cinerin I (0.5%), cinerin II (3.7%),
pyrethrin I (0.5%), pyrethrin II (1.4%), jasmolin I (11%) and jasmolin II (1.5%). Retention times were
18.9, 11.4, 19.1, 11.8, 22.3 and 14.1 minutes respectively.
The pyrethrum extract was subsequently diluted with deionized H2 O, without the need for
surfactants or adjuvants, to the required ppm values of pyrethrins, as specified in the mortality assay
methods. All ppm values quoted in this section are for total pyrethrin content i.e., cinerin I & II,
pyrethrin I & II and jasmolin I & II.
4.4. Mortality Assays

Leaf discs were removed from broad bean leaves using a sterilized 26 mm cork bore. Discs were
then dipped into either negative control (H2 O), low concentration of EPF (1 × 106 CFU mL−1 ), high
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concentration of EPF (1 × 108 CFU mL−1 ), pyrethrum (10 ppm pyrethrins), low dose combination (EPF
at 1 × 106 CFU mL−1 with 10 ppm pyrethrins) or full combination dose (EPF at 1 × 108 CFU mL−1 with
10 ppm pyrethrins). The concentration applied of pyrethrins is lower than the recommended 50 ppm
for aphid control. A lower concentration of 10 ppm was selected from preliminary trials demonstrating
that this elevated mortality but did not lead to 100% mortality, making it a viable candidate to assess
interaction with the fungi.
During the preparation of leaf discs, a 1.5% agar solution (Oxoid Technical Agar No. 2) was
prepared using distilled water. Once the solution was fully mixed, 10 ml was decanted into 29 mL pots
(4.5 cm height × 4 cm diameter) to cool. Once the solution was viscous but not completely set, leaf
discs were embedded into the agar ensuring the edges were sufficiently covered.
A single adult A. fabae was gently removed from plants using a fine paintbrush and placed onto
the center of each leaf disc. A partially mesh lid was placed on each pot preventing aphid escape while
avoiding a build-up of moisture. Pots were placed onto the trays in a Latin square design which was
altered with each replicate to reduce positional bias. Treatments were maintained in a 26 ◦ C room, on
an 12:12 L:D light cycle.
All samples were monitored daily with counts made for offspring which were removed as they
were found, mortality of the adults, and visible formation of hyphae. Monitoring was conducted at the
same time on each day with the same experimenter conducting the observations to ensure consistency.
A total of 20 replicates were performed for each treatment. The experiment was conducted across
two blocks.
4.5. Parasitoid Choice Assays

Behavioral response of A. colemani to treatments of aphid-infested plants (positive control),
uninfested plant (negative control), aphid-infested plant + pyrethrum, aphid-infested plant + EPF,
and aphid-infested plant + pyrethrum + EPF was tested using a dual-choice assay.
Three days prior to each experimental day, bean plants were transferred from glasshouse to the
bioassay room. Broad bean, Vicia faba, Dwarf Sutton variety (Kings seeds, Colchester, UK) were used
for all experiments. All plants were used 3 weeks after germination with between 4 to 5 leaf pairs
developed. Using a fine paintbrush, 50 aphids of mixed ages where transferred from the colony onto
fresh bean plants and left for 72 h to establish plants in the ‘Infested’ treatments. This level is sufficient
to induce production of defensive plant volatiles [45]. Uninfested control plants were maintained in the
same condition with no direct contact with aphids. The pyrethrum and EPF suspension were placed in
separate 1 L spray bottles and sprayed on the plants in a closed arena. All plant leaves were sprayed
both on the top and the underside with pyrethrum or EPF to reflect application of these materials in a
field environment. After administering the treatment, plants were left for 15 min before conducting
the experiments.
In the treatment combining EPF and pyrethrum insecticide, equal amounts of each were sprayed
separately. The order of spraying was alternated between trials. The sprayed plants were then placed
in separate glass vessels to conduct the experiment. All the treatments were tested against the positive
control to make four treatment sets per day.
A glass Y-tube olfactometer (stem 8 cm, arms 8 cm, internal diameter 1 cm and 120◦ angle between
arms) was used to assay parasitoid response to plant treatments in the absence of visual stimuli.
The Y-tube was connected to two, 3 L Kilner jars containing the positive control and the other contained
either an experimental treatment or negative control. The Kilner jars used were modified with an
airtight inlet and outlet fittings, allowing a continuous flow of charcoal filtered air at 200 mL/min
through each vessel to the two arms of the Y-tube olfactometer via Teflon tubing.
Experiments were conducted during active foraging times for the parasitoid (4–9 h after scotophase).
During the experiment, individual A. colemani were released using a 1 ml pipette head connected to
the base of the Y-tube olfactometer and removed immediately after the parasitoid entered the tube.
Each A. colemani was observed for a maximum of 5 min or until it travelled 6 cm up one of the Y-tube
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arms and remained there for 30 s. Wasps that did not enter the Y-tube after 5 min were recorded
as non-responders.
All the four treatments were tested on each experimental day, with the order determined using a
Latin square. The bioassay arena of the Y-tube blocked the entrance of light from all sides except from
the direction of the Y-tube olfactometer arms. In each treatment, the Y-tube arms and odor sources
were swapped after five parasitoids were tested to minimize directional bias or any bias of choice due
to light in one arm. A different Y-tube was used in each experiment, and all Y-tubes were cleaned with
70% ethanol and left to dry before use. Each parasitoid was used only once, reflecting a true biological
replicate. Fifty replicates were completed for each treatment.
4.6. Statistical Analyses
4.6.1. Survival
The first objective of statistical analysis was to determine whether application of pyrethrum
and EPF would result in lower aphid survival than treatment with EPF alone. Differences between
treatment groups were first visualized by plotting Kaplan–Meier survival estimates (Figure 1). A model
with Weibull errors was used to test whether there was an interaction between EPF level (0 CFU
mL–1 , 1 × 106 CFU mL−1 or 1 × 108 CFU mL−1 ) and presence (10 ppm pyrethrins) or absence (0 ppm)
of pyrethrum on aphid survival. The time interval in which the aphid died was entered as the
dependent variable. Aphids which did not die during the experiment were recorded as censored
cases. Independent effects of EPF level and pyrethrum presence or absence were then tested separately.
Differences in survival between individual EPF levels were tested through model simplification
(presence vs. absence of EPF, low (1 × 106 CFU mL−1 ) vs high (1 × 108 CFU mL−1 ) EPF. Significance of
all effects was assessed through χ2 test changes in residual deviance following deletion from the
model [46]. All analyses were performed in R [47–49].
4.6.2. Visible Fungal Growth

A model similar to the survival analysis was applied with Weibull errors was used to determine
whether application of pyrethrum would result in faster hyphae formation of EPF at the three levels
of EPF tested. In this model, time interval in which hyphae were first observed was entered as the
dependent variable. Replicates in which spores were not recorded were entered as censored cases.
EPF level and pyrethrum treatment were entered as factors in the model. An interaction was included
to determine whether the effect of pyrethrum on time until hyphae emergence varied with initial
EPF concentration.
4.6.3. Number of Offspring

A generalized linear model with negative binomial areas [50] was used to determine whether
application of pyrethrum and EPF had significant effects on aphid offspring production. Total number
of offspring produced by each aphid across the experiment was entered as the dependent variable.
EPF level (0 CFU mL−1 , 1 × 106 CFU mL−1 or 1 × 108 CFU mL−1 ) and pyrethrum level (0 ppm pyrethrins
or 10 ppm pyrethrins) were entered as factors in the model. An interaction term was included to
test whether the effect of pyrethrum on number of offspring produced varied with EPF level applied.
Significance of each term was assessed through likelihood ratio tests (χ2 ) following deletion from the
model. Differences between individual factor levels in numbers of offspring produced were assessed
through Tukey’s tests performed on estimated means [51]. Analysis was restricted to the second round
of aphids tested, as none of the aphids in the first round produced offspring.
4.6.4. Parasitoid Dual-Choice Assays

Choice assays using the Y-tube olfactometer data was analyzed using two-sided exact binomial
tests, with an assumption of a 50:50 distribution if the wasps were moving at random. Wasps which
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Plants 2020, 9, 173
made no choice after 5 minutes were excluded from analysis. Tests were conducted using R (v 3.5.1,
Vienna, Austria).
5. Conclusions
Here we show that a combination of the entomopathogenic fungi, Metarhizium anisoplae, and
pyrethrum led to a higher rate of mortality in Aphis fabae than for the individual pesticides when tested
alone. Thus, the combination of these two biopesticides was effective at killing the target pest more
effectively than the individual components with no apparent contraindications, illustrating a novel
approach to compensate for their individual shortcomings; the rapid breakdown of pyrethrum and
slow activation of EPF. The development of fungi on the external cuticle was also observed earlier
when EPF was presented with pyrethrum, which may be key to more rapid establishment in the
population. A surprising finding was that when used alone, the EPF was repellent to the parasitoid
Aphidius colemani, however, when presented in combination with the pyrethrum had no effect on
foraging behavior of the natural enemy. Thus, the combination of EPF and pyrethrum may be better
suited to use in an IPM system that included natural enemies, though timing considerations may be
critical. Combinations of biopesticides that have different mechanisms of action have the potential to
improve the efficacy of the individual components and reduce the build-up of resistance. The additive
effect also suggests that there is potential for applications of pyrethrum at lower doses and so reducing
effects at higher trophic levels or that less refined products could be used at lower production costs to
achieve control. Studies of behavior provide insight into the importance that application technique
and timing play in the effectiveness of this novel combination pesticide technology.
Author Contributions: Conceptualization, P.C.S. and G.M.F.-G.; methodology, G.M.F.-G. and S.J.H.; formal
analysis, D.B.; investigation, S.J.H., J.E. and G.M.F.-G.; writing—original draft preparation, G.M.F.-G. and
J.E.; writing—review and editing, D.B., S.J.H., J.E. and P.C.S.; supervision, G.M.F.-G.; project administration,
G.M.F-G.; funding acquisition, G.M.F.-G. and P.C.S. All authors have read and agreed to the published version of
the manuscript.
Funding: Funding for this project was provided by BBSRC and InnovateUK through the UK-China Agritech
Challenge to PCS and MFG as part of the ‘Environmentally Benign Combination Biopesticides–Transforming Pest
Control in Chinese and UK Agriculture’ and initiated through a grant awarded to MFG by the Higher Education
Innovation Fund (HEIF).
Acknowledgments: We are grateful to Tom Pope of Harper Adams for provision of the Aphis fabae, Botanical
Extracts EPZ Ltd for supplying the pyrethrum, and Real IPM for suppling the Met62 used in these experiments.
All these materials were provided without charge for use in the study.
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27
plants
Article
Extracts of Common Pesticidal Plants Increase Plant
Growth and Yield in Common Bean Plants
Angela G. Mkindi 1 , Yolice L. B. Tembo 2 , Ernest R. Mbega 1 , Amy K. Smith 3,4 , Iain W. Farrell 3 ,
Patrick A. Ndakidemi 1 , Philip C. Stevenson 3,5 and Steven R. Belmain 5, *
1 Department of Sustainable Agriculture, Biodiversity and Ecosystems Management, Centre for Research,
Agricultural Advancement, Teaching Excellence and Sustainability (CREATES), The Nelson Mandela African
Institution of Science and Technology, Box 447 Arusha, Tanzania; angela.mkindi@nm-aist.ac.tz (A.G.M.);
ernest.mbega@nm-aist.ac.tz (E.R.M.); patrick.ndakidemi@nm-aist.ac.tz (P.A.N.)
2 Department of Crop and Soil Sciences, Lilongwe University of Agriculture and Natural Resources,
Bunda, Malawi; ytembo@bunda.luanar.mw
3 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK; AmyKendall.Smith@kew.org (A.K.S.);
I.Farrell@kew.org (I.W.F.); P.C.Stevenson@greenwich.ac.uk (P.C.S.)
4 Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK
5 Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime,
Kent ME4 4TB, UK
* Correspondence: S.R.Belmain@greenwich.ac.uk; Tel.: +44-1634-883761
Received: 10 December 2019; Accepted: 20 January 2020; Published: 23 January 2020
Abstract: Common bean (Phaseolus vulgaris) is an important food and cash crop in many countries.
Bean crop yields in sub-Saharan Africa are on average 50% lower than the global average, which is
largely due to severe problems with pests and diseases as well as poor soil fertility exacerbated by
low-input smallholder production systems. Recent on-farm research in eastern Africa has shown
that commonly available plants with pesticidal properties can successfully manage arthropod pests.
However, reducing common bean yield gaps still requires further sustainable solutions to other
crop provisioning services such as soil fertility and plant nutrition. Smallholder farmers using
pesticidal plants have claimed that the application of pesticidal plant extracts boosts plant growth,
potentially through working as a foliar fertiliser. Thus, the aims of the research presented here were
to determine whether plant growth and yield could be enhanced and which metabolic processes were
induced through the application of plant extracts commonly used for pest control in eastern Africa.
Extracts from Tephrosia vogelii and Tithonia diversifolia were prepared at a concentration of 10% w/v
and applied to potted bean plants in a pest-free screen house as foliar sprays as well as directly to
the soil around bean plants to evaluate their contribution to growth, yield and potential changes in
primary or secondary metabolites. Outcomes of this study showed that the plant extracts significantly
increased chlorophyll content, the number of pods per plant and overall seed yield. Other increases
in metabolites were observed, including of rutin, phenylalanine and tryptophan. The plant extracts
had a similar effect to a commercially available foliar fertiliser whilst the application as a foliar spray
was better than applying the extract to the soil. These results suggest that pesticidal plant extracts
can help overcome multiple limitations in crop provisioning services, enhancing plant nutrition in
addition to their established uses for crop pest management.
Keywords: induced systemic response; foliar fertiliser; rutin; tryptophan; phenylalanine; botanicals
1. Introduction
Common bean (Phaseolus vulgaris) is a strategic crop in low- and middle-income countries, known
for its economic and nutritional benefits [1,2]. Tanzania is among the top 20 producers of common

Plants 2020, 9, 149
bean in the world [3]. However, bean productivity is generally stagnant across much of Africa due
to a number of suboptimal provisioning services such as poor soil fertility and pest damage that are
limiting potential yields [4,5]. Although chemical fertilizers can dramatically increase bean yields, they
are largely unaffordable and unavailable to most smallholder farmers [6] and contribute to reduced
soil stability [7,8], pollution [9] and carbon footprint [10]. Natural soil fertility enhancement through
the use of manure, composts, green mulches, cover crops and crop rotation are not widely used by
smallholder farmers, arguably due to high labour costs and poor local knowledge [11–14].
Sustainable technologies for pest management in legume crops often relies on the breeding
of resistant varieties [15]. However, the use of pesticidal plant extracts in smallholder farming
systems is also an established agro-ecologically sustainable pest control method [16–20]. Although
the economics and cost-benefits of smallholder use of crude plant extracts for pest management are
certainly favourable in many situations [19], uptake and promotion of pesticidal plants could be further
facilitated by increased evidence on potential multiple benefits of their use [21], making their use even
more attractive to smallholder farmers. For example, recent research has shown that the impact of
pesticidal plants on beneficial arthropods such as pollinators and predators, is much less than that
observed when using synthetic pesticides [18]. Research has also demonstrated that other potential
benefits to smallholder use of pesticidal plants could be through direct effects on plant vigour by
functioning as a green fertiliser or through the provision of additional nutrition and inducing systemic
plant responses [22,23]. Very often plants used as pesticides have multiple uses such as providing
fruits, seeds, fibre, timber or in traditional medicines [24–26]. Alternative uses can also include use as
green mulches and cover crops to improve the soil fertility, where previous research points particularly
to the use of Tephrosia vogelii and Tithonia diversifolia [27–30]. This study, therefore, sought to evaluate
the contribution of extracts from T. vogelii and T. diversifolia on the growth, yield and metabolism
of common beans. Evidence from this study could validate farmer observations and increase the
perceived value of using such extracts, thus encouraging wider uptake in smallholder farming systems.
2. Results and Discussions
2.1. Growth and Yield of Common Beans in Response to the Application of Treatments
Extracts were applied to the leaves through foliar spraying or directly to soil as a soil drench in
order to compare the effects on bean plant growth and yield. Significant variation in the growth of
common beans was observed according to treatments where T. vogelii extracts resulted in significantly
higher plant height, number of leaves and branches, leaf area, stem width and leaf greenness. However,
water, water and soap and synthetic pesticide treatments were significantly lower in terms of plant
height number of leaves, number of branches per plant, leaf area, stem width and leaf greenness
(Table S1).
Yield was measured using the number of pods per plant and seed yield per plant (Table 1).
Significantly higher numbers of pods and seeds were recorded in the T. vogelii treatment, followed by
T. diversifolia and the foliar fertiliser for pods per plant and seed yield per plant. The control treatments
(water, water and soap and synthetic pesticide) recorded significantly lower numbers for pods per
plant and seed yield. Both number of pods per plant and seeds per pod showed a significant variation
with respect to the method of application with higher values recorded for the number of seeds per pod
and seed yield per plant when treated by foliar spray compared to when the treatments were applied
to the soil for pod number and seed yield.
29
Plants 2020, 9, 149
Table 1. Effects of foliar fertiliser, synthetic and plant pesticide treatments and application method on
the yield of common beans.
Treatment Applied Number of Pods Per Plants Seed Yield/Plant (g)

Foliar Fertiliser 3.1 ± 0.26 b 2.7 ± 0.33 b
Synthetic pesticide 2.1 ± 0.24 c 1.3 ± 0.19 c
Tephrosia vogelii 4.1 ± 0.23 a 3.8 ± 0.23 a
Tithonia diversifolia 3.1 ± 0.31 b 3.3 ± 0.23 b
Water 1.9 ± 0.23 c 1.5 ± 0.16 c
Water and soap 1.6 ± 0.22 c 1.7 ± 0.11 c
Method of Application
Foliar spray 2.9 ± 0.21 a 2.7± 0.20 a
Soil drenching 2.4 ± 0.16 b 2.1± 0.16 b
2-Way ANOVA (F-Statistics)
Treatment 15.2 *** 29.0 ***
Treatment method 6.7 * 14.8 ***
Treatment * Treatment method 2.0 * 3.1 *
The values presented are means ± SE. *, *** = significant at p ≤ 0.05, p ≤ 0.001 respectively. Means followed by the
same letter in a column are not significantly different.
As the effect was much more pronounced when applied to the leaves compared to the soil, our
data suggest that the plant extracts contribute to plant nutrition as a foliar fertiliser, which may be
particularly useful in smallholder farming systems where soils are often degraded. Furthermore, these
data suggest that previous reports on the use of these pesticidal plants in crop protection [17,18,31] have
maintained crop yield not only by fighting pests, but by functioning as a foliar fertiliser. Contribution
to growth and yield is likely to be related to the addition of nitrogen [32] where T. diversifolia [33,34]
and T. vogelii [35–37] are known to produce nitrogen-rich green biomass.
2.2. Effect of Treatments and Application Method on Common Bean Metabolite Production
Analysis of chlorophyll content, flavonoids and anthocyanins indicated that the T. vogelii treatment
resulted in significantly higher chlorophyll concentration, followed by the foliar fertiliser and
T. diversifolia (Table 2). Lower chlorophyll content was observed in water, water and soap and
the synthetic pesticide. Flavonoid content was highest in T. diversifolia treated plants, followed by
the foliar fertiliser and T. vogelii, and these were significantly different from the water and water and
soap treatments. Pereira et al. [38] reported that chlorophyll content could enhance photosynthesis
rates, which ultimately influences plant vigour. No significant variation was observed in anthocyanin
content across treatments or modes of application suggesting that the influence of treatments on plant
metabolism was specific.
As expected, the commercial foliar fertiliser had a significant effect on metabolite production.
The effect of T. diversifolia on chlorophyll content was supported by previous research by Oke et al. [39].
Leaf samples were further analysed to identify the contribution of treatments on the amounts of
specific metabolites including primary metabolites (phenylalanine and tryptophan) and the secondary
metabolite, rutin. An analysis of variance showed that these metabolites were higher when exposed to
the foliar spray method of application in comparison with soil drenching (Table 3).
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Plants 2020, 9, 149
Table 2. Effect of treatment on the presence of key metabolite groups in common bean.
Chlorophylls Flavonoids Anthocyanins

Treatments
(mg/L) (Abs g DM−1 ) (Abs g DM−1 )
Foliar fertiliser 19.3 ± 1.84 b 2.8 ± 0.28 ab 0.1 ± 0.01 a
Synthetic pesticide 13.7 ± 0.74 c 2.4 ± 0.14 bcd 0.1 ± 0.00 a
Tephrosia vogelii 24.6 ± 1.29 a 2.7 ± 0.23 abc 0.1 ± 0.01 a
Tithonia diversifolia 18.9 ± 0.89 b 3.0 ± 0.16 a 0.1 ± 0.01 a
Water 12.7 ± 0.53 c 2.1 ± 0.17 d 0.1 ± 0.03 a
Water and soap 14.0 ± 0.49 c 2.2 ± 0.15 cd 0.1 ± 0.02 a
Method of Application
Soil drench 15.9 ± 0.89 b 2.5 ± 0.12 a 0.1 ± 0.01 a
Foliar spray 18.5 ± 1.14 a 2.6 ± 0.13 a 0.1 ± 0.01 a
2-Way ANOVA (F-Statistics)
Treatment 27.8 *** 3.4 * 0.6ns
Method of application 12.7 ** 0.5ns 0.4ns
Treatment * Method of application 3.0 * 1.3ns 0.3ns
The values presented are means ± SE. *, **, *** = significant at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 respectively, ns = not
significant. Means followed by the same letter in a column are not significantly different.
Table 3. Two-way Analysis of Variance on the influence of mode of application on the relative
abundance (mg/g dry weight) of phenylalanine, tryptophan and rutin.
Method of Application Phenylalanine Tryptophan Rutin

Foliar spray 43608.3 ± 4557.06 a 45478.3 ± 5450.15 a 15093.8 ± 1675.05 a
Soil drench 26209.9 ± 2127.52 b 26805.8 ± 2566.88 b 9342.5 ± 895.06 b
Two-way ANOVA (F-statistics) 13.4 *** 10.3 ** 12.8 ***
The values presented are means ± SE. **, *** = significant at p ≤ 0.01, p ≤ 0.001 respectively. Means followed by the
Overall, the foliar application was more effective in inducing changes, regardless of treatment
(Figure 1). Foliar application was effective because it facilitated direct contact between the solution
applied and the leaf surface where adsorption takes place [40,41], whereas application to the soil is
indirect [36]. From this study, the production of amino acids induced by T. diversifolia and T. vogelii
was similar to that observed with Neem (Azadirachta indica) where similar metabolic changes were
reported by Sharma [42]. Similarly, Neem extracts applied to tomatoes have been observed to increase
the abundance of several flavonoids through the jasmonate pathway [22].
Primary and secondary metabolites in plants can contribute to the development and growth
of crop plants [22] as well as contribute to plant defence mechanisms [43]. Flavonoids are known
to help a plant relate with other organisms and the environment thereby responding to biotic and
abiotic stress [44,45]. Their contribution to growth is explained by their effect on auxin transport, shoot
growth, root development and nitrogen fixing processes in legumes [46–49]. Examples of flavonoids in
bean plants are kaempferol, quercetin [50,51], and rutin [52]. Flavonoids are also reported to mediate
plant resistance to herbivores [53] thus, their increased occurrence could enhance defence against
antagonists. Amino acids such as phenylalanine and tryptophan are known to contribute to plant
growth and metabolism such as auxin biosynthesis in the rhizosphere [54], growth and nodulation [55].
Hence, applications that increase such metabolites in common beans could be beneficial to provide
sustainable production techniques for bean resistance to pests, growth and yield as reported for ginger
(Zingiber officinale) [56].
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Plants 2020, 9, 149
Figure 1. Relative abundance (mg/g dry weight) of (a) phenylalanine, (b) tryptophan and (c) rutin in
common bean plants when exposed to different experimental treatments.
2.3. Correlations Between Bean Plant Growth Yield Parameters and Common Bean Metabolites
Three principal components (PC1, PC2 and PC3) were retained to explain 87.2% variance of the
dependent variables (Table S1). The criteria for selection were based on a cumulative variance of 70%
and an eigenvalue greater than one. The first principal component accounted for a total variance
of 57.37%, while the second and third components explained 18.3% and 8.7% of the total variance,
respectively. PCA observations of the treatments and their modes of application indicated the plant
extracts applied to the bean plant or the soil were grouped together, implying that their contribution to
bean growth was related (Figure 2a). Regardless of the plant extract species, application to the leaves
had a negative relation with application to the soil. T. vogelii (Foliar spray) and water (Soil drench)
were the treatments showing the highest and lowest influence, respectively. Furthermore, applying
water had a low effect on the bean crop development regardless of the method of application.
Figure 2. Two-dimensional principal component analysis (PCA) of (a) treatments applied using foliar
spray and soil drench methods. Green marks indicate the treatments applied using foliar spray (FS)
while blue marks indicate the application by soil drench (SD) where Tv = T. vogelii; Td = T. diversifolia;
FF = foliar spray; W = water only; W + S = water and soap; S = synthetic; and (b) the covariance
among all growth and metabolite parameters where CC = Chlorophyll content; FL = Flavonoids; AN =
Anthocyanins; PH = Plant height; NL = Number of leaves; NB = Number of branches; LA = Leaf area;
SW = Stem width; LG = Leaf greenness; NPP = Number of pods per plant; and SY = seed yield/plant.
Anthocyanin content correlated with the second principal component, which was different from
the rest of the variables that all correlated with the first principal component (Figure 2b). This difference
32
Plants 2020, 9, 149
is likely to be based on the fact that anthocyanin values were minimal across all the treatments, with
no significant difference observed in influencing bean development across the treatments. The first
principal component’s interpretation showed that yield parameters (number of pods per plant and
seed yield per plant) and chlorophyll content explained more of the variation describing effects of the
treatments. The number of branches showed a positive correlation with key metabolites, e.g., rutin
(0.61), phenylalanine (0.58) and tryptophan (0.63). The PCA correlation matrix, eigenvalues, factor
loadings, and factor scores at p = 0.05 can be found in Tables S2–S6.
3.1. Bean Rearing and Plant Material Preparation

The experiment was carried out in a controlled pest-free glass house at the Nelson Mandela
African Institution of Science and Technology, Arusha, Tanzania (Latitude 3◦ 24 S Longitude 36◦ 47
E, elevation of 1168 masl with a mean annual rainfall of 1200 mm, mean maximum temperature of
21.7 ◦ C and mean minimum temperature of 13.6 ◦ C). Each treatment unit consisted of eight bean plants.
Common bean seeds used for the experiment were of Lyamungo 90 variety, purchased from the Seliani
Agricultural Research Institute. Two seeds were planted in each pot, later thinned to one plant per
each pot, using 2-litre volume pots containing standard potting compost. All pots were arranged in a
complete randomized block design on a bench in the glasshouse, providing even lighting, ventilation,
temperature (25 ± 5 ◦ C) and equal amounts of water per pot.
Pesticidal plant materials (T. vogelii and T. diversifolia) were collected from Lyamungo field
areas, dried under the shade and ground into fine powder using previously reported methods and
locations [18]. T. vogelii and T. diversifolia are among a large group of insecticidal plants that have been
used for decades for pest control [17–19,57]. Positive controls included synthetic pesticide (Karate,
lambda cyhalothrin) and a commercial foliar fertiliser (BioForce, an organic extract from seaweeds
and blue green algae) which were applied according to instructions provided on the label. Pesticidal
plant powders were extracted in soapy water (0.1% soap) to produce an extract solution of 10% (w/v)
following previously reported methods [18]. Negative control treatments were with plain water, and
water with 0.1% soap.
All treatments were applied in two different methods, either as a foliar spray using a hand sprayer
or directly to the soil with a small watering can, ensuring equal amounts were applied to each plant.
The treatments were applied fortnightly from the first week after plant germination until the time of
bean flowering, i.e., a total of four treatment applications.
3.2. Collection of Growth Parameters Data and Leaf Samples for Chemical Analysis
Growth parameters and samples for chlorophyll content and bean leaf chemistry analysis were
collected before bean flowering. Yield parameters were collected close to the maturity of the beans
and the total yield collected after final bean harvesting. The growth parameters that were measured
included plant height, number of leaves, number of branches, main stem width, leaf area and leaf
greenness. Leaf greenness was scored using a scale of 1–5 where 1 was regarded as low greenness and
5 as high greenness using a leaf colour chart as previously reported [58]. Leaf area was determined
from the direct measurements of length as a distance between the base and apex of the leaflet, and the
width between positions of the leaflets. Leaf area was then calculated using the formula described by
Bhatt [59]
LA = 11.98 + 0.06 L × W (1)
where LA = Leaf area’, L = leaf length and W = leaf width.

Plant leaf samples were harvested three days after spraying the beans. Harvesting was done at
the vegetative stage, just before flowering. Four plants from each treatment were randomly selected
from each plant. The leaves were thoroughly washed with distilled water. Two leaves from each plant
33
Plants 2020, 9, 149
were placed in a desiccator with silica gel, desiccated and prepared for phytochemical analysis. The
other two leaves collected from each plant were used for spectrophotometric analysis described below.
3.3. Spectrophotometric Analysis of Key Metabolite Groups in Bean Leaves
3.3.1. Chlorophyll Content Analysis

Chlorophyll concentration was determined through the extraction of chlorophyll from the third
leaf of the growing tip of each plant using Dimethyl Sulphoxide (DMSO) as described by Hiscox,
1980 [60]. This involved placing 100 mg of the middle portion of the leaf in a 15 mL vial containing
7 mL DMSO and incubating at 65 ◦ C for 24 h after which the leaves were completely transparent
signifying chlorophyll extraction. The extracted liquid was transferred to graduated tubes and made
up to a total volume of 10 mL with DMSO and then kept at 4 ◦ C waiting for analysis. To determine the
chlorophyll content, 300 microliters of the sample were transferred into an 86-well plate, where the
absorbance at 645 nm and 663 nm were read using a spectrophotometer (Synergy, Multi-mode reader,
Biotek Instrument Inc. Winooski, VT, USA) against DMSO as a blank. Chlorophyll levels in milligrams
per litre (mg/l) were then calculated using the formula described by Arnon [61].
Total Chl = 20.2 × D645 nm + 8.02 × D663 nm (2)
where Chl = Chlorophyll, D = the Absorbance value at the respective wavelengths obtained from
the spectrophotometer.
3.3.2. Anthocyanins and Flavonoids Analysis

Flavonoids and anthocyanins in bean plant leaves were determined using the method described
by Makoi et al. [62]. Dried and ground bean leaves were used, where 0.1 g of the plant powder was
extracted in 10 mL acidified methanol, made at a ratio of 79:20:1 of MeOH:H2 O:HCl. The extract was
incubated for 72 h in darkness for auto extraction and then filtered through a filter paper (Whatman
#2). Absorbance of the clear supernatant was measured at 300, 530, and 657 nm in a spectrophotometer
(Synergy, Multi-mode reader, Biotek Instrument Inc. Winooski, VT, USA) against acidified methanol
as a standard. Flavonoid concentration was obtained from the measured absorption at 300 nm and
expressed in Abs g DM−1 .
Abs g−1 DM = Abs300 (3)
Anthocyanins were measured by using the formula described by Lindoo and Caldwell [63].
Abs g−1 DM = Abs530 − 1/3 × Abs657 (4)
where Abs = Absorption readings recorded from the spectrophotometer. The resulting concentration
was expressed as Abs g DM−1 .
3.4. HPLC Detection of Primary and Secondary Metabolites

Desiccated beans leaves were powdered using an electric coffee grinder, and 50 mg of the powder
was extracted in methanol (1 mL) and left to stand for 24 h at room temperature before chemical
analysis. Extracts were transferred to Eppendorf tubes and centrifuged for 20 min at 500 rpm. From
this 300 μL supernatant was transferred into HPLC glass vials for separation. The sample analyses
were performed by Liquid Chromatography-Electrospray Ionization Mass Spectroscopy (LC-ESIMS)
and UV spectroscopy using a Thermo Fisher Velos Pro LC-MS. Aliquots of extract were injected directly
onto a Phenomenex (Macclesfield, Cheshire, UK) Luna C18(2) columns (150 × 3.0 mm i.d., 5 um particle
size) and the compounds were eluted using methanol (A), water (B) and acetonitrile containing 1%
formic acid (C) with A = 0%, B = 90% at T = 0 min; A = 90%, B = 0% at T = 20 min and held for 10 min
with C at 10% throughout the analyses. Column temperature was 30 ◦ C with flow rate = 0.5 mL min−1 .
34
Plants 2020, 9, 149
High resolution MS spectra were used to provide additional data for compound identification and
were recorded for a subset of samples using a Thermo LTQ-Orbitrap XL mass spectrometer (Waltham,
MA, United States) with compound separation on an Accela LC system.

The experiment was conducted following the completely randomised block design with eight
replications to assess yield and growth of common beans and four replications to assess the metabolites.
Effects of treatments and their interactions observed were subjected to Analysis of Variance. The means
of treatments and interactions were compared using the least significant difference (LSD) test at a
significant level of p ≤ 0.05. Principal Component Analysis (PCA) was performed to explain potential
covariance between bean plant growth, yield parameters and common bean metabolites. All the
analyses were done using XLSTAT version 2019.2.2.59614 (Addinsoft (2019). XLSTAT statistical and
data analysis solution. Boston, MA, USA. https://www.xlstat.com).
4. Conclusions
In this study, foliar sprays of the pesticidal plants T. vogelii and T. diversifolia enhanced common
bean growth, yield and induced essential metabolites known for facilitating plant growth. Thus,
their use helps to reduce the need for both synthetic pesticides and fertilisers by sustainably reducing
arthropod pests whilst increasing plant nutrition. As soil fertility and crop pests are considered two
of the main problems contributing to the yield gaps of smallholder farmers, using botanical extracts
for crop production can help farmers move towards more sustainable agro-ecological approaches to
crop production, tackling two problems at the same time. Pesticidal plants such as T. vogelii and T.
diversifolia can be obtained cheaply in many African countries. T. vogelii can easily be propagated,
although it should not be cultivated near large bodies of water as the rotenoid compounds can be
harmful to fish. T. diversifolia is widely growing in roadsides and field margins and is considered
invasive in some parts of Africa, therefore, care is also needed when cultivating this plant to keep it
under control. Other commonly used pesticidal plant species may also have beneficial impacts on crop
growth, where further validation is recommended.
Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/2/149/s1,

Table S1: Effects of foliar fertiliser, synthetic pesticides and botanical plants extract on common beans growth.
The values presented are means ± SE. *, **, *** = significant at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 respectively, ns = not
significant. Means followed by the same letter in a column are not significantly different. Tables S2–S6: The
correlation matrix used, eigenvalues, factor loadings, and factor scores.
Author Contributions: Conceptualization, A.G.M., P.C.S., E.R.M., S.R.B., Y.L.B.T., and P.A.N.; methodology,
A.G.M., P.C.S., I.W.F. and S.R.B.; software, P.C.S.; validation, P.C.S.; formal analysis, A.G.M., P.C.S. and A.K.S., and
I.W.F.; investigation, A.G.M., resources, P.C.S. and S.R.B.; data curation, P.C.S., I.W.F. and A.G.M.; writing—original
draft preparation, A.G.M.; writing—review and editing, P.A.N., E.R.M., P.C.S. and S.R.B.; visualization, P.C.S.;
supervision, P.A.N., S.R.B. and P.C.S.; project administration, S.R.B.; funding acquisition, S.R.B. and P.A.N. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by grants from the McKnight foundation to SRB Grant No: 17-070 and the
World Bank to PAN Grant No.5799-TZ.
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38
plants
Article
Fumigant Toxicity in Myzus persicae Sulzer
(Hemiptera: Aphididae): Controlled Release of
(E)-anethole from Microspheres
María J. Pascual-Villalobos 1, *, Manuel Cantó-Tejero 1 , Pedro Guirao 2 and María D. López 3
1 Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor S/N La Alberca,
30150 Murcia, Spain; manuel.canto@carm.es
2 Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, Escuela Politécnica
Superior de Orihuela, Carretera de Beniel Km. 3.2, 03312 Orihuela, Alicante, Spain; pedro.guirao@umh.es
3 Departamento de Producción Vegetal, Facultad de Agronomía, Universidad de Concepción, Campus Chillán,
Avenida Vicente Méndez 595, P.O. Box 537, Chillán 3812120, Chile; lolalopezbelchi@gmail.com
* Correspondence: mjesus.pascual@carm.es
Abstract: (E)-anethole is a phenylpropanoid that is the main compound found in the essential oils
(EOs) of anise and fennel seeds, and either fumigant or direct contact activity of this compound
has been demonstrated against aphids and stored product pests. In this work, solid microspheres
were prepared by three methods—oil emulsion entrapment, spray-drying, and complexed with
β-cyclodextrin. Fumigation activity of each microsphere preparation was tested against the green
peach aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), on pepper leaves. The best insecticidal
activity was with (E)-anethole encapsulated in oil emulsion beads and introduced to aphids as a vapour
over 24 h, with an LC50 of 0.415 μL/L compared to 0.336 μL/L of vapors from free (E)-anethole.
Scanning electron microscopy of the beads revealed a compact surface with low porosity that produced
a controlled release of the bioactive for more than 21 d, whilst most of the volatile was evaporated
within two days if applied unformulated. Spray drying gave spherical particles with the greatest
encapsulated yield (73%) of 6.15 g of (E)-anethole incorporated per 100 g of powder. Further work
will be done on improving the formulation methods and testing the solid microspheres in all aphid
stages scaling up the experimental assay. It is foreseen that nanotechnology will play a role in future
developments of low risk plant protection products.
Keywords: encapsulation; essential oils; botanical active substances; insecticidal activity; aphids;
anise; fennel; oil emulsion entrapment; spray drying
1. Introduction
(E)-anethole [trans-1-methoxy-4-(C1-propenyl) benzene] is an aromatic ether synthesized by
some plants. This phenylpropanoid is the main compound in the essential oil of umbelifers such
as anise (Pimpinella anisum L.) or fennel (Foeniculum vulgare Miller) but it is also present in other
plant families-Schisandraceae-Illicium verum Hook. f, -Rutaceae-Clausena anisata (Willd) Hook f ex
Benth,-Backhousiaceae-Backhousia anisata Vickery and -Magnoliaceae-Magnolia salicifolia (Sieb et Zucc)
Maxim. [1–5].
The Apiaceae (formerly Umbelliferae) family comprises vegetables (celery-Apium graveolens L.,
parsley-Petroselinum sativum L., coriander-Coriandrum sativum L.), herbs, and spices (anise, fennel,
cumin-Cuminum cyminum L.). Aniseeds have long been used to make schnapps like the popular French
pastis, a beverage distilled from anise, liquorice, and fennel seeds macerate.
Fumigant toxicity of anise and cumin essential oils has been reported against the cotton aphid
(Aphis gossypii Glover (Hemiptera: Aphididae) [6]. Vapours of anise essential oil (EO) or its main

Plants 2020, 9, 124
compound (E)-anethole were toxic (LD90 = 0.18 μL/cm2 or 0.14 μL/cm2 respectively) to the bird
cherry-oat aphid (Rhopalosiphum padi L., Hemiptera: Aphididae) in a laboratory bioassay within small
air-tight dishes (2.2 × 2.2 × 1 cm3 ), according to reference [1].
A blend of (E)-anethole, limonene, and fenchone at 880 ppm was toxic (100% mortality) against
Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), a pest of stored cereals (using a fumigant bioassay
1μL/vial of 15 mL at 30 ◦ C in the dark), as reported in reference [7]. Another phenylpropanoid, estragole
(also present in fennel EO) and fenchone were more active against Sitophilus oryzae L. (Coleoptera:
Curculionidae) and Callosobruchus chinensis Fab. (Coleoptera: Bruchidae) than (E)-anethole [8].
(E)-anethole in combination with 1,8-cineole (1:1) was the best regarding fumigant toxicity on the red
flour beetle adults, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), and it was also observed
that heating enhanced the toxicity [9].
Other references in the literature [10–12] point out at direct contact activity of the substances
against aphids and stored products pests (Ephestia kuehniella Zeller, Lepidoptera: Pyralidae). Fennel
EO (with 67.5% of (E)-anethole and 25.5% of fenchone) was more active in M. persicae than anise
(93% (E)-anethole), contact LD50 = 0.06 or 0.43%, respectively, by spraying on aphid-infested cabbage
plants [13].
Solid nanoparticles of monoterpenes (carvacrol, thymol, eugenol) have been obtained using
chitosan, β-cyclodextrin, zeine, modified starch, or polyethylen glycol (PEG) as encapsulating
agents [14,15]. In previous works, beads of linalool were made by an oil emulsion entrapment
method using starch, the encapsulation yield obtained was 86% and the time to release half
of the bioactive exceeded 70 days [16]. Other authors prepared nanoparticles of (E)-anethole by
emulsification and nanoprecipitation using a biodegradable polymer accepted for clinical drug
delivery—polylactic-co-glycolic acid (PLGA) [17]—and after an initial burst release the activity against
Gram+ bacteria lasted for more than four days. Another reference explains the encapsulation of
(E)-anethole in liposomes, that are vesicles in which an aqueous phase is enclosed by a membrane
of phospholipids; in this case, the liposomes were stable at 4 ◦ C and provided a controlled release of
(E)-anethole [18]. An enhancement of the antiaflatoxigenic efficacy of I. verum EO by nanoencapsulation
in gel or lyophilized chitosan nanoparticles has also been reported [2].
Our work focusses on the use of encapsulated EOs as a fumigant system against insect pests
in closed environments. For instance, solid formulations, prepared by emulsification of coriander
and basil EOs in alginate and glycerol and dripping into a calcium chloride solution, were tested
inside funnel traps and were as effective as the insecticide dichlorvos as killing agents for adults of the
Indianmeal moth (Plodia interpunctella Hübner, Lepidoptera: Pyralidae) adults lured [19].
It is hypothesized that fumigant activity of plant volatiles could be exploited to control
phytophagous insects of vegetables grown in greenhouses but this idea has not yet reached commercial
development due to the volatility and low stability of these compounds. The objective of our work was
to formulate (E)-anethole as solid microparticles (by oil emulsion entrapment, spray drying or molecular
inclusion) and test the potential of the vapour released as aphicide on pepper leaves. Experiments were
implemented with the green peach aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), one of the
main pests worldwide attacking fruit trees and vegetables and causing direct damage and transmission
of virus diseases.
2. Results
2.1. Encapsulation of (E)-Anethole

The formulations prepared turned out to be within the micrometric range from 1.7 μm to 4 μm
particle size. Spray drying (SD) gave the greatest encapsulation yield (73%) and loading (6.15 g/100 g
of powder) of (E)-anethole in the capsules, although, by oil emulsion entrapment (OEE), the amount of
bioactive loaded was quite similar (see Table 1).
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Plants 2020, 9, 124
Table 1. Physico-chemical parameters in the microspheres.
Loading
Encapsulation Yield
Formulation Method 1 Dry Sphere Size (μm) (g Monoterpene 100 g−1
(%)
Powder)
SD 4.00 a 6.15 a 73 a
OEE 1.70 b 5.20 a 26 b
MI 3.52 a 1.33 b 14 c
1 SD = Spray-drying, OEE = oil-emulsion-entrapment, MI = molecular inclusion (see Section 4). Samples were
prepared three times and then bulked. Different letters in the same column mean significant differences at (p ≤ 0.05).
The plate in Figure 1A shows the (E)-anethole/β-cyclodextrin inclusion complex (MI) in the
form of irregular particles, therefore this method is less suitable to encapsulate the bioactive product.
On the other hand, spray drying (inlet air temperature of 100 ◦ C) of an emulsion with maltodextrin (SD)
produced spherical particles of all sizes pilled up due to the strong attraction to each other (Figure 1B).
Finally, SEM micrographs (C) and (D) in Figure 1 represent the dry calcium alginate beads (OEE) and
reveal a compact surface with low porosity achieved after using glycerol, the surfactant and a high
percentage of sodium alginate (4%).
(A) (B)
(C) (D)
Figure 1. Scanning Electron Microscopy (SEM) micrographs of nanoparticles obtained by

(A) spray drying (SD), (B) molecular inclusion (MI) with × 100-fold magnification, (C) oil emulsion
entrapment (OEE) with × 100-fold magnification, and (D) oil emulsion entrapment (OEE) with ×
190-fold magnification.
2.2. Fumigant Activity and Controlled Release of (E)-Anethole

