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UNIVERSIDAD POLITÉCNICA DE MADRID
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
INTEGRATION OF PHYSICAL, CHEMICAL AND BIOLOGICAL TACTICS AGAINST INSECT PESTS AND
VIRUS DISEASES IN HORTICULTURAL CROPS
TESIS DOCTORAL
BEATRIZ DÁDER ALONSO
Ingeniera Agrónoma
2015
DEPARTAMENTO DE PRODUCCIÓN VEGETAL: BOTÁNICA Y PROTECCIÓN VEGETAL
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
INTEGRATION OF PHYSICAL, CHEMICAL AND BIOLOGICAL TACTICS AGAINST INSECT PESTS AND
VIRUS DISEASES IN HORTICULTURAL CROPS
Autora: BEATRIZ DÁDER ALONSO
Ingeniera Agrónoma
Directores: ALBERTO FERERES CASTIEL
Dr. Ingeniero Agrónomo
ARÁNZAZU MORENO LOZANO
Dra. en Ciencias
2015
Tribunal nombrado por el Magfco. Y Excmo. Sr. Rector de la Universidad Politécnica de Madrid, el día de de 2015.
Presidente:
Secretario:
Vocal:
Vocal:
Vocal:
Suplente:
Suplente:
Realizado el acto de defensa y lectura de Tesis el día de de 2015 en el Instituto de Ciencias Agrarias del Consejo Superior de Investigaciones Científicas.
EL PRESIDENTE EL SECRETARIO
Fdo.: Fdo.:
LOS VOCALES
Fdo.: Fdo.: Fdo.:
AGRADECIMIENTOS
Alberto Fereres y Aránzazu Moreno, mis directores de Tesis, que confiaron en mí al terminar la
carrera, me dieron un hueco en su grupo de investigación y la oportunidad de hacer dos
estancias en el extranjero. Gracias por vuestra orientación, ayuda y oportunidades
profesionales durante estos años. Aprendo de vosotros cada día.
Elisa Viñuela, mi tutora en la ETSIA, por su inestimable ayuda desde cuando había que realizar
trámites burocráticos hasta en los ensayos de campo en La Poveda.
My supervisors abroad during the two research internships, Dylan Gwynn-Jones in Wales and
Piotr Trebicki in Australia, thank you for receiving me in your labs and letting me do my work
using your facilities. Thank you Piotr for taking me to the Canberra conference. I learnt a lot
from both of you.
María, por echarme mil manos en todos los ensayos y tu organización. Elisa, por tener las
instalaciones tan cuidadas, apoyarme en el montaje de UV y tu ayuda cuando tengo dudas.
Arantxa, y nuestras conversaciones sobre celebrities y trapos, porque no todo es ciencia.
Michele, mi “aberrojo” brasileiro, hemos crecido juntas, te voy a echar tanto de menos. Sara,
por ser tan buena compañera y ayudarme en los muestreos. Casimiro y el Servicio de Ambientes
controlados del ICA, por cuidar mis plantas y tenernos fichados desde tu posición estratégica, sé
que nunca me querrás tanto como a Michele. Andrés y Jaime, os deseo lo mejor en los próximos
años.
Todas las personas con las que trabajé en el ICA durante mi Tesis: Poti -gracias por los
másteres en estadística-, Saioa -gracias por tu ayuda al principio de todo y que ha continuado
con los años-, Marta, Rocco, Víctor, Carmen, Tina, Antonio, Raquel, Vera, Pilar, el lobby
brasileño Lia, Natalie, Mauricelia y Lilian, Alex, Camino, Karla, Sabrina, Laura, Natalia,
Raquel. A todo el personal de La Poveda y al Grupo de Entomología de la ETSIA (Nacho,
Agustín, Fermín, Mar, Flor, Pilar, Andrea, Pedro, Elisa, Ángeles -gracias por tu ayuda cuando
empezábamos el máster TAPAS-), por su ayuda en los ensayos de campo.