Our formulations have good aphicidal potential (Table 2). Free (E)-anethole vapours were fast
and entered the aphids giving the lowest LC50 (0.336 μL/L) after 24 h. Meanwhile OEE formulation
41
Plants 2020, 9, 124
exhibited a LC50 = 0.415 μL/L followed in activity by the SD preparation. Results of vapour toxicity
apply just to the experimental conditions used (2.5 L dessicators plus two pepper leaves and 20 apterous
M. persicae females in each leaf). Overall, the encapsulated (E)-anethole had a LC90 from 0.78 to 3.38 μL/L
after 24 h exposure to the aphids.
Table 2. Lethal Concentrations 1 of vapours of (E)-anethole (μl/L air) to Myzus persicae Sulzer (Hemiptera:
Aphididae), pink clone, after 24 h.
Formulation
LC50 95% CI LC90 95% CI χ2
Method 2
SD 1.292 1.169–1.476 3.383 2.706–4.305 0.487 ns
OEE 0.415 0.416–0.468 0.780 0.675–0.832 23.850 *
Free (E)-anethole 0.336 0.306–0.369 1.043 0.867–1.255 8.572 ns
1 χ2
Probit analysis fitting lethal concentration 50 (LC50 ) and 90 (LC90 ) and confidence intervals. non-significant
(n.s.) or significant (*) at 0.1%. 2 SD = spray drying, OEE = oil emulsion entrapment (see Section 4).
The results are presented in more detail in Figure 2. The graph shows the dose response of the
formulations including the molecular inclusion complexes (MI) for which the lethal concentrations
could not be computed due to the very low mortality values obtained (this is why this treatment is not
included in Table 2). The regression line of free (E)-anethole intercepts the probit = 5 line (that represents
LC50 ) first, indicating the greatest effect at a low concentration, while the OEE formulation intercepts
the probit = 6.28 (that represents LC90 ) first, indicating more effectivity at high doses (Figure 2).
Overall a similar response of the preparations SD, MI, and free (E)-anethole is observed due to the
parallel regression lines; what changes is the amount of product required to produce the same mortality.
Figure 2. Regression lines of probit analysis for mortality against Myzus persicae. OEE = oil emulsion
entrapment, SD = spray drying, MI = molecular inclusion, and free (E)-anethole.
In Figure 3, we can see that the OEE formulate was quite close in toxicity to free (E)-anethole after
24 h, but presumably, the former would have had effects beyond the short period of observation if
evaluated. In this context, MI complexes hardly produced mortality in the short term.
42
Plants 2020, 9, 124
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Figure 3. Mortality (%) in Myzus persicae Sulzer (Hemiptera: Aphididae) after exposure (24 h at 25 ◦ C)
to vapours (μl/L air) of (E)-anethole released from microspheres (OEE = oil emulsion entrapment,
SD = spray drying, MI = molecular inclusion) or free (E)-anethole. Percentages of mortality refer to
total number of insects tested in the six replications per dose and formulation (n = 240).
Such results are explained by different paces at which (E)-anethole is released from the microspheres
(Figure 4). At 15 ◦ C, there are statistically significant differences among all treatments (Figure 4A),
and at 40 ◦ C, there are statistically significant differences between free (E)-anethole and MI but not
between OEE and SD (Figure 4B). The formulation slows down the availability of the toxic vapours
in comparison with the free (E)-anethole particularly under the conditions of the fumigant bioassay
(25 ◦ C and mortality recorded after 24 h). It is foreseen, however, that the toxic vapours would last
several weeks further.
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Figure 4. Cont.
43
Plants 2020, 9, 124
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Figure 4. Release of free (E)-anethole and controlled release from formulated microspheres for 21 d (A)
at 15 ◦ C and (B) at 40 ◦ C. Mean values in the same day with the same letter do not differ significantly
(p > 0.05) using Duncan’s test.
3. Discussion
Plants are a good natural source of (E)-anethole, fennel variations in the Iranian genotypes
accounted for 1.2–88.4% of the EO whilst in anise 78.6–96% are common [1,20–22]. The mode of entry
of the bioactive volatile in the insects is possibly via the respiratory system by inhalation [8,23] but its
mode of action remains unclear. Some publications refer to greater activity when mixtures of volatiles
for example limonene and fenchone [7,13] or 1,8-cineole [9] are applied together with (E)-anethole.
Greater insecticidal fumigant activity against Trichoplusia ni Hübner (Lepidoptera: Noctuidae) of
lemongrass or thyme EOs or the binary mixture of the two main compounds often had better action
than pure compounds [24].
Therefore it is worthwhile to study further the fumigant effect of (E)-anethole in binary mixtures
with monoterpenoids against M. persicae in all insect stages and expand the period of study (to several
days) to provide new data on the advantages of a controlled release to be applied in pest control into
a greenhouse.
The bioassay was done inside air-tight desiccators. Mortality was recorded after 24 h, and once
opened, the concentration of the volatiles inside the desiccators could change; this was the main reason
why we decided to take just one observation. Another reason was to be sure the leaves inside the
desiccator were healthy enough for the aphids to remain alive, but for those affected by the insecticidal
effects of anethole. The bioassay has to be improved for longer periods of observation.
If we compare our results with those of the literature, there is an agreement in the fumigant effect
of EOs containing (E)-anethole. The lethal doses varied depending on the insect pest and the volume
of the chamber used in the assays. The LD50 of fennel EO was 10.3 μL/L in Brevicoryne brassicae L.
(Hemiptera: Aphidae), whereas 2 μL/L of cumin or origanum EOs (with carvacrol, (E)-anethole and
pulegone in the oil) has been reported to cause 100% mortality in A. gossypii [25,26]. Our results of LC50
range from 0.3 to 1.47 μL/L of (E)-anethole (free or encapsulated) against M. persicae. Repellency was
reported for vegetable aphid pests such as M. persicae, A. gossypii, and Macrosiphum euphorbiae Thomas
(Hemiptera: Aphididae) in our previous works with values of RD50 = 0.07–0.09 μL/cm2 for anise and
RD50 = 0.04–0.08 μL/cm2 for (E)-anethole [27,28], and the pure compound was more repellent for the
pink clone of M. persicae and A. gossypii. The efficacy of anise EO by contact applications was greater
44
Plants 2020, 9, 124
against early nymphal instars (first and second nymphs), LD50 = 0.003% v/v, than to late nymphal
instars (third and fourth nymphs), LD50 = 0.017% v/v, of apterous aphids [29]. Newly emerged adults
of T. castaneum were highly susceptible to vapours of (E)-anethole in comparison with sclerotized older
beetles in which concentrations at least of 20 μL/L were required to produce toxic effects [9]. Therefore,
soft-bodied suckling pests such as aphids might be more susceptible to fumigation by EOs than stored
product beetle pests.
Encapsulation offers clear advantages for a bioactive volatile—in addition to avoid releasing the
product all at once—like protection against environmental conditions (light, temperature, oxygen, etc.).
Further work will be done on improving the formulation methods described in this paper where
encapsulation yields have ranged from 14 to 73%; of the three methods tested, OEE and SD are more
promising. It would be of practical use the release of just the required amount of active that causes
high insect mortality (previously calculated for each stage of development) for a prolonged period of
time. Other authors have obtained loadings of 13%, particle size < 180 nm and bactericidal activity
prolonged for more than 4 d from PLGA (E)-anethole nanoparticles [17]. Similarly, PEG nanoparticles
of geranium and bergamot EOs slowed the release of the volatiles down and the residual contact
activity against cockroaches was improved [30]. Polymer based nanoencapsulation of EO is considered
for plant protection products and the type of polymers consist mainly of polysaccharides (chitosan,
alginate and starch), polyesters (PEG) or biodegradable materials such as gum arabic or lecithins.
Plant essential oils are available raw materials, for example: anise EO is obtained from anise fruits
at a yield of 2–6% and its market price is 7–9 €/Kg. We propose that botanicals coming from plants
that have been used as foods or condiments be considered as safe plant protection products. In fact,
the European Food Safety Authority (EFSA) regards them as Low Risk Active Substances (LRAS).
All classes of controlled release systems could be considered as new formulations for insecticide
applications: nanocapsules or microcapsules with polymers, cyclodextrin complexes, solid-lipid
nanoparticles, nanoemulsions or microemulsions, liposomes, and nanogels.
Nanotechnology is an area under development in plant protection and scaling up the experiments
is important to be able to extrapolate the results to applications in agricultural production systems.
4.1. Materials
(E)-anethole (99%), calcium chloride, β-cyclodextrin (98%), maltodextrin and sodium alginate
were obtained from Sigma-Aldrich, whereas glycerol (99.5% pure) was obtained from Labogros, France.
Analytical grade solvents and surfactants (Tween 80) were from Sigma-Aldrich.
4.2. Microsphere Preparation
4.2.1. Beads of (E)-Anethole by Oil-Emulsion-Entrapment (OEE)

Beads were formed by dripping an alginate solution (containing a dispersion of (E)-anethole,
glycerol and Tween 80) into a calcium solution. Diffusion of the calcium in alginate droplets led to their
gelification. The preparation of the internal phase was carried out as follows: (E)-anethole (20 mL) was
dispersed in glycerol (20 mL) and Tween 80 (20 mL). The blend was dispersed in 20 mL of alginate
(40 g/L). This dispersion was dripped into a calcium chloride solution (10 g/100 mL). Beads were
filtered with a wire mesh and finally were dried overnight at room temperature (15 ◦ C). Samples were
prepared three times and then bulked.
4.2.2. Preparation of β-Cyclodextrin/(E)-Anethole Molecular Inclusion (MI) Complexes

A chemical precipitation method was used to prepare β-cyclodextrin/(E)-anethole complexes.
β-cyclodextrin (5 g) was dissolved in 300 mL of water at 55 ◦ C for half an hour, 30 mL of (E)-anethole
were added slowly to the suspension of β-cyclodextrin. The blend was frozen overnight at −20 ◦ C.
45
Plants 2020, 9, 124
The precipitated (E)-anethole/cyclodextrin complex was recovered by lyophilization (24 h) and filtration.
Samples were prepared three times and then bulked.
4.2.3. Spray Drying of (E)-Anethole (SD)

An emulsion of (E)-anethole was mixed with 50 mL of maltodextrin (10%, w/v) then it was stirred
at 300 rpm for 2 h. The emulsion was fed into a laboratory scale dryer (Mini Spray Dryer-B290,
BÜCHI, Flawil, Switzerland) at room temperature with a flow rate of 4 mL min−1 . The inlet and outlet
temperatures were maintained at 100 ◦ C and 60 ◦ C, respectively. The dried powder was collected and
stored in an opaque, air-tight container at 4 ◦ C for further analysis.
4.3. Encapsulation Yield and Loading

The amount of (E)-anethole into the dry microspheres was determined by GC/MS analysis as
follows: 0.5 g of powder was dispersed in 8 mL of distilled water and 4 mL of hexane in 15 mL glass
vials. Vials were heated and stirred in a hot plate at 60 ◦ C for 30 min. The organic phase containing
(E)-anethole was decanted, and the aqueous phase was exhaustively extracted with hexane four times
(4 × 4 mL). These four phases were combined. The hexane was removed using a nitrogen stream.
The quantitative analysis of (E)-anethole was carried out using a model 5890 Series II equipped with
a DB-Waters 30 m × 0.32 mm capillary column coated with a polyethylene glycol film (1 μm thickness)
and an Agilent model 5972 inert mass spectrometry (MS) detector (Agilent, Palo Alto, CA, USA).
The initial oven temperature was held at 60 ◦ C for 1 min. Afterwards, it was increased by 3 ◦ C/min
to 225 ◦ C, with injector at 250 ◦ C, column head pressure at 8.00 psi, helium carrier gas, flow rate of
2.6 mL/min, and splitless with 2 μL of sample injected. The content of (E)-anethole was calculated
according to the area of the chromatographic peak and using linear regression. Prior to the quantification
of monoterpene, the surface (E)-anethole in the formulation was washed.
Encapsulation yield is defined as the ratio between the quantities of (E)-anethole in the capsules
versus the initial amount of (E)-anethole. Loading is defined as the quantity of (E)-anethole per
100 grams of dry microcapsules. SAS version 8.0 for windows (SAS Institute, Inc., USA) was used to
compare mean values of the formulations by a Tukey test at p < 0.05.
4.4. Controlled Release of (E)-anethole through Different Matrix Blends

One gram of dry sample was placed into the vials without sealing. These vials were maintained
in dry conditions at 15 ◦ C and 40 ◦ C in growth chambers (MLR-350H, Sanyo, Japan), and weight loss
was monitored in an analytical balance as a function of time for 21 d. As a control, 1 g of (E)-anethole
was set in a vial to study the weight loss for the same period of time. Data from three replications
were recorded in this assay. Data were statistically analyzed by analysis of variance (ANOVA) using
SPSS (PASW Statistic 18). Duncan’s multiple tests were applied for the calculation of the significant
differences among the controlled release of the blends at the 5% level (p = 0.05).
4.5. Scanning Electron Microscopy (SEM) Analysis

Microspheres were evaluated with SEM JEOL 6100 (SAI, Universidad de Murcia, Spain).
The samples were mounted (both entire structures and cross sections) on specimen stubs with
double sided adhesive tape. The specimens were coated with gold and examined at an accelerating
voltage of 15 kV and a working distance of 20 mm. Topographical images were collected by an image
capture system used for an X-ray detector (INCA, Oxford, UK) at a magnification of 370× and 1000×.
The mean particle diameter, pore diameter, number of pores per unit area and the presence of pores
were recorded. SAS version 8.0 for windows (SAS Institute, Inc., Cary, NC, USA) was used to compare
mean values of the formulations by a Tukey test at p < 0.05.
46
Plants 2020, 9, 124
4.6. Fumigation Bioassay

M. persicae, the green peach aphid, from a laboratory culture (pink clone), maintained at a constant
temperature of 25 ◦ C and a photoperiod of 16:8 h (light:dark) on pepper plants, was used for
the insecticidal experiments. The experimental unit consisted of two pepper leaves, with twenty
apterous females each, placed inside a 2.5 L air-tight desiccator. Each dose was replicated six times,
with 240 insects per dose. The leaf petiole was into an Eppendorf tube with water. The products, either
an amount of powder of the microspheres formulations (range 0–0.1 g/L air), to obtain a dose response,
or the pure free (E)-anethole -pipetted onto a 2.1 cm2 filter paper disk- (range 0–1 μl/L air), were placed
in an unlid petri dish without direct contact with the insects. Therefore, only the volatile toxic effects
were evaluated. The desiccators were maintained at 25 ◦ C and 16:8 photoperiod for 24 h and aphid
mortality was recorded. Controls were prepared exactly the same but the application of the products.
Number of alive and dead aphids was recorded after 24 h. Probit analysis was performed to obtain
LC50 and LC90.
5. Conclusions
Spray drying of an emulsion of (E)-anethole with maltodextrin gave spherical particles with the
greatest encapsulation yield and loading but the beads of (E)-anethole by oil-emulsion entrapment had
better fumigant activity against M. persicae. Most of the free (E)-anethole vapours were available within
2 d of application whilst the preparations prolonged the release period for several weeks and required
at least one week to release 20% of the bioactive depending on the temperature and the formulation, for
instance (E)-anethole complexed with β-cyclodextrin required temperatures over 25 ◦ C to release the
product. Therefore, future experiments should expand the observation period and take into account
the susceptibility of earlier nymphal instars to prove advantages of the practical use of (E)-anethole
encapsulated in the form of microspheres.
Author Contributions: All authors have read and agreed to the published version of the manuscript.
Conceptualization, M.J.P.-V.; methodology and analysis, M.D.L., M.C.-T., and P.G.; writing—original draft
preparation M.J.P.-V.; writing—review and editing, P.G., M.C.-T., M.D.L., and M.J.P.-V.; funding acquisition,
M.J.P.-V. and P.G.
Funding: The authors with to thank the funding received through the research projects FEDER 1420-19 and INIA
RTA2017-00022. Manuel Cantó-Tejero was awarded with a grant (INIA CPD2016-0092) for a predoctoral contract
at IMIDA, Murcia, Spain.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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49
plants
Article
Bioactivity of Common Pesticidal Plants on Fall
Armyworm Larvae (Spodoptera frugiperda)
Kelita Phambala 1 , Yolice Tembo 1 , Trust Kasambala 1 , Vernon H. Kabambe 1 ,
Philip C. Stevenson 2,3 and Steven R. Belmain 2, *
1 Department of Crop and Soil Sciences, Lilongwe University of Agriculture and Natural Resources,
Lilongwe Box 219, Malawi; kelitaphambala@yahoo.com (K.P.); ytembo@bunda.luanar.mw (Y.T.);
tdonga@luanar.ac.mw (T.K.); kabambev@gmail.com (V.H.K.)
Kent ME4 4TB, UK; p.c.stevenson@gre.ac.uk
3 Biological Chemistry and In Vitro Research, Royal Botanic Gardens, Richmond TW9 3AB, UK
* Correspondence: s.r.belmain@gre.ac.uk; Tel.: +44-1634883761
Abstract: The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae) is a recent
invasive pest species that has successfully established across sub-Saharan Africa where it continues
to disrupt agriculture, particularly smallholder cereal production. Management of FAW in its native
range in the Americas has led to the development of resistance to many commercial pesticides before
its arrival in Africa. Pesticide use may therefore be ineffective for FAW control in Africa, so new and
more sustainable approaches to pest management are required that can help reduce the impact of
this exotic pest. Pesticidal plants provide an effective and established approach to pest management
in African smallholder farming and recent research has shown that their use can be cost-beneficial
and sustainable. In order to optimize the use of botanical extracts for FAW control, we initially
screened ten commonly used plant species. In laboratory trials, contact toxicity and feeding bioassays
showed differential effects. Some plant species had little to no effect when compared to untreated
controls; thus, only the five most promising plant species were selected for more detailed study.
In contact toxicity tests, the highest larval mortality was obtained from Nicotiana tabacum (66%) and
Lippia javanica (66%). Similarly, in a feeding bioassay L. javanica (62%) and N. tabacum (60%) exhibited
high larval mortality at the highest concentration evaluated (10% w/v). Feeding deterrence was
evaluated using glass-fibre discs treated with plant extracts, which showed that Cymbopogon citratus
(36%) and Azadirachta indica (20%) were the most potent feeding deterrents among the pesticidal
plants evaluated. In a screenhouse experiment where living maize plants infested with fall armyworm
larvae were treated with plant extracts, N. tabacum and L. javanica were the most potent species at
reducing foliar damage compared to the untreated control whilst the synthetic pesticide chlorpyrifos
was the most effective in reducing fall armyworm foliar damage. Further field trial evaluation is
recommended, particularly involving smallholder maize fields to assess effectiveness across a range
of contexts.
Keywords: botanical pesticide; pesticidal plant; pest management; invasive species; agro-ecological
intensification; sustainable agriculture
1. Introduction
The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) (FAW) is
a polyphagous pest that is invasive and now widely established across sub-Saharan Africa. Although
similar to the native African armyworm, Spodoptera exempta (Walker), FAW is more likely to persist
year-round once established, attacking a much wider range of cereals as well as more than 100 other

Plants 2020, 9, 112
horticultural crops [1]. Native to North and South America, FAW was first reported in West and Central
Africa in 2016 [2] and is now reported in at least 44 African countries [3]. FAW is a heavy foliage feeder
that can cause 100% loss of cereal crops [4]. In the absence of effective control methods, potential maize
yield losses caused by FAW have been estimated between 8.3 and 20.6 million metric tons per year
in just 12 maize-producing countries in Africa. This represents a range of 21–53% of annual maize
production averaged over a three-year period. The value of these losses was estimated at between US
$2481 million and US $6187 million [5].
Current armyworm control relies on the use of synthetic pesticides; however, widespread
over-use and misuse in the Americas have resulted in considerable problems with insecticide
resistance particularly among the carbamates, pyrethroids and organophosphates [6] on which
many African farmers rely. As African farmers have a long history of using plants with pesticidal
properties [7–11], options for developing botanical biopesticides for FAW control may be more realistic
than in other regions [12]. Recent research has evaluated several abundant pesticidal plant species,
confirming that their use in smallholder farming can result in comparable yield to that when using
commercial synthetics, without the severe environmental damage often associated with synthetic
compounds [13–15]. With a need to develop new, effective and agro-ecologically sustainable methods
for controlling FAW in Africa, we set out to screen some of the more promising pesticidal plant
species where considerable knowledge already exists on their abundance, phytochemistry and safe
use. The specific objective of the research presented here was to evaluate potential effects of pesticidal
plants on the larval stage, assessing direct toxicity as well as post-ingestive toxicity and feeding
deterrence. Finally, the most promising pesticidal plant extracts were evaluated in controlled trials
using FAW-infested maize plants to determine whether the plant extracts reduced foliar damage under
cropping conditions.
2. Results and Discussion
2.1. Contact Toxicity and Feeding Bioassays with Ten Pesticidal Plant Species
Water extracts (10% w/v) of ten common pesticidal plants which are regularly used by smallholder
farmers showed variable effects on larval mortality (Figure 1). Tephrosia vogelii Hook.f. and Lantana camara L.
showed very low mortality (<10%) in both feeding and contact toxicity bioassays which was surprising
since previous research on both of these plant species demonstrated high and consistent efficacy against
a range of pest species using the same extraction methods and plant sources and the same biologically
active phytochemicals [13–17]. Low mortality (<40%) was a lso observed with Vernonia amygdalina
Delile followed by Aloe vera (L.) Burm.f. and Trichilia emetica Vahl, despite evidence of efficacy
against other target insect pests [11,18–21]. The most effective plant species were Azadirachta indica
A. Juss., Cymbopogon citratus (DC.) Stapf, Lippia javanica (Burm.f.) Spreng., Nicotiana tabacum L. and
Ocimum basilicum L. which caused at least 50% mortality through at least one bioassay [22]. Although
some plant species had an effect through one application method only (A. indica and L. javanica),
overall, the application method led to comparable effects for most plant species, which is verified
through statistical analyses (Table S1). Other research on the evaluation of botanicals against FAW
in Ethiopia [1], showed N. tabacum to cause 50% mortality to 3rd instars after 72 h exposure, which is
considerably lower than the mortality observed in our bioassay. Further, this work found that A. indica
and six other plant species were more effective than N. tabacum with mortality rates of 75–98%.
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Plants 2020, 9, 112
Figure 1. Mortality of 2nd instars when exposed to extracts of pesticidal plant either topically applied
through a contact toxicity bioassay or through ingestion in a feeding bioassay. Treatments different
from the untreated control (p < 0.05) are indicated by *. Significant differences are presented in Table S1.
2.2. Contact Toxicity and Feeding Bioassays with Five Pesticidal Plant Species
Plant extracts applied to glass fibre discs showed that the five most active plant species from
the previous bioassay caused significantly greater mortality in comparison to the untreated control
(Figure 2a). At the highest concentration (10%), L. javanica (62%) and N. tabacum (60%) exhibited
high larval mortality. The lowest mortality was observed from C. citratus (16%) and O. basilicum
(26%). Although there was a slight dose dependent effect, this was not significant for any of the plant
extracts (p < 0.05; Table S1). Some differences in efficacy were observed in comparison to the first trial
where extracts were prepared in water. Mortality trends between water and methanol extracts were
similar with the exception of L. javanica where no mortality was observed in the feeding bioassay when
applying the water extracts to discs. The reasons for this difference are not clear but may be caused by
differences in methodology, in which water extract was presented on maize leaves to reflect farmer
practices, whereas the methanol extract was a pplied to glass fibre discs to more easily assess potential
effects of extract concentration. However, the differences are more likely due to the extraction efficiency
of the different solvents where hydrophobic compounds with bioactivity were more efficiently extracted
by methanol than water [11,13,16,23]. The lack of a clear dose effect as well as differences in mortality
between the trials using water or methanol could also be caused by differences in larval feeding rates
through feeding deterrence behaviours.
The topical application of plant extracts to FAW larvae showed a strong dose response for four
out of the five plant species (Figure 2b; Table S1), whereas mortality from O. basilicum (4%) was not
significantly different from the untreated control (p < 0.5). As expected, the 10% concentration exhibited
high larval mortality of 50–66% for N. tabacum (66%), L. javanica (66%), A. indica (60%) and C. citratus
(50%). However, the positive control of chlorpyrifos was superior to all plant extracts, causing nearly
100% mortality. Although these extracts were made using methanol, the mortality rates observed were
not significantly different from the application of water extracts in the first trial (p < 0.05).
52
Plants 2020, 9, 112
Figure 2. Mortality of fall armyworm 2nd instars seven days after exposure to five different
concentrations of pesticidal plant extracts when applied through (a) a feeding bioassay and (b) a contact
toxicity bioassay. Treatments differing significantly from the untreated control (p < 0.05) are indicated
by *. Significant differences are presented in Table S1.
All of the five plant extracts used in this trial showed some degree of deterrence (Figure 3).
The most potent feeding deterrents were C. citratus and A. indica. Although mortality appears to
be relatively low with these two species, feeding deterrent compounds could help to reduce crop
damage. In agreement with our observations, other studies demonstrated the deterrent effects of
several Cymbogon species as well as A. indica. In a binary choice test, the essential oils isolated from
C. nardus, C. flexuosus and C. martini exhibited strong antifeedant activity against Acharia fusca and
Euprosterna elaeasa [24]. The antifeedant properties of A. indica are well established, particularly for
a range of lepidopteran pests [25–27].
Figure 3. Antifeedant activity of five plant species extracts fed to fall armyworm 2nd instars, showing
percent of feeding deterrence after 48 h. C = control; T = treated.
Trials evaluating the three most promising pesticidal plant species for their ability to control FAW
larvae on living maize plants showed significant differences in effect among the treatments (Figure 4).
53
Plants 2020, 9, 112
High foliar damage was observed in the negative controls (untreated, water and water plus 0.1%
soap) with mean leaf damage scores of 6.5, 6.3 and 5.4, respectively. The lowest foliar damage score
was observed in N. tabacum treatment (4.6); however, the slightly higher scores for L. javanica (5) and
O. basilicum (5.2) were not significantly different from N. tabacum. The synthetic pesticide, chlorpyrifos,
was the most effective in reducing FAW foliar damage with a mean score of 1.8.
Figure 4. Fall armyworm damage to maize plants when exposed to different treatments over eight
weeks. Boxes represent mean and 95% confidence intervals, tails are max. and min. values, blue crosses
are median values. Significant differences are presented in Table S1.
The observed reduction in foliar damage may be attributed to a combination of toxicity, repellent
and antifeedant effects of the plant extracts, with similar results observed from other studies [1].
The plant extracts did not reduce FAW damage as much as the synthetic pesticide chlorpyrifos, but
most other studies on the use of pesticidal plants show similar lower mortality and damage rates when
using natural pesticides in comparison to synthetic pesticides [13,15]. As most crops can compensate
for some limited pest damage, further studies are required to determine whether these pesticidal plant
treatments are able to maintain yield at comparable levels to synthetic pesticide use, which has been
reported for a number of legume crops [14], cabbages [28,29] and sorghum [30].
3.1. Fall Armyworm Rearing

FAW larvae were collected from maize fields around Mitundu, Lilongwe District, Malawi (latitude
14◦ 11 S longitude 33◦ 46 E, elevation of 1100 metres above sea level (m a.s.l.). To establish a large
colony the larvae were initially reared on portions of young maize leaves; however, once established,
larvae were reared on an established artificial diet. The diet was composed of maize leaf powder,
common bean powder, brewer’s yeast, ascorbic acid, sorbic acid, methyl-p-hydroxybenzoate, vitamin
E capsules, sucrose, formaldehyde and agar [31].
Neonates and 2nd instars were reared in 500 mL plastic containers containing young maize
leaves, which were renewed daily. At the 2nd instar, the larvae were transferred to individual plastic
containers (100 mL) to reduce cannibalism until pre-pupal stage and fed on an artificial diet which was
changed every week until pupation. The pupae were harvested and transferred in Petri dishes lined
with tissue paper and placed in adult emergence cages (mosquito netting around an 18 × 18 × 18 cm
frame). After adult emergence, the moths were fed on honey from a honey-soaked wad of cotton wool
54
Plants 2020, 9, 112
in a container placed in each cage. Eggs laid on filter paper in the cages were removed daily and were
disinfected by dipping them in 10% formaldehyde for 15 min. The eggs were then rinsed thoroughly
with distilled water and dried on filter paper. Thereafter, eggs were placed in small containers until
they hatched to repeat the rearing process [31].
3.2. Plant Material Collection and Extract Preparation

Initial plant screening was carried out with ten pesticidal plant species: A. indica, O. basilicum,
N. tabacum, C. citratus, T. vogelii, A. vera, L. camara, T. emetica, V. amygdalina and L. javanica. Considerable
phytochemical and efficacy knowledge was recorded by our group and others on all the material used
of these species [10,15,17,18,20,24,32–37] which were sourced from the same locations with samples
from four locations around Mitundu, Lilongwe District, Malawi combined to control for potential
chemical variation across space [37,38]. The leaves of all plant species were collected from the wild
from these known locations. Plant materials were shade dried, ground to a fine powder and kept
in cool dark conditions until required. To produce 10% w/v extracts, 100 g of each plant powder were
extracted in 1 L of water containing 0.1% soap for 24 h at room temperature. Thereafter the extracts
were filtered and used immediately in bioassays. This trial included two control treatments, a positive
control of chlorpyrifos and a negative control of water plus 0.1% soap.
Based on these bioassay results, five of the ten plant species were selected for further research:
A. indica, O. basilicum, N. tabacum, C. citratus and L. javanica. In order to improve the extraction efficiency,
the extracts were prepared by weighing 300 g of plant powder into 1.5 L of methanol for 24 h. Extracts
were then filtered and placed in a fume hood for 24 h to allow the methanol to evaporate, leaving
behind the extracted residue. Dried residue was weighed on an analytical scale and then resolubilized
in acetone to make five concentrations of 10%, 5%, 3%, 1% and 0.1% w/v [39]. This trial included two
control treatments, a positive control of chlorpyrifos and a negative control of acetone only.
The evaluation of FAW survival on living maize plants was carried out using the three best plant
species: O. basilicum, N. tabacum and L. javanica. Aqueous extracts were prepared at 10% w/v using
the extraction process described above in the first screening experiment. Four control treatments were
also used in this study: chlorpyrifos as a positive control and three negative controls of no treatment
application, water only and water with 0.1% soap.
3.3. Bioassay Methods

Contact toxicity was performed by means of topical application where 10 μL of the extract were
applied topically on the bodies of the larvae using a 20 μL pipette. The larvae were then individually
placed in plastic bottle tops containing plain artificial diet and covered with foil paper. Chlorpyrifos
48 EC was used as positive control using the manufacturer recommended rate of 20 mL of chlorpyrifos
in 40 L of water while acetone or water was used as the negative control. Each replicate contained
ten 2nd instars, with five replicates per each treatment and concentration evaluated. Final mortality
data were recorded seven days afterwards by counting the number of dead larvae, with mortality data
corrected using Abbott’s formula [40].
The initial feeding bioassay screening ten plant species was carried out by dipping portions of
young maize leaves into each extract, waiting one hour for the extract to dry and then placing five 2nd
instars on the treated leaves to feed, three replicates per treatment. Treated maize leaves were replaced
daily for seven days, with mortality data collected and corrected with Abbott’s formula.
The subsequent feeding bioassay screening the shortlisted five plant species was carried out
using previously reported methods [41]. Aliquots (100 μL) of 0.05 M sucrose in acetone were applied
to individual glass-fibre discs (Whatman 2.1 cm diameter) and left to dry before aliquots (100 μL)
of the plant extracts in acetone were applied to each disc. Once dry, the discs were weighed with
an analytical balance, placed in individual containers and one 2nd instar was introduced with 10
larvae per treatment. Two control treatments were used: sucrose only and chlorpyrifos. After 48 h
the remainder of the disc not eaten by the larvae was weighed and any living larvae were transferred
55
Plants 2020, 9, 112
to plain diet in individual containers. Mortality data were collected seven days from the start
of the trial with mortality data corrected using Abbott’s formula. A feeding deterrence index
was calculated from the weights of control (C) and treated (T) discs using the following formula:
Feeding deterrence = (C − T)/(C + T) × 100 [42].
The final experiment evaluating FAW damage to living maize plants was carried out using maize
variety sc403 planted in pots maintained in a screenhouse. Five seeds were planted per pot and were
later thinned to three. Basal dressing fertiliser of 23:21:0 + 4 s was a pplied at seven days after planting
at a rate of 100 kg/ha while a topdressing fertilizer of urea was a pplied at four weeks after planting at
a rate of 159 kg/ha. All agronomic practices including watering and hand weeding were consistent
across all maize plant pots. Maize plants were infested with 2nd instars at twenty days after seedling
emergence. Each plant was infested with five larvae and the larvae were spaced at different leaf nodes
to avoid cannibalism. Artificial infestation was done early in the morning to avoid exposing the larvae
to harsh environments [31]. After infestation, plant pots were caged individually in cages of size 1.8 m
× 0.6 m × 0.6 m to prevent the movement of larvae from one treatment to another. The experiment was
laid out in a randomized complete block design replicated ten times where the cages acted as blocks.
Hand-held plastic sprayer bottles were used to apply the treatments, ensuring consistent coverage of
each plant. Treatments were first applied 48 h after infestation to allow the larvae to settle down and
establish [43]. Subsequent applications were carried out at seven-day intervals. Foliar damage data
were collected at an interval of seven days beginning from the first day after spraying. Using published
methods [44], FAW foliar damage severity was recorded on an individual plant basis using a scale of
0–9 where 0 means no visible leaf damage, 1 = only pin-hole damage to the leaves, 2 = pin-hole and
shot-hole damage to leaves, 3 = small elongated lesions (5–10 mm) on 1–3 leaves, 4 = midsized lesions
(10–30 mm) on 4–7 leaves, 5 = large elongated lesions (>30 mm) or small portions eaten on 3–5 leaves,
6 = elongated lesions (>30 mm) and large portions eaten on 3–5 leaves, 7 = elongated lesions (>30 cm)
and 50% of leaf eaten, 8 = elongated lesions (30 cm) and large portions eaten on 70% of leaves and
9 = most leaves have long lesions.
3.4. Data Analysis

Experiments were carried out following completely randomised block designs. Effects of
treatments and their interactions observed were subjected to two-way analysis of variance. The means
of treatments and interactions were compared using Tukey’s honest significant difference (HSD) test
at the 95% confidence interval. All the analyses were done using XLSTAT version 2019.2.2.59614
(Addinsoft, 2019); XLSTAT statistical and data analysis solution (Boston, MA, USA). Statistical outputs
are provided in Table S1.
4. Conclusions
Recommendations from this research suggest that some relatively safe pesticidal plant species
could provide an agro-ecologically sustainable pest management option for the exotic invasive FAW
in Africa. Out of the original ten candidate plant species evaluated, four of these merit further
investigation: Azadirachta indica, Ocimum basilicum, Cymbopogon citratus and Lippia javanica. These four
plant species are cosmopolitan and frequently cultivated, so sustainable supplies for large-scale
production would be feasible. Although Tephrosia vogelii did not show significant efficacy in our trials,
further research should be recommended to confirm these results as T. vogelii is being recommended
for fall armyworm control due to is known efficacy against a range of pests. Considerable knowledge
on the chemistry of these plants is reported. Furthermore, O. basilicum, C. citratus and L. javanica
are consumed as spices and teas, whilst Azadirachta indica has well-established low mammalian
toxicity. Despite considerable work on its biopesticidal effects, Nicotiana tabacum is arguably the plant
species with the highest vertebrate toxicity, well-known for the effects of nicotine and related alkaloids.
However, despite potential human toxicity dangers, N. tabacum is being pursued as one of several
potential botanical options for FAW control in several African countries, and thus merits further
56
Plants 2020, 9, 112
investigation regarding its safe use and non-target effects. Evidence from our work and previous
research repeatedly shows that pesticidal plants do not cause mortality rates comparable to synthetic
pesticides. However, the trade-off between lower mortality for lower environmental persistence needs
to be seriously considered, particularly as there is growing evidence that less toxic natural pesticides
can help facilitate natural pest regulation whilst not significantly sacrificing crop yield. The next
step in evaluating the use of pesticidal plants for FAW control in Africa will require systematic trials
under farmer field conditions that can assess their cost-benefits and impact on crop damage and yield
in comparison to commercial synthetic pesticides.

Table S1: Statistical analyses of fall armyworm bioassays evaluating pesticidal plants using two-way analysis of
variance followed by Tukey’s HSD test to separate the means. Means followed by the same letter in an experiment
are not significantly different from each other.
Author Contributions: Conceptualization, Y.T., P.C.S. and S.R.B.; Methodology, K.P., Y.T., P.C.S. and S.R.B.;
Software, K.P. and S.R.B.; Validation, K.P., Y.T., T.K., V.H.K., P.C.S. and S.R.B.; Formal analysis, K.P. and S.R.B.;
Investigation, K.P., Y.T., T.K., V.H.K., P.C.S. and S.R.B.; Resources, K.P., Y.T., V.H.K., P.C.S. and S.R.B.; Data
curation, K.P. and S.R.B.; Writing—Original draft preparation, K.P.; Writing—Review and editing, K.P., Y.T.,
T.K., V.H.K., P.C.S. and S.R.B.; Visualization, K.P. and S.R.B.; Supervision, Y.T., T.K., V.H.K., P.C.S. and S.R.B.;
Project administration, Y.T. and S.R.B.; Funding acquisition, P.C.S. and S.R.B. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the McKnight Foundation, grant number 17-070.
Acknowledgments: The authors are grateful for the contributions of Aubrey Kaphukusi, Ipiana Kayira and
Vincent Kalasanthenga for their technical laboratory and field assistance.
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59
plants
Article
United Forces of Botanical Oils: Efficacy of Neem and
Karanja Oil against Colorado Potato Beetle under
Laboratory Conditions
Kateřina Kovaříková * and Roman Pavela
Group Secondary Metabolites in Crop Protection, Crop Research Institute, Dmovská 507, 161 06 Praha 6-Ruzyně,
Czech Republic; pavela@vurv.cz
* Correspondence: kovarikova@vurv.cz
Received: 14 November 2019; Accepted: 12 December 2019; Published: 14 December 2019
Abstract: Neem and karanja oil are the most promising botanical insecticides in crop protection
nowadays. Given that information about the insecticidal abilities of these oils is lacking, the aim was
to explore the effects of neem and karanja oil binary mixtures. The insecticidal activity of NeemAzal
T/S (Trifolio-M GmbH, Lahnau, Germany) (neem oil), Rock Effect (Agro CS a.s., Česká Skalice,
Czech Republic) (karanja oil), and their binary mixes (at 1:1, 1:2, and 2:1 volume ratios) against the
larvae of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was studied. In our bioassays,
a synergistic effect of the mixtures, which was dose-dependent, was observed for the first time against
this pest. The most effective blend was the 1:1 ratio. Its efficacy was more or less the same as, or even
greater than, the neem oil alone. The LC50 of neem oil two days after application was (0.075 g·L−1 )
and the LC50 of the mixture was (0.065 g·L−1 ). The LC50 of karanja oil was (0.582 g·L−1 ), which
was much higher than the LC50 of neem oil. The LC90 of neem oil five days after application was
(0.105 g·L−1 ) and the LC90 of the mixture was (0.037 g·L−1 ). The LC90 of karanja oil was (1.032 g·L−1 ).
The results demonstrate that it is possible to lower the doses of both oils and get improved efficacy
against CPB larvae; nevertheless, further verification of the results in field conditions is necessary.
Keywords: synergism; neem; karanja; Colorado potato beetle; botanical insecticides
1. Introduction
The Colorado potato beetle (CPB), (Leptinotarsa decemlineata (Say, 1824), Coleoptera:
Chrysomelidae) is one of the most important potato pests. This species is native to North America,
from where it has gradually spread across Europe and Asia [1] alongside the expansion of potato
cultivation. L. decemlineata is now considered to be the most important insect defoliator of potatoes.
Through defoliation, the yield of tubers can be reduced by more than 50% [2]. Moreover, if the
pest appears early and in intense numbers, it can destroy the entire production of a potato growing
operation. The values of “intense numbers” vary from author to author and correspond with economic
(action) thresholds, which were established for optimizing the use of insecticide applications and are
unique to certain conditions. For example, Senanayake and Holliday [3] suggest 0.14 to 0.82 larvae per
plant. Mailloux and Bostanian [4] stated that a measure based on the level of defoliation is better than
one based on pest abundance. These levels were estimated, for example, by Zehnder et al. [5] at 20%
leaf loss for young plants, 30% for plants from early bloom to late bloom, and 60% for plants from late
bloom to harvest for fields in eastern Virginia. Nevertheless, the CPB larval stage is considered to be
the most harmful stage to potato production [4], so we focused our research on the CPB larvae.

Plants 2019, 8, 608
Various non-chemical control methods have been introduced since the 19th century to reduce
the impact of CPB. Crop rotation, trap crops (eggplant), and other agrotechnical practices were
recommended to farmers at that time [6]. In a study by Reed [6], it was concluded that the only two
reliable methods were hand picking and the use of Paris Green (a toxic pigment). Hand picking,
especially before mating, was considered very effective, but impractical on a larger scale. As such,
the majority of alternative methods failed, and the most common method became the use of pesticides.
Although pests have developed resistance to many active ingredients of insecticides, the use of
chemicals remains the most widely used method against CPB to date (although biological controls are
also often applied [7–9]).
According to the Arthropod Pesticide Resistance Database [10], CPB has shown resistance to 56
active ingredients of insecticides to date, among them spinosad [11] and Bacillus thuringiensis [12].
Mota-Sanchez et al. [11] also observed cross resistance to nine other neonicotinoids in an
imidacloprid-resistant adult population of CPB. As of now, resistance has been recorded to most of the
commonly used insecticides, such as pyrethroids, neonicotinoids, diamides, and organophosphates. As
Yamamoto et al. [13] stated, it is possible to delay or prevent resistance development of pests via rotating
insecticides with different modes of action and using certain combinations of insecticides. Barnes et
al. [14] proved that a strategy using mixtures of insecticides is even more effective than the rotation of
insecticides, and this is the very essence of botanical insecticides (BIs), which are complex mixtures of
many functional secondary metabolites [15,16], in contrast to chemical substances, which are often
characterized by one active ingredient, complemented by a number of inactive ingredients to facilitate
the application. Moreover, the excessive use of chemical pesticides harms the environment, non-target
organisms, and humans. By contrast, botanical pesticides are biodegradable and leave no harmful
residues. Unfortunately, the scale of products applicable in organic production is insufficient as well.
It is necessary to search for additional environmentally acceptable substances for effective control of
CPB, and the use of botanical pesticides is a promising possibility. Chaudhary et al. [17] stated that the
use of botanical pesticides is the most efficient means to replace synthetic pesticides, and among those,
extracts and oils are the best choices. Neem and karanja oil, in particular, have great potential for use
in sustainable integrated pest management.
Neem oil is one of the most promising substances in the current approach to pest control. Neem oil
is a product of the Indian neem tree Azadirachta indica (A.) Juss. It possesses a variety of insecticidal
properties, such as repellency [18], antifeedancy, toxicity, and growth disruption, against numerous pest
species [19]. For example, the biochemical effect (growth inhibition, feeding deterrence, oviposition
inhibition) of neem oil against more than 30 Lepidopteran pests [20] has been well documented.
The main active ingredient is azadirachtin, a tetranortriterpenoid, which was isolated from the seeds of
Azadirachta indica by Butterworth and Morgan [21] and is known to disrupt insects’ metamorphosis [22].
Neem oil is a contact insecticide, but even systemic activity has been documented [23]. Pavela et al. [24]
also proved that azadirachtin can be taken up by plant roots and thus affect the population of immature
aphids feeding on the treated plant.
Karanja oil is a product of the seeds of a widespread tropical and subtropical tree called Pongamia
pinnata (L.) Pierre [25]. Karanja oil is rich in furano-flavonoids [26]. Al Muqarrabun et al. [27]
summarized the known attributes of up to 70 flavones and their derivatives that had been isolated
from Pongamia pinnata by various authors. Of these, karanjin, which was first discovered by
Limaye [28], is particularly effective against a large number of insects [29]. The oil and extract
of karanja act as insecticides [30], repellents, antifeedants, and growth regulators [31], and even
oviposition deterrents [32].
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Some insecticides, in combinations, may exhibit greater-than-additive toxicity, but the prediction of
mixture toxicity using a response addition model is not always accurate for active ingredients with different
modes of action [33]. In the case of neem and karanja oils, a synergistic effect was found by Kumar et al. [34]
in bioassays on aphids and mites. Later, this phenomenon was confirmed by Packiam and Ignacimuthu [35]
during experiments on Spodoptera litura. However, although the synergistic effect of insecticides is of great
importance in practice, there is still much to be explored in this research area; for example, application doses
may be reduced (economic and environmental benefits) and, moreover, multi-substance mixtures prevent
the development of resistant populations of pests (anti-resistant strategy).
Although the efficacy of neem extracts on the Colorado potato beetle has already been studied,
the efficacy of karanja oil against this pest has not yet been reported. Similarly, a possible synergistic
effect of binary mixtures of these oils has also not been studied sufficiently to date. Thus the aim of
our research was to evaluate the insecticidal properties of two commercial products (NeemAzal T/S
- Trifolio-M GmbH, Lahnau, Germany, Rock Effect - Agro CS a.s., Česká Skalice, Czech Republic)),
based on Azadirachta indica seed kernel oil and Pongamia pinnata oil, respectively, against the most
destructive stage of CPB and to verify any possible synergistic effects of their binary mixtures in three
different ratios with regard to practical use.
2. Results
The insecticidal activity of both tested BIs against CPB larvae was very good; however, significant
differences in efficacies were found. From the LC50 (LC90 ) of Rock Effect (Agro CS a.s., Česká Skalice,
Czech Republic) (0.582 g·L−1 ) and NeemAzal T/S (Trifolio-M GmbH, Lahnau, Germany) (0.075 g·L−1 ),
it is obvious that karanja oil by itself is significantly less effective against larvae of Leptinotarsa
decemlineata in comparison with neem oil. Complete results are presented in Table 1.
A comparison of the LC values revealed that both BIs showed significant chronic toxicity,
which resulted in an apparent decrease in lethal concentrations over time (Table 1). The LC50
(LC90 ) values for NeemAzal T/S (Trifolio-M GmbH, Lahnau, Germany) were estimated at 0.075 g·L−1
(0.618 g·L−1 ) after 48 h and 0.005 g·L−1 (0.029 g·L−1 ) after 8 days, which is an obviously lower range of
concentrations than the estimate for Rock Effect (Agro CS a.s., Česká Skalice, Czech Republic), where
the LC50 (LC90 ) was 0.582 g·L−1 (1.692 g·L−1 ) after 48 h and 0.259 g·L−1 (0.774 g·L−1 ) after 8 days.
The insecticidal activity of the mixtures was assessed by the activity of the pure BIs. The LC
range of the mixtures was usually within the LC range of NeemAzal T/S (Trifolio-M GmbH, Lahnau,
Germany) and Rock Effect (Agro CS a.s., Česká Skalice, Czech Republic). As such, all the mixtures were
more efficient than Rock Effect (Agro CS a.s., Česká Skalice, Czech Republic) alone. An exception was
found in the case of mixtures after 48 h (acute toxicity), when the LC90 was higher for the mixtures than
for the original substances. The LC values of the mixtures decreased over time, as did the LC values of
the pure BIs. The LC50 (LC90 ) of the mixtures (1:1, 1:2, and 2:1 respectively) were estimated as follows:
0.065–0.001 g·L−1 (1.651–0.011 g·L−1 ), 0.543–0.012 g·L−1 (6.553–0.028 g·L−1 ), and 0.387–0.015 g·L−1
(6.361–0.059 g·L−1 ) 48 h to 8 days after treatment.
The order of the tested BIs according to efficacy (LC) is summarized as follows: mixture (1:1),
NeemAzal T/S (Trifolio-M GmbH, Lahnau, Germany), mixture (1:2), mixture (2:1), and Rock Effect
(Agro CS a.s., Česká Skalice, Czech Republic). The improved efficacy of the mixtures indicates an
obvious synergistic effect.
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Table 1. Toxicity of botanical insecticides (Bis) and their mixtures against Leptinotarsa decemlineata larvae. LC50 and LC90 : concentration causing 50% and 90%
mortality of insects, respectively. CI95 : 95% confidence intervals, insecticide activity is considered significantly different when the 95% CI fails to overlap. Chi =
Chi-square value, significant at p < 0.05 level.
Insecticides Days after Treatment LC50 CI95 LC90 CI95 Chi-Square p-Value
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Neem Azal T/S 2 0.075 ± 0.011 0.066–0.101 0.618 ± 0.215 0.372–1.323 0.988 0.804
5 0.021 ± 0.002 0.011–0.021 0.105 ± 0.011 0.082–0.156 2.031 0.565
8 0.005 ± 0.001 0.003–0.008 0.029 ± 0.004 0.022–0.041 0.007 0.999
Rock Effect 2 0.582 ± 0.093 0.344–0.858 1.692 ± 0.428 1.112–4.319 2.316 0.677
5 0.371 ± 0.071 0.190–0.553 1.032 ± 0.263 0.672–2.813 5.065 0.281
8 0.259 ± 0.054 0.119–0.379 0.774 ± 0.176 0.523–1.165 1.821 0.768
Rock Effect and
2 0.543 ± 0.151 0.367–0.611 6.553 ± 2.441 5.369–7.921 6.591 0.252
NeemAzal T/S 1:2
5 0.021 ± 0.005 0.015–0.031 0.151 ± 0.027 0.094–0.211 5.398 0.369
8 0.012 ± 0.001 0.012–0.017 0.028 ± 0.002 0.024–0.034 0.677 0.954
Rock Effect and
2 0.065 ± 0.016 0.042–0.113 1.651 ± 0.325 1.354–1.923 2.088 0.552
NeemAzal T/S 1:1
5 0.008 ± 0.015 0.001–0.060 0.037 ± 0.023 0.016–0.065 1.507 0.681
63
8 0.001 ± 0.001 0.002–0.026 0.011 ± 0.001 0.007–0.016 0.052 0.996
Rock Effect and
2 0.387 ± 0.052 0.302–0.512 6.361 ± 2.585 3.391–7.182 5.126 0.401
NeemAzal T/S 2:1
5 0.053 ± 0.002 0.012–0.091 0.267 ± 0.085 0.168–0.378 3.771 0.582
8 0.015 ± 0.002 0.011–0.019 0.059 ± 0.007 0.047–0.077 0.336 0.996
Plants 2019, 8, 608
3. Discussion
This experiment involved testing the insecticidal activity of NeemAzal T/S (Trifolio-M GmbH,
Lahnau, Germany) (neem oil), Rock Effect (Agro CS a.s., Česká Skalice, Czech Republic) (karanja oil),
and three binary mixtures. In the course of the experiment, it was observed that the larvae treated with
the oils also showed a reduction in food intake, which resulted in little leaf damage compared to leaves
in the control variant. This effect was not a primary target of the testing; nevertheless, antifeedancy has
previously been reported for karanja oil [36] as well as neem oil [37].
The efficacy of neem oil was very good even at low concentrations such as 0.1–2 g·L−1 in our
bioassays. The efficacy of neem products may vary with respect to concentrations of azadirachtin [38],
and thus some authors have stated lower insecticidal activity against CPB [39,40]. However, that was
not the case in our study, because the commercial product NeemAzal T/S (Trifolio-M GmbH, Lahnau,
Germany) used in the bioassay contains 1% of the purified active ingredient Azadirachtin A, which is
a very potent insect growth inhibitor [41] (reviewed in [19,42]). In the case of karanja oil, higher doses
were needed to achieve the same mortality of CPB larvae as with neem oil. According to the literature,
karanja oil is generally effective at higher doses, from 10 g·L−1 upwards [43–45]. Deshmukh and
Borle [46] also mentioned that karanja oil has some limits for use at the farmer’s level, because its
aqueous suspension is not as effective as that of neem. The higher efficacy of neem oil over karanja oil
was also reported by Biswas et al. [47] in the case of Helicoverpa armigera.
Zehnder and Warthen [37] found that azadirachtin caused mortality, and they also observed
an antifeedant effect in the case of CPB. Feuerhake and Schmutterer [48] tested neem seed extract
formulations and were able to achieve 100% mortality of CPB. Other observed effects of neem oil on
CPB are disruption of egg hatching and larvae molting [49]. The repellent effects of neem oil were even
observed in laboratory as well as in field conditions [50]. Kaethner [51] observed the effect of fitness
reduction in the treated beetles and a lower fecundity of females. Murray et al. [52] confirmed the
oviposition effects of citrus limonoids, which are structurally and functionally similar to azadirachtin.
The effect of karanja oil on CPB has not been studied to date.
With respect to other insect pests, synergistic effects were reported for both neem and karanja oils.
Synergism refers to when the combined effect of two factors is greater than the sum of individual factors.
It can occur between various insecticides [14], insecticides and fungicides [53], insecticides and poor
nutritional conditions [54], and so on. For example, a synergism was found for the combination of neem
oil and Beauveria bassiana on Spodoptera litura Fabricius [55], Tribolium castaneum [56], and aphids [57].
The combination of both oils with some pyrethrins shows synergism as well [36,58]. Synergism was
also observed in the combination of neem and karanja oil by Kumar et al. [34], where both oils were
found to be highly effective individually and also in combination against mites and aphids. In our
case, the most significant synergistic effect was found for the mixture of neem and karanja oil in a
1:1 ratio. In addition, this effect appeared chronically and not acutely, and thus it is possible to use
lower doses of insecticides. A notable observation in this experiment was that the individual neem and
karanja oil treatments were less effective compared to the mixtures. For example, the toxicity (LC90 )
five days after treatment with the 1:1 mixture was almost 3-fold higher than for neem oil alone, and up
to 28-fold higher than for karanja. The other mixtures showed synergism as well, although the effect
was significantly weaker. Packiam and Ignacimuthu [35] came to the same conclusions when testing
various combinations of neem and karanja oils against larvae of Spodoptera litura. Thus, it is clear that
the strength of the synergistic effect is ratio dependent, and this is the first research paper to describe
such a phenomenon in the case of CPB.
Neem and karanja oil seem to provide the perfect solution to all the problems of contemporary
crop protection. Nevertheless, the possible cons should be discussed as well. Because it possesses
a variety of insecticidal properties, even against some beneficial insects in the juvenile stage [59],
the use of neem oil carries a potential risk. Any decision to use neem oil should therefore be made
carefully. The same situation occurs in the case of karanja oil, which is considered a broad-spectrum
insecticide [36]. On the other hand, Koss et al. [60] and Radkova et al. [61] found that beneficial
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arthropods in potato fields were more abundant when botanical and selective pesticides were sprayed
in contrast to when chemical pesticides were used. Neem oil is considered environmentally friendly
because it is free of chloramine, phosphorous, and nitrogen atoms, which are commonly found
in synthetic pesticides. Moreover, using neem oil in the field can prevent some other pests from
ovipositing [62], thus providing improved crop protection, and due to the synergistic effect of the
compounds, a reduced dose is used, significantly reducing toxicity to non-target organisms while also
reducing the cost of applications. Generally, neem products can be recommended for many integrated
pest management (IPM) programs [63]. The effect of an insecticide based on a combination of neem
and karanja oils (PONNEEM# ) has already been tested against the hymenopteran parasitoid wasp
Trichogramma chilonis, which was not affected from applications with up to a 0.5% concentration [35].
The recommended dose for NeemAzal T/S (Trifolio-M GmbH, Lahnau, Germany) in potatoes is 2.5 L
for 300–700 L of water/ha (0.8–0.35% concentration). Because the 1:1 mixture was almost 3 times more
effective, we should theoretically recommend the use of a 0.3–0.1% concentration for field application,
and thus parasitoids should not be affected.
Field efficacy, however, will still need to be verified by a series of tests before its implementation,
because some authors [64] have demonstrated the low stability of neem oil under field conditions
due to photodegradation. This issue can be resolved theoretically via nano and micro encapsulation,
which improves the stability and efficacy of oils exposed to UV light [65]; on the other hand,
this solution is more suitable for soil applications of neem seed oil [66]. Moreover, the cost of such
a product will be affected as well. The greater variability of karanja oil efficacy against CPB larvae
reveals that this compound is less reliable than neem oil, especially when acute toxicity is concerned.
However, Kumar and Singh [36] stated that the persistence of karanja oil is greater than for other
botanical insecticides. In addition, the karanja extract is a highly effective UV absorbent [67] and is
now being used in cosmetics as a component of modern sunscreen preparations, even for humans.
As Wanyika et al. [68] have indicated, stabilization can be developed by adding solar radiation
protectants, and therefore the combination of neem and karanja oils seems to be a perfect match,
because what is missing in one is replaced by the other.
4.1. Insecticides
Two botanical insecticides that are suitable for organic farming were used in the bioassays. The first
one (NeemAzal T/S) (Trifolio-M GmbH, Lahnau, Germany) was tested at five different doses and the
second one (Rock Effect) (Agro CS a.s., Česká Skalice, Czech Republic) was tested at six different doses.
Both substances were tested as contact insecticides.
NeemAzal® T/S (Trifolio-M GmbH, Lahnau, Germany) is a commercial formulation of the seed
kernel extract of the tree Azadirachta indica (A.) Juss., which contains 1% of the purified active ingredient
Azadirachtin A. It is generally used as a 0.3% to 0.5% aqueous solution. In the following text, the term
“neem oil” is used.
Rock Effect (Agro CS a.s., Česká Skalice, Czech Republic) is a commercial formulation of Pongamia
pinnata (L.) Pierre oil. The oil content is declared as 868.5 g·L−1 . It is generally used as a 1% to 3%
aqueous solution. In the following text, the term “karanja oil” is used.
4.2. Insects and Plant Material

Newly enclosed second instar larvae of Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)
were obtained from a colony reared on potato, Solanum tuberosum, cv. Agria, in a climate chamber at
22 ± 1 ◦ C, a relative humidity of 40–60%, and a 16:8 (L:D) photoperiod. The colony was established
from eggs and adults collected from a potato culture at a local field of the Crop Research Institute in
Prague, Czech Republic (50◦ 05 N, 14◦ 18 E, 340 m a.s.l.) in June and August 2019.
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4.3. Laboratory Bioassay

A potato leaf with five to seven leaflets was inserted into floral foam supporting moisture
(Oasis, Belgium) and placed in a plastic box (16 × 11 × 7 cm). Twenty to twenty-five individuals
(L2) of CPB were transferred carefully using a fine brush on each potato leaf. The tested BIs were
dissolved in water right before use. NeemAzal T/S (Trifolio-M GmbH, Lahnau, Germany), Rock Effect
(Agro CS a.s., Česká Skalice, Czech Republic), and their binary mixtures in three ratios (1:1, 2:1, and
1:2, by volume) were tested. For each BI, at least five concentrations that resulted in more than 0%
and less than 100% mortality, based on preliminary assays, were used. The concentration series were
0.1–2 g·L−1 for NeemAzal T/S (Trifolio-M GmbH, Lahnau, Germany), 2–25 g·L−1 for Rock Effect (Agro
CS a.s., Česká Skalice, Czech Republic), 0.05–2 g·L−1 for the 1:1 mixture, 0.1–15 g·L−1 for the 2:1 Rock
Effect:Neem Azal (RE:NA) mixture, and 0.05–5 g·L−1 for the 1:2 (RE:NA) mixture. To obtain the
LC value, the insecticidal activity of the concentration series of each insecticide was tested under
laboratory conditions.
Once the larvae began to feed on the leaves, the prepared solutions were sprayed on both sides of
the leaves using a hand sprayer. The control variant was sprayed with water only. Each variant was
performed in three repetitions. The experiment was run in a climate chamber set at 22 ± 1 ◦ C and 69%
± 6% relative humidity. The mortality rate was evaluated at 48 h, 5 days, and 8 days later. Individuals
unable to right themselves when disturbed were counted as dead.

Abbott’s formula was used to correct the data for control mortality [69]. Probit analysis [70]
was used to estimate the doses needed to cause 50% mortality (LC50 ) and 90% mortality (LC90 ), as
well as the associated 95% confidence limits for each BI, including binary mixtures, using BioStat
(version 5.9.8.5).
5. Conclusions
Because of their natural origin and environmental friendliness, botanical pesticides currently have
great potential. Furthermore, botanical pesticides are also very potent insecticides and, due to their
composition, they can help to fight the global problem of insects developing resistance to insecticides.
Both of the tested BIs (insecticides based on neem oil and karanja oil) were efficient against
L. decemlineata larvae at different concentrations. However, the efficacy of karanja oil against CPB
larvae showed higher variability than for neem oil, on account of the high azadirachtin content of the
commercial neem product and also the slightly different mode of action of both oils. Our experiment
demonstrated a synergistic effect of neem and karanja oils against CPB larvae under laboratory
conditions—one that was ratio-dependent. The most potent mixture (1:1 ratio) was equally, or even
more effective, than neem oil itself. This effect intensified with respect to exposure time and appeared
chronically. Five days after application of the mixture, LC90 values were 3-fold higher for neem oil
and up to 28-fold higher for karanja oil. Due to these results, the recommended field dose of the
mixture was estimated as high as 1.4 L in 500 L of water/ha (0.3%). Nevertheless, further verification
in field conditions of the results achieved by this experiment is necessary in order to reach reliable
conclusions and implement them into practice, and the effect on beneficial and non-target organisms
should be verified.
Author Contributions: Conceptualization: R.P. and K.K.; methodology: R.P. and K.K.; software: R.P.; investigation:
K.K.; data curation: R.P.; writing—original draft preparation: K.K.; writing—review and editing: K.K.;
visualization: K.K.; supervision: R.P.; project administration: K.K.; funding acquisition: R.P.
Funding: This research was funded by the Ministerstvo Zemědělství - Národní Agentura pro Zemědělský
Výzkum, grant number QK1920214.
Acknowledgments: The authors would like to thank the team members for providing the necessary background
for running the experiments.
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plants
Article
Phytochemical Analysis of Tephrosia vogelii across
East Africa Reveals Three Chemotypes that Influence
Its Use as a Pesticidal Plant
Angela G. Mkindi 1 , Yolice Tembo 2 , Ernest R. Mbega 1 , Beth Medvecky 3 , Amy Kendal-Smith 4,5 ,
Iain W. Farrell 4 , Patrick A. Ndakidemi 1 , Steven R. Belmain 4 and Philip C. Stevenson 4,6, *
1 Department of Sustainable Agriculture, Biodiversity and Ecosystems Management, Centre for Research,
Agricultural Advancement, Teaching Excellence and Sustainability (CREATES), The Nelson Mandela African
Institution of Science and Technology, P.O. Box 447 Arusha, Tanzania; angela.mkindi@nm-aist.ac.tz (A.G.M.);
ernest.mbega@nm-aist.ac.tz (E.R.M.); patrick.ndakidemi@nm-aist.ac.tz (P.A.N.)
2 Bunda College, Lilongwe University of Agriculture and Natural Resources-Malawi, P.O. Box 219 Lilongwe,
Malawi; ytembo@bunda.luanar.mw
3 Innovations in Development, Education and The Mathematical Sciences (IDEMS) International, 15 Warwick
Road, Reading, RG2 7AX, UK; bethmedvecky@gmail.com
4 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK; bs16a3s@leeds.ac.uk (A.K.-S.);
i.farrell@kew.org (I.W.F.); s.r.belmain@greenwich.ac.uk (S.R.B.)
5 Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK
Kent ME4 4TB, UK
* Correspondence: P.Stevenson@kew.org
Received: 27 November 2019; Accepted: 10 December 2019; Published: 12 December 2019
Abstract: Tephrosia vogelii is a plant species chemically characterized by the presence of entomotoxic
rotenoids and used widely across Africa as a botanical pesticide. Phytochemical analysis was
conducted to establish the presence and abundance of the bioactive principles in this species across
three countries in East Africa: Tanzania, Kenya, and Malawi. Analysis of methanolic extracts of
foliar parts of T. vogelii revealed the occurrence of two distinct chemotypes that were separated
by the presence of rotenoids in one, and flavanones and flavones that are not bioactive against
insects on the other. Specifically, chemotype 1 contained deguelin as the major rotenoid along with
tephrosin, and rotenone as a minor component, while these compounds were absent from chemotype
2, which contained previously reported flavanones and flavones including obovatin-3-O-methylether.
Chemotype 3 contained a combination of the chemical profiles of both chemotype 1 and 2 suggesting
a chemical hybrid. Plant samples identified as chemotype 1 showed chemical consistency across
seasons and altitudes, except in the wet season where a significant difference was observed for samples
in Tanzania. Since farmers are unable to determine the chemical content of material available care
must be taken in promoting this species for pest management without first establishing efficacy. While
phytochemical analysis serves as an important tool for quality control of pesticidal plants, where
analytical facilities are not available simple bioassays could be developed to enable extension staff
and farmers to determine the efficacy of their plants and ensure only effective materials are adopted.
Keywords: spatial-temporal variation; chemotype 3; deguelin; rotenoids; botanical insecticides
1. Introduction
Tephrosia vogelii Hook. f. (Leguminosae) is a plant species reported to be used widely for its
medicinal, insecticidal, and soil enrichment potential in tropical Africa [1–6]. Specifically, research on
T. vogelii reported medicinal properties such as anti-cancer activity [7–9] and efficacy as an ectoparasite

Plants 2019, 8, 597
treatment for domestic animals including poultry [10–14]. A number of studies have sought to
validate the reported use of T. vogelii as a botanical insecticide under laboratory and field conditions
and have reported its effectiveness for crop protection and reduced impacts on beneficial ecosystem
services [15–17]. Likewise, Tephrosia is reported to have high biomass and is therefore important as
a soil amendment and is compatible with food crops when intercropped in addition to its nitrogen
fixing property [18,19]. Hence, using T. vogelii for small scale farmers may support reduced industrial
fertilizer and synthetic pesticides application all of which bear cost and safety implications.
Deguelin was reported to be the major active compound in T. vogelii occurring in all plant parts
along with the minor components of tephrosin and rotenone [20–23]. However, a previous study
reported that some T. vogelii did not contain rotenoids, and was less effective as an insecticide [15,22].
This highlighted the need to ensure that effective chemotypes of pesticidal plants were available when
promoting their use to farmers to ensure effective control of pests. However, this is challenging in
the absence of suitable local facilities to undertake such quality control and establish variation when
pesticidal plants are harvested, processed, and used locally [24]. Natural variation in the chemistry
of bioactive components in pesticidal plants is reported [25] and can have consequences for use and
ultimately trust in pesticidal plants as an alternative to synthetic inputs by farmers.
Variability in the chemistry of T. vogelii could lead to farmers unknowingly using ineffective
material and influence negatively the wider adoption and commercialization of botanical insecticides.
Small scale farming communities, who are the main beneficiaries of T. vogelii, identify the plant using
locally acquired knowledge through morphological features. However, T. vogelii chemotypes may
not be identified and distinguished morphologically [22]. Likewise, traditional use does not have
the capacity to determine effectiveness prior to use. Phytochemical analysis is an essential tool for
the selection of elite provenances of plant materials [26] and should be used for the identification of
effective T. vogelii provenances prior to propagation. Here we collected samples from local farmers
who had Tephrosia growing in their fields and were using it for some non-food purpose. We sought to
understand the key applications of Tephrosia through a survey of farmers who used the plants. From the
collected samples, we evaluated the presence and concentration of deguelin in T. vogelii leaf materials
across 91 locations in three East African countries to establish the extent of chemotype variation and
identify elite materials for propagation of improved seed material of T. vogelii. Variation of Tephrosia
chemotypes with flower colors, seasons, rainfall, and altitudes were also assessed to establish whether
these traits could be used as markers of effectiveness or for the presence of active chemicals in the plant.
Further recommendations are also presented to help farmers more easily identify bioactive plants for
improved efficacy.
2.1. Status of Use of Tephrosia vogelii by Small Scale Farmers

Eight questions were used to assess the extent of T. vogelii use among farmers in locations where
samples were collected in Tanzania. All interviewed farmers were aware of T. vogelii, commonly known
as “Utupa” (Figure 1.). Farmers reported using T. vogelii mostly for field pest control (59%), fishing
(45%), and storage pest control (36%). Other uses such as, human medication, soil fertility and as mole
repellants and for ectoparasites control were also reported. A few farmers (5%) reported awareness of
the plant from witnessing institutional researchers collecting the plant for use in research and also
planting Tephrosia in research institutions such as the Uyole Agricultural Research—Mbeya and Tanzania
Coffee Research Institute—Kilimanjaro. The specific research activity was not clearly communicated.
Our results indicate that T. vogelii is most widely used for pest control among other uses as
shown in the survey results. Farmers’ responses in this study, align with reports about the use of
T. vogelii in controlling pests in vegetables and in stored products [15] and ectoparasite control in
domestic animals [12,24,27]. The wider use of T. vogelii for small-scale farmers could be associated
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Plants 2019, 8, 597
with previous projects that promoted integrated pest management using T. vogelii and research on soil
improvement [28,29].
Figure 1. Ethno botanical uses of T. vogelii among local small-scale farmers from the six Tanzanian
regions. Data are frequencies of responses on eight key uses of T. vogelii from the sample size (n = 22).
2.2. Phytochemical Analysis of T. vogelii Leaf Samples

Analysis of methanolic extracts of the leaf samples identified 2 chemotypes. Figure S1, shows the
LC-MS chromatograms of chemotype 1 characterised by the presence of rotenoids with corresponding
peaks between 19 and 20 min. These were determined from a comparison of their spectral data to in
house standards [22]. Rotenone was identified from UV (LC-PDA) λmax nm, 301; (MS) m/z, 395.4 [M +
H]+ , while tephrosin was identified from UV (LC-PDA) λmax nm, 272, 300, 314 sh; (MS) m/z, 433.4 [M +
Na]+ and deguelin from UV (LC-PDA) λmax nm, 270, 301, 319; (MS) m/z, 395.4 [M + H]+ . Chemotype 2
with peaks between 18 and 21 min were determined to contain obovatin 3-O-methylether as the major
component from an in house standard and had UV (LC-PDA) λmax nm, 270, 295, 348; (MS) m/z, 337.4
[M + H]+ . Other similar components having ions with m/z = 337 and 367 corresponding to flavones
and flavanones are reported earlier including Z-tephrostachin [22].
We also identified a third chemotype (Figure S1). Chemotype 3 was a chemical hybrid of
chemotypes 1 and 2 showing the presence of both the rotenoids and the flavanones and flavones
reported in 1 and 2 respectively in equivalent quantities. A further finding recorded plants as chemotype
1 but indicated trace quantities of flavones and flavanones from chemotype 2, suggesting that a variety
of potential chemical variants may exist in natural and propagated materials. The analyses were
undertaken on a single leaflet so were not a consequence of sample mixing of chemotypes 1 and 2.
Furthermore individual leaves from the same plant were chemically very similar.
The presence of chemotypes 1 and 2 corresponded with findings [22] in which chemotypes 1
and 2 were first reported. However, here we provide a comprehensive regional assessment of their
occurrence to determine what potential impacts the occurrence of chemotype 2 might have on the
application and uptake of this species for pest control and other uses. Chemotype 3 is a previously
unreported chemotype in this species which we report here. Studies have shown that the production of
new flavonoids such as those found in T. vogelii could be influenced by environmental factors whereby
compounds changed after exposure to conditions such as carbohydrates and light [20]. However,
chemical variation in these varieties is most likely genetic since different chemotypes were first reported
from the same location and in the same soil in adjacent fields [22]. There would therefore be value in
analysing for further chemotypes and determining if the hybrids produce lower quantities of both
compound groups.
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Plants 2019, 8, 597
2.3. Frequency of T. vogelii Chemotypes.

Plant material was collected from specific locations in three countries (Tanzania, Kenya, and
Malawi) where T. vogelii is used and their geographical references are presented in Table S1.
Approximately, 7% of samples were identified as chemotype 3, while 20% were chemotype 2. Most
samples (74%) were chemotype 1. A higher proportion of chemotype 1 was also reported by [15]
in Malawi from the analysis of 12 samples. Table 1, illustrates the proportions of chemotypes by
countries. The abundance in plant materials with chemotype 1 coincided with efficacy studies of T.
vogelii on medicinal [10,13,21,30] and insecticidal properties of rotenoids [15–17,31] which revealed
that rotenoids were the compounds most frequently found in T. vogelii sampled and are responsible for
the plants’ biological activity.
2.4. Spatial Distribution of Plants Samples Chemotypes

In this study, deguelin, the most abundant pesticidal rotenoid in T. vogelii, was used as an
indicator compound and its concentration in the plant was assessed across the study zone. The
chemical composition of T. vogelii was presented with reference to location across the three countries
(Figure 2). Samples collected from Malawi and Kenya contained chemotypes 1, 2, and 3 and were
located in Lilongwe and in 12 Kenyan counties respectively, while only chemotype 1 was recorded
from 14 locations across five regions in Tanzania. The results, from this study present the potential for
understanding the diversity of pesticidal plant chemotypes across a whole region. Local efficacy testing
of pesticidal activity in T. vogelii using a simple assay would potentially be conducted across various
locations where the plants grow to ensure reliable efficacy results for local farmers using the plant.
Figure 2. Spatial variation of T. vogelii chemotypes in Tanzania, Kenya, and Malawi, indicating presence
of Chemotypes 1, 2, and 3. Green bars depict the presence of deguelin while blue marks indicate the
presence of chemotype 2. The purple marks indicates the presence of chemotype 3, a chemical hybrid
of chemotype 1 and 2.
74
Table 1. Summary distribution of chemotype within the study area.
No. of
No. of No. of Mode Frequency Rel. Frequency Proportion
Variables Missing Mode Categories
Observations Categories Frequency Per Category Per Category (%) Per Category
Values
Plants 2019, 8, 597
Overall 91 0 3 Chemotype 1 67 Chemotype 1 67 74 1

Chemotype 2 18 20 0
Chemotype 3 6 7 0
Kenya 57 0 3 Chemotype 1 44 Chemotype 1 44 77 1
Chemotype 2 10 18 0
Chemotype 3 3 5 0
Malawi 20 0 3 Chemotype 1 9 Chemotype 1 9 45 0
Chemotype 2 8 40 0
Chemotype 3 3 15 0
Tanzania 14 0 1 Chemotype 1 14 Chemotype 1 14 100 1
Chemotype 2 0 0 0
Chemotype 3 0 0 0
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Plants 2019, 8, 597
2.5. Spatial Temporal Variation of Chemotype 1 in T. vogelii

Linear regression analysis was performed to test the variation of deguelin content in T. vogelii
based on altitude. The linear regression for Malawi (r2 = 0.178, F = 3.9, df = 18, p = 0.064), Kenya (r2 =
0.03, F = 1.74, df = 56, p = 0.193), dry season in Tanzania (r2 = 0.008, F = 0,096, df = 12, p = 0.762), and
wet season in Tanzania (r2 = 0.122, F = 1.665, df = 12, p = 0.221) showed no significant relationship
between changing altitude and the concentrations of deguelin. Further analysis of data from Tanzania
revealed no significant correlation between rainfall recorded in the wet (r2 = 0.005, F = 0.016, df = 3,
p = 0.9), and dry seasons (r2 = 0.72, F = 7.725, df = 56, p = 0.069). The results concur with findings
reported by [15] although these earlier data were of just a few samples.
Analysis of variance on the samples collected over two seasons in Tanzania showed that there
was no significant variation in the deguelin concentration with locations in the dry season (ANOVA F
= 0.272, df = 8, p = 0.916). In the wet season, however, a significant variation (ANOVA F = 7.092, df = 8,
p = 0.008) was observed (Table 2) where the highest and lowest levels of deguelin were observed in
samples collected from Same and Mbeya districts respectively. Seasonal variation of deguelin was
also reported by [15,32] where higher concentrations occurred in the wet season compared with the
dry season.
Table 2. Spatial and temporal variation of deguelin in T. vogelii from locations in Tanzania. The values
presented are means ± SE. **, = significant at P ≤ 0.01, ns = not significant. Means followed by the
Location Dry Season Deguelin (ppm) Wet Season Deguelin (ppm)

Same 6841 ± 523 a 8756 ± 197 a
Iringa 5644 ± 1202 a 4879 ± 132 bc
Morogoro 5423 ± 1621 a 6229 ± 207 b
Kilimanjaro 5144 ± 682 a 6377 ± 791 b
Mbeya 5699 ± 314 a 3385 ± 196 c
Arusha 5339 ± 139 a 4803 ± 4 bc
One way ANOVA F statistics 0.27 ns 7.09 **
2.6. Association between T.vogelii Flower Color and Chemotypes

One simple morphological feature for identification of T. vogelii and potentially distinguishing
chemotypes is flower color, with colors typically white or purple. In this study, plants that had flowers
at the time of sample collection were recorded along with leaf samples for chemical analysis. Generally,
a high percent of plants with white colored flowers were recorded compared to those with purple
flower. A higher percent of occurrence of white flowers was associated with the presence of chemotype
1. Chemotype 3 was associated with only purple color while a lower percent of white color was
associated with chemotype 2 (Figure 3). Regression analysis showed a strong correlation (r2 = 0.43, F =
22.02, df = 29, p = 0.0001) between chemotype and flower color where chemotype 1 was related to
white and chemotype 2 to purple color. In contrast to earlier findings [22] who found no correlation.
Flower colors could be used as initial tool for identification of chemotypes. However, the fact that
some purple flowers (with smaller percent) were also associated with chemotype 1, the decision to
use a plant for pest control purposes should be guided with simple assays for evidence of effective
chemotypes. Small scale farmers could adopt simpler tests of plant materials against storage pests
such as cowpea weevils (Callosobruchus maculatus) as already done by Belmain et al. [15] although this
was under laboratory setting.
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Plants 2019, 8, 597
Figure 3. The figure above shows (a) a descriptive statistics results showing the presence of purple
and white flowers in Tanzania and Kenya; (b) numerical distribution of T. vogelii flower color with
the chemotype of the plant; and (c) the frequency in percentage (%) of the occurrence of flower color
with chemotype.
2.7. Summary of Indicators for Chemotype Identification

Small-scale farmers require hands-on information to enable them to decide on suitable T. vogelii
materials for pest control. From this study, proposed and tested indicators would be used. Table 3
below highlights what farmers would need to consider while harvesting and using the materials.
Table 3. Tested and proposed options that farmers would need consider to select effective T. vogelii
plant material.
Option Results Reliability for Chemotypes Identification

Tested Options
Elevation No correlation Not reliable
Not reliable: Although wet season enhances higher content of
Season No correlation
bioactive compounds in chemotype 1
Somewhat reliable: Could be used to decide on the chemotype
Flower Color Positive correlation where white flowers are known to be related with chemotype
1. N.B., a few plants with chemotype 1 had purple flowers.
Proposed Options
Reliable: Test assessment of plant (10% leaf powder in small
test container with bruchids), could be a rapid, simple and
Simple assays Report from Belmain et al., 2012
affordable tool. Pesticidal properties of Tephrosia are fast acting
and chemotype could be determined in 48 h.
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Plants 2019, 8, 597
3.1. Analysis of Tephrosia Vogelii Leaf Samples
3.1.1. Samples Collection

Plant leaf samples were collected from farmers’ fields. Farmers identified the specific T. vogelii
plants that were used for controlling crop pests and diseases and for medical uses. In Tanzania,
leaf samples of T. vogelii were collected over two seasons, the wet season and dry season in 14 sites
located across five regions: Arusha, Kilimanjaro, Morogoro, Mbeya, and Iringa. The five regions
were identified after revising T. vogelii collections preserved in the National Herbarium in Tanzania
to identify possible areas where the plants could be growing. Samples were collected in March and
September 2018 during the wet and dry season respectively. In each region, two sites were identified
where samples were collected depending on the availability of the plant at that time.
Herbarium samples were collected and assigned voucher numbers, processed and stored in the
National herbarium. Identified plant in each point of sample collection was used for the two seasons to
justify analysis of variation with seasons. Rainfall data for the particular months of sample collection
were obtained from the Tanzania Meteorological Agency (TMA)—Tanzania. In Malawi, samples were
collected in the Lilongwe area on farmers’ fields. Likewise in Kenya collections were made on farmers’
fields in Kisumu, Homa Bay, Migori, Siaya counties, the western region Kakamega, Busia, Bungoma,
Mumias, and Central Kenyan counties.
A total of 28 samples were collected in Tanzania that included 14 samples for each of the dry and
wet seasons. In Malawi, 20 samples were collected from Lilongwe area between May and November
2018, while in Kenya, a total of 57 samples were collected between February and April 2019. Collected
samples were dried under the shed, packed into plastic zip bags and stored in dark and dry conditions
under ambient temperature before being processed and analyzed.
3.1.2. Survey of Farmers Awareness on the Use of T. vogelii

Twenty two farmers from six Tanzanian regions were interviewed in the survey to determine
the uses of T. vogelii in the household. In order to identify T. vogelii uses with reference to the type
of sample collected, only farmers who owned the plant or neighbors to the famer owning the plants
were interviewed. The selection of farmers therefore did not follow specific social survey protocols for
sample sizes selection.
3.1.3. Sample Analysis

Dried T. vogelii samples were powdered using an electric grinder (SALTER, Model No EK2311ROFB
distributed by UP Global Sourcing, Victoria Street, Manchester, OL9 0DD, UK Made in China). Tephrosia
powder (50 mg/mL) was extracted in methanol. Each extract was left to stand for 24 h at room
temperature before chemical analysis. Plant leaf extracts were transferred to Eppendorf tubes and
centrifuged for 20 min at 5000 rpm. Supernatant (300 uL) was transferred into HPLC vials for analysis.
Extracts were analyzed by liquid chromatography (LC)-Electrospray Ionization Mass Spectroscopy
(ESIMS) and UV spectroscopy using Thermo Fisher Velos Pro LC-MS. Samples (5 μL) were injected
directly on to a Phenomenex Luna C18 (2) column (150 Å~3 mm i.d., 3 μm particle size) at 400 μL min−1
and eluted using a linear gradient of 90:0:10 (t = 0 min) to 0:90:10 (t = 20–25 min), returning to 90:0:10 (t
= 27–30 min). Solvents were water: methanol: 1% formic acid in acetonitrile, respectively. The column
was maintained at 30 ◦ C. Compounds were detected on a Thermo Fisher Velos Pro Dual-Pressure Linear
Ion Trap Mass Spectrometer. Samples were scanned, using FTMS, from m/z 200–600 corresponding to
the range of molecular ions expected in samples of T. vogelii. UV peak area were quantified against a
calibration curve of an authentic in-house standard [22]. The resulting peak areas of deguelin were
measured at a wavelength of 300 nm and arranged in an excel file for statistical analysis.
78
Plants 2019, 8, 597
3.2. Presentation of Data and Sampling Points

Graphical presentation of chemotype and variation in amounts of chemotype 1 in T. vogelii was
performed using ARC GIS, ARCMAP version 10.3.

Analysis of Variance and descriptive statistics, proportion analysis and regression analysis were
performed using XLSTAT version 2019.2.2.59614 (Addinsoft (2019). XLSTAT statistical and data
analysis solution. Boston, MA, USA. https://www.xlstat.com).
4. Conclusions
This study has demonstrated the chemical variation in T. vogelii, across a variety of location types
in three countries of East Africa and revealed considerable variation in chemistry influencing the
bioactivity of plants materials. The study has also highlighted key uses of the plant, hence indicating
its importance to farmers’ livelihoods. Correlation of key factors with the effectiveness of the plant
materials are also discussed along with the identification of options that farmers would consider when
selecting elite materials. From this study we realize the potential of a region wide study that provide
an expanded perspective of plant chemistry for a wider community use and uptake. To mitigate the
variations under local conditions, simple and locally tailored assays, where farmers could test plant
materials against storage pests would provide a rapid assessment tool of plants efficacy. However,
further research on possible propagation strategies that ensure the availability and use of elite materials
as well as investigation of more indicators for chemotype identification is required.

Table S1: Location and chemotype data for samples of T. vogelii in Tanzania, Kenya, and Malawi, Figure S1:
Chromatograms showing: (a) Chemotype 1, presence of deguelin; (b) Chemotype 2 absence of rotenoids; (c)
chemotype 3 signifying presence of chemotypes 1 and 2 suggesting a hybrid, and (d) presence of Chemotype 1
with minimal quantitative presence of chemotype 2.
Author Contributions: Conceptualization, A.G.M., P.C.S., E.R.M., S.R.B., Y.T., and P.A.N.; methodology, A.G.M,
P.C.S., I.W.F., and S.R.B.; software, P.C.S.; validation, P.C.S.; formal analysis, A.G.M., P.C.S., A.K.-S., and I.W.F.;
investigation, A.G.M., B.M.; resources, P.C.S. and S.R.B.; data curation, P.C.S., I.W.F., and A.G.M.; writing—original
draft preparation, A.G.M.; writing—review and editing, P.A.N., E.R.M., P.C.S., S.R.B., and B.M.; visualization,
P.C.S.; supervision, P.A.N. and P.C.S.; project administration, S.R.B.; funding acquisition, S.R.B., P.A.N., and P.C.S.
Funding: This research was funded by grants from the McKnight foundation to SRB and PCS Grant No: 17-070,
GCRF-BBSRC grant BB/R020361/1 grant to PCS and the World Bank to PAN Grant No.5799-TZ.
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Article
Insect Antifeedant Components of Senecio fistulosus
var. fistulosus—Hualtata
Liliana Ruiz-Vásquez 1,2 , Matías Reina 2 , Víctor Fajardo 3 , Matías López 4
and Azucena González-Coloma 5, *
1 Natural Resources Research Center (CIRNA), National University of the Peruvian Amazon (UNAP), Iquitos,
Peru; lilyruizv@gmail.com
2 Institute of Natural Products and Agrobiology (IPNA), Spanish Research Council (CSIC), 38206 Tenerife,
Spain; mreina@ipna.csic.es
3 Faculty of Sciences, University of Magallanes (UMAG), Punta Arenas 01855, Chile; victor.fajardo@umag.cl
4 University Institute of Bio-Organic Antonio González (IUBO), University of La Laguna, 38206 Tenerife,
Spain; mlopez@ull.es
5 Institute of Agricultural Sciences (ICA), Spanish Research Council (CSIC), 28006 Madrid, Spain
* Correspondence: azu@ica.csic.es; Tel.: +34-917-452-500
Received: 22 April 2019; Accepted: 5 June 2019; Published: 15 June 2019
Abstract: From a bioactive methanolic extract of Senecio fistulosus, the antifeedant effects of
the alkaloidal and non-alkaloidal fractions were tested against the insects Spodoptera littoralis,
Myzus persicae and Rhopalosiphum padi, with the non-alkaloidal fraction being antifeedant.
The phytochemical study of the non-alkaloidal fraction of S. fistulosus, resulted in the
isolation of four compounds, two 9-oxo-furanoeremophilanes (1, 2), an eremophilanolide,
1β,10β-epoxy-6-acetoxy-8α-hydroxy-eremofil-7(11)-en-8β,12-olide (3) and a maaliol derivative (4).
The alkaloidal fraction yielded two known pyrrolizidine alkaloids (5, 6). Compounds 1, 3 and 4
are new natural products. Furanoeremophilane 2 was a strong antifeedant against S. littoralis and
maaliane 4 inhibited the settling of M. persicae.
Keywords: Senecio fistulosus; antifeedant; sesquiterpene; pyrrolizidine alkaloid; structure-activity

relationships
1. Introduction
The genus, Senecio (Asteraceae), is distributed worldwide and contains pyrrolizidine alkaloids
(PAs). PAs are toxic to mammals and feeding deterrents for insect herbivores [1]. Compounds present
in the non-alkaloidal fraction of Senecio spp have been described as part of their defense [1–3]. The most
frequent chemical groups found in the non-alkaloidal fraction of Senecio are eremophilane-type
sesquiterpenes of the furanoeremophilane and eremophilanolide type [1]. Some of these compounds
have insect antifeedant, acaricidal, fungicidal, cytotoxic, phytotoxic, antioxidant, anti-inflammatory, and
antimicrobial effects [1–6] and have been proposed as being an important part of Senecio defense [1–5].
In Chile, the genus Senecio is abundant (~210 species) [7]. There are several reports
on eremophilane sesquiterpenes from Chilean Senecio species with defensive properties [1–3].
The species, Senecio fistulosus, grows from the western area of Patagonia to central Chile
and it is used in folk medicine for its effects on the heart [8,9]. A previous study
on the phytochemistry of S. fistulosus, from the central region of Chile, reported the
presence of furanoeremophilanes 4α-hydroxy-6β-angeloxy-10βacetoxy-9-oxo-furanoeremophilane
and 4α-hydroxy-6β-angeloxy-9-oxo-furanoeremophilane [10], but there are no reports on the defensive
chemistry of this species.

Plants 2019, 8, 176
In this work, the authors studied the chemical defenses of S. fistulosus var. fistulosus from the
Magallanic region, containing a large number of the Chilean Senecio species and subspecies distributed
in the Patagonic Cordillera and the coastal areas [7].
From a bioactive methanolic extract of S. fistulosus, the alkaloidal and non-alkaloidal fractions
were tested against the insects Spodoptera littoralis (Boisd), Myzus persicae (Sulzer) and Rhopalosiphum
padi, with the non-alkaloidal being antifeedant. Two furanoeremophilanes (1, 2), one eremophilanolide
(3) and maaliol derivative (4) have been isolated from the non-alkaloidal fraction, with compounds 1, 3
and 4 being reported as natural products for the first time. Additionally, two pyrrolizidine alkaloids (5,
6) were isolated from the alkaloidal fraction.

Extracts of S. fistulosus (methanolic, MeOH, non-alkaloidal and alkaloidal) were tested against
the phytophagous insects Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi. The MeOH
extract showed a significant effect on M. persicae (SI = 78 ± 4%; EC50 = 1.69 μg/cm2 , 0.69–4.15, 95%
confidence limits—CL), the non-alkaloidal extract was active on S. littoralis (FI = 76 ± 6%) and M.
persicae (SI = 75 ± 6%; EC50 = 2.25 μg/cm2 , 1.19–4.24, 95% CL). The alkaloidal extract showed moderate
activity against S. littoralis (SI = 62 ± 6%) and R. padi (SI = 69 ± 6%), indicating that S. fistulosus defense
chemistry is mainly due to compounds present in the non-alkaloidal fraction, as previously suggested
for PA producing plants [2–4,11].
The phytochemical study of the non-alkaloidal fraction of S. fistulosus resulted in the
isolation of four compounds, two 9-oxo-furanoeremophilanes (1, 2) [10,12], an eremophilanolide,
1β,10β-epoxy-6-acetoxy-8α-hydroxy-eremofil-7(11)-en-8β,12-olide (3) and a maaliol derivative (4).
The alkaloidal fraction yielded two known pyrrolizidine alkaloids (5, 6) [13,14] (Figure 1).
Figure 1. Chemical structures of compounds 1–6.
Compounds 1, 3 and 4 are described here for the first time as natural products. A previous
study on S. fistulosus reported the presence of furanoeremophilanes 4α-hydroxy-6β-angeloxy-10β-
acetoxy-9-oxo-furanoeremophilane and 4α-hydroxy-6β-angeloxy-9-oxo-furanoeremophilane [10].
The difference in furanoeremophilane composition could be related to the different origin of the
plant populations studied (Magallanes versus the central region of Chile).
The structural elucidation was carried out based on their 1 H and 13 C NMR spectra including (1D)
and (2D) (COSY, HSQC, HMBC and NOESY) experiments, X-ray diffraction, as well as its physical,
spectrometric (EIMS and HREIMS) and comparison with the chemical bibliography reported for
similar compounds.
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Plants 2019, 8, 176
Compound 1 was isolated in crystalline form. Its infrared (IR) spectrum showed absorption bands
at 3452, 1748, 1720 and 1679 cm−1 attributable to a hydroxyl, ester, and carbonyl groups. Its HR-EI-MS
showed a molecular-ion peak at m/z 404.1838 (M+ , calculated for C22 H28 O7 , 404.1835) and a major
fragment in the upper part of the spectrum at 345 (3%) [M-OCOCH3 ]+ . The 1 H and 13 C NMR spectra
of compound 1 (Table 1) showed signals of an olefinic proton at δ(H) 7.41 (br s, J = 1.3, H-C(12)) and
one methyl group on a double bond at δ(H) 1.91 (d, J = 1.1, Me(13)), indicating the presence of a furan
ring with a methyl group at C(11). The chemical shifts of signals δ(H) 0.98 (s), 1.17 (d, J = 7.5) assigned
to Me(14) and Me(15), indicated a cis-decalin system [10]. Signals at δ(H) 3.98 (br s) were assigned to a
proton on a hydroxyl group, and signals at δ(H) 5.92 (qq, J = 1.5, 7.2, H-C(3 )), 1.88 (dq, J = 1.6, 7.4,
Me(4’)), 1.55 (quint., J = 1.5 Hz, Me(5’)); δ(C) 126.6 (s, C(2 )), 140.5 (d, C(3 )), 15.8 (q, C(4 )), 19.9 (q,
C(5 )) corresponded to an angelate group. The chemical shift at δ(H) 7.03 (s) was attributed to Hα -(6),
a geminal proton of an acetate group [δ(H) 2.19 (s, OCOCH3 ); δ(C) 20.9 (q) and 170.9 (s)]. The HMBC
experiment showed correlations between H-C(1) with C(3), C(5), C(10) and C(1 ), which allowed for
the location of the angelate group at C(1). Correlations of the OH proton with C(5), C(9) and C(10)
confirmed the location of the hydroxyl group at C(10). Correlation of Hα -C(6) with C(4), C(8), C(11),
C(14) and OCOCH3 located the acetate group at C(6) (Figure 2). The remaining correlations were in
agreement with the proposed structure. The relative stereochemistry of 1 was established by a NOESY
experiment (Figure 3). Hβ -C(1) gave a positive NOE effect with the Hβ -C(2) and Hβ -C(3) signals,
confirming the α-configuration of the angelate group. In the same way, Hα -C(6) presented a NOE effect
with protons Hα -C(4) and Me(13), establishing the configuration of the acetate group as C-6β. The NOE
effect of H-C(3 ) with Me(4) /Me(5 ) and the chemical shift of Me(5 ) (δ(C) = 20.6 ppm) suggested the
Z-geometry for the double bond of the H-C(2´)/H-C(3 ) of the angelate group. The molecular structure
of 1 was confirmed by X-ray diffraction (Figure 3), resolved by direct methods with SIR97, and was
established as 1α-angeloyloxy-6β-acetoxy-10β-hydroxy-9-oxo-furanoeremophilane.
The HR-EI-MS of compound 2, showed a molecular-ion peak at m/z 346.1785 (M+ , calculated for
C20 H26 O5 , 346.1780), and its IR spectrum showed the presence of absorption bands at 3446, 1733, 1716
and 1699 cm−1 attributable to hydroxyl, ester, and carbonyl groups. The analysis of 1 H and 13 C NMR
spectroscopic data of 2 (Table 1) indicated the presence of a trans-decalin based on the chemical shift
at δ(H) 0.88 (s, Me(14)), and by comparison with published data. Therefore, the structure of 2 was
confirmed as 1α-hydroxy-3α-angeloyloxy-10α H-9-oxo-furanoeremophilane, previously isolated from
Senecio smithii, [12].
Table 1. 1 H (500 MHz) and 13 C (125 MHz) NMR data of compounds 1 and 2 in CDCl3 .
1 2
Position δH in ppm, δH in ppm,
δC in ppm δC in ppm
Multiplicity, J (in Hz) Multiplicity, J (in Hz)
1β 4.84 br s 74.5 d 4.25 ddd (4.8, 9.7, 11.7) 65.5 d
2α 2.30 m 1.50 q (11.8)
20.7 t 39.1 t
2β 1.64 m 2.47 m
3α 1.40 m -
23.9 t 71.4 d
3β 2.32 m 4.90 dt (4.6, 11.5)
4α 1.65 m 32.3 d 1.84 m 46.9 d
5 - 50.2 s - 43.9 s
6α 7.03 s 2.52 d (16.6)
68.6 d 35.9 t
6β - 2.71 d (16.6)
7 - 139.4 s - 138.1 s
8 - 145.9 s - 146.5 s
9 - 186.9 s - 188.7 s
10β - 79.8 s 2.42 d (9.8) 60.6 d
11 - 121.8 s - 121.7 s
12 7.41 br s 146.9 d 7.41 s 145.8 d
13 1.91 d (1.1) 8.3 q 1.99 d (1.0) 7.8 q
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Table 1. Cont.
1 2
δC in ppm δC in ppm
14 0.98 s 15.5 q 0.88 s 14.3 q
15 1.17 d (7.5) 16.0 q 0.98 d (6.7) 10.5 q
1’ - 165.7 s - 167.6 s
2’ - 126.6 s - 128.1 s
3’ 5.92 qq (1.5, 7.2) 140.5 d 6.05 qq (1.4, 7.2) 137.8 d
4’ 1.88 dq (1.6, 7.4) 15.8 q 1.97 dq (1.5, 7.2) 15.8 q
5’ 1.55 quint. (1.5) 19.9 q 1.88 quint. (1.5) 20.6 q
OCOCH3 2.19 s 20.9 q - -
OCOCH3 - 170.9 s - -
OH-10 3.98 br s - - -
Figure 2. HMBC correlations observed for compound 1.
Figure 3. NOESY and ORTEP of compound 1.
The HR-EI-MS of compound 3 showed a molecular ion peak at m/z 322.1425 (M+ , calculated for
C17 H22 O6 , 322.1416) and fragments in the upper part of the spectrum at m/z 262 (100%) [M-CH3 COOH]+ .
Signals for seventeen carbon atoms were observed in its 13 C NMR spectrum. Their multiplicities were
analyzed by a DEPT experiment which determined four methyl, three methylenes, three methines
and six quaternary carbons. The 1 H and 13 C NMR spectra of 3 showed signals at δ(H) 3.18 (d, J = 4.6,
H-C(1)); δ(C) 62.7 (d, C-(1)) and 60.9 (s, C-(10)), attributed to chemical shifts characteristic of an epoxide
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Plants 2019, 8, 176
group at C-(1)-C-(10); signals at δ(H) 2.20 (s, OCOCH3 ); δ(C) 20.9 (q, OCOCH3 ) and 170.6 (s, OCOCH3 ),
corresponding to an acetate group at C(6), a signal for a geminal proton of an acetate group at δ(H) 5.92
(q, J = 1.8); δ(C) 73.8 (d, C(6)); and a signal at δ(H) 1.87 (d, J = 1.8); δ(C) 8.2 (q) corresponding to a Me(13).
The HSQC and HMBC experiments (Table 2) confirmed the presence of an eremophilanolide skeleton
and the localization of the epoxide and acetate groups, respectively. The relative stereochemistry of 3 was
determined by a NOESY experiment (Figure 4). The positive NOE effect of Hα -(6) with the methyl Me(13)
signal was consistent with a β configuration of the γ-lactone, which agrees with the observed homoalilic
coupling constant J6–13 = 1.8 [15,16] with an angle between the two bonds of about 90◦ . Therefore, an
α-configuration for the hydroxyl group at C(8) was determined. The observed NOE effect of Hα -(1)
with Hβ -(9) confirmed the β-configuration of the epoxide. Compound 3 was identified based on its
spectroscopic data as 1β,10β-epoxy-6β-acetoxy-8α-methoxy-eremofil-1(10),7(11)-diene-12,8β-olide,
previously obtained by epoxidation and subsequent acetylation of the compound 6β-hydroxy-
8α-methoxy-eremophil-1(10),7(11)-dien-12,8β-olide, isolated from S. magellanicus [2].
Table 2. 1 H (500 MHz) and 13 C (125 MHz) NMR data of compounds 3 and 7 * in CDCl3 .
3 7*
δ G in ppm
C
G δC in ppm
1β 3.18 d (4.6) 62.7 d 3.14 d (4.3) 63.0 d
2α 1.96 dd (6.8, 10.8) 2.04 m
20.3 t 21.0 t
2β 2.04 dd (5.9, 10.5) 2.21 m
3α 1.36 dc (3.5, 9.4) 1.38 m
23.9 t 24.2 t
3β 1.63 m 1.61 m
4α 1.62 m 32.5 d 1.62 m 33.0 d
5 - 43.4 s - 43.5 s
6α 5.92 c (1.8) 73.8 d 5.69 t (1.7) 74.3 d
7 - 155.0 s - 153.9 s
8 - 101.3 s - 104.4 s
9α 1.79 d (13.6) 1.80 d (13.6)
43.4 t 43.6 t
9β 2.31 d (13.6) 2.27 d (13.6)
10 - 60.9 s - 61.0 s
11 - 124.6 s - 126.8 s
12 - 170.8 s - 170.9 s
13 1.87 d (1.8) 8.2 q 1.92 d (1.2) 8.6 q
14 1.09 s 14.5 q 1.09 s 14.5 q
15 1.04 d (7.2) 16.1 q 1.03 d (7.0) 16.5 q
OMe-8 - - 3.23 s 50.9 q
OCOCH3 2.20 s 20.9 q 2.21 s 21.0 q
OCOCH3 - 170.6 s - 170.3 s
* Source: Reina et al. [2].
Figure 4. NOESY of compound 3.
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Compound 4 was isolated as a colorless oil. Its HR-EI-MS mass spectrum showed a molecular-ion
peak at m/z 220.1831 (M+ , calculated for C15 H24 O, 220.1827) and fragments at 205 (55%) [M-CH3 ]+ and
187 (19%) [M-CH3 + H2 O]+ . The presence of three tertiary methyl groups at δ(H) 1.04 (s, Me(13)), 1.06
(s, Me(12)) and 1.28 (s, Me(15)) were observed in the 1 H and 13 C NMR spectra (Table 3). An HSQC
experiment showed their correlations with carbons at δ(C) 16.5 (q, Me(13)), 28.8 (q, C(12)), and 26.2
(q, C(15)), and signals from two hydrogens of a cyclopropane at δ(H) 0.47 (dd, J = 9.6, 11.4, H-C(6));
δ(C) 30.1 (d, C(6)) and 0.71 (ddd, J = 6.1, 9.5, 11.4, H-C(7)); δ(C) 27.7 (d, C(7)) [16]. Additional signals
attributable to two protons at δ(H) 4.69 (t, J = 1.6, Ha –C(14)) and 4.63 (q, J = 1.7 Hb -C(14)); a δ(C) 106.4
(t) signal assigned to an exocyclic methylene and a proton signal at δ(H) 2.20 (m, Hβ -C(10)); δ(C) 53.6
(d, C(10)) were observed. The latter signal presented HMBC correlations at δC 153.6 (t, C-(14)), 39.1
(t, C(2)) and 54.6 (d, C(5)), suggesting the location of the exocyclic methylene at C-1. The remaining
signals were assigned by analysis of the 1D and 2D NMR spectra and by comparing these data with
similar compounds. The structure of 4 was identified as the maaliol derivative (+)-1(14)-en-maaliol [17]
which has not been previously isolated as a natural product.
Table 3. 1 H (500 MHz), 13 C (125 MHz) and HMBC NMR data of compound 4 in CDCl3 .
Position δH in ppm, Multiplicity, J (in Hz) δC in ppm HMBC