Silvia Rondon and Phyllis Weintraub, my external reviewers, besides reading my Thesis as part
of the paperwork, you took the time to point out valuable suggestions that contributed to improve
it.
Dylan, Sara, Ana and Alan, thank you for your hospitality during my first international and
rainy experience. Piotr & family, Audrey, Lucy, Helena, Simone and Isaac, thank you so much
for making me so welcome in Australia. Coping with distance was easier although I was in the
other side of the world.
Inés y Guti, Lorena y Alber, que no saben de qué va esto, pero precisamente eso es lo que los
hace geniales.
María, hermana con la que comparto vidas paralelas y las decepciones investigadoras.
Mi centro: tío, abuela, tía, papá y mamá, Rodri, Javi, nunca podré devolveros todo lo que me
dais cada día, os quiero.
Susana, cada día más.
INDEX
ACRONYMS AND ABBREVIATIONS i
RESUMEN v
1. INTRODUCCIÓN v
2. METODOLOGÍA vii
3. RESULTADOS xi
4. DISCUSIÓN xviii
SUMMARY xxiii
CHAPTER 1. INTRODUCTION 1
1.1. VEGETABLE PRODUCTION IN PROTECTED ENVIRONMENTS 1
1.2. INSECT PESTS 2
1.3. PLANT VIRUSES 5
1.4. INTEGRATED PEST MANAGEMENT 7
1.4.1. BIOLOGICAL CONTROL 8
1.4.2. PHYSICAL CONTROL 9
1.4.2.1. INSECTICIDE-TREATED NETS 9
1.4.2.2. UV-ABSORBING PLASTIC COVERS 10
1.5. EFFECTS OF UV RADIATION ON PLANTS 10
1.6. EFFECTS OF UV RADIATION ON PESTS, VIRUSES AND BENEFICIALS 11
CHAPTER 2. OBJECTIVES 13
CHAPTER 3. MATERIALS AND METHODS 15
3.1. EXPERIMENTAL SITES 15
3.2. PLANT DEVELOPMENT 15
3.3. INSECT REARING 16
3.4. GLASSHOUSE FACILITIES 19
3.5. VIRUS INOCULATION AND DETECTION 19
3.6. PHOTOSELECTIVE COVERS 21
3.7. LONG LASTING INSECTICIDE-TREATED NETS (LLITNs) 22
3.8. SPATIAL ANALYSIS 23
3.9. GENERAL STATISTICS 25
CHAPTER 4. SPATIO-TEMPORAL DYNAMICS OF VIRUSES ARE DIFFERENTIALLY AFFECTED BY PARASITOIDS DEPENDING ON THE MODE OF TRANSMISSION 27
ABSTRACT 27
4.1. INTRODUCTION 27
4.2. OBJECTIVE 29
4.3. MATERIALS AND METHODS 29
4.3.1. EXPERIMENTAL DESIGN 29
4.3.2. STATISTICAL METHODS 32
4.3.3. SPATIAL ANALYSIS 32
4.4. RESULTS 33
4.4.1. EFFECT OF Aphidius colemani ON APHID DISPERSAL AND THE SPREAD OF Cucumber mosaic virus 33
4.4.2. EFFECT OF Aphidius colemani ON APHID DISPERSAL AND THE SPREAD OF Cucumber aphid-borne yellows virus 39
4.5. DISCUSSION 44
CHAPTER 5. FLIGHT BEHAVIOUR OF VEGETABLE PESTS AND THEIR NATURAL ENEMIES UNDER DIFFERENT UV-BLOCKING ENCLOSURES 49
ABSTRACT 49
5.1. INTRODUCTION 50
5.2. OBJECTIVE 51
5.3. MATERIALS AND METHODS 51
5.3.1. EXPERIMENTAL DESIGN 51
5.3.2. STATISTICAL METHODS 53
5.4. RESULTS 54
5.4.1. PHOTOSELECTIVE COVERS 54
5.4.2. ABILITY TO LEAVE THE RELEASE PLATFORM 54
5.4.3. SHORT TUNNELS – DISPERSAL OF PESTS 56
5.4.4. SHORT TUNNELS – DISPERSAL OF NATURAL ENEMIES 58
5.4.5. LONG TUNNELS – DISPERSAL OF PESTS 59
5.4.6. LONG TUNNELS – DISPERSAL OF NATURAL ENEMIES 61
5.5. DISCUSSION 61
CHAPTER 6. IMPACT OF UV-A RADIATION ON THE PERFORMANCE OF APHIDS AND WHITEFLIES AND ON THE LEAF CHEMISTRY OF THEIR HOST PLANTS 67