1 - 153.6 s -
2a 2.42 ddd (1.3, 6.3, 13.3) C-1, C-4, C-10, C-14
39.1 t
2b 2.05 m
3a 1.77 m C-2, C-4
41.9 t
3b 1.56 d
4 - 81.1 s -
5α 1.32 m 54.6 d C-1, C-4, C-6, C-10, C-11
6β 0.47 dd (9.6, 11.4) 30.1 d C-4, C-8, C-11, C-13
7β 0.71 ddd (6.1, 9.5, 11.4) 27.7 d C-5, C-11, C-13
8a 1.98 m -
24.9 t
8b 1.01 m
9a 1.90 m C-5, C-10
26.9 t
9b 1.63 m
10β 2.20 m 53.6 d C-1, C-2, C-5, C-6, C-14
11 - 20.4 s -
12 1.06 s 28.8 q C-7, C-6, C-11, C-13
13 1.04 s 16.5 q C-7, C-6, C-11, C-12
14a 4.69 t (1.6) C-2, C-10
106.4 t
14b 4.63 q (1.7)
15 1.28 s 26.2 q C-3, C-4, C-5
Two unsaturated pyrrolizidine alkaloids (PAs) were isolated from the alkaloidal fraction,
9-O-angeloylpetasinecine (hectorine 5), and rosmarinine (6). These alkaloids were identified by
comparison of their spectral data (1 H a 13 C NMR and EIMS) with previous reports [13,14].
The antifeedant effects of compounds 1, 2 and 4 are shown in Table 4. Furanoeremophilane 2 was
a strong antifeedant to S. littoralis (EC50 = 0.64 μg/cm2 ) while the maaliane 4 affected M. persicae (EC50
= 0.97 μg/cm2 ). Antifeedant furanoeremophilanes have been described in Senecio species such as S.
magellanicus (against M. persicae and S. littoralis) [2] and S. otites (against M. persicae and R. padi) [11,18].
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Table 4. Antifeedant activity of S. fistulosus compounds 1, 2, 4.
S. littoralis M. persicae
Compound EC50 (μg/cm2 ) b EC50 (μg/cm2 ) b
%FI (50 μg/cm2 ) a %SI b (50 μg/cm2 ) a
1 65 ± 6 * 52 ± 7
2 83 ± 6 * 0.64 (0.36-1.16) 52 ± 7
4 64 ± 7 * 90 ± 3 * 0.97 (0.71–1.32)
a %FI/%SI = [1 − (T/C)] × 100, where T and C are the consumption/settling of treated and control leaf disks,
respectively. b Effective antifeedant dose (EC50 ) and 95% confidence (lower, upper). * p < 0.05, Wilcoxon paired test.
Furanoeremophilanes are less abundant in Senecio than eremophilanolides. Therefore, the studies
on their structure-activity relationships (SAR) are limited. Table 5 shows a compilation of the available
information on the SAR of these structures, including the results presented in this work. The active
compounds against the aphid M. persicae are characterized by the absence of substituents in C-1, C-3 and
C-10, regardless of the substituent in C-6 (8–10, 11, 12). The presence of β-OH/C-1 and the α-OAng/C-3
group (compound 2) resulted in an important antifeedant activity against S. littoralis. In addition, the
C-6 substitution pattern together with the C-1/C-10 unsaturation determined post-ingestion effects on
S. littoralis [11].
Table 5. Antifeedant structure-activity relationships of Senecio furanoeremophilanes against S. littoralis

and M. persicae.
Substituent S. littoralis M. persicae

Compound
C-1 C-3 C-6 C-10 c
%FI (EC50 ) d %SI c (EC50 ) d
1 α-OAng H2 β-OAc β-OH 65.0 52.0
2 α-OH α-OAng H2 α-H 83.0 (0.64) 52.0
8a Δ1 H2 β-OAng Δ10 32.0 67.0
9a Δ1 H2 β-OH Δ10 62.0 71.0
10 a Δ1 H2 β-OCOCH2 CH3 Δ10 45.0 75.0
11 b H2 H2 β-OAc α-H 51.0 74.0 (21.9)
12 b H2 H2 β-OTigl α-H 65.0 74.0 (12.2)
aCompounds 8–10 from Domínguez et al. [11]. b Compounds 11, 12 from Reina et al. [2]. c %FI / %SI values at
50μg/cm2 . d Effective antifeedant dose (μg/cm2 ).
Maalianes have been isolated from a range of organisms, such as liverworts, marine sponges,
soft corals and bacteria, however, they are not abundant in nature. A small amount of biological
activity has been reported and includes fish toxicity, in vitro antimalarial activity, cytotoxicity and
antimicrobial [19]. This is the first report on the insect antifeedant effects of a maaliane sesquiterpene.
PAs 5 and 6, with necines of the rosmarinecine and petasinecine type (1,2-saturated base),
were isolated from the alkaloidal extract of S. fistulosus. The role of PAs as plant defenses against
phytophagous insects has been widely documented [20], however, this alkaloidal extract showed
moderate-low antifeedant activity (62 ± 6%FR against S. littoralis and 69 ± 6%SI against R. padi).
PAs with unsaturated retronecines are potentially more toxic than rosmarinecine and petasinecine
type (1,2-saturated base) PAs [21]. For example, rosmarinine with a petasinecine, did not form
hepatotoxic reactive pyrrole intermediates [22,23] and cytotoxic assays have demonstrated a higher
toxicity of retronecine and otonecine PAs compared with platynecine PAs [24]. Therefore, PAs 5 and 6
have a low risk of associated toxicity.
3.1. General
For column chromatography (CC), Si-gel (107734, 107741, and 107749, Merck) and Sephadex
LH-20 (Sigma–Aldrich) were used. For TLC chromatography, Si-gel (105554 and 105715; Merck)
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plates were used and visualized with óleum solution (sesquiterpenes) and Dragendorff’s reagent
(alkaloids). The prep. HPLC chromatography was carried out on a Beckman 125P system equipped
with an Ultrasphere semiprep column (10 × 250 mm) and a UV/visible diode array detector 168.
Optical rotations were determined at 20 ◦ C on a Perkin-Elmer 343 Plus polarimeter. IR Spectra were
recorded in CHCl3 on a Perkin Elmer 1600 spectrophotometer. NMR spectra were recorded on a
pulsed-field gradient Bruker Advance II-500 MHz spectrometer (solvent as internal standard CDCl3 ,
at δH 7.26 and δC 77.0) and the Bruker software was used for DEPT, 1 H, 1 H-COSY (Homonuclear
correlation spectroscopy), NOESY (Nuclear Overhauser Effect Spectroscopy), HSQC (Heteronuclear
single quantum coherence spectroscopy) and HMBC (Heteronuclear Multiple Bond Correlation).
EI and HR-EI-MS spectra were recorded in m/z on a Micromass Autospec spectrometer.
3.2. Extraction and Isolation

Aerial parts of S. fistulosus (Asteraceae), identified by Orlando Dollenz, were collected in Sierra
Baguales (March 2009, Punta Arenas, Magallanes, Chile,) during the flowering period. A voucher
specimen (# 7569) has been deposited in the Herbarium of the Patagonian Institute, Magallanes
University (UMAG), Punta Arenas, Chile.
Grounded dried aerial plant parts (2.50 kg) were extracted with MeOH (20 L) at room temperature
for a week to give a crude MeOH extract (190.5 g, 7.62% yield of plant dry weight). The MeOH
extract (157.6 g) was treated with a f H2 SO4 0.5 M and CH2 Cl2 (1:1) solution. Zinc dust was used
to reduce the aqueous phase under continuous stirring (4–6 hours) and then filtered, basified (30%
NH4 OH, pH = 8–9) and extracted with CH2 Cl2 (236.0 mg of alkaloids, 9.4 × 10−3 %). The organic
phase, dried over anhydrous Na2SO4 (non-alkaloidal fraction, 9.0 g, 0.36%), was chromatographed
on a SiO2 vacuum-liquid chromatography column (VLC) and eluted with a hexane/EtOAc/MeOH
gradient to give seven fractions. Fr-0 (hexane 100%, 360.7 mg), Fr-1, (hexane/EtOAc 95:5%, 2.2 g), Fr-2
(hexane/EtOAc 90:10%, 1.7 g), Fr-3 (hexane/EtOAc 75:25%, 1.1 g), Fr-4 (hexane/EtOAc 50:50%, 966.6
mg), Fr-5 (EtOAc 100%, 478.2 mg), Fr-6 (MeOH 100%, 2.6 g). Fr-1 (2.2 g, 8.8 × 10−2 %) was further
chromatographed on a CC Sephadex LH-20 column, CC silica gel and semi-preparative normal-phase
HPLC eluted with an isocratic mixture of hexane/EtOAc at 3 ml/min flow rate to give compound
4 (16.1 mg, 6.4 × 10−4 %). Fr-2 (1.7 g) was chromatographed on CC Sephadex LH-20, CC silica gel,
circular chromatography and semi-preparative normal phase HPLC eluted with an isocratic mixture
of hexane/EtOAc at a flow rate of 3 ml/min to give compounds 1 (41.0 mg, 1.6 × 10−3 %), 2 (29.9 mg,
1.2 × 10−3 %) and 3 (3.5 mg, 1.4 × 10−4 %).
The alkaloidal fraction was submitted to neutral alumina CC, eluted with an EtOAc/MeOH
gradient and PTLC (20 × 20 cm, 0.25 mm) to give compounds 5 (1.3 mg; 5.2 × 10−5 %) and 6 (0.9 mg;
3.6 × 10−5 %).
3.2.1. α-Angeloyloxy-6β-acetoxy-10β-hydroxy-9-oxo-furanoeremophilane (1)
Colorless crystal, mp 127–130 ◦ C (hexane/EtOAc); [α]20 D −28.2 (c, 0.82, CHCl3 ). IR (CHCl3 ) νmáx .:
3452, 1748, 1720, 1679, 1232 cm−1 . EI-MS: 404 (1, M+ ), 345 (3), 260 (20), 262 (12), 178 (55), 136 (4), 91 (4),
83 (100), 57 (8), 55 (39). HR-EI-MS: 404.1838 (M+ , C22 H28 O7 ; calculated for 404.1835). For 1 H and 13 C
NMR data see Table 1.
3.2.2. α-hydroxy-3α-angeloyloxy-10αH-9-oxo-furanoeremophilane (2)
White amorphous solid; [α]20 D −37.5 (c, 0.59, CHCl3 ). IR (CHCl3 ) νmáx .: 3446, 1733, 1716, 1699,
1456 cm−1 . EI-MS: 346 (3, M+ ), 246 (9), 228 (10), 213 (18), 191 (8), 163 (100), 135 (9), 105 (5), 91 (13), 83
(19), 77 (7), 55 (24). HR-EI-MS: 346.1785 (M+ , C20 H26 O5 ; calculated for 346.1780). For 1 H and 13 C NMR
data see Table 1 [12].
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3.2.3. β,10β-epoxy-6β-acetoxy-8α-hydroxy-eremophil-7(11)-en-8β,12-olide (3)
Colorless oil; [α]20 −1

D −72.8 (c, 0.25, CHCl3 ). IR (CHCl3 ) νmáx .: 3392, 1771, 1749, 1717 cm . EI-MS:
+
322 (0.4, M ), 298 (2), 280 (3), 262 (100), 244 (7), 216 (6), 142 (100), 124 (50), 95 (63). HR-EI-MS: 322.1425
(M+ , C17 H22 O6 ; calculated for 322.1416). For 1 H and 13 C NMR data see Table 2.
3.2.4. (+)-1(14)-en-maaliol (4)
Colorless oil; [α]20

D +4.0 (c, 0.78, CHCl3 ). IR (CHCl3 ) νmáx .: 3421, 2930, 2868, 1653, 1457, 1375, 1152,
913, 889, 668 cm−1 . EI-MS: 220 (5, M+ ), 205 (55), 187 (19), 177 (12), 162 (24), 159 (34), 147 (36), 133
(29), 121 (38), 119 (60), 105 (75), 93 (78), 91 (100), 79 (93), 69 (85). HR-EI-MS: 220.1831 (M+ , C15 H24 O;
calculated for 220.1827). For 1 H and 13 C NMR spectral data see Table 3.
3.2.5. 9-O-angelylpetasinecine (hectorine) (5)
Colorless oil; [α]20 +

D −62.86 (c, 0.07, CHCl3 ). EI-MS: 239 (9, M ), 222 (9), 190 (3), 188 (11), 140 (61),
122 (9), 111 (9), 83 (100), 70 (11), 68 (6), 55 (27). HR-EI-MS: 239.1512 (M+ , C13 H21 NO3 ; calculated for
239.1521). 1 H NMR (CDCl3 , 500 MHz): δH 2.51 (1H, m, Hα -C(1)), 4.23 (1H, t, J = 3.9 Hz, Hα -C(2)), 3.42
(1H, dd, J = 4.0, 13.0 Hz, Hα -C(3)), 3.03 (1H, d, J = 13.0, Hz, Hβ -C(3)), 3.50 (1H, t, J = 8.5 Hz, Hα -C(5)),
2.98 (1H, m, Hβ -C(5)), 1.84 (1H, m, Hα -(6)), 2.06 (1H, m, Hβ -C(6)), 1.80 (1H, m, Hα -C(7)), 1.99 (1H,
m, Hβ -C(7)), 3.90 (1H, m, Hα -C(8)), 4.73 (1H, dd, J = 10.0, 11.5 Hz, Hd -C(9)), 4.15 (1H, dd, J = 4.9,
11.5 Hz, Hu -C(9)), 6.15 (1H, cc, J = 1.5, 7.2 Hz, H-(3’)), 1.99 (3H, d, J = 7.0 Hz, H-C(4’)), 1.90 (3H, quint.,
J = 1.6 Hz, H-C(5’)). 13 C-NMR: δ(C) 46.3 (d, C-1), 73.2 (d, C-2), 61.6 (t, C-3), 56.9 (t, C-5), 27.2 (t, C-6),
27.9 (t, C-7), 66.7 (d, C-8), 60.3 (t, C-9), 169.2 (s, C-1’), 127.4 (s, C-2’), 139.8 (d, C-3’), 16.1 (q, C-4’), 20.7 (q,
C-5’) [13].
3.2.6. Rosmarinine (6)
As a white resin; [α]20 +

D −51.1 (c, 0.09, CHCl3 ). EI-MS: 353 (5, M ), 282 (2), 227 (4), 180 (6), 156 (43),
154 (87), 138 (100), 122 (32), 98 (27), 82 (86), 81 (21), 55 (41). HR-EI-MS: 353.1835 (M+ , C18 H27 NO6 ;
calculated for 353.1838). 1 H NMR (CDCl3 , 500 MHz): δ(H) 2.54 (1H, m, Hα -C(1)), 4.26 (1H, m, Hβ -C(2)),
3.15 (1H, dd, J = 7.3, 11.2 Hz, Hα -C(3)), 2.94 (1H, dd, J = 7.9, 11.3 Hz, Hβ -C(3)), 3.33 (1H, t, J = 8.9 Hz,
Hα -C(5)), 2.60 (1H, m, Hβ -C(5)), 2.08 (1H, m, Hα -C(6)), 2.27 (1H, m, Hβ -C(6)), 5.08 (1H, t, J = 2.9 Hz,
Hα -C(7)), 3.71 (1H, dd, J = 3.4, 7.8 Hz, Hα -C(8)), 4.89 (1H, dd, J = 5.4, 12.6 Hz, Hd -C(9)), 4.11 (1H,
dd, J = 1.1, 12.6 Hz, Hu -C(9)), 1.80 (1H, m, Hβ -C(13)), 2.27 (1H, m, Ha -C(14)), 1.96 (1H, m, Hb -C(14)),
1.34 (3H, s, Hα -C(18)), 0.97 (3H, d, J = 6.7 Hz, Hα -C(19)), 5.80 (1H, c, J = 7.1 Hz, H-(20)), 1.85 (3H, dd,
J = 1.5, 7.2 Hz, H-C(21)). 13 C-NMR: δ(C) 49.1 (d, C-1), 69.3 (d, C-2), 60.9 (t, C-3), 53.5 (t, C-5), 34.6 (t,
C-6), 75.2 (d, C-7), 69.6 (d, C-8), 62.3 (t, C-9), 180.7 (s, C-11), 77.6 (s, C-12), 38.0 (d, C-13), 39.7 (t, C-14),
132.7 (s, C-15), 167.6 (s, C-16), 25.8 (q, C-18), 11.9 (q, C-19), 134.9 (d, C-20), 15.3 (q, C-21) [14].
3.2.7. Crystal Structure Analysis

Intensity data, for both compounds, were collected at 293 K on an Oxford Diffraction Supernova
dual Atlas CCD diffractometer, using Cu Kα (λ = 1.5418 Å) radiation. Data collection, cell refinement
and data reduction were performed with the CrysAlisPRO [25] set of programs. The structure was
solved by direct methods using SIR97 [26]. Refinements were performed with SHELXL-97 [27] using
full-matrix least squares, with anisotropic displacement parameters for all the non-hydrogen atoms.
The H-atoms were placed at calculated positions with C-H distances 0.95-1.00 Å and refined using a
riding model. Calculations were mainly performed with WinGX [28] and molecular graphics were
computed with PLATON [29].
X-ray crystal data: C22 H28 O7 , Mw = 404.44, orthorhombic, space group, P21 21 21 , Z = 8,
a = 8.7493(2), b = 13.4234, c = 37.0309(11) Å; V = 4349.1(2) Å3 , μ(Cu Kα) = 0.76 mm−1 , ρcalc = 1.23 g.cm−3 ;
S = 1.06, final R indices: R1 = 0.0678 and Rw = 0.1860 for 7134 observed from 8339 independent
and 15715 measured reflections (θmax = 70.99, I > 2σ(I) criterion and 536 parameters); maximum and
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minimum residues are and 0.30 and −0.24 e.Å−3 respectively. There are two independent molecules in
the asymmetric unit with minor conformational differences between them. The absolute structure is
based on the refinement of the Flack [30] (Flack 1983), x = 0.0 (3), parameter against 3610 CuKα Bijvoet
pairs. The Hooft [31] analysis yielded y = 0.06(8) and P2 (true) = 1.000.
Crystallographic data (excluding structure factor tables) has been deposited with de Cambridge
Crystallographic Data Center as supplementary publications no. CCDC1455588. Copies of the data can
be obtained free of charge on application to The Director, CCDC, 12 Union Road, Cambridge CB1EZ,
UK ((Fax: Int. + (1223) 336 033); e-mail: deposit@ccdc.cam.ac.uk)).
3.3. Insect Bioassays

S. littoralis, M. persicae and R. padi colonies were reared on an artificial diet [32], bell pepper
(Capsicum annuum) and barley (Hordeum vulgare) plants, respectively. The plants are grown from seeds
in pots with commercial substrate. The plants for rearing aphids are infected regularly (bell pepper
plants with 4 leaves, barley plants of 10 cm length). The insect colonies and host plants were maintained
at 22 ± 1 ◦ C, > 70% relative humidity with a photoperiod of 16:8 h (L:D) in a growth chamber.
Antifeedant bioassays: The upper surface of C. anuum and H. vulgare leaf disks or fragments
(1.0 cm2 ) were treated with 10 μl of the test substance. The crude extracts and products were tested at
an initial dose of 100 or 50 μg/cm2 respectively. Five Petri dishes (9 cm diam.) or twenty ventilated
plastic boxes (2 × 2 cm) with two newly molted S. littoralis L6 larvae (≤24 h) or ten apterous aphid
adults (24–48 h old) each were allowed to feed at room temperature for S. littoralis (<2 h) or in a
growth chamber for the aphids (24 h, environmental conditions as above). Each experiment was
repeated 2-3 times (SE < 10%) and terminated when the consumption of the control disks reached
65–75% for S. littoralis or after 24 h for aphids. The leaf disk area consumed was measured on
their digitalized images (Image J, http://imagej.nih.gov/ij). Settling was measured by counting the
number of aphids settled on each leaf fragment. Feeding or settling inhibition (%FI or %SI) was
calculated as % FI/%SI = [1 − (T/C) × 100], where T and C are the consumption/settling of treated and
control leaf disks, respectively. The antifeedant effects (% FI/SI) were analyzed for significance by the
nonparametric Wilcoxon signed-rank test. Extracts and compounds with an FI/SI ≤ 75% were further
tested in a dose-response experiment (3–4 serial dilutions) to calculate their relative potency (EC50 , the
effective dose to give a 50% feeding/settling reduction) from a linear regression analysis (% FI/SI on
Log-dose) [33].
4. Conclusions
Senecio fistulosus is characterized by their content in sesquiterpenes (furanoeremophilanes,
eremophilanolides and maaliane type) and pyrrolizidine alkaloids. The antifeedant properties
of ethanolic, non-alkaloidal, alkaloidal extracts and compounds have been studied. Most of the
insect antifeedant effects were found in the ethanolic and non-alkaloidal extracts, containing mainly
sesquiterpenes with low amounts of PAs. The isolated furanoeremophilanes sesquiterpenes type had
structure-dependent antifeedant effects. In addition to their antifeedant action, these sesquiterpenes
could play a role in insect-plant interactions.
Author Contributions: Conceptualization, A.G.-C., M.R. and V.F.; funding acquisition, A.G.-C.; investigation,
A.G.-C., L.R.-V., M.L. and M.R.; methodology, A.G.-C., L.R.-V. and M.R.; resources, V.F.; writing—original draft,
A.G.-C., M.L. and M.R.; writing—review & editing, A.G.-C. and L.R.-V.
Funding: This work has been supported by grants CTQ2015-64049-C3-1-R, (MINECO/FEDER), UMAG
027103-026703 (Dirección de Investigación, Chile) and a JAEPRE-DOC-CSIC predoctoral fellowship to L.R.V.
Conflicts of Interest: The authors declare no conflicts of interest.
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93
plants
Review
Opportunities and Scope for Botanical Extracts and
Products for the Management of Fall Armyworm
(Spodoptera frugiperda) for Smallholders in Africa
Naomi B. Rioba 1, * and Philip C. Stevenson 2,3
1 School of Agriculture and Biotechnology, University of Kabianga, Kericho P.O. Box 2030-20200, Kenya;
naomirioba@kabianga.ac.ke
2 Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, UK;
p.c.stevenson@gre.ac.uk
3 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
* Correspondence: naomirioba@kabianga.ac.ke; Tel.: +254-720-361-466
Received: 16 January 2020; Accepted: 2 February 2020; Published: 6 February 2020
Abstract: Fall Armyworm (FAW) (Spodoptera frugiperda) is a polyphagous and highly destructive
pest of many crops. It was recently introduced into Africa and now represents a serious threat to
food security, particularly because of yield losses in maize, which is the staple food for the majority
of small-scale farmers in Africa. The pest has also led to increased production costs, and threatens
trade because of quarantines imposed on produce from the affected countries. There is limited
specific knowledge on its management among smallholders since it is such a new pest in Africa.
Some synthetic insecticides have been shown to be effective in controlling FAW, but in addition to
the economic, health and environmental challenges of pesticide use insecticide resistance is highly
prevalent owing to years of FAW management in the Americas. Therefore, there is a need for the
development and use of alternatives for the management of FAW. These include plant-derived
pesticides. Here we review the efficacy and potential of 69 plant species, which have been evaluated
against FAW, and identify opportunities for use among small-scale maize farmers with a focus on
how pesticidal plants might be adopted in Africa for management of FAW. The biological activities
were diverse and included insecticidal, insectistatic (causing increased larval duration), larvicidal,
reduced growth and acute toxicity (resulting in adverse effects within a short time after exposure).
While most of these studies have been conducted on American plant taxa many South American
plants are now cosmopolitan weeds so these studies are relevant to the African context.
Keywords: biopesticides; botanicals; corn; insects; pests; prospects
1. Introduction
Fall Armyworm (FAW) (Spodoptera frugiperda Hurst) (Lepidoptera: Noctuidae) is a highly
polyphagous pest having been reported on more than 80 species in 23 families [1] including cotton
(Gossypium hirsutum L.) (Malvales: Malvaceae), corn (Zea mays L.) (Cyperales: Poaceae) and many
other grass crops [2]. Originally native and restricted to the Americas, FAW was recorded for the first
time in Africa in 2016 [3] and now it has spread to over 30 countries in Africa.
These invasive populations are now well established and causing severe destruction to important
crops that underpin the livelihoods of many farmers across Africa [4], due to the variety of host plants
and the favorable environment and climate. The pest has characteristics that means it presents a
wider-reaching threat to Africa [3]. For example, in comparison with the African armyworm (Spodoptera
exempta), FAW larvae have unique mouthparts with notched cutting edges, enabling it to feed on flora
that are rich in silica content. More so, the older larvae feed on the younger ones and can dominate the

Plants 2020, 9, 207
competitors of the same species and others of different species within the same genus hence ensuring
its survival [5]. FAW has raised greater concern among farmers than related African Spodoptera species
because it causes especially severe damage to maize, feeding on virtually all parts of the plant leading
to considerable damage, and sometimes results in total crop failure [6].
Sustainable approaches to managing this new African pest should ideally be integrated, tailored
and appropriate for smallholders with mixed cropping farming systems and reduced input costs.
While the use of chemical pesticides dominates existing approaches [7], several alternative control
options exist and are being considered including resistant varieties [8–10], biological control [11,12],
crop management practices [13,14], plant diversity [14], and mechanical methods [15]. However, none
of these methods has yet delivered a viable option for effective control of FAW, hence the search for
alternative approaches including those from plant extracts and their products. Some pesticidal plants
and botanical insecticides are effective and their use could reduce reliance on synthetic insecticides
since they have lower non-target impacts and could even boost growth [16–19]. Here we review
existing research on plant extracts that have been evaluated for the management of FAW with the aim
of identifying those with potential for use by small-scale farmers in Africa, or informing approaches
to identifying and evaluating untested native African plant taxa since pesticidal plants are already
used as crudely produced products among smallholder farming communities in Africa with notable
success [20–22]. While one recent study has specifically sought to evaluate African plant taxa for
activity against FAW [23], most of the studies reviewed here have been conducted on South American
taxa but many of these species are now cosmopolitan weeds so are relevant to the African context. For
example, Ageratum conyzoides L. is a widely used plant for a multitude of uses in Africa including pest
control but originates from South America where it has been evaluated for efficacy against FAW [24,25].
Similarly, Dysphania (syn. Chenopodium) ambrosioides (L.) Mosyakin & Clemants, has been shown to be
biologically active vs FAW [26,27] but is also considered for use in Africa [28], while species such as
Corymbia (syn. Eucalyptus) citriodora (Hook.) K.D. Hill & L.A.S.Johnson are widespread in both Africa
and America but non-native and have been evaluated for activity against FAW [29].
2. Opportunities and Potential of Botanical Extracts and Products

Interest in using plant extracts for pest control is increasing since these can: 1) reduce the cost
of production of the crop, 2) reduce the environmental damage and non-target effects, and 3) reduce
dependence on synthetic insecticides [30,31]. There are many researchers studying insecticidal plants
for the control of FAW with several reporting promising results, although many do not since they do
not establish the chemical basis of activity or store any reference specimens [32]. Some of the pesticidal
plant species that have been shown to be effective in the management of FAW are presented in Table 1.
FAW larvae ingesting maize leaves treated with the essential oil of Ageratum conyzoides were killed
with 70% mortality caused at the concentration of 0.5%. The essential oil contained precocene as the
major active component (87%) [24]. This finding is highly relevant to the African context where this
plant grows widely on farmland. This means that it is easily available to farmers. It has already been
used to control lepidopteran and other pests by some small holders in Africa. It has been shown to
have reduced non-target effects on natural enemies of pests [85].
Ruta graveolens, Cymbopogon citratus, Zingiber officinale, Malva sylvestris, Petiveria alliaceae,
Bacharis genisterlloides and Artemisia verlotorum were also shown to cause mortality for caterpillars of
FAW [36], but active components in these plants were not identified, meaning that the work has limited
value in the efforts to develop new approaches for FAW control unless more research is conducted to
identify the active compounds that are responsible for the biological activity.
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Plants 2020, 9, 207
Table 1. Plant species that have been evaluated for their activity against Fall Armyworm (FAW) and
potential for use in its management.
Family Plant Species Action Refs

Dysphania (syn.
Amaranthaceae Chenopodium) ambrosioides L. Mortality, decreased pupal weight [26,27]
Mosyakin & Clemants
Anacardiaceae Schinus molle L. High mortality [26]
Decreased pupa weight, increased
Annonaceae Annona squamosa L. [33]
larval mortality
Apiaceae Foeniculum vulgare Mill. Sublethal effects [34]
Calotropis procera (Aiton) W.T. Decreased pupa weight, increased
Apocynaceae [33]
Aiton larval mortality
Growth regulating activity,
increased developmental period,
Asparagaceae Yucca periculosa Baker insecticidal activity, reduced [35]
pupation survival, reduced insect
growth
Asteraceae Ageratum conyzoides L. Insecticidal (70% mortality) [24]
Baccharis genistelloides (Lam.)
Asteraceae Mortality [36]
Pers.
Artemisia verlotiorum
Asteraceae Mortality [36]
Lamotte
Roldana barba-johannis (DC.)
Asteraceae Insecticidal [37]
H. Rob. & Brettell
Longer time for pupation and
Gutierrezia microcephala emergence of adults, severe toxicity
Asteraceae [38]
DC. A. Gray against adults, insect growth
inhibitory activity
Asteraceae Lychnophora ericoides Mart. Egg mortality [39]
Trichogonia villosa (Spreng.)
Asteraceae Egg mortality [39]
Sch. Bip. Ex Baker
Lychnophora ramosissima
Asteraceae Larvicidal [39]
Gardner
Vernonia holosenicea Mart. Ex
Asteraceae 87% mortality [39]
DC.) L.
Antifeedant, insecticidal,
Asteraceae Senecio salignus DC. [40]
juvenomimetric activity
Antifeedant effect causing 50%
reduction of larval weight, 40–80%
Asteraceae Tagetes erecta L. [41]
pupal mortality, 48–72% larval
mortality
Myrtillocactus geometrizans Insect growth regulating, larvicidal,
Cactaceae [41]
Mart. Ex Pfeiff delayed pupation
Alters biochemical profile of larvae,
Cymbopogon winterrianus
Cardiopteridaceae diminished reproduction, [42,43]
Jowitt.
reproductive failure
Caricaceae Carica papaya L. 90% mortality [44–46]
Maytenus disticha (Hook. F)
Celastraceae Insecticidal activity [47]
Urb.
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Table 1. Cont.

Celastraceae M. boaria (Molina) Insecticidal activity [47]
Ipomoea murucoides Roem. 46.16% mortality, reduced larval
Convovulaceae [48,49]
And Schult weight increased pupation time
Insecticidal and insectistatic,
Euphorbiaceae Ricinus communis L. [50,51]
larvicidal, growth inhibition
Euphorbiaceae Jatropha curcas L. High mortality [26]
Antifeedant to larva, synergistic
Euphorbiaceae Jatropha gossypiifolia L. [52]
with pesticide
58.5% mortality, reduced larva and
Euphorbia pulcherrima Willd.
Euphorbiaceae pupae weight, increased larva [53,54]
Ex Klotzsch
period, reduced egg viability
Low fertility and fecundity, low
viability of eggs, larval growth
Leguminosae Copaifera langsdorffii Desf. [55]
reduction, inhibited trypsin activity,
egg abnormalities
Leguminosae Militia ferruginea Hochst. High mortality [26]
Toxicity, non-preference,
Lamiaceae Ocimum basilicum L. [23,56,57]
knockdown
Lamiaceae Ocimum gratissimum L. Sublethal effects [58]
Lamiaceae Salvia keerlii Benth. Insecticidal [59]
Insecticidal, insectistatic, increased
Lamiaceae Salvia ballotiflora Benth. larval and pupal duration, reduced [59,60]
pupa weight
Lamiaceae Salvia connivens Epling Insecticidal, insectistatic [59]
Antifeedant, insecticidal,
Lamiaceae Salvia microphylla Kunth [40]
juvenomimetric activity
Malvaceae Malva sylvestris L. Mortality [36]
Reduced larval feeding, reduced
Meliaceae Melia azedarach L. larval growth, synergistic with [52,61]
pesticide
Trichilia pallens C. de
Meliaceae Mortality [62,63]
Candolle
Meliaceae Trichilia pallida Sw. Mortality [63]
Larval mortality, growth reduction,
Meliaceae Cedrela salvadorensis Standl. inhibited larval growth, reduced [64]
pupal weights and adult emergence
Larval mortality, growth reduction,
Meliaceae Cedrela dugessi S. Watson inhibited larval growth, reduced [64]
pupal weights and adult emergence
Meliaceae Melia abyssinica High mortality [26]
Meliaceae Trichilia pallida Sw. No egg deformities [65]
Reduced insect growth, increased
development period, mortality, low
Azadirachta indica
Meliaceae egg laying, antifeedant activity, [24,26,65–75]
Juss.
growth regulating activity, mortality,
larvicidal
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Plants 2020, 9, 207
Table 1. Cont.

Monimiaceae Peumus boldus Molina 75% mortality [76]
Low feeding ratio, (antifeedant
Moringaceae Moringa oleifera Lam. [77]
activity) mortality
Myrtaceae Eucalyptus citriodora Hook Growth regulating activity [29]
Eucalyptus staigeriana F.
Myrtaceae Sublethal effects [34]
Muell. Ex Bailey
Myrtaceae Eucalyptus globulus Labill. High mortality [26]
Myrtaceae Siphoneugena densiflora Berg 100% larval mortality [78]
Petiveriaceae Petiveria alliacea L. Mortality [36]
Phytolaccaceae Phytolacca dodecandra L’Herit. High mortality [26]
Piperaceae Piper tuberculatum Jacq. Insecticidal [79]
Affects spermatogenis and egg
Piperaceae Piper hispidinervum C. DC. [79]
laying
Cymbopogon citratus (DC.)
Poaceae Mortality [36]
Stapf
Poaceae Cymbopogon flexuosus Steud. Toxic, insecticidal activity [80]
Poaceae Cymbopogon nardus L. [33]
larval mortality
Poaceae Zea diploperennis L. High larval survival [81]
Rhamnaceae Zizyphus joazeiro Mart. [33]
larval mortality
Morinda citrifolia Decreased pupa weight, increased
Rubiaceae [33]
L. larval mortality
Psychotria goyazensis Mull. Reduced hatching rate, Egg
Rubiaceae [82]
Arg. mortality
Rutaceae Ruta graveolens L. Mortality [36]
Rutaceae Citrus limon L. Antifeedant [83]
Magonia pubescens Decreased pupa weight, increased
Sapindaceae [33]
A. St.-Hil. larval mortality
Sapindaceae Talisia esculenta Rsdlk. Mortality [61]
Sapindaceae Sapindus saponaria L. Mortality [61]
Solanaceae Nicotiana tabacum L. High mortality [23,26]
Verbenaceae Lantana camara L. High mortality [26]
Verbenaceae Vitex polygama High mortality [84]
Zingiberaceae Zingiber officinale L. Mortality [36]
The essential oil of Cymbopogon flexuosus was reported to be lethal to FAW (LC50 = 1.35 mg Ll−1 )
when supplemented in to an artificial diet at 2.25, 2.5 and 4 mg Ll−1 concentrations and 18.85 h median
lethal time (LT50 ). The insecticidal activity of citral was not significantly different to the essential oil,
suggesting that citral, a compound of this essential oil caused insecticidal effects of the Cymbopogon
flexuosus essential oil to FAW [80].
Moringa oil induced a lower feeding ratio expressed as the ratio of consumed area of treated
leaf discs to consumed area of untreated (control) leaf discs and highest total corrected mortality
percentage of FAW. This study concluded that at 10% concentration, Moringa oils can be used as a
botanic insecticide in the management of FAW. Saponifiable components of the Moringa oils comprised
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Plants 2020, 9, 207
of oleic acid (74.2%) and palmitic acid (7.16%). However, the LC50 of moringa oil, unsaponifiable
and saponifiable matters were 1.9%, 3.4% and 7.6% respectively, indicating that saponifiable matter
was less effective against FAW larvae [77]. This therefore, means that there is no need for separation
and identification of the moringa oil components for application in FAW control. Farmers should be
advised to apply whole Moringa oil to benefit from the synergistic effects of the components therein.
Linalool showed potential in controlling FAW through non-preference, knockdown and toxicity
effects on FAW larvae [56,57]. More than 80% of the essential oil of Ocimum basilicum consisted of
linalool suggesting that this is the main active component [86]. More recently this species has been
evaluated against FAW in Africa as part of a study focused on plants that were either native or widely
grown in Malawi [23]. Another species investigated in this study included Tephrosia vogelii, a rotenoid
producing and widely used species for pest control in Africa but this was not active suggesting a
level of tolerance in FAW to the insecticidal rotenoids occurring in this species [23,87]. Another South
American plant which grows widely as an invasive weed in Africa where it has been shown to have
biological activity against insects [88] and used widely as a pesticide is Tithonia diversifolia but again
this species was not active [23]. The most promising plant species based on their low mammalian
toxicity, abundance and bioactivity against FAW identified through this work were Lippia javanica,
Ocimum basilicum and Cymbopogon citratus which showed various activities including anti-feedancy
and increased mortality. These three species are consumed as spices and teas so are far safer than
synthetics [23]. C. citratus has also been shown in studies elsewhere to be effective against FAW. For
example, it was been reported that sub-lethal doses of citronella oil altered the biochemical profile
of FAW larvae causing damage to their reproductive histophysiology and resulted in diminished
reproduction or reproductive failure [42]. The citronella-treated midgut of FAW larvae displayed
modifications to the epithelium such as increased periodic acid-Schiff positive granules, columnar cell
extrusion, cytoplasmic protrusions and pyknotic nuclei [42]. This study showed further that there
was an increase in regenerative cells, which aided successive renewal of the epithelium. Trophocytes
which are the main cell type of the fat body, once exposed to citronella, had reduced amounts of
proteins, glycogen, and lipids. The fat bodies also showed distended vacuoles and mitotic bodies.
This implies that citronella oil acts by causing changes in the morphology of the midgut and reducing
stored resources in the fat body, limiting insect reproduction and survival.
FAW larvae feeding activity was reduced when treated with 1% and 10% methanolic extract of
Melia azedarach seed. Other effects were slowed caterpillar growth due to ingestion of toxic substances
present in M. azedarach [52,61], extended pupation time, small pupae and deformed moths. While
native to Indomalaya and Australasia Melia azaderach grows widely in South America and Africa as
well, so this study is highly relevant to the African context although there is some concern about the
toxicity of the plant [89].
The ethanolic extract of Poinsettia (Euphorbia pulcherima) leaves obtained during the vegetative
and reproductive phase was evaluated against FAW. The extracts were fed to the FAW larvae after
mixing with artificial diet. Administered at 0.5 and 1% concentrations, the extracts increased the larval
period, reduced larval and pupae weight as well as egg viability and resulted in greater larval mortality.
It was further noted that the extract prepared from leaves that were in the reproductive phase of the
plant effectively reduced the FAW population. Cold aqueous extract of E. pulcherrima also resulted in
58.5% mortality of FAW [53; 54] affecting Neonotonia wightii (perennial soybean).
Trials were done on the bioactivity of aqueous plant extracts of Calotropis procera, Jatropha curcas,
Cymbopogon nardus (citronella), Zyzyphus joazeiro, ‘noni’, Morinda citrofolia, Magonia pubescens and
Annona squamosa and showed that the consumption of leaves impregnated with different plant extracts
increased larval mortality and significantly decreased pupal weight. The Annona squamosa treatment
had the most effective insecticide activity against FAW. However, no identification of the phytochemicals
responsible for these activities was done making the exploitation of these data difficult [33].
Methanolic extracts of leaves, bark and fruit peel of Copaifera langsdorffii resulted in low FAW egg
viability [79]. Findings of this study showed that the methanolic extracts from leaves and fruit peel
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Plants 2020, 9, 207
added to the artificial diet of 2nd instar FAW had several effects including reduced larval growth,
long development duration, lower fertility and fecundity of adults as well as augmented mortality.
The aeropylar and micropylar regions of the eggs had abnormalities. The insect feces were high in
protein as reflected by repressed trypsin activity in the in vitro test. They suggested that C. langsdorffii
presented the greatest potential for use as alternative bioinsecticide for control and management
of FAW.
The effects of aqueous extracts of Talisia esculenta and Sapindus saponaria on the FAW at 8 and
14 days of development led to increased larval mortality at 63.15% and 26.71% for S. saponaria and
T. esculenta, respectively [61]. The extract of T. saponaria was the most promising for the control of FAW.
This might be because their seeds high in fat content, yielding a similarly fatty extract with adjuvant
capacity thus facilitating fixing and distribution of the extract on the leaves of maize hence increasing
the insecticide action. There remains, however, the need to determine the insecticidal compounds in
the plants on whose basis new natural insecticidal products could be produced or improvements to
the extraction could be made.
A study under laboratory conditions and conducted on the biological activity of boldus (Peumus
boldus Molina) water extract against FAW and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) [76]
showed that FAW was the most susceptible with 75% mortality at seven days when exposed to 8%
w/w of P. boldus extract and had an LC50 value of 2.31 mL kg−1 . Again, no chemistry was undertaken
making the usefulness of this data questionable.
The bioactivity of Ipomoea murucoides methanolic extracts and fractions on FAW were evaluated
by incorporating the extracts into a meridic diet at concentration of 2 mg mL−1 . These were then fed to
FAW larvae (1st instars) [48]. After seven days, crude leaf extracts caused up to 46.16% mortality (leaf
extract LC50 = 2.692 mg mL−1 ). Other effects were reduced larval weight, increased pupation time and
in the time to attain adulthood. No influence was noted for the number of eggs. Despite the fact that
the partly purified fraction caused no toxicity to FAW, the greatest effect was on reduced larval weight,
augmented pupation time and time to attain adulthood with an influence on number of eggs.
An earlier study on a methanolic extracts of I. murucoides calli [49] reported that it induced a
higher (95%) neonate larvae mortality than was reported by [48]. This difference was explained by the
fact that in [48], the leaf extracts contained a large amount of chlorophyll (that is lacking in calli) which
masked the compounds and therefore inhibited their activity.
A study reported in [37] investigated how tocotrienols and hydroquinones from Roldana
barba-johanis affected the growth of insects. The major compounds obtained from the aerial parts
methanol extract were sargachromenol, sargahydroquinoic acid and sargaquinoic acid. These
compounds and their associated methylated and acetylated derivatives exhibited insect growth
regulatory and insecticidal activities against the FAW. The most biologically active phytochemicals
were sargachromenol, sargahydroquinoic acid and sargaquinoic acid in the order of abundance. These
compounds and the acetylated form of this mixture resulted in negligible effects. When used at 5.0 and
20.0 ppm in diets, they caused substantial inhibitory effects on FAW larvae with insecticidal activity
ranging between 20 and 35 ppm.
Eucalyptus citriodora Hook (Myrtaceae) contained eucalyptin in methanol extract of leaves along
with naringenin, chrysin, apigenin, quercetin, and luteolin, oleanolic acid, ursolic acid, betulinic acid
and composite mixtures of flavonoids and triterpenes that were not identified [29]. These compounds
exhibited insecticidal and insect growth regulatory and antifeedant activities, against FAW and the
Yellow Mealworm (Tenebrio molitor) (Coleoptera:Tenebrionidae).
The sublethal effects of the essential oils of Foeniculum vulgare, Ocimum gratissimum and Eucalyptus
staigeriana on FAW have been reported [34]. The essential oils caused reduced larval and pupal weights,
increased larval and pupation periods, reduced oviposition period and adult survival although there
were variations in effects. The essential oil of O. gratissimum had the greatest effects across the tested
doses. These insecticidal effects could have been as a result of essential oil components like limonene,
geranial, (E)-anethole, eugenol and α-pinene in the essential oils. This provides an opportunity for
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researchers to explore other plants with these compounds with the aim of incorporating them in the
pool of plants that provide promising outcomes for managing FAW in Africa.
Methanol extracts of Yucca periculosa bark yielded 4, 4 -dihydroxystilbene, resveratrol and 3, 3 , 5,

5 -tetrahydroxy-4-methoxystilbene. These compounds showed growth regulatory effects against the
FAW. The most active compound was 3, 3 , 5, 5 -tetrahydroxy-4-methoxystilbene which was active at
3 μg g−1 in diets [35]. However, the utilization of Y. periculosa (Agavaceae) is limited due to its local use
as a source of firewood. In addition, the leaves of this plant are used for making handicrafts while the
flowers are utilized as food. At 25.0 ppm concentration, the methoxy stilbene and methanolic extract of
Y. periculosa caused 100% mortality of larvae. Most importantly, the methoxy stilbene and methanolic
extract of Y. periculosa proved to be more active than gedunin and the methanolic extract of Cedrela
salvadorensis with LC50 values of 5.4 ppm and 7.18 ppm, respectively. They also indicated that there was
a decrease in the percentage of larvae attaining pupation across treatments as compared to the control.
Survival of the pupae was reduced to 0.05 at 25 and 50 ppm for the methoxy stilbene and methanolic
extract, respectively. The percentage of adult emergence showed further impacts at the pupal stage
with resveratrol, the methoxy stilbene, methanolic extract of Y. periculosa, gedunin and methanolic
extract of C. salvadorensis with 0.0%, 27.0%, 18.0%, 13.0%, and 8.0% of emergence, respectively at 25,
10, 10, 25 and 25 ppm. The methoxy stilbene and the methanolic extract of Y. periculosa with Relative
Growth Index (RGI) values of 0.25 and 0.45 at 10 and 15 ppm gave the greatest outcome. The effects
of resveratrol, the methoxy stilbene and methanolic extract of Y. periculosa did not differ from that
of gedunin but had greater potency than the methanolic extract of C. salvadorensis [64). This finding
presents these plants as having potential for further development for use against FAW.
The aerial portions of Gutierrezia microcephala yielded four oxyflavones, which were tested for
activity against neonate larvae of FAW [38]. The flavone, a clerodane, its methyl ester, methanolic
and n-hexane extracts caused a major delay in the time taken to attain pupation and adult emergence.
Severe toxicity against FAW adults and insect growth inhibition were also reported [38].
Maytenus disticha aerial parts and Maytenus boaria seeds were evaluated to determine their
effects on the FAW [47]. Several β-dihydroagarofurans were isolated including 9-benzoyloxy-
1,2,6,8,15-pentaacetoxy-dihydro-β-agarofuran-(1) and 9-furanoxy-1,6,8-triacetoxy-dihidro- β-agarofuran
and their insecticidal activities compared to ethanol extracts from A. indica and M. azedarach [65]. There
was a 58% and 100% growth inhibition at 16 and 80 ppm, respectively. This suggested that agarofurans
and MeOH and hexane/EtOAc extracts from M. disticha and M. boaria, respectively, have potential for
use as a biopesticide against FAW.
Extracts of A. indica and M. azedarach caused significant larval deaths, slowed the growth rate of
larva and lengthened pupation time. The influence of 9-furanoxy-1,6,8-triacetoxy-dihidro-β-agarofuran
and hexane/EtOAc extract on FAW was comparable to that of limonoids such as gedunin and
cedrelone [90]. The action of these compounds was comparable to toosendanin, which is a commercially
available biopesticide, suggesting that there is potential for researchers to harness these plants and
produce products that can assist in controlling FAW.
Ricinus communis has been identified as a potentially important pesticidal plant owing to its
insecticidal properties. Some fatty acids obtained from the aqueous extracts of caster plant have shown
insecticidal and insectistatic activity against FAW. For example, linoleic acid, palmitic acid and stearic
acid show biological activity against FAW [46] while linolenic acid was reported to have insecticidal
and insectistatic activities against FAW [51].
Castor oil and vicinine which can be extracted from seeds or leaves of R. communis were active
against FAW, however, the seed extract was more potent [50]. The two test substances were associated
with the effects observed for FAW. The half maximum larvae viability concentration (LVC50 ) was
0.38 × 103 ppm for the vicinine, 0.75 × 103 ppm for methanol extract of seeds, 1.97 × 103 ppm for ethyl
acetate seed extract, 2.69 × 103 ppm for castor oil, 4.83 × 103 ppm for a methanol extract of leaves and
10.01 × 103 ppm for a hexane extract of leaves. Bioactivity in castor plants is particularly relevant to
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the African context as this plant is cosmopolitan and grows abundantly adjacent to farmland in many
parts of Africa.
Trichilia pallida leaf and branch extracts when applied at very low concentrations of ≤ 0.0008%
were shown to have no effects on eggs and larvae of FAW [63]. Although less diverse species of the
genus Trichilia also occur in Africa such as T. emetica indicating the potential for using knowledge from
American studies to inform the use of African species.
Insecticidal activity of Salvia spp. has also been reported on FAW and Spodoptera littoralis
(Lepidoptera:Nuctuidae) [59]. The extracts from Salvia keerlii and Salvia ballotiflora were shown to have
modest insecticidal action (LV50= 1527 and 1685 μg mL−1 , respectively. On the other hand, the extract
of S. ballotiflora increased the larval and pupal stages by 5.2 and 2.9 days, respectively and caused a
decrease in the pupal weight by 13.2%. Furthermore, Salvia microphylla showed insecticidal activity
against FAW (LC50 = 919 ppm) [59]. The bioactivity of the essential oil of S. ballotiflora at 1000, 600,
400, 120 and 80 μg mL−1 led to reduced viability of larva which was 0%, 5%, 10%, 10%, and 20%,
respectively [59]. They also reported extended duration of the larval stage by 30.5, 8.0, 5.5 and 5.5 days
at 600, 400, 120 and 80 μg mL−1 compared with the control. The pupation period was extended by
1.6 days at 400 μg mL−1 . Moreover, the reduction in weight of the pupae decreased by 52%, 39%, 29%
and 29% at 600, 400, 120 and 80 μg mL−1 , respectively, in relation to the control.
S. microphylla contains palmitic acid, oleic acid and Y-sitosterol which have been associated with its
activities against FAW [40]. Furthermore, they pointed out that there was a possibility to use of Senecio
salignus and Salvia microphylla extracts for controlling FAW as they produce bioactive compounds
that are antifeedants [91]. Salvia species are abundant in Africa including the South American exotic
species Salvia suaveolens thus this species may be worthy of investigation to identify similar activities.
FAW eggs died at a rate of 97.7% one day after being exposed to extracts of Lychnophora ericoides
and Trichogonia villosa [39]. Thus only 2.3% of the eggs hatched being a very low percentage to sustain
populations that can cause damage.
Citrus-derived limonoids have been implicated in reduced feeding activity in insect pests. They
include limonin, nomlin and abacunone and their semisynthetic products. Limonoids from Citrus
limon have exhibited similar effects on FAW [83]. Citrus crops are also grown widely in Africa so
further work on by-products of the peel from the fruit processing sector may provide opportunities for
bioactive plant compounds in Africa.
The biological activity of extracts from various plant parts of wild and in-vitro plants of Piper
tuberculatum on the 3rd instars of FAW in Brazil have been studied [78]. The dichloromethane (DCM):
methanol (2:1) and ethanol extracts of leaves and stems and boiling water extracts of leaves, stems
and spikes of P. tuberculatum showed no effects on FAW 3rd instars across the dosage. However, the
DCM: methanol (2:1) and ethanol extracts of mature spikes from wild and DCM: methanol (2:1) extract
of in vitro plants were reported to have exhibited potential insecticidal activity on the 3rd instars of
FAW. This result suggests that there is a potential for direct use of P. tuberculation mature spike of
EtOH extracts that would allow farmers to utilize their locally brewed alcoholic drinks as extraction
solvents. It would also mean that using in vitro techniques, the respective bioactive compounds
can be biologically synthesized in large quantities using in-vitro cell suspension cultures [92]. This
may require adequate and well-equipped laboratories most of which are out of reach for the farming
support and commercial systems in Africa. P. tuberculatum has palmitic and oleic acids which could be
responsible for the reduced viability of the larvae at 33.3% and 48.5%, respectively with a concentration
of 1600 ppm.
The main components identified in Carica papaya seed were oleic acid (45.97%), palmitic acid
(24.1%) and stearic acid (8.52%) [44]. When evaluated against FAW the viability of the larvae was
reduced to 33.3% for oleic acid, 48.5% for palmitic acid and 62.5% for stearic acid at 1600 ppm. Single
fatty acids in C. papaya possessed greater potential to kill the insect pest compared to the chloroform
extract. Amongst the three, palmitic acid was the most active.
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A high mortality of FAW was reported with extracts of Jatropha curcas, Militia ferruginea,
Phytolacca dodecandra, Scinus molle, Melia abyssinica, Nicotiana tabacum, Lantana camara,
Chenopodium ambroides, Azadirachta indica and Jatropha gossypifolia [26]. This is the first report
where these plant species were evaluated against FAW in Africa-Ethiopia. Similar activities were
reported for A. indica and N. tabacum against FAW supporting these earlier findings [23].
The neem tree, A. indica can control many pest species including FAW [24,70,93,94]. The
deleterious properties of neem oils and extracts on pests are associated with the content of limonoids
like azadirachtin which is a highly complex and effective molecule [69]. Azadirachtin, is freely
decomposable, selective, non-mutagenic causing minimal harm to mammals and the environment
and could present an excellent option for controlling FAW [67]. For example, egg laying by female
FAW was about 50% lower on the neem treated than on untreated cloth [67]. However, this substance
has limitations such as being highly costly, it cannot be synthesized chemically and has to be purified
using expensive and sophisticated methods. It can be produced from large quantities of seasonally
available seeds [94] so may not be so well suited to small holders in Africa. The main components
occur in the seeds and even for a “low-tech” processing method require considerable effort to extract
them. One additional problem with the use of neem is that the main active components including the
various azadirachtin related structures are highly UV labile so may low residual effects in the field [95].
There is no standardization and control of quality in neem-based preparations manufactured in Brazil
an indicator that it may not possible to reproduce the desired effects of the insecticide [96]. To increase
effectiveness, controlled-release preparations of insecticides by polymeric encapsulation [97,98] has
been done. Encapsulation of neem oil and extracts into films or polymeric walls shelters the active
component and permits controlled release stopping the loss of unstable compounds and increasing
their stability in the environment [95]. Although again this approach may be beyond the needs and
scope of smallholders but illustrates technologies in development to improve persistence in the field
for botanicals.
In another study neem seed cake extract was more active (LC50 = 0.13%) than leaf extract
(LC50 = 0.25%) [73]. This was because of the higher amounts of azadirachtin the most effective of the
toxic tetranortriterpenoids, because 90% of azadirachtin is more intense in the neem cake after pressing
the seeds [74]. Farmers often use Neem leaves when seed is unavailable. However, the concentration
of the active constituents is very low in leaves such that it has low and potentially no efficacy so
promoting the use of Neem leaves should be discouraged as poor efficacy may negatively influence
farmer opinions about the value of plants as alternatives to synthetics. Additionally, the bioassay
indicated a static effect on the growth of FAW caterpillars, as most of them exhibited their exuviae in
the terminal part of the body, incompletely releasing them as expressed by [69] as it limits the ability
of the insects to feed by affecting the physiological functioning of ecdysis and in cellular processes,
eventually causing insect death. This process takes some time and that is why comparatively, there is
low larval mortality and high pupal mortality [99].
Zea diploperennis was evaluated against FAW and indicated that methanol extract and residual
fiber of the plant adversely influenced the size of pupae. The aqueous extract caused 100% of larval
cumulative mortality [81].
An extract was obtained from the roots and aerial parts of M. geometrizans using methanol as
a solvent. Its components were peniocerol, macdougallin and chichipegenin and the mixtures of
peniocerol and macdougallin. They all exhibited insect growth regulatory and insect killing activities
against FAW [100].
3. Future Prospects
The plant species reviewed above provide an illustration of the extent of work undertaken to
identify new pest management options from plants. These plants have been shown to have biological
activity against FAW through various modes of action If these initial indications of activities are to be
translated to the African context then not only are the bioactivity of extracts in the laboratory required
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but also the chemistry of these activities needs to be determined and the materials tested in field
conditions using tailored approaches to extraction that are appropriate for small scale farmers.
The plants reviewed had a variety of modes of action in controlling FAW including induction
of low feeding ratio through the action of oleic acid (74.2%) and palmitic acid (7.16%) [44], repellent
effects, severe toxicity, non-preference and knockdown effects by linalool from Ocimum basilicum.
Citronella oil changes the chemical profile of FAW larvae, affecting reproductive and cell physiological
parameters causing reduced reproduction and sometimes reproductive failure. It is also associated
with changed epithelium that has cytoplasmic projections, columnar cell extrusion, pyknotic nuclei
and increased periodic acid-schiff positive granules. Citronella oil caused morphological changes
of the midgut and reduction of stored resources in the fat body, which may adversely affect insect
reproduction and survival. It has been further reported that reduced feeding after ingestion of Melia
azedarach caused starvation. This in addition to ingestion of toxic substances from M. azedarach [60].
Leaf extracts at vegetative and reproductive phase of Poinsettia (Euphorbia pulcherrima) increased
larval period, reduced the weight of larvae and pupae egg viability [54]. The methanolic extracts of
Copaifera langsdorffii leaves, bark of fruits and fruit peels resulted in low egg viability, reduction in
larval growth, prolonged period of development, increased mortality, lowered fertility and fecundity
of adults, abnormalities in the aeropylar and micropylar regions, increased excretion of protein in the
insect feces and invitation of trypsin activity [79].
There is adequate evidence as indicated by the research findings presented in this review, that
there are numerous opportunities for the use of botanical extracts in the management of FAW. However,
exploitation of these opportunities is limited because the potential for use may face challenges attributed
to the following:
1. Despite there being numerous plant products many are unstable upon application because they
are UV labile. This means they may need more frequent application incurring greater costs in
time. However, as they are non-persistent, they are potentially less damaging to the environment
particularly non-target insects [17,18,99,101].
2. African smallholder farmers are not economically endowed to buy the botanical pesticides as
has been the case for other farm inputs [102,103]. This therefore means that farmers will be
encouraged to self-harvest these plant materials [104,105] and use them as crudely produced
products as reported earlier [21,22].
3. There are different modes of action, which are determined by the stage of growth of both maize
and the FAW raising the issue of exposure period, effectiveness, mode of application and method
of extraction. However different modes of action could help to reduce the build-up of resistance
in the pest where used in combination.
4. The opportunities maybe limited in scope where the products are not standardized for
reproducibility and scale-up and this will require uniformity of the chemistry for the plant
material and they likely need propagation [87,106]. More so, surprisingly few have been
evaluated under field conditions [26]. This is a major oversight of the work as it means there
is little evidence that any of the biological activities translate to a real-world setting. Field
evaluations provide options to engage with farmers and determine effects on yield and determine
non-target effects as undertaken recently in the African context [17,85,101].
5. Some of the plant materials tested may not be available for use by the farmers, for example the
use of citrus seeds at farm level may not be attainable because it may not be feasible in terms of
availability of the seeds. However, there may be good opportunities for propagation, and this
would likely overcome some of the challenges of chemical variation across plant populations
and provide consistency, which may otherwise be lacking when plants are harvested from the
wild [106].
6. All the studies conducted failed to include economic viability for the tested plant extracts. This
is closely linked to sustainable availability of the plant materials which is key driver for farmer
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adoption. Most of the plant materials used in these studies were wild harvested and this may not
be self-sustaining unless efforts are made to commercialize the promising plant products or at
least determine the economic viability of their use compared to alternatives including the use of
synthetics [18,85].
4. Recommended Research Areas for Further Studies

1. Evaluate different extraction methods with the aim of documenting the most appropriate for
adoption by small-scale maize farmers.
2. Investigate modes of action of different products based on part used, pure compounds and
mixtures across the different growth stages of maize plant and FAW.
3. Conduct field evaluations of these plants and potentially determine any benefits of combining
materials that could deliver different mechanisms of activity to address issues of insect resistance.
4. Investigate standardization to increase the scope of reproducibility and adoption especially
through propagation.
5. Conduct research on approaches for upscaling, commercialization and sustainability of the
botanical extracts and products
6. Testing activity of pure compounds from extracts to determine which components are active
allowing the evaluation of variability across materials and improving methods for optimizing
extraction and [87,107,108].
Author Contributions: Original draft preparation, N.B.R.; review and editing, P.C.S. All authors have read and
agreed to the published version of the manuscript.
Funding: The contribution from PCS was funded by a grant from the McKnight foundation to PCS Grant
No: 17-070.
Acknowledgments: Authors acknowledge Peter Opala who assisted in editing the manuscript for his
valuable input.
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plants
Review
The Phytochemical Composition of Melia volkensii
and Its Potential for Insect Pest Management
Victor Jaoko 1,2,3, *, Clauvis Nji Tizi Taning 1 , Simon Backx 2 , Jackson Mulatya 3 ,
Jan Van den Abeele 4 , Titus Magomere 5 , Florence Olubayo 5 , Sven Mangelinckx 2, *,
Stefaan P.O. Werbrouck 1 and Guy Smagghe 1
1 Department of Plants and Crops, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium;
tiziclauvis.taningnji@ugent.be (C.N.T.T.); stefaan.werbrouck@ugent.be (S.P.O.W.);
guy.smagghe@ugent.be (G.S.)
2 SynBioC, Department of Green Chemistry and Technology, Ghent University, Coupure Links 653,
B-9000 Ghent, Belgium; simon.backx@ugent.be
3 Kenya Forestry Research Institute, P.O Box 20412-00200 Nairobi, Kenya; jmulatya@kefri.org
4 Better Globe Forestry, P.O Box 823-00606 Nairobi, Kenya; jan@betterglobeforestry.com
5 Department of Plant Science and Crop Protection, University of Nairobi, P.O Box 30197-0010 Nairobi, Kenya;
magomere.titus@ku.ac.ke (T.M.); olubayo@uonbi.ac.ke (F.O.)
* Correspondence: victor.jaoko@ugent.be (V.J.); sven.mangelinckx@ugent.be (S.M.);
Tel.: +254-722157414 (V.J.); +32-9-264-59-51 (S.M.)
Abstract: Due to potential health and environmental risks of synthetic pesticides, coupled with their
non-selectivity and pest resistance, there has been increasing demand for safer and biodegradable
alternatives for insect pest management. Botanical pesticides have emerged as a promising alternative
due to their non-persistence, high selectivity, and low mammalian toxicity. Six Meliaceae plant species,
Azadirachta indica, Azadirachta excelsa, Azadirachta siamens, Melia azedarach, Melia toosendan, and Melia
volkensii, have been subject to botanical pesticide evaluation. This review focuses on Melia volkensii,
which has not been intensively studied. M. volkensii, a dryland tree species native to East Africa,
has shown activity towards a broad range of insect orders, including dipterans, lepidopterans and
coleopterans. Its extracts have been reported to have growth inhibiting and antifeedant properties
against Schistocerca gregaria, Trichoplusia ni, Pseudaletia unipuncta, Epilachna varivestis, Nezara viridula,
several Spodoptera species and other insect pests. Mortality in mosquitoes has also been reported.
Several limonoids with a wide range of biological activities have been isolated from the plant, including
volkensin, salannin, toosendanin, trichilin-class limonoids, volkendousin, kulactone among others.
This paper presents a concise review of published information on the phytochemical composition
and potential of M. volkensii for application in insect pest management.
Keywords: Meliaceae; Melia volkensii; botanical pesticide; limonoid; insect pest; antifeedant;
growth inhibitor
1. Introduction
The continuous and indiscriminate use of synthetic pesticides in crop protection has led to an
increase in pest resistance, health and environmental concerns [1]. This has led to a renewed interest
in natural products as alternative sources for insect pest control [1]. One of the most promising
options is the use of secondary metabolites produced by plants, many of which are toxic to a wide
spectrum of insect pests [2]. Plant extracts can offer a solution to insect pest control because they are
environmentally friendly, easily biodegradable, and are target-specific [3].
The Meliaceae plant family has been reported to produce a wide range of compounds,
including flavonoids, chromones, coumarins, benzofurans, mono-, sesqui-, di-, and triterpenoids,

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but tetranortriterpenoids with a β-substituted furanyl ring at C17α are the best known for the
production of limonoids [4]. Limonoids are known for a range of biological activities, including
insect antifeedant and growth-regulating properties and antibacterial properties [4]. Alkaloids are
rarely isolated from Meliaceae [4]. Reviews on the Meliaceae plant family have been reported in the
literature. The use of Meliaceae plant extracts as potential mosquitocides have been reviewed, and
Azadirachta indica A. Juss (Indian neem tree) is reported as a potential plant for the control of vector
mosquitoes [5]. Reviews on the chemical constituents of the genus Melia reported the isolation of
terpenoids, steroids, alkaloids, flavonoids, anthraquinones with a wide range of biological activities
including antiviral, pesticidal, inhibition of iNOS, antitumor, antibacterial and antifungal activities [6,7].
A phytopharmacological review of Melia azedarach Linnaeus (chinaberry) has been reported outlining
its use in folk medicine having antifertility, antiviral, cytotoxic, antibacterial, immunomodulatory,
repellent, antifeedant, antilithic and anthelmintic activity from various parts of the plant [8,9]. A review
on A. indica has reported its use in agriculture for application as manure, fertilizer, soil conditioner,
fumigant, and as botanical pesticide [10]. Melia volkensii (Gurke) has also been identified as one of the
pesticidal plants in Africa [11]. Another review has explored the phytochemical and antimicrobial
activities of the Meliaceae family [12]. Detailed information on commercially available neem products
developed for agricultural pest control has also been reviewed [13].
Several plant species of the Meliaceae have shown promising bioactivity against a variety of
insects [3]. Their insect growth regulatory and antifeedant properties against many insect pests have
made them emerge as a potent source of insect control products [14]. Six species have been subjected
to botanical pesticide evaluation; these include A. indica (Indian neem tree), Azadirachta excelsa Jack
(Philippine neem tree), Azadirachta siamens Valeton (Siamese neem tree), M. azedarach (chinaberry),
Melia toosendan Siebold and Zucc., and M. volkensii [13]. However, research has concentrated mostly on
A. indica (neem tree) and M. azedarach (chinaberry) [15]. Azadirachtin, a commercial biopesticide, and
other limonoids isolated from A. indica, have been effective growth regulators and feeding deterrents
for a wide range of insect species [16]. Azadirachtin targets the corpus cardiacum in insects, which in
turn affects neuroendocrine activity and turnover of neurosecretion [17]. Extracts from M. azedarach
have also shown antifeedant activity against the juvenile and adult Xanthogaleruca luteola Muller (elm
leaf beetles) and mortality against its larvae [16]. Fruit extracts from M. azedarach are also effective
against Napomyza lateralis Fallen (agromyzid leafminers) and Trialeurodes vaporariorum Westwood
(whiteflies) [16]. Toosendanin, a limonoid constituent of M. azedarach which has been commercialized
in China, is an effective growth inhibitor against Ostrinia nubilalis Hübner (European corn borer),
effective repellent against Pieris brassicae Linnaeus (cabbage moth) and an oviposition deterrent against
Trichoplusia ni Hübner (cabbage looper) [16]. Toosendanin is reported to be mainly active against
lepidopteran pests and is less active than azadirachtin [18].
M. volkensii, a dryland tree species native to East Africa has, however, not been intensively
studied [16]. It is a tall tree (15–25 m), shown in Figure 1, which grows in semi-arid areas of Kenya,
Tanzania, Ethiopia, and Somalia at altitudes of between 350 to 1700 m above sea level [19]. The tree,
like other meliaceous plants, is fast growing and produces fruits after 4–5 years [19]. It remains
green for most of the year and is prized by farmers for its termite-resistant timber. It is intercropped
with food crops, used for shade, firewood, and livestock fodder [19]. Several chemical compounds
occur only in M. volkensii. These include: 1-O-cinnamoyltrichilin, meliavolkinin, 1,3-diacetylvilasinin,
meliavolkin, volkensin, volkensinin, 12β- and 6β-hydroxykulactone, meliavolkenin, meliavolin,
meliavolen, melianinone, meliavolkensin A and B, melianin C, (E)- and (Z)-volkendousin, meliavosin,
2-9-epoxymeliavosin [6]. M. volkensii seed kernel extracts have more insect growth inhibitory and acute
lethal toxicity than azadirachtin-containing fractions from neem seed kernel extracts [20]. It has been
reported that when M. volkensii dried fruit powder and residual fruit cake obtained after extraction
with ethanol are used as goat feed, their growth and performance are not negatively affected, indicating
that both fruit powder and its cake could be used as safe ruminant feed supplement [21]. Its use as a
fodder crop underscores its safety in mammals [20], and traditionally, it is used for the treatment of
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diarrhea, pain, skin rashes, and eczema [22]. Aqueous extracts of M. volkensii have also traditionally
been used to control ticks and fleas in goats [19]. M. volkensii offers a key indigenous tree species that
can be used to mitigate against desertification in arid and semi-arid lands [23], while also offering a
high economic potential for the rural community in these regions [24]. This paper presents a concise
review of published information on the phytochemical composition and potential application of M.
volkensii in insect pest management.
Figure 1. Melia volkensii and its various parts: (a) 10-year old M. volkensii plantation, (b) leaves, (c) seeds,
(d) fruits and (e) nuts [23].
2. Biological Activity of Melia volkensii Extracts Against Insects

Crude fruit extracts from M. volkensii have been reported to pose activity towards a broad
range of insect orders including Diptera, Lepidoptera, Coleoptera among others [19] as shown in
Table A1 (Appendix A). The methanolic fruit extracts were first reported to have antifeedant effects
against Schistocerca gregaria Forsk. (desert locusts) [25]. Repellency effect, decreased mobility, retarded
development and reduced fecundity were observed against S. gregaria when seed extract was applied
to their preferred host plants mainly Schouwia thebaica Webb, Fagonia olivieri DC (fagonbush plant) and
Hyoscyamus muticus Linnaeus (Egyptian henbane) in a field trial experiment [26]. Although the mode
of action of the extracts is still unknown, it is postulated that the active compounds in M. volkensii
extracts could affect hormone levels in S. gregaria larvae [27]. In fifth-instar nymphs of S. gregaria,
80% mortality was recorded 48 hours after injection with crude ethanolic and methanolic extracts
at a concentration of 30 μg/g of the insect [19]. When sprayed on third- to fifth-instar S. gregaria,
M. volkensii and neem oil have been reported to cause mortality of up to 91% and 92%, respectively,
after 14 days in a comparative study [26]. In contrast to synthetic pesticides, these botanicals do not
have a knock-down effect, but their slow response is similar to inhibitors of chitin synthesis [26].
Antifeedant and larval growth inhibitory effects of fruit extracts have been observed in Trichoplusia
ni Hübner (cabbage looper) and Pseudaletia unipuncta Haworth (true armyworm) [25,28]. Crude seed
extracts are also an effective growth inhibitor against T. ni (dietary EC50 = 7.6 ppm) and feeding
deterrent (DC50 = 0.9 μg/cm2 ) [29]. Prolonged exposure to M. volkensii extracts has been observed to
lead to a decrease in antifeedant response when tested against T. ni implying that the insect could
develop tolerance to the extracts [30]. However, when tested against Plutella xylostella Linnaeus
(diamondback moth) and P. unipuncta, there was no significant decrease in feeding deterrent response
to the extracts following continuous exposure [31]. It has been postulated that triterpenoids from
seed kernels of M. volkensii are responsible for the insecticidal activity in T. ni [11]. Comparative
efficacy has been observed with M. volkensii extracts, other Meliaceae plant extracts (A. indica, A. excelsa,
M. azedarach, and Trichilia americana Sessé & Mocino) and commercial botanical insecticides (ryania,
pyrethrum, rotenone and essential oils of rosemary and clove leaf) when tested against T. ni and
P. unipuncta [32].
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M. volkensii fruit extracts when tested at concentrations ranging from 1 to 50 μg/μL showed
feeding deterrence, growth disruption and mortality against Nezara viridula Linnaeus (stink bug),
a polyphagous pest which attacks a variety of crops, including nuts, corn, cotton, grains and
tomatoes [16]. The disruption of the molting process led to eventual mortality in N. viridula [16].
Furthermore, deformities and malfunctions like shortened or missing antennae, legs failing to detach
from the exuvium, absent or shortened hemelytra, notching, and lack of symmetry have been observed
in N. viridula when exposed to fruit extracts, with 10 μg/μL causing malformation in up to 85.70%
of surviving adults [16]. A delay of the imaginal molt was observed in immature Coranus arenaceus
Walker even though there were no deformities in resultant adults after topical application of the M.
volkensii extracts at 1, 5, and 10 μg/μL [16].
When applied to cabbage leaf disks in a choice bioassay, M. volkensii fruit extract showed potent
antifeedant properties against Epilachna varivestis Mulsant (Mexican bean beetle) [16]. Growth inhibition
has also been observed in P. unipuncta (dietary EC50 = 12.5 ppm) with refined seed extracts to the leaf
discs in a choice bioassay [29]. The seed extracts also showed feeding deterrent effects on third-instar
larvae of P. unipuncta and P. xylostella, and adults of E. varivestis (DC50 = 10.5, 20.7 and 2.3 μg/cm2 ,
respectively) [29]. In fact, M. volkensii seed extracts have been recorded to have stronger antifeedant
activity compared to pure allelochemicals: digitoxin, cymarin, xanthotoxin, toosendanin, thymol and
trans-anethole against P. unipuncta, P. xylostella and E. varivestis [29]. When applied to Spodoptera litura
Fabricius, neem, rotenone, M. volkensii extract, toosendanin, Annona squamosal L. extract and pyrethrum
at 1% concentration recorded larval growth (% relative to control) of 4.1, 97.5, 26.2, 48.3, 61.4, and
56.6%, respectively after 96 h in a comparative study [1].
Dried M. volkensii fruit extracts have shown growth-inhibiting activity against Aedes aegypti
Linnaeus (yellow fever mosquito) larvae at 2 μg/mL in water, whilst recording high mortality during
the molting and melanization process with LC50 of 50 μg/mL in 48 h [13]. At a high dose (100 μg/mL),
the extracts caused acute toxicity, while at a low dose, the lethal effect took a long time, indicating the
presence of compounds with an acute toxic effect at a high concentration and a growth-inhibiting effect
at a low concentration [20]. Growth inhibiting and disrupting effects in A. aegypti could be a result of
synergistic effects of a plethora of limonoid compounds or a single active compound exerting these
effects [20].
A column chromatography-purified fraction of M. volkensii fruit kernel extract showed growth-inhibiting
activity against Anopheles arabiensis Giles with an LC50 of 5.4 μg/mL in 48 h [13]. Mortality
(LC50 of 34.72 μg/mL in 48 h) and oviposition deterrence was observed in second-instar larvae
of Culex quinquefasciatus Say (Southern house mosquito) when treated with refined methanolic fruit
extracts [33]. The granular formulation of M. volkensii fruit acetone extract showed S- and U-shaped
postures and frequent stretching in C. quinquefasciatus; such postures and stretching are a characteristic
of mosquito larvae reared in M. volkensii fruit extract [34]. The test granules also caused 86% mortality in
third- and fourth-instar larvae of C. quinquefasciatus within 36 h [34]. Acetone extracts from M. volkensii
seeds have recorded growth inhibitory effects and equal toxicity (LD50 of 30 μg/mL) for larvae and
pupae of C. pipiens f. molestus Forskål (London underground mosquito) [17]. M. azedarach seed extracts
recorded lower toxicity (LD50 of 40 μg/mL) while pure azadirachtin A recorded higher toxicity (LD50
of 1–5 μg/mL) against C. pipiens when compared with M. volkensii extracts [17]. The water solubility of
the acetone seed extract from M. volkensii may indicate the presence of saponins as toxic principles
thus making it an interesting candidate for application against aquatic insects such as mosquitoes and
other vectors of diseases [17].
3. Phytochemistry and Insect Bioactivity of Melia volkensii

Insect antifeedants have been found in major classes of secondary metabolites—alkaloids, phenolics,
and terpenoids [35]. However, it is in the terpenoids that the greatest number and diversity of
antifeedants, and the most potent, have been found. Most well-documented antifeedants are
triterpenoids [35]. Effective insect antifeedants have been isolated from various parts of M. volkensii,
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as shown in Figure 2 and Table A2 (Appendix B), although azadirachtin, the major ingredient in
neem seeds, does not occur in M. volkensii. This indicates that insect control bioactivity is, therefore,
based on other compounds than azadirachtin [25]. It is postulated that the major active compound in
M. volkensii fruit is more lipophilic than azadirachtin [20]. Botanical antifeedants are easily degraded
after application thereby causing little environmental impact [36].
O OH O O O O
O O
O
O O O O O
O O O O
O OH O O O O
O O O
volkensin (1) salannin (2) 2',3'-dihydrosalannin (3)
O
O
O O
O
O O
O
O
O O OH
O
O
O
1-detigloyl-1-isobutylsalannin (4) 1D,3D-diacetylvilasinin (5)
Figure 2. Chemical structures of compounds isolated from Melia volkensii with antifeedant and
growth-inhibition activity against insects.
The insect antifeedants volkensin (1) and salannin (2) have been isolated from seed extracts of
M. volkensii [37]. Additionally, volkensin (1) and salannin (2) were isolated from the whole fruits of
M. volkensii [37]. Volkensin (1) has shown antifeedant activity against Spodoptera frugiperda Smith (fall
armyworms) larvae with an ED50 of 3.5 μg/cm2 [19]. Salannin (2) has also shown antifeedant activity
against insect pests such as Acalymma vittata Fabricius (striped cucumber beetle), Musca domestica
Linnaeus (housefly), Epilachna varivestis Mulsant (Mexican bean beetle), Heliothis virescens Fabricius
(tobacco budworm), S. frugiperda and Spodoptera littoralis Boisduval (cotton leafworm) [38]. Salannin
(2) has also been reported to cause feeding suppression against larvae of Earias insulana Boisduval
(Egyptian stemborer), weight reduction (59%–89%) in Cnaphalocrocis medinalis Guenee (rice leafroller)
and reduction in activities of acid phosphatases (ACP), alkaline phosphatases (ALP) and adenosine
triphosphatases (ATPase), implying that gut enzyme activities were affected. 2’,3’-Dihydrosalannin
(3), 1-detigloyl-1-isobutylsalannin (4) and 1α,3α-diacetylvilasinin (5) have also been isolated from the
plant [7].
M. volkensii seed extracts, extracted in cold water, have been reported to contain unsaturated
fatty acids (oleic acid (6), linoleic acid (7) and gadoleic acid (8)) and saturated fatty acids (palmitic
acid (9), stearic acid (10) and arachidic acid (11)) as shown in Figure 3 [39]. Fatty acids with at least
18 carbon atoms have been found to synergistically enhance insecticidal activity of insecticides [40].
Oleic acid (6), linoleic acid (7), linolenic acid, and ricinoleic acid have enhanced insecticidal activity of
organophosphates and carbamates when applied against sucking insects and defoliating insects [40].
Other chemical compounds that have been isolated from various parts of M. volkensii are shown
in Figure 4. Toosendanin (12), which has been isolated from the root bark of M. volkensii [22], has been
reported to be an effective growth inhibitor against O. nubilalis, an effective repellent against P. brassicae
and an oviposition deterrent against T. ni [16]. 1-Cinnamoyltrichilinin (13) has shown antifeedant
activity towards S. littoralis having minimum antifeedant concentration (MAC) value of 1000 mg/L
and a significant antibacterial activity against Porphyromonas gingivalis ATCC 33277 with minimum
inhibitory concentration (MIC) value of 15.6 μg/mL [7]. Nimbolin B (14) has been reported to have
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antifeedant activity against several Spodoptera species (S. exigua, S. eridania and S. littoralis) [7]. There
was a clear-cut structure-activity relationship when trichilin-class limonoids (1-cinnamoyltrichilinin 13,
1-acetyltrichilinin 15, 1-tigloyltrichilinin 16) were tested against Spodoptera eridania Stoll (Southern
armyworm) where the 12α-OH function was the most potent, followed by 12β-OH, 12-desoxy,
and 12α-acetoxy groups in order of decreasing potency [7]. The 12-OH functionality could be
necessary for maximum bioactivity in trichilin-class limonoids (13, 15, 16) [7]. 2,19-oxymeliavosin 17,
which has weak activity with marginally significant selectivity for breast cancer cell line (MCF-7),
has also been isolated from the root bark of M. volkensii [41]. Ohchinin-3-acetate (18), isolated from
methanolic extract of M. volkensii fruits [42], and meliantriol (19), both insect antifeedants have also been
reported [15]. Meliantriol has exhibited moderate cytotoxicity against human epidermoid carcinoma
of the nasopharynx (KB), multidrug-resistant (KB-C2), and breast cancer cell line (MCF-7) [43].
O O
OH OH
oleic acid (6) linoleic acid (7)
O O
OH OH
gadoleic acid (8) palmitic acid (9)
O O
OH OH
stearic acid (10) arachidic acid (11)
Figure 3. Chemical structures of saturated and unsaturated fatty acids isolated from Melia volkensii.
O O O O
O OH
O O O O
O
O
OH O
O O
O O
O O O O
O OH O OH O
O
O
OH
toosendanin (12) 1-cinnamoyltrichilinin (13) nimbolin B (14)
O O O O
O O O O
O O
O O
O O HO
O OH O OH HO
O O
2,19-oxymeliavosin (17)
1-acetyltrichilinin (15) 1-tigloyltrichilinin (16)
H OH
O
O O O HO
H
O O OH
H
O
O O H
O HO
H
ohchinin-3-acetate (18) meliantriol (19)
Figure 4. Further chemical structures of compounds isolated from Melia volkensii with antifeedant and
growth-inhibition activity against insects.
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4. Further Phytochemical Composition and Biological Activity of Melia volkensii

Other compounds have also been isolated from M. volkensii with different biological activities.
These include volkensinin, as isolated from ethanolic extracts of M. volkensii root bark [44], which showed
weak bioactivity in the brine shrimp lethality test BST (LC50 = 57 μg/mL) and weak cytotoxicity against
six human tumor cell lines with ED50 values of 27.90, 28.35, 33.56, 29.55, 8.43, and 28.51 μg/mL in A-498
(human kidney carcinoma), PC-3 (prostate adenocarcinoma), PACA-2 (pancreatic carcinoma), A-549
(human lung carcinoma), MCF-7 (human breast carcinoma), and HT-29 (human colon adenocarcinoma),
respectively [44]. Toosendanin has activity against Escherichia coli Migula and Aspergillus niger Tiegh.
with respective minimum inhibitory concentration (MIC) values of 12.5 and 6.25 μg/mL [22]. Melianin
B, isolated from the root bark of M. volkensii, showed cytotoxicity against six human solid tumor
cell lines: A-549, MCF-7, HT-29, A-498, PACA-2, and PC-3 [45]. Bioactivity-guided fractionation of
M. volkensii root bark led to the isolation of meliavolkenin which showed moderate cytotoxicity against
three human tumor cell lines with a respective ED50 value of 10.33 μg/mL, 4.30 μg/mL, and 0.67 μg/mL
in A-549, MCF-7, and HT-29 cells [46]. The bioactive apotirucallane triterpenes meliavolkensin A
and meliavolkensin B, both isolated from the root bark of M. volkensii [47], have shown cytotoxicity
against human colon tumor cell lines H-29 (human colon adeno-carcinoma) with ED50 values of
0.49 μg/mL and 0.25 μg/mL, respectively [47]. (E)-volkendousin, isolated from M. volkensii root
bark, also showed activity against six human tumor cell lines (A-549, MCF-7, HT-29, A-498, PACA-2
and PC-3) [48]. Meliavolin, marginally cytotoxic against human tumor cell lines with an ED50 of
11.25 μg/mL, 0.57 μg/mL and 6.65 μg/mL in A-549, MCF-7 and HT-29 cells, respectively [49], has been
isolated from M. volkensii root bark following activity-directed fractionation with brine shrimp test [49].
Kulactone was isolated from root bark of M. volkensii and exhibited significant activity against E. coli and
A. niger with a respective minimum inhibitory concentration (MIC) value of 12.5 and 6.25 μg/mL [22].
Bioactivity-guided antimycobacterial investigations against Mycobacterium tuberculosis Zopf resulted in
the isolation of 12β-hydroxykulactone, 6β-hydroxykulactone and kulonate from M. volkensii seeds with
MIC values of 16 μg/mL, 4 μg/mL, and 16 μg/mL, respectively [50]. Meliavolkin has shown anticancer
activity against three human tumor cell lines: A-549 (ED50 = 0.57 μg/mL), MCF-7 (ED50 = 0.26 μg/mL),
and HT-29 (ED50 = 0.12 μg/mL) [7]. Other limonoids isolated from M. volkensii include 3-episapelin,
meliavolen, melianinone [4], and nimbolin B [51] and all have shown selectivity for the colon cell line
HT-29 [51]. Other compounds, which have been isolated from M. volkensii include scopoletin [22],
melianin C and meliavolkinin [7], methyl kulonate and 2,19-epoxymeliavosin [6], nimbolidins C-E [12].
However, their activity against insects has not been reported in literature.
5. Conclusions
Extracts and pure compounds isolated from M. volkensii have proved to be effective insect
antifeedants and growth inhibitors. Extensive research has been done on mosquito control using
M. volkensii; however, more research needs to be done on insect pests of agricultural importance.
M. volkensii has no reported adverse effect on the environment or mammals, making it a potential
botanical pesticide for the biosafe application in integrated pest management. The availability of
renewable resources from the tree, such as fruits, stem bark, and leaves makes this plant a potential
candidate for insect control with minimal interference on the plant. In this regard, M. volkensii could be
further exploited as a source of natural insecticide.
Author Contributions: Conceptualization—G.S., S.P.O.W., J.M., T.M., F.O. and J.V.d.A.; investigation—V.J., S.B.,
C.N.T.T., G.S., S.M., S.P.O.W., F.O.; resources—S.P.O.W. and G.S.; writing—original draft preparation—V.J.;
writing—review and editing—G.S., S.P.O.W., S.M., C.N.T.T., S.B., J.M., T.M., F.O. and J.V.d.A.; supervision—G.S.,
S.M., S.P.O.W., F.O., C.N.T.T.; project administration—S.P.O.W., F.O., T.M.; funding acquisition—G.S., S.P.O.W.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by VLIR-UOS. Grant number KE2018TEA465A103.
Acknowledgments: The authors thank VLIR-UOS for the financial support.
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Appendix A
Table A1. Melia volkensii as a botanical pesticide for insect pest control.
Target Insect * Order Biological Activity Plant Part Used Reference