ABSTRACT 67
6.1. INTRODUCTION 68
6.2. OBJECTIVE 70
6.3. MATERIALS AND METHODS 70
6.3.1. EXPERIMENTAL DESIGN 70
6.3.2. PLANT BIOCHEMICAL ANALYSIS 74
6.3.2.1. SECONDARY METABOLITES 74
6.3.2.2. SOLUBLE SUGARS 75
6.3.2.3. FREE AMINO ACID AND PROTEINS 75
6.3.2.4. PHOTOSYNTHETIC PIGMENTS 76
6.3.3. STATISTICAL METHODS 76
6.4. RESULTS 76
6.4.1. PLANT GROWTH 76
6.4.2. INSECT RESPONSES 77
6.4.3. PLANT BIOCHEMICAL RESPONSES 79
6.4.3.1. SECONDARY METABOLITES 79
6.4.3.2. SOLUBLE SUGARS 82
6.4.3.3. FREE AMINO ACID AND PROTEINS 83
6.4.3.4. PHOTOSYNTHETIC PIGMENTS 84
6.5. DISCUSSION 86
CHAPTER 7. CONTROL OF INSECT VECTORS AND PLANT VIRUSES IN PROTECTED CROPS BY NOVEL PYRETHROID-TREATED NETS 91
ABSTRACT 91
7.1. INTRODUCTION 92
7.2. OBJECTIVE 93
7.3. MATERIALS AND METHODS 94
7.3.1. LABORATORY EXPERIMENTS 94
7.3.2. DETERMINATION OF THE INSECTICIDE CONCENTRATION OF LLITNs 95
7.3.3. EFFICACY OF LLITNs IN FIELD CONDITIONS AGAINST APHIDS AND WHITEFLIES 96
7.3.4. EFFECT OF BIFENTHRIN-TREATED NETS ON THE APHID PARASITOID Aphidius colemani 97
7.3.5. STATISTICAL METHODS 98
7.3.6. SPATIAL ANALYSIS 98
7.4. RESULTS 98
7.4.1. EFFICACY OF LLITNs AGAINST APHIDS IN LABORATORY TRIALS 98
7.4.2. EFFICACY OF LLITNs AGAINST WHITEFLIES IN LABORATORY TRIALS 99
7.4.3. EFFICACY OF LLITNs IN FIELD CONDITIONS AGAINST APHIDS AND WHITEFLIES 102
7.4.4. EFFECT OF BIFENTHRIN-TREATED NETS ON THE APHID PARASITOID Aphidius colemani 107
7.5. DISCUSSION 107
CHAPTER 8. GENERAL DISCUSSION 111
CONCLUSIONS 119
REFERENCES 121
i
ACRONYMS AND ABBREVIATIONS
C: Distance to crowding
CABYV: Cucumber aphid-borne yellows virus
CIPAC: Collaborative International Pesticides Analytical Council
cm: Centimeters
CMV: Cucumber mosaic virus
CSIC: Consejo Superior de Investigaciones Científicas (Spanish National Research Council)
cv.: Cultivar
D: Distance to regularity
DAS-ELISA: Double Antibody Sandwich Enzyme-Linked ImmunoSorbent Assay
df: Degrees of freedom
Dr.: Doctor
ETSIA: Escuela Técnica Superior de Ingenieros Agrónomos (College of Agricultural
Engineering)
g: Grams
HPLC: High Pressure Liquid Chromatography
Ia: Index of aggregation
IBERS: Institute of Biological, Environmental & Rural Sciences
ICA: Instituto de Ciencias Agrarias (Institute of Agricultural Sciences)
IPM: Integrated Pest Management
ITNs: Insecticide Treated Nets
kb: Kilobase
kg: Kilograms
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KJ: Kilojoules
LC-MS: Liquid Chromatography-Mass Spectrometry
m: Meters
mg: Miligrams
min: Minutes
MIP: Manejo Integrado de Plagas
MJ: Megajoules
mM: Micromolar
mm: Milimeters
nm: Nanometers
PAR: Photosynthetically Active Radiation (400-700 nm)
rpm: Revolutions per minute
s: Seconds
SADIE: Spatial Analysis by Distance IndicEs
t: Metric ton
UPM: Universidad Politécnica de Madrid (Polytechnic University of Madrid)
UV: Ultraviolet radiation (200-400 nm)
UV-A: Ultraviolet-A radiation (315-400 nm)
UV-B: Ultraviolet-B radiation (280-315nm)
UV-C: Ultraviolet-C radiation (100-280 nm)