Desert locust, Antifeedant, repellency,
Orthoptera Fruit [19,25,26]
Schistocerca gregaria growth inhibition, mortality
Antifeedant, growth
Cabbage looper, Trichoplusia ni Lepidoptera Fruit, seed [25,28–30]
inhibition, mortality
True armyworm,
Lepidoptera Antifeedant, growth inhibition Fruit, seed [11,25,28,29,31]
Pseudaletia unipuncta
Diamondback moth,
Lepidoptera Antifeedant Fruits [29,31]
Plutella xylostella
Antifeedant, growth
Stink bug, Nezara viridula Hemiptera Fruit [16]
disruption, mortality
Coranus arenaceus Hemiptera Growth inhibition Fruit [16]
Mexican bean beetle,
Coleoptera Antifeedant, growth inhibition Seed [16,29]
Epilachna varivestis
Yellow fever mosquito,
Diptera Growth inhibition, mortality Fruit [13,20]
Aedes aegypti
Anopheles arabiensis Diptera Growth inhibition Fruit kernel [13]
Southern house mosquito, Oviposition deterrence,
Diptera Fruit [13,33,34]
Culex quinquefasciatus mortality
London underground
mosquito, Culex pipiens Diptera Growth inhibition, mortality Seed [17]
molestus
* Non exhaustive list of potential target insect pests.
Appendix B
Table A2. Phytochemical investigation of Melia volkensii.
Plant Part
Compound * Biological Activity Reference
Isolated From
Antifeedant against fall armyworms,
Volkensin Seed, fruit [19,37]
Spodoptera frugiperda
Antifeedant and weight reduction against
Acalymma vittata, Musca domestica, Epilachna
Salannin Seed, fruit varivestis, Heliothis virescens, Spodoptera [7,37,38]
frugiperda, Earias insulana, Cnaphalocrocis
medinalis and Spodoptera littoralis
Growth inhibitor and oviposition deterrent
Toosendanin Root bark against Ostrinia nubilalis, Pieris brassicae, [16,22]
Trichoplusia ni
Meliantriol Not reported Antifeedant [15]
Unsaturated fatty acids (oleic acid,
linoleic acid and gadoleic acid); Synergistic enhancement of
Seed [39,40]
saturated fatty acids (palmitic acid, insecticidal activity
stearic acid and arachidic acid)
1-cinnamoyltrichilinin Not reported Antifeedant against Spodoptera littoralis [7]
1-tigloyltrichilinin Not reported Antifeedant against Spodoptera eridania [7]
1-acetyltrichilinin Not reported Antifeedant against Spodoptera eridania [7]
Antifeedant against Spodoptera species.
Nimbolin B Not reported [7,51]
(exigua, eridania and littoralis)
Ohchinin-3-acetate Fruit Antifeedant [42]
* Non exhaustive list of compounds present in M. volkensii.
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Review
Botanicals Against Tetranychus urticae Koch Under
Laboratory Conditions: A Survey of Alternatives for
Controlling Pest Mites
Ricardo A. Rincón 1,2 , Daniel Rodríguez 1, * and Ericsson Coy-Barrera 2, *
1 Biological Control Laboratory, Universidad Militar Nueva Granada, Cajicá 250247, Colombia
2 Bioorganic Chemistry Laboratory, Universidad Militar Nueva Granada, Cajicá 250247, Colombia
* Correspondence: daniel.rodriguez@unimilitar.edu.co (D.R.); ericsson.coy@unimilitar.edu.co (E.C.-B.);
Tel.: +57-6500000 (ext. 3269) (D.R.); +57-6500000 (ext. 3270) (E.C.-B.)
Received: 1 July 2019; Accepted: 3 August 2019; Published: 7 August 2019
Abstract: Tetranychus urticae Koch is a phytophagous mite capable of altering the physiological
processes of plants, causing damages estimated at USD$ 4500 per hectare, corresponding to
approximately 30% of the total cost of pesticides used in some important crops. Several tools
are used in the management of this pest, with chemical control being the most frequently exploited.
Nevertheless, the use of chemically synthesized acaricides brings a number of disadvantages, such as
the development of resistance by the pest, hormolygosis, incompatibility with natural predators,
phytotoxicity, environmental pollution, and risks to human health. In that sense, the continuous
search for botanical pesticides arises as a complementary alternative in the control of T. urticae Koch.
Although a lot of information is unknown about its mechanisms of action and composition, there are
multiple experiments in lab conditions that have been performed to determine the toxic effects of
botanicals on this mite. Among the most studied botanical families for this purpose are plants
from the Lamiaceae, the Asteraceae, the Myrtaceae, and the Apiaceae taxons. These are particularly
abundant and exhibit several results at different levels; therefore, many of them can be considered as
promising elements to be included into integrated pest management for controlling T. urticae.
Keywords: Tetranychus urticae; resistance; botanical pesticides; acaricide; integrated pest management
1. Introduction
One of the most important pests in commercial crops worldwide is the polyphagous, two-spotted
spider mite, Tetranychus urticae Koch. This mite is able to alter the physiological processes of plants,
reducing the area of photosynthetic activity and causing the abscission of leaves in severe infestations [1].
The cost of damages caused by this pest in crops such as beans, citrus, cotton, avocado, apples, pears,
plums, and many other horticultural and ornamental crops are estimated at over USD$ 4500 per
hectare. Such costs correspond to 30% of the total cost of pesticides in crops of ornamental flowers.
This constitutes a spending of almost 62% of the global market value on T. urticae Koch control based
on data of 2008 [2]. The main tools used to control this pest are chemically synthesized acaricides.
However, this mite is known to generate a resistance to these chemicals in a short period of time [3].
In addition, when the T. urticae Koch is exposed to sublethal pesticide levels, this mite has the ability to
increase its reproduction rate, thus its populations increase in a shorter time [4]. Furthermore, many of
the active ingredients in pesticide formulations are incompatible with the T. urticae Koch’s natural
predators; consequently, when they are applied to crops, they suppress populations of predators that
can contribute to the decrease of phytophagous mites [5].
Courtesy of the above-mentioned issues—together with problems related to environmental
contamination, the risk for human and animal health, and phytotoxicity—it is necessary to complement

Plants 2019, 8, 272
the control of T. urticae Koch with tools other than chemically-synthesized acaricides, such as biological
control and the use of botanical pesticides (plant extracts), a growing alternative for the control of this
pest. From the perspective of locating new options for the control of two-spotted spider mites, the use
of botanical pesticides represents a useful tool with minimal detrimental effects on the environment,
a low residuality, a slight induction of resistance due to its complex matrix, and with fewer harmful
effects on human health when compared to those of the chemically-synthesized acaricides. Therefore,
in the present review, a survey is presented based on some characteristics of T. urticae Koch behavior
in the presence of toxic substances. In addition, this review builds upon other studies in order to
determine the biological activity of some botanical pesticides on the phytophagous mite T. urticae Koch
under laboratory conditions.
2. Characteristics of T. urticae
The T. urticae Koch is the most abundant and the most widely distributed species of the genus
Tetranychus. This genus presents a confusing taxonomy due to partial reproductive incompatibilities
that have been found in some populations. It is known that, in certain cases, these incompatibilities are
caused by species of bacteria from the genus Wolbachia [6].
The individuals of the T. urticae Koch are characterized by having two spots on their back (dorsal
idiosome), green or brown coloration, and white or yellow colored legs [4]. They present sexual
dimorphism, as males are smaller than females [4]. An important feature of this species is that it is
is able to form a web on the plants in which it grows [4]. These mites feed initially on the leaves
of the lower part of the plants, but they can later colonize the rest of them as the population grows.
The damage they cause is observed in the form of chlorotic spots and, in some cases, the tanning of
leaves and defoliation [4].
2.1. The Biology of T. urticae Koch

The life cycle of the family Tetranychidae includes the stages of egg, larva, protonymph,
deutonymph, and adult [7], between each of which a quiescent state usually occurs. Their eggs are
round, white, or translucent, and the duration of their cycle depends on the temperature, the relative
humidity, and the host plant in which they develop. Under temperature conditions between 25 and
30 ◦ C, the T. urticae Koch can complete its cycle between three and five days [8,9]. The eggs are
approximately 0.13 mm in diameter. The larvae are spherical or oval in shape, generally greenish
yellow with three pairs of legs, and their size is approximately 0.16 mm in length. The protonymphs
have an oval shape and a pale green color. They are distinguished from larvae by having four pairs
of legs, and their length is approximately 0.2 mm. In the case of deutonymphs, they reach a length
of approximately 0.3 mm and have a yellow or light brown color. At this stage, two dark brown
spots usually appear on the dorsal level. On the other hand, adults have a globular or oval shape
and range from pale green to reddish yellow in color; adults present two red or dark brown spots
on the idiosome. Males are smaller than females, with lengths of 0.4 and 0.5 mm, respectively [7,10].
This species is arrhenotokous [8], which increases the probability that a female will mate with her
offspring. According to some authors, its high genetic variability allows it to adapt quickly and
decreases its probability of expressing deleterious mutations [9].
2.2. Characteristics of Resistance of T. urticae Koch to Acaricides

The T. urticae Koch is a widespread polyphagous pest that attacks more than 1100 different
plant species [9,11], making it one of the main phytosanitary problems for many crops. This trait is
owing (among other reasons) to its capacity for quickly generating resistance to synthetic acaricidal
products [12]—from two to four years of new active ingredients [9]—even after a few applications of
the active ingredient [11].
This resistance capacity to pesticides of the T. urticae Koch has encouraged some researchers to
carry out several studies regarding their genetic characteristics in response to the pressure generated
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by the use of acaricides. Such is the case of Grbić et al. (2011) [9], who carried out a deep analysis of
the T. urticae Koch genome. They found that more than 10% of their genome comprises transposable
elements (9.09 Mb). In the same study, they also observed the presence of several families of genes
involved in digestion, detoxification, and transport of xenobiotic compounds with a unique composition.
Eighty-six genes encode for cytochromes P450, a group of 32 genes encode for glutathione S-transferases
(GST) (12 of these are believed to be unique to vertebrates), and 39 genes encode for drug-resistant
proteins of the ABC transporters type (ATP-binding cassette). This repertoire of transporter proteins
greatly exceeds the number presented by crustaceans, insects, vertebrates, and nematodes.
All these detoxifying enzymes are closely related to the resistance of T. urticae Koch, but this is not
the only mechanism used by these mites to counteract the effect of xenobiotics. A set of mutations in
the action points of pesticides is another way they are able to mitigate the effect of these compounds.
Demaeght et al. (2014) [13] reported a resistance case for this species when there was a mutation in
quitin synthase 1, which is the target enzyme of etoxazole. Additionally, because of its similarity to the
mechanism of action of hexythiazox and clofentezine, this mutation can cause a cross-resistance to these
products. Table 1 shows an example of the effects of 10 different acaricides on four different populations
of T. urticae Koch in the state of Pernambuco (Brazil) [14]. This information demonstrates the ability
of this pest to counteract the effects of different active ingredients, showing variable responses to the
same compounds in different regions.
Table 1. The resistance of different populations of T. urticae Koch—from the state of Pernambuco
(Brazil)—to 10 different acaricides. Adapted from Ferreira et al. [14].
Acaricide Population N◦ LC50 (mg/L) * LC95 (mg/L) * RR50

Petrolina II 426 6.6 70 1
Piracicaba 484 10.7 105 1.6
Diafenthiuron
Brejão 340 4053 93,708 619
Bonito 401 7732 133,440 1180
Piracicaba 484 0.6 8.3 1
Petrolina II 455 5.4 101 9.9
Milbemectin
Bonito 373 357 3726 650
Brejão 380 384 2386 700
Piracicaba 315 22 341 1
Petrolina II 469 87 1929 4
Fenpyroximate
Bonito 378 3246 10,014 200
Brejão 387 4343 16.234 150
Piracicaba 424 1.3 9.3 1
Petrolina II 481 2.8 20.1 2.2
Clorfenapyr
Brejão 477 735 4157 570
Bonito 524 4652 94.598 3600
Piracicaba 401 16.4 1590 1
Petrolina II 547 37.5 370.870 2.3
Spirodiclofen
Bonito 538 6401 127.750 390
Brejão 414 6586 56.390 400
Piracicaba 465 0.83 436 1
Fenbutatin Petrolina II 538 1.72 1093 2.1
oxide Bonito 477 293 52.892 350
Brejão 459 1705 197.990 2048
Piracicaba 472 6.5 28 1
Petrolina II 397 15 66 2.3
Propargite
Bonito 395 291 990 45
Brejão 391 622 4410 96
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Table 1. Cont.
Acaricide Population N◦ LC50 (mg/L) * LC95 (mg/L) * RR50

Piracicaba 416 2938 64.871 1
Petrolina II 404 4370 100.510 1.5
Hexythiazox
Brejão 440 12,700 605.400 4.3
Bonito 415 1384 381.630 4.7
Piracicaba 418 373 18.404 1
Petrolina II 487 487 17.752 1
Spiromesifen
Brejão 467 1388 42.781 3.7
Bonito 470 3201 90.424 8.6
Petrolina II 613 0.0011 0.033 1
Piracicaba 714 0.0084 0.066 8
Petrolina I 676 0.036 0.205 34.4
Abamectin Gravatá 787 0.041 1.66 39.3
Goiânia 584 1.79 27.9 1716
Brejão 610 118 3000 113.532
Bonito 693 326 3397 295.270
* LC50 : the mortality-causing concentration of 50% of the test population. LC95 : the mortality-causing concentration
of 95% of the test population. N◦ : the number of mites used in the trial. RR50 : the resistance proportion between the
resistant population and the susceptible one at LC50 .
Function of Detoxifying Enzymes

Cytochrome P450 has been extensively investigated, as it is the most important group of detoxifying
proteins in arthropods [15]. This enzyme group has been linked to cases of resistance in the common
fly, Musca domestica L., with resistance to those furanocoumarins produced by a host plant in Papilio
polyxenes Fabricius [15], and in cases of resistance to abamectins in T. urticae Koch [16]. One of
the characteristics of this group of proteins in arthropods is their inducibility over time, which is
proportional to the consumption of certain toxic compounds from the plants that serve them as
food. Such is the case of the Spodoptera frugiperda Smith. In this species, it was demonstrated that,
when consuming a diet containing indole-3-carbinol, in time and with the increase in the concentration
of this compound, there was an increase in the production of P450 enzymes [15].
Another group of proteins that is important in the response to xenobiotics is the
Glutathione-S-Transferases (GST) family. Among these proteins, two enzymes belonging to the
delta class—tuGSTd10 and tuGSTd14—and one of the mu class—tuGSTm09—are present in T. urticae
Koch. They are strongly associated with mite resistance to the active ingredient abamectin [17].
Similarly, Pavlidi et al. (2017) [18], through molecular docking analysis and implementation of
HPLC-MS, deduced that the active ingredient cyflumetofen and its de-esterified metabolite could be
transformed by the enzyme TuGSTd05 in the same mite species.
On the other hand, Merzendorfer (2014) [19] and Dermauw and Van Leeuwen (2014) [20]
mentioned the presence of 104 genes belonging to subfamilies of ABC genes in T. urticae Koch.
This number is higher than that of other different species such as Homo sapiens L., Apis mellifera L.,
Drosophila melanogaster Meigen, Anopheles gambiae Giles, Bombyx mori L., Tribolium castaneum Herbst,
Pediculus humanus L., Daphnia pulex Leydig, Caenorhabditis elegans Maupas, and Saccharomyces cerevisiae
Meyen ex EC Hansen, demonstrating its importance within this species. This group of genes has also
been related to the development of elytra and wings in some insects and to the transport of certain
drugs of hydrophobic origin. The type of transport of compounds of these proteins has been elucidated
through models constructed by crystallography, for which it is known that they act as proteins of
import, export, or as flipases [19].
2.3. Relationship Between Resistance and the Host Plant

The different mechanisms of resistance presented by the T. urticae Koch suggest that these
adaptations may not be due exclusively to the pressure generated from the use of pesticides.
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This question was asked by Dermauw et al. (2012) [21], who made an interesting finding when
studying the transcriptome of resistant and susceptible strains of the T. urticae Koch in the presence of
different host plants.
In that study, they demonstrated that a susceptible strain of this phytophagous mite was capable
of expressing diverse deactivated genes when it was relocated from a bean to a tomato as its host
plant [21]. In addition, the number of expressed genes that are related to the generation of resistance
increased considerably, going from 13 genes—expressed after two hours from host plant change—
to 1206 genes after five generations. On the other hand, they compared the transcriptome of two
resistant strains and that of the susceptible strain developed in the tomato. They also found that both
mite strains shared the expression of a significant number of genes related to resistance (Figure 1).
This seems to indicate that there is a strong relationship between the resistance mechanisms developed
by the T. urticae Koch and its host plants. These mechanisms may be similar to those developed by this
species to face exposure to different pesticides.
Figure 1. A graphical representation of the study performed by Dermauw et al. (2012) [21]. (a) represents
the transcriptional changes in the susceptible London strain of the T. urticae Koch when changing host
plant. (b) represents the number of genes expressed in two resistant strains and the susceptible London
strain of T. urticae Koch after 5 generations from the relocation to another host plant. The scheme was
constructed by R.A. Rincón for this review from the data published by Dermauw et al. (2012) [21].
Evidence of the resistance capacity of this phytophagous mite is shown in Table 2. A list of
important pest arthropod species is shown, reporting the number of active ingredients to which they
developed resistance until the year 2012 [22,23]. The list is led by the T. urticae Koch, a species that
showed a reported resistance to 93 active ingredients until that moment.
Owing to the large number of reports of resistance existing for the T. urticae Koch, some studies have
provided important information and promising aspects in terms of understanding the resistance with
promising results. Such is the case of the research conducted by Demaeght et al. (2013) [24] concerning
cross-resistance. They studied two T. urticae Koch strains that were resistant to Spirodiclofen—an active
ingredient belonging to group 23 of the IRAC (i.e., inhibitors of acetyl CoA carboxylase). Although
strains appeared to be strongly resistant to this ingredient, they had a very low cross-resistance to
spirotetramat and spirodiclofenenol. This information could serve as a base for the understanding of
some routes of resistance-generation in this phytophagous mite, because they demonstrated that the
spirodiclofen detoxification route affects—at least partially—all of the tetranic and the tetronic acid
derivatives in the T. urticae Koch.
In the same study, Demaeght et al. (2013) discarded resistance to spirodiclofen by active site
mutations after aligning the sequences of active sites from target proteins with BlastP [24]. However,
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when microarrays were made to express the genome of the studied strains and subsequently compared,
they found similarities in several genes expressed among the spirodiclofen resistant strains, which were
identified as P450 family proteins, carboxylesterases, glutathione S-transferases, transport proteins,
lipocalins, and several proteins without homology in the available databases. This fact demonstrated
that this detoxifying route is strongly related to the response of the T. urticae Koch to this ingredient.
On the other hand, Kwon et al. (2012) [25] detected a fitness decrease of T. urticae Koch strains that
demonstrated Monocrotophos resistance. Although the presence of more than one mutation increased
the resistance up to 1165-fold, these modifications in genes significantly decreased the catalytic capacity
of acetyl cholinesterase, thus gene overexpression seems to be necessary in order to compensate for
deficiency acquired by resistance-conferring mutations to the acaricide.
Table 2. A list of pest arthropods based on the reported number of active ingredients resistance and
the number of reported cases per species—adapted from Van Leeuwen et al. (2010, 2012) [22,23].
The information for the species Plutella xylostella L., Myzus persicae Sulzer, Leptinotarsa decemlineata Say,
Blatella germanica L., and Panonychus ulmi Koch correspond to the cases reported up to 2010.
Number of Active Cases of

Species Taxonomy Kind of Pest
Ingredients Resistance
Tetranychus urticae Koch Acari: Tetranychidae Crop 93 389
Plutella xylostella L. Lepidoptera: Plutellidae Crop 81 437
Myzus persicae Sulzer Hemiptera: Aphididae Crop 73 320
Leptinotarsa decemlineata Say Coleoptera: Chrysomelidae Crop 51 188
Musca domestica L. Diptera: Muscidae Urban 53 266
Blatella germanica L. Blattodea: Blatellidae Urban 43 213
Rhipicephalus microplus
Acari: Ixodidae Cattle 43 158
Canestrini
Helicoverpa armigera Hubner Lepidoptera: Noctuidae Crop 43 639
Bemisia tabaci Gennadius Hemiptera: Aleyrodidae Crop 45 428
Panonychus ulmi Koch Acari: Tetranychidae Crop 42 181
Varroa destructor
Acari: Varroidae Bees parasite 2 10
Anderson y trueman
Ixodes scapularis Say Acari: Ixodidae Cattle 0 0
Culex pipiens L. Diptera: Culicidae Disease vector 36 161
Culex quinquefasciatus Say Diptera: Culicidae Disease vector 32 256
Tribolium castaneum Herbst Coleoptera: Tenebrionidae Stored-grain pest 32 113
Aedes egypti egypti L. Diptera: Culicidae Disease vector 24 267
Spodoptera frugiperda Smith Lepidoptera: Noctuidae Crop 16 25
Pediculus humanus L. Phthiraptera: Pediculidae Disease vector 9 59
Anopheles gambiae Giles Diptera: Culicidae Disease vector 3 39
Manduca sexta L. Lepidoptera: Sphingidae Crop 3 4
Rhodnius prolixus Stal Hemiptera: Reduviidae Disease vector 3 3
Anopheles darlingi Root Diptera: Culicidae Disease vector 1 2
Linepithema humile Mayr Hymenoptera: Formicidae Urban 2 2
3. Control Strategies for T. urticae Koch

In agricultural crops, the main pest control method used is the spraying of solutions based on
chemically synthetic products such as insecticides and acaricides [26]. Although this method has
been effective in some cases for T. urticae Koch control, it has also demonstrated serious limitations
and disadvantages, especially due to T. urticae Koch’s high reproductive potential. This peculiarity
encourages farmers to use acaricides in larger volumes and doses, causing high levels of toxic waste in
fruits, the development of resistant populations, the intoxication of mammals, and the destruction of
beneficial organisms [23,27,28].
Another strategy used for T. urticae Koch management is biological control. Among the predators
of this pest are some mites of the family Phytoseiidae. Within this family, two predators stand
out—the Neoseiulus californicus McGregor and the Phytoseiulus persimilis Athias-Henriot. These mites
are characterized by consuming a large number of prey at adequate conditions and having high
reproductive rates and a capacity for rapid development [4]. Other natural predators that are less
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commonly used for the control of this mite are the beetle Stethorus punctillum Weise (Coccinellidae)
and the Conwentzia psociformis Curtis (Neuroptera: Coniopterygidae)—which are found naturally
in Spain [29]—purely to mention some of the predators of this phytophagous species. Additionally,
the fungus Neozygites floridana Weiser and Muma has also exhibited significant control over the T. urticae
Koch, but some difficulties in cultivation have hampered its use [30]. However, other fungi such as
the Lecanicillium lecanii Zimmermann and the Beauveria bassiana Bals.-Criv., as well as the bacterium
Bacillus thuringiensis Berliner, have been commercially used for the management of the two-spotted
spider mite with positive effects.
3.1. Other Methods for T. urticae Koch Control

As complementary strategies, these mites are controlled in some crops through the application of
water washings and the manual massaging of the affected leaves using water and soap in order to
remove the mites from the plant, kill them mechanically, and break their webs. Within the strategies
used for controlling the T. urticae Koch, biopesticides based on plant extracts or phytochemicals
are considered to be another alternative to chemically-synthesized acaricides [31,32], which have
also emerged as a complement to traditional management. This has allowed the development of
commercial products with formulations based on substances of natural origin, such as CinnAcar® ,
Biodie® and PHC Neem® , which are produced from compounds and mixtures isolated from plant
extracts. As an example for this case, they have demonstrated compatibility with the natural
predator Tamarixia radiata Waterston (Hymenoptera: Eulophidae)—parasitoid of the Diaphorina citri
Kuwayama (Hemiptera: Psyllidae)—thus these formulations may constitute excellent alternatives to
be included into integrated management programs (IPM) of the so-called “Asian citrus psyllid” [33].
Therefore, the fact that these botanical pesticides are compatible with natural predators becomes
an advantage in the control of pests and constitutes an additional tool that can be used in integrated
pest management strategies.
An essential prerequisite for success when using extracts as a control strategy for pests is their
compatibility with other management strategies. Within the context of the IPM, a relevant issue is the
evaluation of how this type of product can affect biological control agents. In the particular case of
the T. urticae Koch, a question arises about how phytoseiid mites that have been successfully used as
a control strategy could be affected—a topic that has been explored by different researchers. Among
the botanical pesticides, probably the most used are the neem derivatives, a trend that is also present
in the case of the T. urticae Koch. A moderate reduction in female survival and fecundity in response
to Azadirachtin use on P. persimils Athias-Henriot was reported by Duso et al. (2008) [34], although
a positive shift in favor of the predator in terms of the predator–prey interaction can be inferred,
since azadirachtin was more toxic to the T. urticae Koch. A moderate effect was also reported by Spollen
and Isman (1996) [35], who found a maximum mortality of 14% in P. persimils Athias-Henriot adults
sprayed with neem extract. Although variables such as egg eclosion, the mean number of eggs laid per
female, and differences in preference between treated or untreated leaves were not found, the authors
concluded that neem-derived insecticides could be effective and safe. Neem pesticides have exhibited
few negative impacts on the phitoseids N. californicus McGregor [36], Euseius alatus De Leon [37],
and Phytoseiulus macropilis Banks [36,37]. On the other hand, the negative effects of NeemAzal-T/S
in terms of its potential impact on populations of the predator Metaseiulus occidentalis Nesbitt were
reported by Yanar (2019) [38], who recommended the use of low concentrations of this product in cases
where the M. occidentalis is a relevant component of IPM. Regarding another plant species used to
obtain botanicals, crude extracts of Artemisia judaica L. exhibited acaricidal bioactivity against T. urticae
Koch in terms of LC50 , while its negative impacts upon P. persimils Athias-Henriot were clearly lower,
suggesting that such extracts are compatible with the predaceous mite [39]. Similarly, a promising
toxic effect of the Melissa officinalis L. on T. urticae Koch has been reported, along with an LC50 for
the N. californicus McGregor, which is comparatively extremely lower. Undertaking compatibility
evaluations between extracts and natural predators is essential, since there is no reason to generalize
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slight or innocuous effects of these products on said beneficial organisms. Commercial formulations
and application rates similar to those used by farmers are needed in order to obtain results with more
predictive value in respect of those expected in the field. Sublethal effects will also be a subject of
relevant research in the future, because, although many of the evaluations that demonstrate little or
no effect on the natural predators have been carried out in adults, the sublethal effects could raise
compatibility issues that are not evident when restricting evaluations to adult individuals [40].
3.2. The Use of Plant Extracts for the Control of T. urticae Koch in The Field
There are many studies regarding the use of plant extracts for the control of the T. urticae Koch.
Although many of these trials have delivered successful results, others have not demonstrated the level
of expected control over this mite species. For that reason, a greater understanding of the mechanisms
of action presented by molecules that demonstrate biological activity on these mites and the way in
which these molecules interact is highly required. In addition, the toxic effects of such molecules are
generated in many cases by the presence of several toxic compounds contained in the same extract,
which act in a synergistic manner. An understanding of these factors will help to foster a broader
understanding of the use of this tool in the control of the two-spotted spider mite. Further studies
must take into account the results of the studies developed thus far, which have delivered promising
results, not just in terms of the toxic effects demonstrated on these mites, but also in terms of sublethal
effects such as low fecundity and repellency.
3.2.1. Methods for the Evaluation of Extracts under Laboratory Conditions

The methods of testing the effects of extracts are very much the same as those used thus far for
evaluations of chemical compounds. An important prerequisite for making appropriate evaluations is
having a susceptible population of individuals. Generally, this population can be obtained by rearing
individuals that have not been exposed to any type of chemical substance with a possible acaricidal
effect for a considerable number of generations [5,41–43]. Additionally, the origin and the type of the
selected plant material must be clearly defined in order to ensure the repeatability of results. Hence,
correct taxonomic classification, location, season of the year, time of day, phenological stage, and organs
to be collected and processed in order to obtain the extracts affect the particular composition of the
tested botanical and influence the acaricidal activity [5,44–46]. Finally, the type of preparation and the
extracting protocol are also crucial steps for obtaining a standardized mixture of plant-based compounds,
which would be the source of effective botanical-based acaricidal or repellent agents. Thus, solid–liquid
(S–L) extractions—i.e., the selected plant material directly enters into contact with the extracting
solvent during a defined period through a continuous (maceration) or discontinuous (percolation or
Soxhlet) procedure—are the most commonly used method for obtaining different types of extracts,
depending on the polarity of the extracting solvent. In order of polarity, water, water/ethanol mixture
(hydroalcoholic), ethanol/methanol, chloroform, ethyl acetate, and hexane are the most commonly
used solvents for extractions. Other types of preparations are essential oil (usually obtained by steam
distillation) or low-polar/volatile extracts (afforded by hydrodistillation, supercritical fluid extraction,
microwave or ultrasound-assisted hydrodistillation, among others) [47]. The physicochemical nature
of these naturally occurring compounds, which are present in the preparation (extract or essential oil),
is the critical prerequisite information required to identify the extracting procedure. The purification or
the isolation of the active principles requires several steps, usually using preparative techniques such as
column chromatography under a bioguided fractionation strategy—although the isolated compounds
might be separately assessed after a conventional purification protocol. In any case, these efforts could
affect upon acaricidal rather than repellent activities to facilitate mite control, but this choice depends
of the aims of use. Essential oils often exhibit repellent activity in comparison to extracts, owing to
their volatile nature.
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3.2.2. Bioassays
The purpose of bioassays is to determine the effect of a given agent on the physiology of
an organism, which, in the context of acari research, is generally associated with determining the
toxicity of a chemical compound—or resistance to it—either in the field or in laboratory conditions [48].
Repeatability of results, practical facilities, and conditions as similar as possible to those under which
the acaricide will be used are desirable [49]. In the case of mites, a small size and fast movement
are aspects conditioning the bioassay design. The main aspects of some common bioassays used for
the evaluation of botanicals on T. urticae Koch adults (generally females) and their advantages and
disadvantages are described below.
Slide Dip Methods

An initial method was described by Voss (1961) [50] as part of an acaricide screening procedure.
Double-sided Scotch® tape is adhered on one of its sides to a microscope slide. It is important to avoid
bubbles or empty spaces between the tape and the glass, because deposits of the substance under
evaluation could be formed, which could affect the test results [51]. After this, the mites must be affixed
to the other side of the tape by the dorsal part of the hysterosoma. A fine brush is usually used to
transfer individuals to the tape. The slides are then dipped into the solution containing the toxicant for
5 s [48,51] and, after this, are placed on a paper towel. It is important to remove any excess of liquid with
filter paper. After this, the slides are placed on trays covered with slightly moistened disposable towels,
which must then be taken into controlled conditions. The mortality criterion in the different methods is
usually an absence of movement when the individual is gently prodded with a fine brush. High control
mortalities due to desiccation and an absence of food are common in this method for times of evaluation
greater than 24 h, limiting the accuracy of the response parameters. Furthermore, individuals are
exposed to toxicants in an artificial substrate, and in some cases, problems distinguishing alive and
dead mites arise [48]. Despite such problems, this method has been used repeatedly to determine
the effect of botanicals on adults of the T. urticae Koch, in some cases considering evaluation times
of 24 h [52], but in other cases employing higher evaluation times [41,52–57]. An advantage of this
method is that the results obtained are highly reproducible. It can also be modified by employing
a spray tower to supply the toxicant, which allows for a better coverage [58].
Petri Dish Methods

The main variant is the Petri Dish Residue-Potter Tower Method (PDR-PT), in which the bottom
and the top inner surfaces of a Petri dish are sprayed with the toxicant using a Potter Tower and
allowed to dry for around 30 min at room temperature. After this, the individuals are transferred to
the dishes using a fine brush. For this method, high mortalities after 48 h have been reported, thus it is
advisable to restrict the evaluation time to shorter periods, such as 24 h [59]. The petri dish methods
have been used in some cases for the evaluation of botanicals against T. urticae Koch [3,60].
Leaf Disc Methods

For this kind of bioassay, leaf discs of variable diameter (approximately 20 mm) are cut from leaves
of several plant species, such as beans [1,43,46], peaches [48], or roses [3], and placed upside down in
a Petri dish containing moistened cotton wool when bean or rose leaf discs are used or a semi-solid
agar pad in the case of peach leaf discs. A variable number of adults (between five and 20) must be
transferred to each leaf disc using a fine brush. The experimental assembly is maintained without
disturbance for at least one hour before the spraying of the toxicant is performed. This spraying can be
performed using an airbrush, provided the required distance—as well as the number of drops/cm2 and
the pressure—may be adequately standardized [61], which corresponds to the basic Leaf Disc Direct
Method (LDD). This method can be improved employing the Potter spray tower, derivating in the
so-called “Leaf Disc Direct-Potter Tower Method” (LDD-PT) [48]. This device was developed by C.
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Potter from the Rothamsted Experimental Station [62], and it is recognized as a reference standard
for making chemical sprays under laboratory conditions, since it enables the achievement of an even
deposition of spray in the target area. The LDD and the LDD-PT methods can also be used to evaluate
the effect of a residual film of the toxicant on adults placed on a sprayed surface (such as a leaf disc in
this instance). In this case, the procedures are named “Leaf Disc Residue Method” (LDR) and “Leaf Disc
Residue-Potter Tower Method” (DR-PTM) [48]. Both a direct spray and the residual film are intended
to evaluate the toxic effects generated by contact between the individual mites and the test substance.
After the spray, Petri dishes are kept uncovered for around 30 min, which allows for the drying of the
leaf disc surface. They are then covered and placed under controlled conditions. Generally, mites that
cannot walk a distance equivalent to their body length are considered dead. Since the leaf disc method
implies the presence of the natural substrate of spider mites, it can be considered to have a greater
similarity with field conditions than the slide dip or the petri dish methods. However, one drawback
is the escape of individuals. This problem intensifies when the toxicant requires a prolonged time
to act or when it should be ingested in the feeding process. The fate of individuals that escape is
uncertain, thus the most advisable procedure is to discard them in the analysis; to consider them as
part of the mortality rate would not be justifiable [48]. An alternative approach is the development of
methods that do not allow for the escape of individuals, as proposed by Bostanian et al. [63]. In their
setup, a large leaf disc (50 mm in diameter) is placed upside down and tightly fitted to the bottom
of a plastic Petri dish of the same diameter, thus it occupies the whole dish. The base of each petri
dish contains thinly moistened cotton wool (1.5 mm in thickness) to prevent desiccation. A circular
window of 28 mm is cut in the top of the Petri dish to facilitate air circulation and avoid condensation.
For bioassays involving tetranychyds, they recommend covering the window with a 40 μm polyester
mesh screen to avoid run-off. The edges of the Petri dish bottoms are wrapped with masking tape
to ensure a strong grip on the top, preventing the escape of individuals. A small hole in the lower
half of the Petri dish allows the petiole to protrude outwards, where it must be covered with a wet
cloth. This method enables observations for a period as long as nine days, which makes it suitable for
slow- and fast-acting reduced-risk toxicants. A different variant of the leaf disc method is the Leaf
Disc-Residue Dipping Method (LDR-D), in which the leaf discs are dipped into the solution containing
the toxicant [64]. Although estimations of lethal concentrations obtained by this method are less precise
when compared to the LDD-PT, this fact could be explained by an uneven distribution of residues on
the leaf surface. The leaf disc methods have been widely used in several trials of botanicals against
T. urticae Koch [5,40,54–62].
Leaf Absorption Method

In this method, the leaf is placed in some kind of recipient containing the toxicant solution in
order to allow the absorption by the leaf for an adequate period (usually around 72 h). The leaf is then
located in a Petri dish containing agaropectin to prevent desiccation, and the mites are transferred onto
the leaf, where they are allowed to feed by 24 h, and mortality is then evaluated. The design of the
experimental unit should consider alternatives to prevent the escape of mites, as discussed in the leaf
disc method [42,63].
Whole Plant Direct Method

The purpose of this kind of bioassay is to evaluate the direct effect of toxicants under conditions
as similar as possible to those of the field. Young bean plants with 2–3 leaves can be used, and the
apical part of the plant must be removed to prevent the appearance of new leaves, which have not
received the treatment. Adult females are then placed in the plants long enough before performing the
application in order to allow oviposition. Alternatively, a specific number of immature stages and
adults can be placed in the plants. The spray of the toxicant is made using an atomizer, considering
an application volume similar to the required under crop conditions. The number of eggs, nymphs,
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and adults is recorded at predefined evaluation times, usually between 5 and 10 days [65,66]. This
method has been also used to evaluate repellency [67].
Filter Paper Difussion Methods (Fumigant Bioassays)

This kind of assay is designed to evaluate the fumigant action of toxictants, thus it is essential to
avoid any direct contact between individuals and the toxicant. The setup is similar to that employed in
the leaf disc method, but the bottoms of the Petri dishes are covered using a tight-fitting lid with a fine
wire sieve. The toxicant is applied to filter papers, which should be allowed to dry before being placed
over the wire sieve [68]. In some cases, the paper is attached to the downside of the lid with a small
quantity of solid glue that should not affect individuals [42].
3.3. Studies Carried Out for the Control of T. urticae Koch from Plant Extracts Grouped by Plant Families
The investigations carried out, which focused on the effects of biopesticides on T. urticae Koch,
have led to the identification of a large number of plant extracts with acaricidal, repellent, and deterrent
properties. Below are descriptions of some species—grouped by plant families—whose plant extracts
have been used in laboratory studies that have exhibited their biological activity on the two-spotted
spider mite (the information is summarized and complemented in Table A1 in the Appendix A).
3.3.1. Family Amaranthaceae

This family has aroused interest in different areas such as traditional and alternative medicine,
given the properties that have been identified in some of the species that comprise it. Such is
the case of Achyranthes aspera L., whose secondary metabolites have antinociceptic activity [69],
or Chenopodium ambrosioides Mosyakin et Clemants, which has toxic effects that have been studied in
some human parasites [70].
Due to these toxic effects, Hiremath et al. (1995) [71] evaluated the acaricidal effect of the extract
of this plant. They compared the activity of the methanolic extracts obtained from 21 different species
of African plants against T. urticae Koch adults using the leaf immersion method. Among the most
active extracts, the whole plant of Celosia trigyna Linn. exhibited the highest biological activity, causing
mortality rates between 40% and 60% of evaluated mites.
Chiasson et al. (2004) [41] also evaluated the acaricidal effects of a species of this family.
They studied the effect of an emulsifiable concentrate—obtained from Chenopodium ambrosioides
Mosyakin et Clemants essential oil—on adults and eggs of the T. urticae Koch and the Panonychus
ulmi Koch and compared it with the effect obtained from the use of commercially available products.
The products were applied with an airbrush on females that were placed on microscope slides with
glue. In the case of eggs, the application was made on the eggs previously laid by females on leaf discs
located within Petri dishes. Thus, a dose of 0.5% produced a mortality of 94.7% in females, which was
higher than that obtained from the Neem extract (22.1%). Otherwise, hatching was diminished on days
five and nine after application. This hatching effect was lower in treatments with Neem, Abamectin,
and insecticide soap. A lower effect was observed for an ethanolic extract from seeds of Chenopodium
quinoa Willd. on adult females and nymphs of this mite, exhibiting an LD50 of 1.24% w/v [72].
Two years later, Shi et al. (2006) [52] evaluated the effect of Kochia scoparia (L.) Schrad extract
on T. urticae Koch, T. cinnabarinus Boisdu-Val, and T. viennensis Zacher using three different solvents
for extracting the compounds contained in the plant material: methanol, chloroform, and petroleum
ether. The mortality trials were carried out using three different methodologies: (1) the slide dip
method measuring mortality after 24 h of immersion, (2) the LDD-PT, and (3) the leaf absorption
method. Using these methodologies, the highest mortality of the T. urticae Koch was obtained with the
chloroform-soluble extract, which exhibited a 78.86% average mortality and an LC50 of 0.88 using the
dipping method, in which mites were glued to an adhesive tape.
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3.3.2. Family Amaryllidaceae

This family is studied widely due to its potential uses in the control of human diseases [73] as well
as its antitumor [74] and insecticides properties [75]. Abbassy et al. (1998) [76] determined the LC50 of
the alkaloidal extract, the ethanolic extract, and the essential oil of the bulb of the ornamental plant
Pancratium maritimum L. (Amaryllidaceae) on the T. urticae Koch, whose values were 0.2%, 0.36%, and
1.5%, respectively.
The insecticidal properties demonstrated by various studies led Attia et al. (2011) [77] to expose
adult T. urticae Koch females to different concentrations of garlic extract (Allium sativum L.).
These concentrations ranged between 0.46 and 14.4 mg/L using the Potter Tower application. After the
bioassays, they determined the LD50 and the LD90 , whose values were 7.49 and 13.5 mg/L, respectively.
On the other hand, they concluded that fecundity was reduced by using the concentrations of 0.36
and 0.74 mg/L. Geng et al. (2014) [78] measured the toxicity by the contact and the repellency of the
garlic extract at 20, 10, 5, 2.5, and 1.25 g/L. From these tests, they found that treatment with 20 g/L
caused a 76.5% mortality rate on mites at 48 h after its application. Additionally, with the obtained
data, they calculated the regression equation of toxicity as Y = 1.3 x + 3.9. They were also able to
determine the LD50 value, which corresponded to 7.2 g/L. Furthermore, the repellencies were found to
be 95.6% and 65.2% at extract concentrations of 10 g/L and 20 g/L, respectively.
3.3.3. Family Annonaceae

Within this group of plants, the presence of several important secondary metabolites involved in
the communication of arthropods and plants’ defenses against the attack of pests has been identified [79].
However, Ohsawa et al. (1991) [80] obtained negative results when using Annona glabra L. seed extract
on T. urticae Koch eggs. During their experiment, they dissolved 10 mg of the extract in acetone (1 mL)
and applied 0.5 mL of the solution to a bean leaf where the eggs were laid. After this, they noticed that
the extract demonstrated no impact on mortality rates, deterrence in feeding, or mite growth.
Pontes et al. (2007) [44] also demonstrated the acaricidal activity of the essential oils of this
family of plants, but in this case, they used the species Xilopia serícea A.St.-Hil., which was evaluated
on T. urticae Koch. Using gas chromatography–mass spectrometry (GC–MS), they identified the
compounds present in both leaves and fruits, finding mostly monoterpenes and sesquiterpenes.
When comparing their acaricidal activity, they concluded that the essential oils of the leaves exhibited
a greater toxicity than those obtained from the fruits.
3.3.4. Family Apiaceae

Plants of this family are widely used within the diet of different human communities [81], although
their nature is so varied that many species have been used as pesticides and repellents [82]. For example,
Choi et al. (2004) [42] tested the essential oils of 53 plants to determine their acaricidal potential on
T. urticae Koch eggs and adults. Among these oils, the highest toxicity was exhibited by species of the
family Apiaceae—i.e., Carum carvi L.—since a 100% mortality rate of adult mites was obtained. To carry
out this study, the researchers conducted bioassays by diffusion on filter paper, avoiding any direct
contact between the oil and the mites. The tests were developed in a plastic container (4.5 × 9.5 cm) at
a concentration of 14 × 10−3 μL/mL air.
Tsolakis and Ragusa (2007) [83] studied the effect of a mixture of essential oils from the C. carvi L.
with potassium salts of fatty acids on the T. urticae Koch and one of its predators, Phytoseiulus persimilis
Athias-Henriot. This combination proved to be very selective, since it generated a mortality rate of
83.4% in T. urticae Koch females compared to a 24% mortality rate in P. persimilis Athias-Henriot females.
Besides, the product also caused a decrease in the intrinsic growth rate of the phytophagous mite
while having no effect on the growth rate of the predator. Approximately four years later, this same
essential oil was tested by Han et al. (2010) [68] on the same species of mite. In this case, by using
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mortality bioassays by vapor phase to evaluate fumigant effect (see section of Myrtaceae Family),
they established an LD50 of 22.4 μg/cm3 air.
Among works carried out with plants of this family, Attia et al. (2011) [43] showed that the Deverra
scoparia Coss. & Durieu essential oil has an acaricidal effect and decreases the fecundity of the T. urticae
Koch. In the same study, they isolated the components of the oil and tested them individually on
the pest, obtaining the highest toxicities with the compounds α-pinene, Δ3 -carene, and terpinen-4-ol.
Amizadeh et al. (2013) [84] also decided to evaluate the effect on two species of this family of the
inhalation of essential oils. For this purpose, they carried out tests to determine the fumigant activity of
Heracleum persicum Desf. Ex. Fisch. essential oils and Foeniculum vulgare Mill. seeds on adult females and
eggs of the T. urticae Koch. The LD50 s were 3.15 μL/L and 1.53 μL/L for females and eggs treated with
Heracleum persicum Desf. Ex. Fisch. essential oil, respectively, and 5.75 μL/L and 1.17 μL/L for females
and eggs treated with Foeniculum vulgare Mill. essential oil, respectively. Other essential oils obtained
from Apiaceae plants having acaricidal activity on T. urticae Koch were Cuminum cyminum L. (seeds)
and Ferula gumosa Boiss (leaves), showing LD50 values of 3.74 and 6.52 μL/L air, respectively [85,86].
On the other hand, Pavela (2015) [65] tested acaricidal and ovicidal effects of the methanolic
extract of Ammi visnaga (L.) Lamarck seeds on T. urticae Koch. The efficacy in terms of adult mortality
rates increased over time, with LD50 s (after 72 h from the time of application) estimated at 17, 10,
and 98 μg/cm2 for the extract and its two major compounds, khellin and visnagin (furanochromenes),
respectively. Moreover, the extract and the two isolated furanochromenes inhibited the development
of eggs and caused their mortality, with LD50 s of 13.3, 0.5, and 1.8 μg/cm2 for the extract, the visnagin,
and the khellin, respectively. The application of the extract to leaves infested with T. urticae Koch
achieved a reduction of the number of individuals in all stages of development. The concentration
of 10 mg/mL showed the highest efficacy, which was 98.5% on the tenth day since the application.
The terpenes isofuranodiene and germacrone, isolated from Smyrnium olusatrum L. inflorescences,
also exhibited toxicity on this mite (LD50 s = 1.9 and 42.7 μg/mL, respectively) [87].
3.3.5. Family Asteraceae