µL: Microliters
µm: Micrometers
µmol: Micromol
vi: Index of clustering in patches
vj: Index of clustering in gaps
X: Index of spatial association
iii
W: Watts
iv
v
RESUMEN
1. INTRODUCCIÓN
Actualmente, la gestión de sistemas de Manejo Integrado de Plagas (MIP) en cultivos hortícolas
tiene por objetivo priorizar los métodos de control no químicos en detrimento del consumo de
plaguicidas, según recoge la directiva europea 2009/128/CE ‘Uso Sostenible de Plaguicidas’
(OJEC, 2009). El uso de agentes de biocontrol como alternativa a la aplicación de insecticidas es
un elemento clave de los sistemas MIP por sus innegables ventajas ambientales que se utiliza
ampliamente en nuestro país (Jacas y Urbaneja, 2008). En la región de Almería, donde se
concentra el 65% de cultivo en invernadero de nuestro país (47.367 ha), MIP es la principal
estrategia en pimiento (MAGRAMA, 2014), y comienza a serlo en otros cultivos como tomate o
pepino. El cultivo de pepino, con 8.902 ha (MAGRAMA, 2013), tiene un protocolo semejante al
pimiento (Robledo et al., 2009), donde la única especie de pulgón importante es Aphis gossypii
Glover.
Sin embargo, pese al continuo incremento de la superficie de cultivo agrícola bajo sistemas MIP,
los daños originados por virosis siguen siendo notables. Algunos de los insectos presentes en los
cultivos de hortícolas son importantes vectores de virus, como los pulgones, las moscas blancas
o los trips, cuyo control resulta problemático debido a su elevada capacidad para transmitir virus
vegetales incluso a una baja densidad de plaga (Holt et al., 2008; Jacas y Urbaneja, 2008).
Las relaciones que se establecen entre los distintos agentes de un ecosistema son complejas y
muy específicas. Se ha comprobado que, pese a que los enemigos naturales reducen de manera
beneficiosa los niveles de plaga, su incorporación en los sistemas planta-insecto-virus puede
desencadenar complicadas interacciones con efectos no deseables (Dicke y van Loon, 2000;
Jeger et al., 2011). Así, los agentes de biocontrol también pueden inducir a que los insectos
vectores modifiquen su comportamiento como respuesta al ataque y, con ello, el grado de
dispersión y los patrones de distribución de las virosis que transmiten (Bailey et al., 1995; Weber
et al., 1996; Hodge y Powell, 2008a; Hodge et al., 2011).
Además, en ocasiones el control biológico por sí solo no es suficiente para controlar
determinadas plagas (Medina et al., 2008). Entre los métodos que se pueden aplicar bajo
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sistemas MIP están las barreras físicas que limitan la entrada de plagas al interior de los
invernaderos o interfieren con su movimiento, como pueden ser las mallas anti-insecto (Álvarez
et al., 2014), las mallas fotoselectivas (Raviv y Antignus, 2004; Weintraub y Berlinger, 2004;
Díaz y Fereres, 2007) y las mallas impregnadas en insecticida (Licciardi et al., 2008; Martin et
al., 2014).
Las mallas fotoselectivas reducen o bloquean casi por completo la transmisión de radiación UV,
lo que interfiere con la visión de los insectos y dificulta o impide la localización del cultivo y su
establecimiento en el mismo (Raviv y Antignus, 2004; Weintraub, 2009). Se ha comprobado
cómo su uso puede controlar los pulgones y las virosis en cultivo de lechuga (Díaz et al., 2006;
Legarrea et al., 2012a), así como la mosca blanca, los trips y los ácaros, y los virus que estos
transmiten en otros cultivos (Costa y Robb, 1999; Antignus et al., 2001; Kumar y Poehling,
2006; Doukas y Payne, 2007a; Legarrea et al., 2010). Sin embargo, no se conoce perfectamente
el modo de acción de estas barreras, puesto que existe un efecto directo sobre la plaga y otro
indirecto mediado por la planta, cuya fisiología cambia al desarrollarse en ambientes con falta de
radiación UV, y que podría afectar al ciclo biológico de los insectos fitófagos (Vänninen et al.,
2010; Johansen et al., 2011). Del mismo modo, es necesario estudiar la compatibilidad de esta
estrategia con los enemigos naturales de las plagas. Hasta la fecha, los estudios han evidenciado
que los agentes de biocontrol pueden realizar su actividad bajo ambientes pobres en radiación
UV (Chyzik et al., 2003; Chiel et al., 2006; Doukas y Payne, 2007b; Legarrea et al., 2012c).
Otro método basado en barreras físicas son las mallas impregnadas con insecticidas, que se han
usado tradicionalmente en la prevención de enfermedades humanas transmitidas por mosquitos
(Martin et al., 2006). Su aplicación se ha ensayado en agricultura en ciertos cultivos al aire libre
(Martin et al., 2010; Díaz et al., 2004), pero su utilidad en cultivos protegidos para prevenir la
entrada de insectos vectores en invernadero todavía no ha sido investigada. Los aditivos se
incorporan al tejido durante el proceso de extrusión de la fibra y se liberan lentamente actuando
por contacto en el momento en que el insecto aterriza sobre la malla, con lo cual el riesgo
medioambiental y para la salud humana es muy limitado. Los plaguicidas que se emplean
habitualmente suelen ser piretroides (deltametrina o bifentrín), aunque también se ha ensayado
dicofol (Martin et al., 2010) y alfa-cipermetrina (Martin et al., 2014). Un factor que resulta de
vital importancia en este tipo de mallas es el tamaño del poro para facilitar una buena ventilación
del cultivo, al tiempo que se evita la entrada de insectos de pequeño tamaño como las moscas
blancas (Bethke y Paine, 1991; Muñoz et al., 1999). Asimismo, se plantea la necesidad de
estudiar la compatibilidad de estas mallas con los enemigos naturales. Es por ello que en esta
Resumen
vii
Tesis Doctoral se plantea la necesidad de evaluar nuevas mallas impregnadas que impidan el
paso de insectos de pequeño tamaño al interior de los invernaderos, pero que a su vez mantengan
un buen intercambio y circulación de aire a través del poro de la malla.
Así, en la presente Tesis Doctoral, se han planteado los siguientes objetivos generales a
desarrollar:
1. Estudiar el impacto de la presencia de parasitoides sobre el grado de dispersión y los
patrones de distribución de pulgones y las virosis que éstos transmiten.
2. Conocer el efecto directo de ambientes pobres en radiación UV sobre el comportamiento
de vuelo de plagas clave de hortícolas y sus enemigos naturales.
3. Evaluar el efecto directo de la radiación UV-A sobre el crecimiento poblacional de
pulgones y mosca blanca, y sobre la fisiología de sus plantas hospederas, así como el
efecto indirecto de la radiación UV-A en ambas plagas mediado por el crecimiento de
dichas planta hospederas.