There have been numerous studies carried out with species from this group to evaluate their
acaricidal activity. First, Chiasson et al. (2001) [45] evaluated the essential oils of two plant species
known as potential pesticides—Artemisia absinthium L. and Tanacetum vulgare L.—to determine their
acaricidal activity against the T. urticae Koch. The oils were obtained via a microwave-assisted process
(MAP), distillation in water (DW), and by direct distillation with steam (DDS), and their relative
toxicities were tested by direct contact. All oils were tested at 1%, 2%, 4%, and 8% as emulsions
prepared in water with 9% denatured ethanol and 0.32% Alkamul EL-620 as emulsifier, and mite
mortality was evaluated after 48 h.
The three oils of A. absinthium L. were toxic to the T. urticae Koch; however, there were differences
in their levels of toxicity. For example, the oil extracted by MAP and DW methods caused 52.7% and
51.1% mortality in the mites, respectively, while the oil obtained by DDS produced a mortality rate
of 83.2%. Consequently, the LC50 of the oil extracted by DDS was lower (0.043 mg/cm2 ) than those
obtained by MAP (0.134 mg/cm2 ) and by DW (0.130 mg/cm2 ). The extracts of T. vulgare L. obtained
by DW and DDS exhibited greater acaricidal activity than the extract prepared by the MAP method.
At a concentration of 4%, oils delivered mortality rates of 60.4%, 75.6%, and 16.7%, respectively.
The chemical analysis of the extracts of T. vulgare L. indicated that the compound p-thujone is the
major compound in the oil (>87.6%) and probably contributes significantly to its acaricidal activity.
Additionally, the acetone extract from leaves of Artemisia judaica L. exhibited an LD50 of 0.56 μg/mL
against adult females [39].
Trials that have shown acaricidal activity within this family have also identified important
compounds in essential oils that may play a role in the toxic activity against the T. urticae Koch. One of
these cases was developed by Attia et al. (2012) [46], who identified the terpinen-4-ol compound in
the Santolina africana Jord. & Fourr. essential oil. This compound was the most abundant component
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(54.96%) within the study. They evaluated the acaricidal activity of the S. africana Jord. & Fourr. and
the Hertia cheirifolia (L.) Kuntze essential oils, with positive impacts upon the mortality rates of the
T. urticae Koch and important effects in the reduction of oviposited eggs.
In another study, this same group of researchers tested the effect of the Chrysanthemum coronarium L.
essential oil on the T. urticae Koch and produced mortality rates of 88% and 93% on larvae and adult
females, respectively [88]. In the same year, another paper was published by Afify et al. (2012) [89],
who tested the acaricidal activity of Chamomilla recutita L. extract on the T. urticae Koch. The LD50 values
obtained for adults and eggs in this study were 0.65% and 1.17%, respectively. In this study, the authors
identified the main compounds of C. recutita L. by means of gas chromatography–mass spectrometry.
The most predominant compounds were α-bisabolol oxide (35.25%) and trans-β-farnesene (7.75%).
The essential oil from the aerial part of Achillea mellifolium L. showed LD50 values of 1.208% v/v and
1.801 μL/L air when evaluated through leaf dipping and fumigation, respectively. The GC–MS chemical
profile of this oil was mainly composed of piperitone (12.8%) and p-cymene (10.6%) [64].
However, not all studies using species from this plant family obtained satisfactory results in terms
of the T. urticae Koch. For example, extracts obtained from Artemisia absinthium L.—known insecticides
and acaricides used throughout the world to control aphids—demonstrated weak activity upon the
T. urticae Koch, as reported by Aslan et al. (2005) [90]. Similarly, Derbalah et al. (2013) [91] found that
the extract of castor leaves (Artemisia cinae O. Berg & C.F. Schmidt ex.Plajakov) exhibited low toxicity
against the T. urticae Koch, with an LD50 of 1326.53 ppm. Similarly, Pavela et al. (2016) [92] studied the
effect of the methanolic extract taken from leaves of the Tithonia diversifolia Hemsl. on T. urticae Koch
and its ethyl acetate fraction in order to measure acute and chronic toxicity as well as its inhibitory
effects on oviposition. In acute toxicity trials, mortality did not exceed 50%, even for the highest dose
evaluated (150 μg/cm3 ). On the other hand, in the chronic toxicity tests on the fifth day after application,
the LD50 of the methanolic extract was 41.3 μg/cm3 , and the LD90 was 98.7 μg/cm3 . However, the two
extracts caused inhibition in the oviposition of mites.
3.3.6. Family Boraginaceae

A low polar extract from roots of Onosma visianii Clem. demonstrated significant chronic
toxicity and oviposition inhibition on T. urticae Koch adult females (LD50 = 2.6 μg/mL). Eleven
naphthoquinone-type related compounds were isolated and structurally elucidated [93]. Although all
isolated derivatives exhibited effects against this mite, isobutylshikonin and isovalerylshikonin were
found to be the most active isolated compounds (LD50 s = 2.69 and 1.06 μg/mL, respectively).
3.3.7. Family Burseraceae

Several species belonging to this family exhibit anti-inflammatory properties [94]. They are
considered to be anticarcinogenic agents with antimalarial, antidiarrheal, and antifever properties and
uses as insecticides [95], antimicrobials, and antioxidants [96] (among others) for disease treatment [95].
However, some studies have pursued applications in agriculture, specifically for the management
of important pests. In that respect, Pontes et al. (2007) [97] studied the acaricidal and the repellent
effects of the Protium bahianum Daly plant resin oil on the T. urticae Koch by fumigant tests. For this,
they kept mites in leaf discs of Canavalia ensiformis (L.) DC. inside 9 cm Petri dishes as test chambers.
Each chamber had a strip stuck on the inner side that was saturated with different amounts and
concentrations of the oil (5, 10, 15, 20, and 25 μL, corresponding to 2, 4, 6, 8, and 10 μL/L of air,
respectively). They evaluated the fresh resin oil and the old resin oil separately. Results showed that
the fumigant effect of the oil in both cases increased with concentration and exposure times and had
mortality rates of 79.6% and 59.0% after 72 h for the old and the new resin oils, respectively. Regarding
the deterrent effect of oviposition, the fresh resin oil presented an increased activity, with only 14 eggs
oviposited at 72 h at a concentration of 10 μL/L of air. In repellency tests, only fresh resin oil showed
positive effect against mites.
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3.3.8. Family Cannabaceae

Although this family of plants is recognized for its various pharmaceutical uses, little has been
studied about its effects as an insecticidal and an acaricidal agent. Among the studies that have been
performed, Yanar et al. (2011) [60] used the extract obtained from the flower buds of Humulus lupulus L.
on T. urticae Koch adults at 5% (adhesive tape method) and at 50% (residual film method). Using the
adhesive tape methodology (in which 1 mL of solution was applied to the tape left for 4 to 5 h to dry,
and 20 adult females were then placed on it), the mortality rate after 24 h was 67.84% ± 2.52%. On the
other hand, with the residual film methodology (in which the extract was applied to a 90 mm Petri
dish, distributed homogeneously, and left for 2 to 4 h to dry before the addition of 20 T. urticae Koch
adult females), the mortality observed after 24 h was 56.37% ± 0.99%. The acaricidal effect against this
mite of an essential oil from panicles of hemp (Cannabis sativa L.) was also evaluated, exhibiting 83.28%
of mortality on adult females at 0.10% [98].
3.3.9. Family Caryophyllaceae

The acaricidal effect of an aqueous extract from roots of Saponaria officinalis was evaluated against all
developmental stages of T. urticae Koch [66]. The lowest sensitivity was found for adults (LD50 = 0.31%
w/v), while eggs revealed the highest sensitivity (LC50 = 1.18% w/v). Oviposition was also inhibited by
this extract (LC50 = 0.91% w/v).
3.3.10. Family Combretaceae

There are several plant species of this group on which acaricidal activity studies of the T. urticae
Koch have been carried out—the majority of them successfully. An example of this is the study
performed by Hiremath et al. (1995) [71], who compared the activity of the methanolic extracts
obtained from 21 different species of African plants against adults of the T. urticae Koch using the leaf
immersion method. Among the results found, the Combretum micronthum G. Don. and the Piloitigma
vetilicolin whole plant extracts demonstrated effects on the rates of T. urticae Koch mortality of between
40% and 60%.
3.3.11. Family Convolvulaceae

There are few studies on the T. urticae Koch that involve this plant family, with plants of the genera
Convolvulus and Ipomaea being the most used. Chermenskaya et al. (2010) [99] studied the effect of
the species Convolvulus krauseanus Regel. and Schmalh. on three species of pest arthropods, among
which was the T. urticae Koch. From this study, which gathered the effect of extracts from 123 plant
species, they concluded that the C. Kraseanus Regel. and Schmalh. roots extract was one of the two that
showed the highest miticidal effect [together with the Ailanthus altissima (Mill.) Swingle leaf extract,
Simarubaceae], causing a mortality rate of 95.6% after seven days from the application (using the
immersion method).
3.3.12. Family Cupressaceae

Essential oils from two plants of this family were evaluated against adult females of T. urticae
Koch in the same study [55]. Oil from leaves of Cupressus macrocarpa Hartw. ex Gordon had an LD50
of 5.69 μL/L air, whereas Thuja orientalis L. leaves resulted in an LD50 of 7.51 μL/L air. The main
compounds in these essential oils were β-citronellol (35.92%) and α-pinene (35.49%), respectively.
3.3.13. Family Euphorbiaceae

The species of this family have not been well studied in terms of their pesticide properties. One of
the works carried out in this area was that of Dang et al. (2010) [100], who investigated the effect of
the dried root extract of Euphorbia kansui S.L. Liou ex S.B. Ho on the T. urticae Koch, as well as that
of two of its compounds separately: 3-O-(2,3-dimethylbutanoyl)-13-ododecanoilingenol (compound
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1) and 3-O-(2 E, 4 Z-decadienoyl)-ingenol (compound 2). Concerning the extract, they found that it
generated mortality rates of 27% and 55% at concentrations of 3 and 5 g/L, respectively. When testing
the two compounds obtained by fractionation and evaluating them on mites, they determined that
compound 1 caused mortality rates of 45% and 59% when applied at 500 and 1000 mg/L, respectively.
In contrast, compound 2 showed no acaricidal activity during the study.
On the other hand, in 2015, Numa et al. (2015) [61] published a study in which they tested the
susceptibility of T. urticae Koch females to the Cnidoscolus aconitifolius (Mill) I.M. Johnst. leaf extract
using the leaf immersion methodology merged with direct application using an airbrush. In this
study, they determined that a dose of 2000 μg/mL was the only one that did not show differences in
the positive control (based on chlorfenapyr as the active ingredient). This dose could be the most
appropriate for an extract formulation based of this plant during its potential use in the control of pests
in agricultural crops, taking into account the fact that it caused a 92% rate of mortality of mite females
in the trials.
3.3.14. Family Fabaceae

This family is well known as an aspect of human diets throughout the world. Several studies
have been carried out to evaluate the effects of their plant extracts on arthropods with very varied
results. These include the study performed by Hiremath et al. (1995) [71], who compared the activity of
methanolic extracts obtained from 21 different species of African plants against adults of the T. urticae
Koch using the leaf immersion method. The most active extracts were those obtained from the leaves,
the fruits, and the whole plant of Prosopis chinensis (Molina) Stuntz, which caused mortality rates
between 61% and 80% for the leaf extract and higher than 80% in the case of the extracts obtained from
the fruits and the whole plant. The plant oil of Millettia pinnata L. showed an LD50 of 0.004% on adult
females after four days of testing [101].
3.3.15. Family Gramineae (Poaceae)

Although this family is made up of nearly 10,000 plant species, studies involving the effect of its
plant extracts on the T. urticae Koch have focused on only some of the 55 species that make up the
Cymbopogon genus [102]. In one of these cases, Choi et al. (2004) [42] included the oil from Cymbopogon
nardus (L) Rendle within the 53 essential oils that they evaluated on the T. urticae Koch. This oil
showed a positive result, causing a mortality rate greater than 90% on adults of this phytophagous
mite. In a study of another species of genus Cymbopogon, Han et al. (2010) [68] examined the effect
of Citronella Java oil on the T. urticae Koch, evaluating its fumigant effect. To do this, they took
disc-shaped bean leaves and placed them on moistened cotton contained in Petri dishes together with
T. urticae Koch adult mites. On each Petri dish, a mesh cover was placed and placed over this was filter
paper moistened with the essential oil. Under these conditions, the LD50 found was 22.5 μg/cm3 .
3.3.16. Family Lamiaceae

The effects of plant extracts and essential oils from the species that make up this family have
been the most studied on the phytophagous mite T. urticae Koch. Among the studies reported in
the literature are, for instance, those from the species Rosmarinus officinalis L. and Salvia officinalis L.
The essential oils of these plants demonstrated effective control over populations of the T. urticae Koch
and a decrease in the number of oviposited eggs when concentrations increased [53]. In a similar way,
Choi et al. (2004) [42] performed trials using the S. officinalis L. essential oil on the same species of mite,
obtaining an adult mortality rate of 82%. In the same study, they included another species from the
family Lamiaceae—Mentha spicata L.—from which they obtained the essential oil that was evaluated
on the T. urticae Koch. As a result, the mortality rate of these arthropods in the adult stage was 81%.
On the other hand, Rasikari et al. (2005) [103] carried out a screening of the leaf extracts of
67 species of plants belonging to the Lamiaceae family. They were evaluated on the T. urticae Koch,
which were applied by direct contact with the Potter Tower to bean leaves kept in Petri dishes with
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cotton. From the extracts tested, 14 had a moderate to acute toxic effect on mites. From these, extracts
obtained from the plants Clerodendrum traceyi F. Muell., Premna serratifolia L., Ceratanthus longicornis
(F.Muell.) G. Taylor, Plectranthus habrophyllus P.I. Forst, and Plectranthus sp. Hann caused a 100%
mortality rate, whereas the extracts of Gmelina leichardtii F.Muell. & Benth, Premna acuminata R. Br.,
Viticipremna queenslandica Munir, Plectranthus diversus S.T. Blake, Plectranthus glabriflorus P.I. Forst, and
Plectranthus suaveolens S.T. Blake caused mortality rates that were between 90% and 99%.
In 2006, a study performed by Miresmailli et al. (2006) [104] was published. In that investigation,
they tested the effect of the R. officinalis L. essential oil on the T. urticae Koch. For that, they took two
different populations of mites, one from bean plants and another from tomato plants. For the tests,
they used five different concentrations (2.5, 5, 10, 20, 40, and 80 mL/L) of the essential oil diluted in
methanol and water (70:30 v/v). In order to evaluate the mortality rates of mites, they took 3 mm
disc leaves within Petri dishes, to which they applied 20 μL of the treatment solution. Once dried
at room temperature, they placed five adult females on the leaves and kept them at a temperature
of 26 ± 2 ◦ C, a relative humidity (RH) between 55% and 60%, and a photoperiod of 16:8 (light:dark).
From these assays, they determined that the LC50 for the females maintained on bean plants was 10
mL/L, while for the females kept on tomato plants, it was 13 mL/L. Moreover, with a concentration of
20 mL/L, a mortality of 100% of females produced in bean plants was obtained, whereas a 40 mL/L
concentration was necessary before females on the tomato plants reached total mortality (100%).
Additionally, Miresmailli et al. [104] identified the components of R. officinalis L. essential oil using
GC–MS by column chromatography and tested them individually on the T. urticae Koch. In the case of
mites reared on bean plants, two compounds revealed a significant toxicity—1,8-cineol and α-pinene
(with 88% ± 4.8% and 32% ± 4.8% mortality, respectively)—whereas for mites raised on tomato plants,
the same two compounds were those that revealed a significant toxicity. The resulting values were
80% ± 6.2% and 72% ± 4.8% for 1,8-cineol and α-pinene, respectively.
In a similar study, Çalmaşur et al. (2006) [105] tested the effect of the vapors of three essential oils
from Micromeria fruticosa L., Nepeta racemosa L., and Origanum vulgare L. on nymphs and adults of the
T. urticae Koch and adults of the Bemisia tabaci Gennadius, finding the highest mortality rates (96.7%,
95%, and 95%, respectively, for T. urticae Koch, and 100% for B. tabaci Gennadius) when using doses
of 2 μL/L of air at 12 h of exposure. Han et al. (2010) [68] also studied several essential oils obtained
from species of this family. To do this, they evaluated its fumigant effects on the T. urticae Koch and,
as a result, obtained LD50 s of 22.7, 22.8, 23.7, 38.8, 39.5, and 63.7 μg/cm3 for Thymus vulgaris L., Mentha L.
piperita, Mentha pulegium L., Mentha spicata L., Ocimum basilicum L., and Salvia officinalis L., respectively.
In 2012, Afify et al. (2012) [89] tested the acaricidal activity of Majorana hortensis Moench extract
on the T. urticae Koch. The LD50 values obtained for adults and eggs in the trial were 1.84% and 6.26%,
respectively. In the study, they identified the main compounds of M. hortensis Moench by means of
gas chromatography–mass spectrometry as terpinen-4-ol (23.86%), p-cymene (23.40%), and sabinene
(10.90%)—the main compounds for this species. In the same year, Attia et al. (2012) [88] tested the
effect of the essential oil of Mentha pulegium L. on the T. urticae Koch, obtaining a mortality rate of 91%
in larvae and adult females. The same essential oil was evaluated by Choi et al. (2004) [42] on the same
mite species, in which a mortality rate higher than 90% was obtained. Within the same experiment,
they analyzed the effect of the essential oil of the Mentha piperita L., in which the mortality rate also
exceeded 90%. On the other hand, Amizadeh et al. (2013) [84] studied the fumigant effect of the
essential oil obtained from leaves of the Satureja sahendica Bornm. on eggs and adult females. The LD50
obtained for females was 0.98 μL/L, while it was of 0.54 μL/L for eggs.
3.3.17. Family Meliaceae

The insecticidal properties of plants belonging to the family Meliaceae have been studied
extensively [106]. For this reason, Ismail (1997) [107] evaluated the relative toxicity of the extracts of
Melia azedarach L. and some synthetic acaricides against recently hatched larvae of the T. urticae Koch
and third-instar larvae of the predatory beetle, Stethorus gilvifrons Mulsant. The methanolic extract
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of the plant was the most effective among the tested products, followed by the extracts of acetone
and petroleum ether. The toxicity of the plant material obtained was less active against the predator
compared to the effect it had on the two-spotted spider mite, in which a decrease in fecundity was
also observed. The study of the joint action of the products also revealed a strong synergy in the
bromopropylate mixture with the methanolic extract of the M. azedarach L. Interestingly, this mixture
demonstrated no effect on the predator.
In a similar way, Brito et al. (2006) [37] tested the toxicity of different commercial products
based on one of the plants with the highest pesticide potential, the Neem (Azadirachta indica A. Juss.).
It was tested not only on the T. urticae Koch but also on its predators, Euseius alatus DeLeon and
Phytoseiulus macropilis Banks. In this study, they found that the formulation of the product Neemseto
(1%) was the one that obtained the best result on the T. urticae Koch by topical contact. In the same
way, they tested the product at different concentrations (0.25%, 0.5%, and 1.0%) and found that the
product had a repellent effect on T. urticae Koch and E. alatus DeLeon; however, it did not affect the
P. macropilis Banks. Additionally, the Neemseto exhibited an important reduction in T. urticae Koch
fecundity, but on the predatory mites, a significant decrease was only observed when mites were
exposed to the highest concentrations. This shows that this product can be a promising option for the
management of the two-spotted spider mites within integrated pest management schemes given its
relative compatibility with predatory mites.
3.3.18. Family Myrtaceae

T. urticae Koch toxicity studies involving these plants have had varying results. First, Choi et al.
(2004) [42] determined that the Eucalyptus citriodora Hook’s essential oil is capable of causing a mortality
rate of more than 90% on T. urticae Koch adults. This essential oil was also tested by Han et al.
(2010) [68] on the same mite species using the vapor-phase mortality bioassay; they found similar
fumigant activity results to those obtained previously [42]. The test performed consisted of placing
3 cm diameter bean leaf discs on wet cotton inside Petri dishes, each with 20 adult mite individuals [68].
On each Petri dish, they installed a mesh cover on which a filter paper moistened with the essential oil
at the evaluated concentrations was placed (after drying for two minutes). From these experiments,
they estimated an LD50 of 19.3 μg/cm3 .
On the other hand, they also wanted to evaluate the fumigant effect of Syzygium aromaticum
(L.) Merr. & L.M. Perry essential oil. Within the study, they found an LD50 value of 23.6 μg/cm3 on
T. urticae Koch adults. In 2011, Afify et al. [108] tested the activity of six extracts of Syzygium cumini (L.)
Skeels at three different concentrations (75, 150, and 300 μg/mL) on the T. urticae Koch. The highest
mortality rates were obtained with the ethanolic extract (98.5%), followed by the hexane extract (94%)
and the ether-ethyl acetate extract (90%). The LD50 values obtained were 85, 101, 102, and 98 μg/mL,
respectively. The same group of researchers in 2012 conducted a study to measure the acaricidal
activity of Eucalyptus sp. on the same mite [89]. The LD50 values obtained for adults and eggs in the
assay were 2.18 and 7.33 μg/mL, respectively.
In 2013, Amizadeh et al. [84] also tested the fumigant effect of some essential oils of this family,
including those obtained from leaves and fruits of the Eucalyptus microtheca F. Muell. on both eggs
and adult females of the T. urticae Koch. For the tests, mites were placed on bean leaf discs laid in
plastic containers in which an oil-impregnated filter paper was held without coming into direct contact
with leaf discs or mites. The LD50 s on the adult females were 1.52 μL/L and 5.7 μL/L for the extracts
of leaves and fruits, respectively, while for eggs, they were 0.56 μL/L and 2.36 μL/L for leaf and fruit
extracts, respectively.
3.3.19. Piperaceae Family

There have been few studies carried out concerning the effects of extracts of species of the
Piperaceae family on the T. urticae Koch—particularly considering the fact that they have focused
on very few species of the genus Piper, which has more than 1000 species [109]. One of those
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studies was developed by Araújo et al. (2012) [110], who reported acaricidal and repellent activity
of the essential oils obtained from Piper aduncum L. leaves and its components separately on the
T. urticae Koch. The repellent activity was attributed to the components (E)-nerolidol, α-humulene,
and β-caryophyllene, while the toxicity was attributed to β-caryophyllene. The extracts and their
components exhibited a better performance in fumigation than in contact.
3.3.20. Family Ranunculaceae

In general terms, the toxicity studies of extracts of these plants used on the T. urticae Koch have not
been very satisfactory. A case demonstrating this is the study conducted by Derbalah et al. (2013) [91],
which found that the black cumin seeds (Nigella sativum L.) extract showed a low toxic effect on the
T. urticae Koch, with an LD50 of 708.57 ppm. However, some species of this family—such as Aconitum
soongaricum Stapf and Clematis orientalis L.—have shown toxic effects on the T. urticae Koch with
mortality rates ranging between 50% and 80% of mites [99].
3.3.21. Family Rutaceae

In 2005, Tewary et al. [111] tested two concentrations (5000 and 10,000 ppm) of the Zanthoxylum
armatum DC. leaf extract on the arthropods H. armigera Hübner, P. xylostella L., T. urticae Koch,
and A. craccivora Koch, with mortality rates of 46% at 10,000 ppm in the H. armigera Hübner, 42% at
10,000 ppm in the P. xylostella L., 36% and 39% at 5000 and 10,000 ppm, respectively, in the T. urticae
Koch, and 30% and 65% at 5000 and 10,000 ppm, respectively, in the A. craccivora Koch. On the other
hand, Attia et al. (2012) [88] also included plants of the Rutaceae family, since they proved the effect
caused by the essential oil of Haplophyllum tuberculatum (Forssk.) A. Juss. on the T. urticae Koch,
obtaining a mortality of 93%.
Da Camara et al. (2015) [67] demonstrated that essential oils obtained from the epicarp of
pear orange fruits (Citrus sinensis Osbeck var. Pera) and the lime orange (Citrus aurantium L.) had
repellent effects against the T. urticae Koch, with very similar repellency results to those obtained with
eugenol. Using mass spectrometry, 27 compounds were idenitified both in C. sinensis Osbeck and in
C. aurantium L., which corresponded to 98.1% and 98.9% of the total constituents of the two extracts,
respectively. This demonstrated that the major compound in the two essential oils was d-limonene.
Within this study, the authors determined that all the identified compounds were responsible for
the repellency.
3.3.22. Family Santalaceae

Within this family, Roh et al. (2011) [112] studied the effect of Santalum L. sp. essential oil on
the T. urticae Koch using the leaf immersion method. Through this methodology, they found that the
mortality rate of mites was 87.2% ± 2.9%. Additionally, they noticed an oviposition decrease of 89.3%
on leaves treated with oil. Subsequently, they evaluated a mixture of α and β–Sandalool—the two
main compounds of Santalum L. sp.—on the T. urticae Koch and obtained a mortality of 85.5% ± 2.9%
and a decrease of 94.7% in fecundity.
3.3.23. Family Scrophulariaceae

The toxic effects of Scrophulariaceae plants on the T. urticae Koch have been less studied
than plant species of other groups. Within the investigations carried out in this regard,
Khambay et al. (1999) [113] studied the effect of two compounds of Calceolaria andina Benth extract
with recognized insecticidal activity—2-(1,1-dimethylprop-2-enyl)-3-hydroxy-1,4-naphthoquinone
(compound 1) and 2-acetoxy-3-(1,1-dimethylprop-2-enyl)-1,4-naphthoquinone (compound 2)—on 29
pest species, including the T. urticae Koch. The LD50 s for this species were 80 ppm and 30 ppm for
each compound, respectively. The two cases were evaluated using the micro-immersion method.
Additionally, they performed the same test on individuals from a population that showed resistance
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to chlorpyrifos and bifenthrin using the same compounds of C. andina Benth extract, thus obtaining
LD50 s of 44 ppm and 33 ppm for compounds 1 and 2, respectively.
3.3.24. Family Simarubaceae

The toxicity of plant extracts from species of this family on the tetraniquid mite T. urticae Koch
have not been well studied. Among the studies accomplished, Latif et al. (2000) [114] tested the
extract from Quassia sp. aerial parts on this mite at a concentration of 10,000 ppm, finding acaricidal
activity. Subsequently, they identified the quassinoid Chaparinone compound and tested it separately,
obtaining an LC50 of 47 ppm. Chermenskaya et al. (2010) [99] evaluated extracts from 123 different
plant species on the T. urticae Koch, Frankliniella occidentalis Pergande and Shizaphis graminum Rondani,
using the leaf immersion method. Within these extracts, one that demonstrated a high acaricidal effect
was obtained from the Ailanthus altissima (Mill.) Swingle leaves, which caused a mortality rate of 97.4%
after 7 days of evaluation.
3.3.25. Family Solanaceae

Although most studies involving plant extracts tested on the T. urticae Koch have focused on
assessing the effects on mortality and fecundity, those involving the Solanaceae family have been mostly
dedicated to determining the repellent effects of certain extracts. Such is the case of the study conducted
by Snyder et al. (1993) [115]. They isolated dihydrofarnesoic acid as one of the phytoconstituents
in trichomes of Lycopersicon hirsutum Dunal, and its repellent effect on the phytophagous mite was
then evaluated. For this purpose, 10 μL of the extract was applied to a filter paper separated by
1.5 cm from another similar filter, which was impregnated with 10 μL of hexane. Once the solvent
was evaporated, a strip of filter paper was positioned to connect the two filter papers, and a mite was
placed in the middle of the paper bridge to evaluate its displacement preference. This process was
performed with approximately 40 adult females. According to the obtained results, they concluded that
dihydrofarnesoic acid exhibited a repellent activity against the T. urticae Koch. Similarly, Antonious et al.
(2006) [116] also evaluated toxic and repellent effect of the fruit extracts of Capsicum chinense Jacq.,
Capsicum frutescens L., Capsicum baccatum L., Capsicum annuum L., and Capsicum pubescens Ruíz & Pav.
In their results, they determined that the highest mortality rate (45%) occurred when using the extract
of the C. annuum L., while the extracts of the fruits of the C. baccatum L. and C. annuum L. caused
repellence on mites.
Extracts of leaves and seeds of the Datura stramonium L. were used by Kumral et al. (2009) [5]
to evaluate their acaricidal, repelling, and deterrent effects on oviposition over T. urticae Koch adults at
167.25 mg/L and 145.75 mg/L (for leaves and seeds, respectively). For these tests, they used a Potter
Tower in order to place the mites on leaf discs contained in Petri dishes. These concentrations caused
98% and 25% of the mortality, respectively, for the two concentrations after 48 h of application. Through
a simple logistic regression analysis, they determined that an increase in the leaf extract dose caused
a significant increase in mite mortality, while the effect of increasing the dose of the seed extract was
not significant. Based on Probit analysis, they estimated that the lethal dose (LD50 ) with the leaf extract
was 70.59 mg/L. According to the Pearson X2 test, they concluded that mites showed a strong tendency
to flee from areas treated with leaf and seed extracts to untreated areas.
3.3.26. Verbenaceae Family

In this family, a highlighted study was conducted by Cavalcanti et al. (2010) [117], in which they
carried out a characterization of the essential oils of the Lippia sidoides Cham. (Verbenaceae) by GC–MS
and tested their acaricidal activity on T. urticae Koch females. They concluded that the compounds
thymol and carvacrol—as well as the essential oil of L. sidoides Cham.—showed a promising miticidal
activity against this mite.
141
Plants 2019, 8, 272
3.4. Additional Studies with Isolated Compounds Obtained after Plant Extract Fractionation
As with essential oils and plant extracts, a considerable number of their isolated constituents have
also been tested on the T. urticae Koch. For example, Lee et al. (1997) [118] studied the insecticidal
and the acaricidal effects of several monoterpenes and their possible phytotoxicity in maize plants
that served as hosts of the Diabrotica virgifera virgifera LeConte, T. urticae Koch, and Musca domestica L.
Twenty-nine compounds belonging to different chemical classes were tested against the T. urticae Koch
by means of the leaf immersion method.
These tests used: the alcohols carveol, carvomentenol, citronellol, geraniol, 10-hydroxygeranol,
isopulegol, linalool, menthol, perilyl alcohol, aterpineol, and verbenol; the phenols carvacrol,
eugenol, and thymol; the ketones (−)-carvone, (+)-carvone, (+)-fenchone, menthone, pulegone,
tuyone, and verbenone; the aldehydes citral and citronellal; citronelic acid; ether 1,8-cineol; and the
hydrocarbons limonene, α-terpinene, and y-terpinene.
All compounds were tested in water with Triton X-100 as a wetting agent at 10,000 and 1000 ppm,
and the activity was evaluated 24, 48, and 72 h after the treatment. The toxicity varied depending on
the concentrations and the exposure times. All of the monoterpenes tested—except for 1,8-cineole,
10-hydroxygeraniol, aterpineol, verbenol, and verbenone—caused a 100% mortality rate at the
highest concentration after 24 h. However, carvacrol was the most effective compound in the lowest
concentrations, followed by citronellol.
On the other hand, geraniol produced a 100% rate of mortality, while its 10-hydroxy geraniol
analogue exhibited a 0% mortality rate. During the trial, a longer exposure time increased acaricidal
effects. Alternately, the most effective monoterpenoids (carvacrol, carvomenthenol, carvone, citronellol,
eugenol, geraniol, perilyl alcohol, 4-terpineol, thymol) were evaluated separately in more detailed
tests. From these compounds, carvomentenol and 4-terpineol demonstrated greater acaricidal activity
(LC50 s = 59 and 96 ppm, respectively).
In another study, Martínez et al. (2005) [119] examined the effect of azadirachtin at 64 and
128 ppm on different biological parameters of the T. urticae Koch, such as longevity, fecundity, fertility,
and offspring development. The tests were performed on bean leaf discs in Petri dishes using the
Potter Tower. The results found that this compound affected mortality and fecundity but exhibited
no effects on fertility and offspring development. In a later analysis of life table, they determined
that, with the application of azadirachtin at 80 ppm, the adult survival rate was reduced to 50%.
Duso et al. (2008) [34] also tested the toxicity of Azadirachtin on the T. urticae Koch. In that case,
the micro-immersion bioassay methodology was implemented using a concentration of 4.5 g of active
ingredient/L on T. urticae Koch females. For those conditions, the mortality rate obtained was 86.49%.
Similarly, Han et al. (2011) [120] tested some constituent compounds of the Eucalyptus citriodora
Hook extract and other plants on resistant and susceptible acaricidal T. urticae Koch females. Among
them, those that showed the highest toxicity were menthol (LD50 of 12.9 μg/cm3 ) and citronellium
acetate (LD50 of 16.8 μg/cm3 ), evaluated on females susceptible to acaricides. Other compounds such
as β-citronellol, citral, geranyl acetate, and eugenol also demonstrated a high toxic activity, with LD50 s
between 21.7 μg/cm3 and 24.6 μg/cm3 . When comparing the mortality results obtained for both
susceptible and acaricide-resistant mites, the researchers estimated that they were very similar to each
other and therefore evidenced that the mechanisms of action of the components of the essential oil and
of the synthetic acaricides are different and do not present processes that promote cross-resistance.
One year later, Akhtar et al. (2012) [121] studied the effect of eight quinones on the T. urticae
Koch—Myzus persicae Sulzer, Myzocallis walshii Monell, and Illinoia liriodendri Monell—using the leaf
immersion method. The compound plumbagine was the one that exhibited the greatest activity on
the mite, with an LC50 of 0.001%. Marčić and Međo (2014) [122] also performed experiments with
secondary metabolites from plants. In their study, they tested a combination of oximatrin and psoralen
(0.2% and 0.4%, respectively) on the T. urticae Koch and measured acute toxicity and repellency.
The applications were made on bean leaves with a Potter Tower, and the subsequently calculated
LD50 s were 55.49, 52.68, 6.88, 13.03, and 8.8 μL/L for eggs, females that had not oviposited, larvae,
142
Plants 2019, 8, 272
protonymphs, and deutonymphs, respectively. Additionally, they noticed that, in preferential tests
on the leaves, the mites tended to be located in the middle of the untreated leaf, at which point the
oviposition was greater.
The same authors also tested compounds from the Neem extract (azadirachtin-A) on females of
the two-spotted spider mite [123]. For this case, they introduced bean leaf discs inside Petri dishes
with moistened cotton and made applications of the product using a Potter Tower in the middle of the
leaf. They concluded that females preferred to be located in the middle of the leaf not treated with the
product and, in the same way, they observed that oviposition was higher in females that were located
in the untreated areas.
4. Conclusions
In conclusion, 458 records of plant species from 67 plant families (listed in this survey) have
repellent or acaricidal effects against the T. urticae Koch under laboratory conditions. The efficacy is
available at different levels depending on species, extractions (extract or essential oils), plant parts
used, and concentrations of test extract/essential oil. Among the most studied botanical families
for this purpose are plants from Lamiaceae, Asteraceae, Myrtaceae, and Apiaceae taxons. Extracts
from species including Celosia Trygina L., Cassia mimosoides L., Clome viscosa L., Boscia senagalensis
(Pers.) Lam. Ex. Poir., Cobretum micranthum G. Don, Ipomaea asarifolia (Desr.) Roem. and Schult.,
Cnidoscolus aconitifolius (Mill) I.M. Johnst., Azadirachta indica A. Juss., Syzygium cumini (L.) Skeels,
Papaver rhoeas L., Plantago major L., Ailanthus altissima (Mill.) Swingle, and Capsicum annuum L. exhibited
better acaricidal properties with efficacies between 90% and 100% at a concentration range between
0.2% and 1%—comparable to some commercial acaricides. LD50 values can be found below 20 μg/mL or
5 μL/L air. Thus, botanical-based preparations can be a good source of effective acaricidal preparations
either as extracts or as essential oils. Although the information herein presented only concerns a basic
screening of the acaricidal efficacy of botanicals at laboratory (in vitro) levels, several plants could
be considered for future research on field evaluations or as sources of acaricide compounds. In this
sense, several compounds such as azadirachtin, 10-hydroxygeraniol, terpineols, verbenol, verbenone,
carvacrol, plumbagine, linalool, and citral, among others, have been isolated as bioactive acaricidal
compounds. In future studies, attention may be focused on acaricidal activity rather than on repellent
properties to facilitate two-spotted mite control. However, formulations and application rates similar
to those used by farmers must be assessed in order to achieve more predictive results in further field
experiments. Sublethal effects must also be relevant in future research, since those effects could produce
other subsequent problems or benefits in the control of mites. Finally, more compatibility studies
and phytotoxicity as well as extract stability, extraction standardization, and field formulations are
required to ensure good results on integrated pest management programs for T. urticae Koch control
using effective botanicals.
Author Contributions: Conceptualization, R.A.R., D.R., E.C.-B.; methodology, R.A.R.; validation, R.A.R.; formal
analysis, R.A.R., D.R., E.C.-B.; investigation, R.A.R.; resources D.R., E.C.-B.; data curation, R.A.R., D.R., E.C.-B.;
writing—original draft preparation, R.A.R.; writing—review and editing, D.R., E.C.-B.; supervision, D.R., E.C.-B.;
project administration, E.C.-B.; funding acquisition D.R., E.C.-B.
Funding: This research was funded by the Vicerrectoria de Investigaciones at UMNG, grant number
INV-CIAS-1788-validity 2016.
Acknowledgments: Authors thank Universidad Militar Nueva Granada (UMNG) for the financial support
through the project INV-CIAS-1788.
143
Appendix A
Table A1. Compilation of reported studies using plant extracts and essential oils against T. urticae Koch under laboratory conditions.
T. urticae Identified
Family Plant Species Source Concentration Bioassaya Effect on T. urticae Koch Ref.
Koch Stage Compounds
Plants 2019, 8, 272
Amaranthaceae Amaranthus viridis L. Whole plant extract 5000 ppm G Adults Mortality between 40 and 60% - [108]
Amaranthaceae Amaranthus viridis L. Whole plant extract 2500 ppm G Adults Mortality between 40 and 60% - [108]
Amaranthaceae Blepharis linariifolia Pers. Whole plant extract 5000 ppm G Adults Mortality between 61 and 80% - [108]
Amaranthaceae Blepharis linariifolia Pers. Whole plant extract 2500 ppm G Adults Mortality between 61 and 80% - [108]
Amaranthaceae Blepharis sp. Whole plant extract 5000 ppm G Adults More than 80% of mortality - [108]
Amaranthaceae Blepharis sp. Whole plant extract 2500 ppm G Adults Mortality between 61 and 80% - [108]
Amaranthaceae Celosia Trygina L. Whole plant extract 5000 ppm G Adults More than 80% of mortality - [108]
Amaranthaceae Celosia Trygina L. Whole plant extract 2500 ppm G Adults More than 80% of mortality - [108]
Chenopodium
Emulsifiable
Amaranthaceae ambrosioides Mosyakin 0.50% A,C Adults and eggs 94.7% of mortality - [41]
Concentrate
et Clemants
Chenopodium quinoa 6–9% w/v Adult females Mortalities ranged
Amaranthaceae Seeds extract E,F - [89]
Willd. [1.24% w/v (LD50 )] and nymphs from 30% to 99%
Kochia scoparia (L.) 98.13% (chloroform
Amaranthaceae - A,E,H Adult females 92.58% of mortality - [52]
Schrad. extraction)
Mortalities of 65% (larvae) and
144
Amaryllidaceae Allium cepa L. Essential oil - D Larvae and adults. - [88]
67% (adults)
Amaryllidaceae Allium cepa L. Peel fruit extract 1% G Adult females Mortality between 0 and 20% - [99]
Allium galanthum
Amaryllidaceae Whole plant extract 1% G Adult females Mortality between 20 and 50% - [99]
Kar. & Kir.
Amaryllidaceae Allium obliquum L. Whole plant extract 1% G Adult females Mortality between 50 and 80% - [99]
Amaryllidaceae Allium sativum L. - 7.2 g/L A,G Adult females LD50 - [78]
Amaryllidaceae Allium sativum L. Bulb extract 7.49 and 13.5 mg/L E,F Adult females LD50 and LD90 (respectively) - [77]
Amaryllidaceae Allium sativum L. Essential oil - D Larvae and adults - [88]
61% (adults)
Alkaloidal ethanolic
0.2%. 0.36% and 1.5%
Amaryllidaceae Pancratium maritimum L. extract and bulb - LD50 - [76]
respectively
essential oil
Ungernia severtzovii
Amaryllidaceae Root extract 1% G Adult females Mortality between 20 and 50% - [99]
Regel
Anacardiaceae Cotinus coggygria Scop. Essential oil - D Larvae and adults - [88]
58% (adults)
Anacardiaceae Cotinus coggygria Scop. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Anacardiaceae Pistacia lentiscus L. Essential oil - D Larvae and adults - [88]
23% (adults)
Annonaceae Annona glabra L. Seed extract 1000 ppm D,G Eggs No effects - [80]
Table A1. Cont.
T. urticae
Family Plant Species Source Concentration Bioassaya Effect on T. urticae Koch Identified Compounds Ref.
Koch Stage
Cananga odorata (Lam.)
Annonaceae Essential oil 0.1% G Adult females 24.2% of mortality - [112]
Plants 2019, 8, 272
Hook.F. & Thomson

α-pinene (0.41% leaves,
17.18% fruits), β-pinene
Leaves and fruits (45.59% fruits), cubenol
Annonaceae Xilopia sericea A.St.-Hill. 4.08 μL/L C Adult females LD50 [44]
essential oils (57.43% leaves), myrcene
(9.13% fruits), between
others
Apiaceae Ammi visnaga Seed extract 17 μg/cm2 D,I Eggs LD50 Kheline and visnagine [65]
19 × 10−3 μL/mL
Apiaceae Carum carvi L. Essential oil J Adults 100% of mortality - [42]
of air.
Apiaceae Carum carvi L. Essential oil 22.4 μg/cm3 C,J Adults LD50 - [68]
570 ppm of
Essential oils mixed
essential oil and
Apiaceae Carum carvi L. with Fatty acid E Adults 83.4% of mortality - [83]
2478 ppm of
potassium salts
potassium salts
Flowers and Mortalities of 95.18% and
Apiaceae Conium maculatum L. 10–50% A,B Adult females - [60]
leaves extract 81.11%, respectively
145
19 × 10−3 μL/mL
Apiaceae Coriandrum sativum L. Essential oil J Adults 92% of mortality - [42]
of air
Essential oil α-pinene (29.1%), limonene
Apiaceae Cuminum cyminum L. 3.74 μL/L air C,J Adult females LD50 [85]
from seeds (22%), 1,8-cineole (17.9%)
Mortalities of 5% (larvae)
Apiaceae Daucus carota L. Essential oil - D Larvae and adults - [88]
and 3% (adults)
Deverra scoparia 1.79 and α-pinene, Δ3 -carene and
Apiaceae Essential oil E Young females LD50 and LD90 , respectively [43]
Coss. & Durieu 3.2 mg/L terpinen-4-ol
Deverra scoparia Mortalities of 98% (larvae)
Apiaceae Essential oil - D larvae and adults - [88]
Coss. & Durieu and 97% (adults)
6.98 and Eggs and adults, β-pinene (50.1%), α-pinene
Apiaceae Ferula gumosa Boiss. Essential oil C,J LD50 [86]
6.52 μL/L air respectively (14.9%), δ-3-carene (6.7%)
5.75 μL/L
Seed essential
Apiaceae Foeniculum vulgare Mill. (females), J Eggs and adults LD50 - [84]
oil vapors
1.17 μL/L (eggs)
Apiaceae Foeniculum vulgare Mill. Essential oil 1.17% E Adults LD50 - [83]
Heracleum persicum Fruit essential 3.15 μL/L (females)-
Apiaceae J Eggs and adults LD50 - [84]
Desf. Ex. Fisch. oils vapors 1.53 μL/L (eggs)
Table A1. Cont.
Heracleum persicum
Apiaceae Essential oil 1.53% E Adults LD50 - [83]
Plants 2019, 8, 272
Desf. Ex. Fisch.