4. Caracterización de diversas mallas impregnadas en deltametrina y bifentrín con
diferentes propiedades y selección de las óptimas para el control de pulgones, mosca
blanca y sus virosis asociadas en condiciones de campo. Estudio de su compatibilidad
con parasitoides.
2. METODOLOGÍA
Los experimentos de la presente Tesis Doctoral se llevaron a cabo en el Instituto de Ciencias
Agrarias (ICA) (Madrid, España), en la estación experimental de La Poveda-CSIC (Madrid,
España), y en los campos de prácticas de la Escuela Técnica Superior de Ingenieros Agrónomos
de la Universidad Politécnica de Madrid (ETSIA-UPM). Parte de los ensayos correspondientes
al Capítulo 6 se desarrollaron en la Universidad de Aberystwyth (IBERS, Reino Unido), en
colaboración con el Dr. Dylan Gwynn-Jones, en el marco de una Estancia Breve financiada por
el Ministerio de Economía y Competitividad.
Para ello, se realizaron ensayos en condiciones controladas de laboratorio, invernadero y campo
utilizando especies vegetales y poblaciones de insectos criados en las instalaciones del ICA
según los protocolos del Grupo de Investigación Insectos Vectores de Patógenos de Plantas
(IVPP). Las especies vegetales utilizadas en esta Tesis Doctoral fueron cinco especies hortícolas
de gran valor económico en el área mediterránea: pepino (Cucumis sativum L.), pimiento
(Capsicum annuum L.), berenjena (Solanum melongena L.), tomate (Solanum lycopersicum L.) y
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melón (Cucumis melo L.). Se criaron cuatro especies de insectos plaga de cultivos hortícolas y
dos enemigos naturales ampliamente comercializados para el control biológico en invernaderos.
En concreto, se trabajó con los pulgones Myzus persicae (Sulzer) y Aphis gossypii Glover, la
mosca blanca Bemisia tabaci (Gennadius) y la polilla Tuta absoluta (Meyrick). En cuanto a los
enemigos naturales, se utilizaron el parasitoide Aphidius colemani Viereck y el sírfido
depredador Sphaerophoria rueppellii (Weidemann).
El primer objetivo de la Tesis Doctoral tuvo como finalidad estudiar el efecto de un parasitoide
sobre dispersión y distribución del pulgón A. gossypii, y la incidencia y distribución espacial de
dos virus, el Virus del mosaico del pepino (CMV, Cucumovirus) y el Virus del amarilleo de las
cucurbitáceas (CABYV, Polerovirus), transmitidos de manera no persistente y persistente,
respectivamente, por el pulgón A. gossypii. Para ello, se realizaron ensayos en jaulones de 1 m3
en los invernaderos del ICA. Se liberaron 100 pulgones y 5 parasitoides hembra sobre una planta
de pepino infectada con virus colocada en el centro del jaulón, y se evaluó su posición y
densidad en las 48 plantas colindantes a la planta fuente, así como la infección viral a corto y a
largo plazo (2 y 7 días para CMV, y 7 y 14 días para CABYV, respectivamente). El análisis
espacial de los datos se realizó mediante el procedimiento SADIE, estudiando los patrones de
distribución del vector y las virosis así como la asociación entre ambos agentes.
Como segundo objetivo, se estudió el comportamiento de vuelo de tres plagas clave de cultivos
hortícolas, el pulgón M. persicae, la mosca blanca B. tabaci y la polilla del tomate T. absoluta, y
dos enemigos naturales, el parasitoide A. colemani y el sírfido S. rueppellii, dentro de jaulones
de 1 metro de longitud revestidos con un amplio espectro de mallas fotoselectivas con distintos
niveles de absorción de radiación UV (2-83%) y PAR (54-85%) (O, G, A y P) (Figura 1.a).
Además se incluyeron dos testigos, una malla sin propiedades fotoselectivas (T) y un film de
plástico (PL) que bloqueaba el 98% de la radiación UV y permitía la difusión del 85% de luz
visible. Los ensayos se llevaron a cabo las primaveras de 2011 y 2012 al aire libre en dos
localizaciones, los campos de prácticas de la ETSIA y la estación experimental de La Poveda-
CSIC, respectivamente. Los insectos se liberaron en tubos desde una plataforma suspendida en el
aire con el fin de que tuvieran que iniciar el vuelo para poder desplazarse por el interior de los
jaulones. Se colocaron dianas a diferentes distancias de la plataforma de vuelo de acuerdo a cada
insecto: trampas amarillas para evaluar las capturas de pulgón y mosca blanca, plantas de tomate
para la oviposición de la polilla, plantas de pepino infestadas con A. gossypii para el parasitismo
de A. colemani, y plantas de pimiento infestadas con M. persicae para la oviposición del sírfido
(Figura 1.b). El segundo año se mejoró el diseño experimental en base a los resultados obtenidos
Resumen
ix
durante el primer año, aumentando la longitud del jaulón a 2 metros (Figura 1.c). Las dianas se
colocaron más separadas entre sí y a mayor distancia de la plataforma de vuelo (Figura 1.d).
Figura 1. Diseño experimental de los ensayos sobre la capacidad de vuelo de insectos en el interior de jaulones revestidos con materiales de distintas propiedades ópticas durante los años 2011 (a, b) y 2012 (c, d). Las letras T, P, A, O, G y PL corresponden a las abreviaturas de los distintos materiales testados (a, c). Se muestra una vista cenital de los jaulones con la disposición de la plataforma de liberación y las dianas (b, d; números 1 a 4).
En el tercer objetivo se investigó el efecto directo de la radiación UV-A sobre la eficacia
biológica (“fitness”) del pulgón M. persicae y la mosca blanca B. tabaci, y el efecto indirecto
mediado por sus plantas hospederas, pimiento y berenjena, crecidas con diferentes niveles de
radiación UV-A. Además, se investigó la respuesta directa de dichas plantas hospederas. Para
ello, las plantas crecieron en el interior de jaulones en los invernaderos del ICA, uno de ellos
cubierto con un film de plástico que bloqueaba la radiación UV-A, y el otro con un film testigo
transparente que permitía la transmisión de dicha radiación. Las plantas se cultivaron desde su
germinación hasta el final del ensayo bajo los dos tipos de ambiente lumínico, denominados
UVA- y UVA+ (Figura 2). Tras esta primera exposición a la radiación UV-A, y cuando las
plantas alcanzaron un estado fenológico de 10 hojas verdaderas en pimiento y 4 hojas en
berenjena, la mitad de las plantas crecidas bajo cada régimen lumínico se intercambió al régimen
contrario. Además, parte de las plantas se infestaron con formas inmaduras de los insectos
fitófagos objeto de estudio, el pulgón M. persicae y la mosca blanca B. tabaci, con el fin de
seguir su desarrollo y evolución. Al mismo tiempo, se tomaron muestras foliares para estimar los
parámetros fisiológicos de las plantas. A continuación, las plantas fueron sometidas a un nuevo
periodo de exposición a radiación UV-A empleando los mismos jaulones descritos anteriormente
(Figura 2). Se determinaron los parámetros de ciclo de vida del insecto (tiempo de desarrollo,
fertilidad, fecundidad y tasas de crecimiento), al tiempo que se volvió a tomar muestras foliares
x
para determinar los parámetros fisiológicos de las plantas al término del ensayo. La longitud
total del tallo y la superficie foliar se midieron semanalmente durante todo el ciclo del cultivo.
Se analizó el contenido en fenoles, azúcares, aminoácidos, proteínas y pigmentos fotosintéticos
(clorofilas y carotenoides) de las hojas recogidas.