Isolation of
1.9 and 42.7 μg/mL,
LD50 (chronic toxicity after 5 isofuranodiene and
Apiaceae Smyrnium olusatrum L. Inflorescence extract respectively for D Adult females [87]
days) germacrone,
isolated compounds
separately evaluated
Vinca erecta
Apocynaceae Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Regel & Schmalh
Apocynaceae Vinca minor L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Araceae Arum korolkovii Regel Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Calotropis gigantea
Asclepiadaceae Leaf extract 5000 ppm G Adults Mortality between 61 and 80% - [71]
W.T. Aiton
Calotropis gigantea
Asclepiadaceae Leaf extract 2500 ppm G Adults Mortality between 40 and 60% - [71]
W.T. Aiton
1.208% v/v (leaf
Essential oil from dipping) and Piperitone (12.8%),
Asteraceae Achillea mellifolium L. G,J Adult females LD50 [64]
aerial part 1.801 μL/L air p-cymene (10.6%)
(fumigation)
146
Asteraceae Achillea millefolium L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Acroptilon repens (L.)
Asteraceae Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
DC.
Ajania fastigiata
(C. Winkler) Poljakov
Anaphalis rosea-alba
Krasch.
Asteraceae Anthemis nobilis L. Essential oil 19 × 10−3 μL/mL of air J Adults 69% of mortality - [42]
Mortalities of 92.37% and
Asteraceae Anthemis vulgaris L. Flower extract 7–50% A,B Adult females - [60]
92.34%, respectively
Asteraceae Anthemis vulgaris L. Leaf extract 13–50% A,B Adult females - [60]
Asteraceae Artemisia absinthium L. Essential oil 19 × 10−3 μL/mL of air J Adults 97% of mortality - [42]
Asteraceae Artemisia absinthium L. Essential oil 0.043 mg/cm2 A - LD50 - [45]
Asteraceae Artemisia absinthium L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Artemisia aschurbajewii
C. Winkl.
Artemisia cinae O. Berg &
Asteraceae Leaf extract 1326.53 ppm A,B Adult females LD50 - [60]
C.F. Schmidt ex. Pljakov
Table A1. Cont.
Artemisia compacta
Plants 2019, 8, 272
Fisch. Ex. Besser

Asteraceae Artemisia dracunculus L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Leaves (acetone
Asteraceae Artemisia judaica L. 0.56 μg/mL C,G Adult females LD50 - [39]
extract)
Asteraceae Artemisia panciflora Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Asteraceae Artemisia vulgaris L. Leaf extract 15-50% A,B Adult females - [60]
Asteraceae Artemisia vulgaris L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Asteraceae Calendula officinalis L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
α-basabolol oxide
(35.25%), Trans
Asteraceae Chamomilla recutita L. Essential oil 0.65–1.17% C Adults ggs LD50 [89]
β-farersene
(7.76%)
Asteraceae Chrisanthemum coronarium L. Essential oil - D Larvae and adults - [88]
93% (adults)
Asteraceae Handelia trichopylla Heimerl Aerial part extract 1% G Adult females Mortality between 0 and 20%. - [99]
LD50 and side-effect over
147
Asteraceae Hertia cheirifolia (L.) Kuntze Essential oil 3.43 mg/L E Adult females [46]
fecundity
Asteraceae Hertia cheirifolia (L.) Kuntze Essential oil - D Larvae and adults - [88]
89% (adults)
Hieracium dschirgalanicum
E. Nikit.
Asteraceae Inula helenium L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Asteraceae Jurinea capussi Franch. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Lamyropappus schakaptaricus
Knorr & Tamamsch.
Asteraceae Matricaria chamomilla L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Matricaria matricarioides
(Less.) Porter
Asteraceae Matricaria recutita L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Pseudoglossanthis litwinowii
(Tzvel.) R. Kam.
Pyrethrum alatavicum
O. & B. Fedtsch.
Pyrethrum branchanthemoides
R. Kam. & Lazkov
Pyrethrum
cinerariifolium Trev.
Table A1. Cont.
T. urticae
Koch Stage
Pyrethrum sovetkinae
Plants 2019, 8, 272
Kovalevsk
Pyrethrum sussamyrense
Asteraceae Root extract 1% G Adult females Mortality between 0 and 20% - [99]
Lazkov
Santolina africana LD50 and side-effect over
Asteraceae Essential oil 2.35 mg/L E Adult females Terpinen-4-ol (54.96%) [46]
Jord. & Fourr. fecundity
Santolina africana Mortalities of 77% (larvae) and
Asteraceae Essential oil - D Larvae and adults - [88]
Jord. & Fourr. 68% (adults)
Senecio saposhnikovii
Krasch et. Schipcz.
Seriphidium herba-album Mortalities of 54% (larvae) and
Asteraceae Essential oil - D Larvae and adults - [88]
(Asso) Sojak 37% (adults)
Asteraceae Tagetes minuta L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Tanacetopsis ferganensis
Kovalevsk
Tanacetopsis setacea
Kovalevsk
Tanacetopsis submarginata
148
Kovalevsk
Tanacetum boreale
Fisch. Ex. DC.
Tanacetum pseudoachillea
C. Winkl.
Asteraceae Tanacetum vulgare L. Essential oil 4% A - 75.6% of mortality - [45]
Asteraceae Thitonia diversifolia Hemsl. Methanolic extract 150 μg/cm3 D Adult females Mortality less than 50% - [92]
Tripleurospermum inodorum
Sch. Bip.
Asteraceae Xanthium strumarium L. Fruit extract 9-50% A,B Adult females - [60]
Asteraceae Xanthium strumarium L. Leaf extract 11-50% A,B Adult females - [60]
Berveridaceae Berberis iliensis Popov Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Bignonianceae Jacaranda obtusifolia Bonpl. Leaf extract 0.06% C,G Adult females Mortality of 64.4% [124]
Boraginaceae Echium vulgare L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Shikonin derivatives
LD50 (chronic toxicity after (naphthoquinones),
Boraginaceae Onosma visianii Clem. Root extract 2.6 μg/mL D Adult females [93]
5 days) i.e., isobutylshikonin
and isovalerylshikonin
Table A1. Cont.
Armoracia rusticana G.
Plants 2019, 8, 272
Brassicaceae Gaertn., B. Mey. & Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Scherb.
Barbarea vulgaris
Brassicaceae Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
W.T. Aiton
Brassicaceae Capsella bursa-pastoris L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Brassicaceae Cardaria repens Schrenk Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Brassicaceae Lepidium latifolium L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Burseraceae Boswellia carterii Birdw. Essential oil 0.1% G Adult females 24.8% of mortality - [112]
Commiphora myrrha
Burseraceae Essential oil 0.1% G Adult females 22.8% of mortality - [112]
(Nees) Engl.
Fresh and old resin Mortalities of 79.6% (fresh
Burseraceae Protium bahianum Daly - J Adult females - [97]
essential oils resin) and 59% (old resin)
Caesalpiniaceae Cassia mimosoides L. Leaf extract 5000 ppm G Adults More than 80% of mortality - [71]
Caesalpiniaceae Cassia mimosoides L. Leaf extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Caesalpiniaceae Cassia occidentalis L. Whole plant extract 5000 ppm G Adults Mortality between 61 and 80% - [71]
Caesalpiniaceae Cassia occidentalis L. Whole plant extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Caesalpiniaceae Cassia tora L. Whole plant extract 5000 ppm G Adults More than 80% of mortality - [71]
149
Caesalpiniaceae Cassia tora L. Whole plant extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Codonopsis clematidea
Campanulaceae Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Schrenk
β-myrcene
Essential oil from (18.5%),
Cannabaceae Cannabis sativa L. 0.10% G Adult females 83.28% of mortality [98]
panicles trans-caryophyllene
(35.6%)
Aerial part and root
Cannabaceae Cannabis sativa L. 1% G Adult females Mortality between 50 and 80% - [99]
extracts
Cannabaceae Humulus lupulus L. Flower extract 5-50% A,B Adult females - [60]
Cappandaceae Clome viscosa L. Whole plant extract 5000 ppm G Adults More than 80% of mortality - [71]
Cappandaceae Clome viscosa L. Whole plant extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Boscia senagalensis (Pers.)
Capparidaceae Leaf extract 5000 ppm G Adults More than 80% of mortality - [71]
Lam. Ex. Poir.
Boscia senagalensis (Pers.)
Capparidaceae Leaf extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Lam. Ex. Poir.
Caprifoliaceae Sambucus nigra L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
0.31% (eggs),
1.18% (adulst), Eggs, adults and
Caryophyllaceae Saponaria officinalis L. Root extract I LD50 - [66]
0.91% oviposition
(oviposition) w/v
Table A1. Cont.
Caryophyllaceae Silene sussamyrica Lazkov Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Plants 2019, 8, 272
Seed and bark

Chenopodiaceae Anabasis aphylla L. 1% G Adult females Mortality between 50 and 80% - [99]
extracts
Anthochlamis tianschanica
Chenopodiaceae Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Iljin
Chenopodium álbum (L.) Flower and leaf Mortalities of 96.99% and
Chenopodiaceae 8–50%. A,B Adult females - [60]
Mosc. Ex. Moq. extracts 91.15%, respectively
Cobretum micranthum
Combretaceae Whole plant extract 5000 ppm G Adults More than 80% of mortality - [71]
G. Don
Cobretum micranthum
Combretaceae Whole plant extract 2500 ppm G Adults More than 80% of mortality - [71]
G. Don
Combretum glutinosum
Combretaceae Leaf extract 5000 ppm G Adults More than 80% of mortality - [71]
Perr. Ex. DC.
Combretaceae Leaf extract 2500 ppm G Adults More than 80% of mortality - [71]
Perr. Ex. DC.
Combretaceae Stem extract 5000 ppm G Adults Mortality between 61 and 80% - [71]
Perr. Ex. DC.
150
Combretaceae Stem extract 2500 ppm G Adults Mortality between 40 and 60% - [71]
Perr. Ex. DC.
Combretaceae Guiera senegalensis J.F. Gmel. Leaf extract 5000 ppm G Adults More than 80% of mortality - [71]
Combretaceae Guiera senegalensis J.F. Gmel. Leaf extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Combretaceae Guiera senegalensis J.F. Gmel. Stem extract 5000 ppm G Adults Mortality between 61 and 80% - [71]
Combretaceae Guiera senegalensis J.F. Gmel. Stem extract 2500 ppm G Adults Mortality between 40 and 60% - [71]
Combretaceae Piloitigma vetilicolin Whole plant extract 5000 ppm G Adults More than 80% of mortality - [71]
Combretaceae Piloitigma vetilicolin Whole plant extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Convolvulaceae Convolvulus arvensis L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Convolvulus krauseanus
Convolvulaceae Root extract 1% G Adult females Mortality between 80 and 100% - [99]
Regel. & Schmalh
Ipomaea asarifolia (Desr.)
Convolvulaceae Whole plant extract 5000 ppm G Adults More than 80% of mortality - [71]
Roem. & Schult.
Ipomaea asarifolia (Desr.)
Convolvulaceae Whole plant extract 2500 ppm G Adults Mortality between 40 and 60% - [71]
Roem. & Schult.
Convolvulaceae Ipomaea sp. L. Whole plant extract 5000 ppm G Adults More than 80% of mortality - [71]
Convolvulaceae Ipomaea sp. L. Whole plant extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Table A1. Cont.
T. urticae
Koch Stage
Cupressus macrocarpa
Cupressaceae Leaf extract 5.69 μL/L air A Adult females LD50 β-citronellol (35.92%) [55]
Plants 2019, 8, 272
Hartw. ex Gordon
Cupressaceae Cupressus sempervirens L. Essential oil 0.1%. G Adult females 28.9% of mortality - [112]
Cupressaceae Juniperus communis L. Essential oil 0.1%. G Adult females 42.6% of mortality - [112]
Cupressaceae Juniperus phoenicea L. Essential oil - D Larvae and adults - [88]
56% (adults)
Cupressaceae Thuja orientalis L. Leaf extract 7.51 μL/L air A Adult females LD50 α-pinene (35.49%) [55]
Elaeagnaceae Elaeagnus angustifolia L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Equisetaceae Equisetum arvense L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Euforbiaceae Jatropha curcas L. Leaf extract 0.06% C,G Adult females Mortality of 63.3% - [124]
7-O-β-D-[2”,6”-bis(4-
hydroxy-E-cinnamoyl)]
Chrozophora oblongifolia Whole plant 312.72 and Adult females glucopyranoside, apigenin
Euphorbiaceae G LD50 [125]
(Delile) Spreng. extract 206.91 ppm and larvae 7-O-ß-D-glucopyranoside
isolated from butanol
fration
Cnidoscolus aconitifolius
Euphorbiaceae Leaf extract 2000 μg/mL C,G Adult females 92% of mortality - [61]
(Mill) I.M. Johnst.
151
Euphorbia ferganensis
Euphorbiaceae Root extract 1% G Adult females Mortality between 0 and 20% - [99]
B. Fedtsch.
3-O-(2,3-
dimethylbutanoyl)-13-
Euphorbia kansui Mortalities of 27% and 55%,
Euphorbiaceae Root extract 3-5 g/L C Adult females dodecanoylingenol y [100]
S.L. Liou S.B. Ho respectively
3-O-(2 E,4 Z-
decadienoyl)-ingenol
Fabaceae Acacia cyanophylla Lindl. Essential oil - D Larvae and adults - [88]
26% (adults)
Amnopiptanthus nanus
Fabaceae Pod extract 1% G Adult females Mortality between 50 and 80% - [99]
(M. pop) Cheng
Fabaceae Bowdichia virgilioides Kunth Leaf extract 0.06% w/v C,G Adult females Mortality of 64.4% [126]
Fabaceae Gleditschia spp. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Fabaceae Glycirrhisa uralensis L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Hedysarum cephalotes Whole plant
Fabaceae 1% G Adult females Mortality between 20 and 50% - [99]
Franchet extract
Hedysarum
Fabaceae daraut-kurganicum Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Sultanova
Fabaceae Hymenaea courbaril L. Leaf extract 0.06% w/v C,G Adult females Mortality of 59.4% [126]
Table A1. Cont.
Fabaceae Medicago minima L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Plants 2019, 8, 272
Fabaceae Melilotus officinalis L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Fabaceae Millettia pinnata L. Laef oil 0.004% C Adult females LD50 (after 4 days) - [101]
Fabaceae Oxytropis rosea Bunge Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Fabaceae Sophora korolkovii Koehne. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Sophora secundiflora (Ortega) Mortalities of 68% (larvae) and
Fabaceae Essential oil - D Larvae and adults - [88]
Lag. Ex. DC. 61% (adults)
Fabaceae Vicia cracca L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
terpinen-4-ol
Geraniaceae Pelargonium graveolens L’Her Leaf extract 12.27 μL/L air A Adult females LD50 [55]
(20.29%)
19 × 10−3 μL/mL
Geraniaceae Pelargonium graveolens L’Hér. Essential oil K Adults 100% of mortality - [42]
of air.
Geraniaceae Pelargonium graveolens L’Hér. Essential oil - D Larvae and adults - [88]
70% (adults)
Geraniaceae Pelargonium roseum Willd Essential oil 0.1%. G Adult females 30% of mortality - [112]
Gramineae Chrysopogon zizanioides (L.) Essential oil 18.82 μg/mL J Adult females LD50 - [127]
19 × 10−3 μL/mL
Gramineae Cymbopogon citratus (DC.) Stapf Essential oil J Adults 100% of mortality - [42]
of air.
152
Gramineae Cymbopogon citratus (DC.) Stapf Essential oil 0.1%. G Adult females 17.8% of mortality - [112]
Cymbopogon flexuosus (Nees ex
Gramineae Essential oil 17.23 μg/mL J Adult females LD50 - [127]
Steud.) W. Watson
Cymbopogon Martini (Roxb.) W. 19 × 10−3 μL/mL
Gramineae Essential oil J Adults 67% of mortality - [42]
Watson of air
19 × 10−3 μL/mL
Gramineae Cymbopogon nardus (L) Rendle Essential oil J Adults 99% of mortality - [42]
of air
Gramineae Cymbopogon nardus (L) Rendle Essential oil 22.5 μg/cm3 C,J Adults LD50 - [68]
Cymbopogon winterianus Jowitt
Gramineae Essential oil 0.1% G Adult females 27.6% of mortality - [112]
ex. Bor
Leaf and flower Mortalities of 91.43% and
Gramineae Lolium perenne L. 6-50% A,B Adult females - [60]
methanolic extracts 93.5%, respectively
Guttiferae Hypericum perforatum L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Iridaceae Iris sogdiana Regel. Leaf extract 1% G Adult females Mortality between 0 and 20% - [99]
Adult females and
Juglandecaea Juglans regia L. Leaf extract 12% v/w C,G Mortality between 83 and 90% - [128]
nymphs
Lamiaceae Acinos thymoides (L.) Moench Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Table A1. Cont.
Lamiaceae Ajuga australis R.Br. Leaf extract 1% C - Mortality between 20 and 49% - [103]
Plants 2019, 8, 272
Lamiaceae Callicarpa pedunculata R.Br. Leaf extract 1% C - Mortality between 20 and 49% - [103]
Ceratanthus longicornis
Lamiaceae Leaf extract 1% C - 100% of mortality - [103]
(F.Muell.) G. Taylor
Clerodendrum floribundum
Lamiaceae Leaf extract 1% C - Mortality between 50 and 89% - [103]
R.Br.
Clerodendrum inerme
(L.) Gaertn.
Clerodendrum tomentosum
(Vent.) R.Br.
Clerodendrum traceyi
F. Muell.
Lamiaceae Faradaya albertissii F. Muell. Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Faradaya splendida F. Muell. Leaf extract 1% C - Mortality between 50 and 89% - [103]
Lamiaceae Glossocarya calcicola Domin. Leaf extract 1% C - Less than 20% of mortality - [103]
Glossocarya
hemiderma Benth.
Gmelina leichardtii
153
(F.Muell.) Benth
Hemiandra australis
B.J. Conn.
Lamiaceae Hemiandra leiantha Benth. Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Hemiandra pungens R.Br. Leaf extract 1% C - Mortality between 20 and 49% - [103]
Lamiaceae Hemigenia humilis Benth. Leaf extract 1% C - Mortality between 20 and 49% - [103]
Lamiaceae Hemigenia sericea Benth. Leaf extract 1% C - Less than 20% of mortality - [103]
Hemigenia
Lamiaceae Leaf extract 1% C - Less than 20% of mortality - [103]
westringioides Benth.
Lamiaceae Hyssopus officinalis L. Aerial part extract 1% G Adult females Less than 20% of mortality - [99]
Lamiaceae Hyssopus officinalis L. Essential oil 0.1%. G Adult females 28.1% of mortality - [112]
Lachnostachys eriobotrya
(F. Muell.) Druce
1,8-cineole, camphor,
Lamiaceae Lavandula angustifolia Mill. Leaf extract 4.93 μL/L J Adult females LD50 [129]
β-pinene
Essential oil from linalool (37.8%),
Lamiaceae Lavandula latifolia Medik. twigs with leaves 0.20–0.25% v/v A,C Adult females Mortality between 95 and 100% 1,8-cineole (24.9%), [56]
andflowers camphor (18.7%)
19 × 10−3 μL/mL
Lamiaceae Lavandula officinalis Chaix Essential oil J Adults 97% of mortality - [42]
of air
Table A1. Cont.
Lavandula officinalis Mortalities of 38% (larvae) and
Lamiaceae Essential oil - D Larvae and adults - [88]
Plants 2019, 8, 272
Chaix 41% (adults)

Lamiaceae Lavandula vera DC. Essential oil 0.1% G Adult females 26.1% of mortality - [112]
Leonorus turkestanicus V.
Lamiaceae Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Krecz. & Kupr.
Lamiaceae Lycopus australis R. Br. Leaf extract 1% C - Mortality between 20 and 49% - [103]
terpinen-4-ol (23.86%),
Majorana hortensis Adults and eggs
Lamiaceae Essential oil 1.84–6.26% C LD50 p-cymene (23.40%) [89]
Moench (respectively)
and sabinene (10.90%)
Lamiaceae Melissa officinalis L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Lamiaceae Menta spicata L. Essential oil 15 μL/L C,J Eggs LD50 carvone (68.5%) [130]
Lamiaceae Mentha arvensis L. Aerial part extract 1% G Adult females Mortality between 80 and 100% - [99]
Lamiaceae Mentha longifolia L. Essential oil 11.08 μg/mL J Adult females LD50 - [127]
19 × 10−3 μL/mL
Lamiaceae Mentha piperita L. Essential oil J Adults 100% of mortality - [42]
of air
Lamiaceae Mentha piperita L. Essential oil 22.8 μg/cm3 C,J Adults LD50 - [68]
Lamiaceae Mentha piperita L. Essential oil 0.1%. G Adult females 23.7% of mortality - [112]
Lamiaceae Mentha piperita L. Essential oil 15.86 μg/mL J Adult females LD50 - [127]
154
19 × 10−3 μL/mL
Lamiaceae Mentha pulegium L. Essential oil J Adults 100% of mortality - [42]
of air
Lamiaceae Mentha pulegium L. Essential oil 23.7 μg/cm3 J Adults LD50 - [68]
Lamiaceae Mentha pulegium L. Essential oil - D Larvae and adults - [88]
91% (adults)
19 × 10−3
μL/mL
Lamiaceae Mentha spicata L. Essential oil J Adults 100% of mortality - [42]
of air
Lamiaceae Mentha spicata L. Essential oil 38.8 μg/cm3 C,J Adults LD50 - [68]
carvone (59.4%),
essential oil
Lamiaceae Mentha spicata L. 7.53 μL/L air C,J Adult females LD50 limonene (9.8%), [85]
from leaves
1,8-cineole (7.4%)
Lamiaceae Mentha sylvestris L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Microcorys capitata
(Bartl.) Benth.
Lamiaceae Microcorys sp. Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Micromeria fruticosa L. Essential oil vapors 2 μL/L of air J Adults and nimphs 96.7% of mortality - [105]
Lamiaceae Micromeria fruticosa L. Essential oil vapors 2 μL/L J Adults and nimphs 96.7% of mortality - [105]
Table A1. Cont.
Lamiaceae Nepeta racemosa L. Essential oil vapors 2 uL/L of air J Adults and nimphs 95% of mortality - [105]
Plants 2019, 8, 272
Lamiaceae Nepeta racemosa L. Essential oil vapors 2 μL/L J Adults and nimphs 95% of mortality - [105]
Lamiaceae Ocimum basilicum L. Essential oil 19 × 10−3 μL/mL of air J Adults 88% of mortality - [42]
Lamiaceae Ocimum basilicum L. Essential oil 39.5 μg/cm3 C,J Adults LD50 - [68]
Lamiaceae Ocimum basilicum L. Essential oil 0.1% G Adult females 21% of mortality - [112]
linalool
Lamiaceae Ocimum basilicum L. Essential oil 0.6 μL/L C,J Adult females LD50 [130]
(65.7%)
Lamiaceae Origanum majorana L. Essential oil 19 × 10−3 μL/mL of air J Adults 92% of mortality - [42]
Lamiaceae Origanum majorana L. Essential oil 0.1% G Adult females 7.1% of mortality - [112]
pulegone
Lamiaceae Origanum vulgare L. Essential oil 8.52 μL/L air A Adult females LD50 [55]
(77.45%)
Lamiaceae Origanum vulgare L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Lamiaceae Origanum vulgare L. Essential oil vapors 2 uL/L of air J Adults and nimphs 95% of mortality - [105]
Lamiaceae Origanum vulgare L. Essential oil vapors 2 μL/L J Adults and nimphs 95% of mortality - [105]
Otostelgia olgae
(Regel.) Korsch.
Pityrodia bartlingii
(Lehm.) Benth.
155
Pityrodia verbascina
(F. Muell.) Benth.
Plectranthus actites
P.I. Forst.
Plectranthus alloplectus
S.T. Blake
Plectranthus amoenus
P.I. Forst
Plectranthus apreptus
S.T. Blake
Plectranthus argentatus
S.T. Blake
Plectranthus cremnus
B. J. Conn.
Plectranthus diversus
S.T. Blake
Plectranthus fasciculatus
P.I. Forst.
Table A1. Cont.
Lamiaceae Plectranthus foetidus Benth. Leaf extract 1% C - Less than 20% of mortality - [103]
Plants 2019, 8, 272
Plectranthus glabriflorus
P.I. Forst
Lamiaceae Plectranthus gratus S.T. Blake Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Plectranthus graveolens R.Br. Leaf extract 1% C - Mortality between 50 and 89% - [103]
Plectranthus habrophyllus
P.I. Forst
Lamiaceae Plectranthus Koonyum Range Leaf extract 1% C - Mortality between 50 and 89% - [103]
Lamiaceae Plectranthus leiperi P.I. Forst. Leaf extract 1% C - Mortality between 50 and 89% - [103]
Lamiaceae Plectranthus mirus S.T. Blake Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Plectranthus nitidus P.I. Forst. Leaf extract 1% C - Mortality between 50 and 89% - [103]
Lamiaceae Plectranthus omissus P. I. Forst. Leaf extract 1% C - Mortality between 50 and 89% - [103]
Lamiaceae Plectranthus parviflorus Willd Leaf extract 1% C - Mortality between 50 and 89% - [103]
Plectranthus scutellarioides (L.)
R.Br.
Lamiaceae Plectranthus sp. buchanans Fort Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Plectranthus sp. Hann Tableland Leaf extract 1% C - 100% of mortality - [103]
Lamiaceae Plectranthus sp. Pinnacle Leaf extract 1% C - Less than 20% of mortality - [103]
156
Lamiaceae Plectranthus spectabilis S.T. Blake Leaf extract 1% C - Less than 20% of mortality - [103]
Plectranthus suaveolens
S.T. Blake
Lamiaceae Pogostemon cablin Benth. Essential oil 0.1% G Adult females 20.3% of mortality - [112]
Lamiaceae Premna acuminata R.Br. Leaf extract 1% C - Mortality between 90 and 99% - [103]
Lamiaceae Premna serratifolia L. Leaf extract 1% C - 100% of mortality - [103]
Lamiaceae Prostanthera incisa Benth. Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Prostanthera lasianthos Labill. Leaf extract 1% C - Mortality between 20 and 49% - [103]
Prostanthera nivea A. Cunn. Ex.
Benth.
Lamiaceae Prostanthera rotundifolia R.Br. Leaf extract 1% C - Less than 20% of mortality - [103]
Table A1. Cont.
T. urticae
Koch Stage
Prostanthera spinosa
Plants 2019, 8, 272
F. Muell.
Prostanthera stricta
R.T. Baker
1,8-cineole (26.7%),
camphor (17.5%),
α-pinene (18.6%),
camphene (11.8%),
myrcene (9%), bornyl
Mortalities of 15%, 79%, 100%
0.10, 0.15, 0.20, Adult females and acetate (4%), β-pinene
Lamiaceae Rosmarinus officinalis L. Essential oil A,C and 100% for females, [53]
and 0.25%. eggs (2.8%), humulene
respectively
(0.5%), borneol (1.8%),
β-caryophyllene (1.5%),
linalool (1%),
Verbennone (0.9%),
α-terpineol (0.8%)
Lamiaceae Rosmarinus officinalis L. Essential oil - D Larvae and adults - [88]
53% (adults)
157
1,8-cineole and
α-pinene (mortalities of
Lamiaceae Rosmarinus officinalis L. Essential oil 10 mL/L D - LD50 88 ± 4.8% and [104]
32 ± 4.8%, respectively
with each compound)
Lamiaceae Rosmarinus officinalis L. Essential oil 0.1% G Adult females 11.7% of mortality - [112]
Aerial part
Lamiaceae Salvia desertorum 1% G Adult females Mortality between 0 and 20% - [99]
extract
Lamiaceae Salvia fruticosa Mill. Leaf extract 3.77 μL/L J Adult females LD50 [129]
19 × 10−3 μL/mL
Lamiaceae Salvia officinalis L. Essential oil J Adults 100% of mortality - [42]
of air
Table A1. Cont.
T. urticae
Koch Stage
α-tujone (42.5), 1,8-cineole
Plants 2019, 8, 272
(10.3%), camphor (11%),

α-pinene (6.7%), camphene
(6.5%), β-tujone (6.6%),
0.10%. 0.15%. Adult females and 100% of female mortality in all myrcene (1.4%), bornyl
Lamiaceae Salvia officinalis L. Essential oil A,C [53]
0.20% and 0.25% eggs concentrations acetate (0.7%), β-pinene
(3.4%), humulene (2.4%),
viridiflorol (2.2%), borneol
(2%), β-caryophyllene
(1.5%), cymene (1%)
Lamiaceae Salvia officinalis L. Essential oil 63.7 μg/cm3 C,J Adults LD50 - [68]
Lamiaceae Salvia officinalis L. Essential oil - D Larvae and adults - [88]
57% (adults)
19 × 10−3 μL/mL
Lamiaceae Salvia sclarea L. Essential oil J Adults 61% of mortality - [42]
of air
Lamiaceae Salvia sclarea L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Lamiaceae Salvia sclarea L. Essential oil 0.1%. G Adult females 71% of mortality - [112]
Salvia vvedenskyi E.
158
Lamiaceae Root extract 1% G Adult females Mortality between 50 and 80% - [99]
Nikit.
0.98 μL/L
Satureja sahendica Essential
Lamiaceae (females), J Eggs and adults LD50 - [84]
Bornm. oil vapors
0.54 μL/L (eggs)
Satureja sahendica
Lamiaceae Essential oil 0.54% E Adults LD50 - [83]
Bornm.
Lamiaceae Scutellaria mollis R. Br. Leaf extract 1% C - Mortality between 50 and 89% - [103]
Stachys tschatkalensis
Knorr.
Teucrium racemosum R.
Br.
Thymbra capitata (L.) Mortalities of 61% (larvae) and
Lamiaceae Essential oil - D Larvae and adults - [88]
Cav. 52% (adults)
19 × 10−3 μL/mL
Lamiaceae Thymus vulgaris L. Essential oil J Adults 93% of mortality - [42]
of air
Lamiaceae Thymus vulgaris L. Essential oil 22.7 μg/cm3 C,J Adults LD50 - [68]
Lamiaceae Thymus vulgaris L. Essential oil 0.1% G Adult females 62.2% of mortality - [112]
Vitex lignum-vitae
Schauer
Viticipremna
queenslandica Munir
Table A1. Cont.
Westringia eremicola A.
Plants 2019, 8, 272
Cunn. Ex. Benth.

Lamiaceae Westringia glabra R.Br. Leaf extract 1% C - Less than 20% of mortality - [103]
Lamiaceae Westringia saxatilis B.J. Conn Leaf extract 1% C - Less than 20% of mortality - [103]
Westringia viminalis
B.J. Conn & Tozer
Lamiaceae Ziziphora clinopodioides Lam. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Cinnamomum
Lauraceae Essential oil 0.1% G Adult females 23.6% of mortality - [112]
zeylandicum Blume
Lauraceae Laurus nobilis L. Leaf extract 17–50% A,B Adult females - [60]
Lauraceae Laurus nobilis L. Essential oil - D Larvae and adults - [88]
46% (adults)
Licaria puchury-major (Mart.) safrole (38.8%),
Lauraceae Essential oil 30.8 μg/mL J Adult females LD50 [131]
Kosterm. 1,8-cineole (21.7%)
Lilliaceae Convallaria majalis L. Root extract 1% G Adult females Mortality between 0 and 20% - [99]
Malvaceae Abutilon theophasti Medic. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Whole plant
159
Malvaceae Corchorus sp. 5000 ppm G Adults Mortality between 61 and 80% - [71]
extract
Whole plant
Malvaceae Corchorus sp. 2500 ppm G Adults Mortality between 61 and 80% - [71]
extract
Whole plant
Malvaceae Hybiscus sp. 5000 ppm G Adults More than 80% of mortality - [71]
extract
Whole plant
Malvaceae Hybiscus sp. 2500 ppm G Adults - - [71]
extract
Whole plant
Malvaceae Malva pusilla Sm. 1% G Adult females Mortality between 50 and 80% - [99]
extract
Meliaceae Azadirachta indica A. Juss. Commercialformulation 1%. C,D Adult females 97.5% of mortality - [37]
Meliaceae Azadirachta indica A. Juss. Leaf extract 5000 ppm G Adults Mortality between 61 and 80% - [71]
Meliaceae Azadirachta indica A. Juss. Leaf extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Meliaceae Melia azedarach L. Fruit extract 14–50% A,B Adult females - [60]
Meliaceae Melia azedarach L. Essential oil - D Larvae and adults - [88]
75% (adults)
Acetone and
petroleum ether
Meliaceae Melia azedarach L. - C Larvae Lethal and fecundity effects - [107]
methanolic
extracts
Prosopis chinensis
Mimosaceae Leaf extract 5000 ppm G Adults More than 80% of mortality - [71]
(Molina) Stuntz
Prosopis chinensis
Mimosaceae Leaf extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
(Molina) Stuntz
Table A1. Cont.
Prosopis chinensis
Mimosaceae Stem extract 5000 ppm G Adults Mortality between 61 and 80% - [71]
Plants 2019, 8, 272
(Molina) Stuntz
Prosopis chinensis
Mimosaceae Stem extract 2500 ppm G Adults Mortality between 40 and 60% - [71]
(Molina) Stuntz
Prosopis chinensis
Mimosaceae Fruit extract 5000 ppm G Adults More than 80% of mortality - [71]
(Molina) Stuntz
Prosopis chinensis
Mimosaceae Fruit extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
(Molina) Stuntz
Callistemon viminals
Myrtaceae Leaf extract 40.66 μL/L air A Adult females LD50 1,8-cineole (71.77%) [55]
(Sol. ex Gaertn.) G. Don
Eucalyptus Mortalities of 62.61% and
Myrtaceae Leaf extract 18–50% A,B Adult females - [60]
camaldulensis Dehnh. 55.57%, respectively
Eucalyptus Mortalities of 51.91% and
Myrtaceae Flower extract 20–50% A,B Adult females - [60]
camaldulensis Dehnh. 47.15%, respectively
19 × 10−3 μL/mL
Myrtaceae Eucalyptus citriodora Hook Essential oil J Adults 100% of mortality - [42]
of air
Myrtaceae Eucalyptus citriodora Hook Essential oil 19.3 μg/cm3 Adults LD50 - [68]
Eucalyptus ghomphocephala Mortalities of 60% (larvae) and
160
Myrtaceae Essential oil - D Larvae and adults - [88]
A. Cunn. Ex. DC. 34% (adults)
19 × 10−3 μL/mL
Myrtaceae Eucalyptus globulus Labill. Essential oil J Adults 89% of mortality - [42]
of air
Myrtaceae Eucalyptus globulus Labill. Essential oil 0.1% G Adult females 19.7% of mortality - [112]
Essential 1.52 μL/L
Eucalyptus microtheca
Myrtaceae oil vapors from (females), 5.7 μL/L J Eggs and adults LD50 - [84]
F. Muell.
fruits and leaves (eggs)
Eucalyptus microtheca Fruits and leaves 0.56% (leaves),
Myrtaceae E Adults LD50 - [83]
F. Muell. essential oils 2.36% (fruits)
1,8-Cineole (31.96%),
Myrtaceae Eucalyptus oleosa L. Essential oil 2.42 μL/L air J Adult females LD50 α-pinene (15.25%), [132]
trans-anethole (7.32%)
Eucalyptus radiata
Myrtaceae Essential oil 0.1% G Adult females 27.9% of mortality - [112]
Sieber ex. DC.
1,8-cineole (28.57%),
Myrtaceae Eucalyptus torquata L. Essential oil 3.59 μL/L air J Adult females LD50 α-pinene (15.74%), [132]
globulol (13.11%)
Myrtaceae Eucapyptus sp. Essential oil 2.18–7.33% C Adults and eggs LD50 - [89]
Bud and leaf 19 × 10−3 μL/mL Mortalities of 80% (buds) and
Myrtaceae Eugenia caryophyllata Thunb. J Adults - [42]
essential oils of air 66% (leaves)
Table A1. Cont.
Myrtaceae Eugenia caryophyllata Thunb. Essential oil 23.6 μg/cm3 C,J Adults LD50 - [68]
Plants 2019, 8, 272
Melaleuca alternifolia Maiden

& Betche ex. Cheel
Myrtaceae Melaleuca leucadendron L. Essential oil 0.1% G Adult females 23.5% of mortality - [112]
Melaleuca viridiflora
Sol. Ex. Gaertn.
Myrtaceae Myrtus communis L. Essential oil - D Larvae and adults - [88]
47% (adults)
Pimenta racemosa 19 × 10−3 μL/mL
Myrtaceae Essential oil J Adults 60% of mortality - [42]
(Mill.) J.W. Moore of air
eugenol (78.5%),
Syzygium aromaticum (L.) Essential oil from
Myrtaceae 6.13 μL/L air C,J Adult females LD50 β-caryophyllene [85]
Merr. & L.M. Perry flower buds
(13.8%)
Syzygium aromaticum (L.)
Merr. & L.M. Perry
Mortalities of 98.5%
Ethanolic, hexane
75. 150 and (ethanolic extract), 94% (hexane
Myrtaceae Syzygium cumini (L.) Skeels and ether ethyl C Adult females - [108]
300 μg/mL extract) and 90% (ether-ethyl
161
acetate extracts
acetate extract)
Nitrariaceae Peganum harmala L. Essential oil - D Larvae and adults - [88]
12% (adults)
Nitrariaceae Peganum harmala L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Bougainvilleae
Nyctaginaceae Leaf extract 5000 ppm G Adults Mortality between 61 and 80% - [71]
spectabilis Willd.
Bougainvilleae
Nyctaginaceae Leaf extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
spectabilis Willd.
Papaveraceae Chelidonium majus L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Papaveraceae Papaver pavoninum Schrenk. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Papaveraceae Papaver rhoeas L. Essential oil - D Larvae and adults - [88]
34% (adults)
Papaveraceae Papaver rhoeas L. Aerial part extract 1% G Adult females Mortality between 80 and 100% - [99]
Papaveraceae Roemeria refracta DC. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Cedrus atlantica (Endl.)
Pinaceae Essential oil 0.1% G Adult females 12.4% of mortality - [112]
Manetti ex. Carriére
Picea schrenkiana
Pinaceae Leaf extract 1% G Adult females Mortality between 50 and 80% - [99]
Fisch & Mey.
Pinaceae Pinus sylvestris L. Essential oil 0.1% G Adult females 50.4% of mortality - [112]
Table A1. Cont.
Mortality effect [(E)-nerolidol,
(E)-nerolidol,
Plants 2019, 8, 272
Leaf essential oil α–Humulene and

Piperaceae Piper aduncum L. - J - α–Humulene and [109]
compounds β-caryophyllene)] and
β-caryophyllene
repellence (β-caryophyllene)
Piperaceae Piper nigrum L. Essential oil 0.1% G Adult females 22.8% of mortality - [112]
Plantaginaceae Globularia alypum L. Essential oil - D Larvae and adults - [88]
2% (adults)
Plantaginaceae Plantago major L. Aerial part extract 1% G Adult females Mortality between 80 nd 100% - [99]
Limonium
Plumbaginaceae Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
tianschanicum Lincz.
Polygonaceae Polygonum aviculare L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Polygonum
Polygonaceae Root extract 1% G Adult females Mortality between 0 and 20% - [99]
toktoquilicum Lazkov
Polygonaceae Rumex acetosa L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Primulaceae Anagallis arvensis L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Ranunculaceae Aconitum soongaricum Stapf Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Ranunculaceae Adonis parviflora Fisch. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Ceratocephallus testiculata
162
Ranunculaceae Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
(Crantz.) Bess.
Ranunculaceae Clematis orientalis L. Seed extract 1% G Adult females Mortality between 50 and 80% - [99]
Ranunculaceae Clematis songarica Bge. Seed extract 1% G Adult females Mortality between 20 and 50% - [99]
Ranunculaceae Nigella sativum L. Seed extract 708.57 ppm G Adult females LD50 - [91]
Ranunculaceae Ranunculus polyanthemus L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Rosaceae Geum urbanum L. Aerial part extract 1% G Adult females Mortality between 0 and 20% - [99]
Rosaceae Padus avium Mill. Leaf extract 1% G Adult females Mortality between 0 and 20% - [99]
Leaves, flower Eggs and adult
Rosaceae Prunus laurocerasus L. 12% v/w A,C Mortality between 37 and 100% - [54]
and seed extract females
Whole plant
Rubiaceae Boirerio radiata 5000 ppm G Adults Mortality between 40 and 60% - [71]
extract
Whole plant
Rubiaceae Boirerio radiata 2500 ppm G Adults Mortality between 40 and 60% - [71]
extract
Rubiaceae Galium verum L. Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
Adult females and 49 and 66% of mortality,
Rubiaceae Gardenia jasminoides J. Ellis Fruits extract 10000 ppm I,J - [133]
nymphs respectively
19 × 10−3 μL/mL
Rutaceae Citrus aurantium L. Essential oil J Adults 68% of mortality - [42]
of air
Rutaceae Citrus aurantium L. Essential oil - D Larvae and adults - [88]
55% (adults)
Fruit epicarp Repellent effect due to all 27
Rutaceae Citrus aurantium L. 1% I,J Adult females d-limonene [67]
essential oil identified compounds
Table A1. Cont.
Citrus aurantium L.
Rutaceae Essential oil 0.1% G Adult females 21.4% of mortality - [112]
Plants 2019, 8, 272
var. Armara
19 × 10−3 μL/mL
Rutaceae Citrus bergamia Risso & Poit. Essential oil J Adults 87% of mortality - [42]
of air
Rutaceae Citrus bergamia Risso & Poit. Essential oil 0.1% G Adult females 11% of mortality - [112]
Rutaceae Citrus limon (L.) Burm. F. Essential oil 0.1% G Adult females 34.9% of mortality - [112]
Rutaceae Citrus paradisi Macfad Essential oil 6.96 μL/L air A Adult females LD50 limonene (74.29%) [55]
Rutaceae Citrus paradisi Macfad. Essential oil 0.1% G Adult females 30.6% of mortality - [112]
19 × 10−3 μL/mL
Rutaceae Citrus sinensis Osbeck Essential oil J Adults 61% of mortality - [42]
of air
Fruit epicarp Repellent effect due to all 27
Rutaceae Citrus sinensis Osbeck 1% I,J Adult females d-limonene [67]
essential oil identified compounds
Rutaceae Citrus sinensis Osbeck Essential oil 0.1% G Adult females 45.6% of mortality - [112]
Haplophyllum tuberculatum Mortalities of 94% (larvae) and
Rutaceae Essential oil - D Larvae and adults - [88]
(Forssk.) A. Juss. 93% (adults)
Rutaceae Ruta chalepensis L. Essential oil - D Larvae and adults - [88]
61% (adults)
5000 and 10000 Mortalities of 36% and 39%,
163
Rutaceae Zanthoxylum armatum DC. Leaf extract G Adults - [111]
ppm respectively
87.2% of mortality and
Santalaceae Santalum sp. Essential oil 0.1% G Adult females - [112]
fecundity decrease
2-(1,1-dimethylprop-2-
enyl)-3-hydroxi-1,4-
Two extract naphthoquinone and
Scrophulariaceae Calceolaria andina Benth 80 and 30 ppm G - LD50 [113]
compounds 2-acetoxy-3-(1,1-
dimethylprop-2-enyl)-
1,4-naphthoquinone
Scrophulariaceae Verbascum thapsus L. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Ailanthus altissima
Simarubaceae Leaf extract 1% G Adult females Mortality between 80 and 100% - [99]
(Mill.) Swingle
10000 ppm and 47
Chaparinone
Simarubaceae Quassia sp. Aerial part extract ppm C - LD50 (chaparinone) [114]
quasinoid
(chaparinone)
Solanaceae Capsicum annuum L. Aerial part extract 1% G Adult females Mortality between 80 and 100% - [99]
Solanaceae Capsicum annuum L. Fruit extract - B,J Adult females 45% of mortality - [116]
Solanaceae Capsicum baccatum L. Fruit extract - B,J Adult females Repellent effect - [116]
Solanaceae Capsicum chinense Jacq. Fruit extract - B,J Adult females Repellent effect - [116]
Solanaceae Capsicum frutescens L. Fruit extract - B,J Adult females Repellent effect - [116]
Table A1. Cont.
167.25 (leaves) and
Seed and leaf Mortalities of 98% (leaves) and
Plants 2019, 8, 272
Solanaceae Datura stramonium L. 145.75 C,D Adults - [5]

extracts 25% (seeds)
(seeds) mg/L
Solanaceae Hyoscyamus niger L. Aerial part extract 1% G Adult females Mortality between 50 and 80%. - [99]
Dihidrofarnesoic
Solanaceae Lycopersicon hirsutum Dunal - - J Adult females Repellent activity [115]
acid
Flower and leaf Mortalities of 69.88% and
Solanaceae Solanum nigrum L. 12–50% A,B Adult females - [60]
extracts 79.36%, respectively
Solanaceae Solanum nigrum L. Fruit extract 19–50% A,B Adult females - [60]
Solanaceae Solanum nigrum L. Leaf extract 1% G Adult females Mortality between 20 and 50% - [99]
Solanaceae Solanum nigrum L. Leaf extract 279.69 μg/mL C,G Adult females LD50 after 72 h - [134]
Whole plant
Sterculiaceae Waltheria indica L. 5000 ppm G Adults Mortality between 61 and 80% - [71]
extract
Whole plant
Sterculiaceae Waltheria indica L. 2500 ppm G Adults Mortality between 40 and 60% - [71]
extract
Styracaceae Styrax officinalis L. Seed cover extract 16–50% A,B Adult females - [60]
164
Styracaceae Styrax officinalis L. Seed extract 21–50% A,B Adult females - [60]
Angelica tschimganica
Umbelliferae Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
(Korov.) B. Tikhom.
Umbelliferae Conium maculatum L. Seed extract 1% G Adult females Mortality between 20 and 50% - [99]
Umbelliferae Dorema microcarpum Korov. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Umbelliferae Ferula foetida (Bunge) Regel. Root extract 1% G Adult females Mortality between 0 and 20% - [99]
Ferula foetidissima
Umbelliferae Root extract 1% G Adult females Mortality between 20 and 50% - [99]
Regel. & Schmahl.
Ferula inciso-serrata M. Aerial part and
Umbelliferae 1% G Adult females Mortality between 0 and 20% - [99]
Pimen. & Baranova root extracts
Umbelliferae Heracleum dissectum Ledeb. Aerial part extract 1% G Adult females Mortality between 20 and 50% - [99]
Mediasia macrophylla (Regel.
Umbelliferae Aerial part extract 1% G Adult females Mortality between 50 and 80% - [99]
& Schmahl.) M. Pimen.
Umbelliferae Prangos lipskyi Korov Root extract 1% G Adult females Mortality between 80 and 100% - [99]
Urticaceae Urtica pilulifera L. Essential oil - D Larvae and adults - [88]
46% (adults)
Valerianaceae Valeriana officinalis L. Aerial part extract 1% G Adult females Mortality between 20 and 50%. - [99]
Verbenaceae Lantana camara L. Essential oil - D Larvae and adults - [88]
1% (adults)
carvacrol (48.31%),
Verbenaceae Lippia origanoides H.B.K. Essential oil 25.1 μg/mL J Adult females LD50 p-cymene (9.11%), [135]
thymol (8.78%)
Table A1. Cont.
0.01 μL/L (extract).
Plants 2019, 8, 272
0.001 μL/L (thymol). Thymol,

3.02 μL/L (p-cymene). p-cymene,
Verbenaceae Lippia sidoides Cham. Essential oil J Adult females LD50 [117]
0.08 μL/L β-caryophyllene
(β-caryophyllene) and and carvacrol
0.036 μL/L (carvacrol)
Elettaria cardamomum
Zingiberaceae Essential oil 19 × 10−3 μL/mL of air J Adults 87% of mortality - [42]
(L.) Maton
Zingiberaceae Zingiber officinale Rosc. Essential oil 0.1% G Adult females 11.9% of mortality - [112]
Balanites aegyptiaca (L.)
Zygophyllaceae Leaf extract 5000 ppm G Adults More than 80% of mortality - [71]
Delile
Balanites aegyptiaca (L.)
Zygophyllaceae Leaf extract 2500 ppm G Adults Mortality between 61 and 80% - [71]
Delile
aThis column includes the bioassays used in each study, which are coded as follows: A = slide dip, B = petri dish, C = leaf disc direct (LDD); D = leaf disc residue (LDR); E = leaf disc
direct potter tower (LDD-PT); F = leaf disc residue potter tower (DR-PTM); G = leaf disc residue dipping (LDR-D); H = leaf absorption test; I = whole plant direct; J = filter paper
difussion (fumigant).
165
Plants 2019, 8, 272
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Pesticidal Plants

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Pesticidal Plants

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Pesticidal Plants

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Special Issue Editors

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

ISBN 978-3-03928-788-8 (Pbk)

Cover image courtesy of Philip C. Stevenson.

About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Pesticidal Plants” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

G. Mandela Fernández-Grandon, Steven J. Harte, Jaspher Ewany, Daniel Bray and

Marı́a J. Pascual-Villalobos, Manuel Cantó-Tejero, Pedro Guirao and Marı́a D. López

Kateřina Kovařı́ková and Roman Pavela

Liliana Ruiz-Vásquez, Matı́as Reina, Vı́ctor Fajardo, Matı́as López and

Naomi B. Rioba and Philip C. Stevenson

Ricardo A. Rincón, Daniel Rodrı́guez and Ericsson Coy-Barrera

Philip C. Stevenson, Steven R. Belmain, Murray B. Isman

Received: 21 February 2020; Accepted: 29 February 2020; Published: 4 March 2020

Plants 2020, 9, 324; doi:10.3390/plants9030324 1 www.mdpi.com/journal/plants

2.1. Stem Eﬀects

Powdered Stem Germination Root Length Shoot Length

2.2. Inﬂorescence Eﬀects

Inﬂorescence Mean Shoot

2.3. Root Eﬀects

Powdered Root Germination Root Length Shoot Length

2.4. Seed Eﬀects

Germination Root Length Shoot Length

2.5. Polyphenol Content in L. multiﬂorum Extracts

4. Materials and Methods

4.1. Plant Material

4.2. Aqueous Extract Bioassay

4.3. Plant Part Powder Bioassay

4.4. Seedling Growth Parameter and Germination Indices

Germinated Seed Number

where D is the number of days from the beginning of germination.

4.5. Determination of Polyphenolic Content

4.6. Statistical Analysis

Received: 25 December 2019; Accepted: 29 January 2020; Published: 1 February 2020

Abstract: Sustainable agricultural intensiﬁcation employs alternatives to synthetic insecticides for

Keywords: biopesticide; organic pesticide; Y-tube olfactometer; pyrethrum; parasitoid;

Plants 2020, 9, 173; doi:10.3390/plants9020173 14 www.mdpi.com/journal/plants

2.1. Aphid Mortality Assay

2.1.2. Hyphal Growth on Insect Surface

2.1.3. Number of Oﬀspring

2.2. Parasitoid Dual-Choice Assays

Figure 4. Parasitoid, A. colemani, responses to treatments in a Y-tube olfactometer. N = 50 for

1. Eﬃcacy of pyrethrum and EPF would be enhanced when presented in combination.

4. Materials and Methods

4.1. Insect Rearing

4.2. Entomopathogenic Fungi

4.3. Preperation of Pyrethrum

4.4. Mortality Assays

4.5. Parasitoid Choice Assays

4.6. Statistical Analyses

4.6.2. Visible Fungal Growth

4.6.3. Number of Oﬀspring

4.6.4. Parasitoid Dual-Choice Assays

Received: 10 December 2019; Accepted: 20 January 2020; Published: 23 January 2020

Plants 2020, 9, 149; doi:10.3390/plants9020149 28 www.mdpi.com/journal/plants

2. Results and Discussions

Treatment Applied Number of Pods Per Plants Seed Yield/Plant (g)

Chlorophylls Flavonoids Anthocyanins

Method of Application Phenylalanine Tryptophan Rutin

3. Materials and Methods

3.1. Bean Rearing and Plant Material Preparation