Figura 2. Diagrama temporal de los ensayos sobre el efecto directo e indirecto de la radiación UV-A en insectos fitófagos y sus plantas hospederas. Se muestran los cuatro tratamientos (T1: UVA+/UVA+, plantas crecidas con UV-A durante todo el ciclo; T2: UVA+/UVA-, plantas crecidas con UV-A antes de la introducción de los insectos y sin UV-A tras dicha introducción; T3: UVA-/UVA+, plantas crecidas sin UV-A antes de la introducción de los insectos y con UV-A tras dicha introducción y T4: UVA-/UVA-, plantas crecidas sin UV-A durante todo el ciclo), la fecha de introducción de los insectos para el estudio de su eficacia biológica y las dos tomas de muestra de material vegetal. Las flechas que parten desde los tratamientos UVA+ y UVA- se refieren al momento en el que la mitad de las plantas crecidas bajo cada régimen lumínico se intercambió al régimen contrario.
Finalmente, en el cuarto objetivo de la Tesis Doctoral se realizó una selección de las mallas
impregnadas en los insecticidas piretroides deltametrina y bifentrín más eficaces para controlar
los insectos plaga M. persicae, A. gossypii y B. tabaci y sus virosis asociadas en base a ensayos
de laboratorio destinados a evaluar diferentes tamaños de poro y dosis de plaguicidas
incorporados a la malla. Se emplearon tubos de vidrio de 4 cm de diámetro donde se intercaló la
malla problema. En la parte inferior del tubo se liberaron los insectos y en la parte superior se
colocó una hoja diana con el fin de que los insectos atravesaran la malla. Se evaluó el número de
insectos capaces de atravesar la malla y asentarse sobre la hoja diana y el número de insectos
muertos en el tubo, a las 6 horas en pulgones y 24 horas en mosca blanca. La eficacia de dos
mallas impregnadas en bifentrín con poros de 0.46 y 0.29 mm2 que presentaron buenos
resultados en condiciones de laboratorio se evaluó en sendos ensayos de campo en los
invernaderos dobles tipo túnel (8 x 6.5 x 2.6 metros) de la estación experimental de La Poveda-
Introducción de insectos
Segunda toma de muestras foliares
Siembra Primera toma de muestras foliares
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Resumen
xi
CSIC durante el otoño de 2011 y 2013. En ellos, se colocaron unas secciones laterales de 4.3 x
3.5 metros con la malla impregnada o sin tratar, según tratamiento. El resto del túnel estuvo
cubierto de malla estándar que impedía el paso de insectos. En el interior de los túneles se
dispuso un cultivo de pepino y en el espacio entre el túnel interior y el exterior se plantaron
pimientos infectados con los virus CMV y CABYV sobre los que se liberaron las plagas A.
gossypii y B. tabaci. Los muestreos de las plagas se realizaron semanalmente, efectuando un
muestreo de presencia/ausencia en todas las plantas de cada módulo y conteos de densidad de
insecto en 11 plantas marcadas de cada módulo. Al final del ciclo, se analizó el porcentaje de
infección de ambos virus. El análisis espacial de los datos se realizó mediante el procedimiento
SADIE. La compatibilidad de una malla impregnada en bifentrín con el parasitoide A. colemani
se probó en ensayos de campo paralelos en módulos similares.
3. RESULTADOS
Los resultados del primer objetivo indicaron que, a corto plazo (2 días), la presencia del
parasitoide A. colemani favoreció la dispersión de A. gossypii, tal y como se confirmó con el
patrón no asociado entre virus y vector en presencia de los parasitoides (Figura 3). Esto ocasionó
una mayor incidencia de CMV en las plantas adyacentes a la planta fuente de virus (Figura 4).
Sin embargo, a largo plazo (7 días) no existieron diferencias en la incidencia viral de CMV en
presencia y ausencia de parasitoides. La tasa de transmisión de CMV en presencia de
parasitoides se mantuvo en un nivel similar al obtenido a corto plazo, mientras que el incremento
de la transmisión en el caso del tratamiento control puede ser explicado por la propia capacidad
colonizante del pulgón (Figura 4).
En cuanto a CABYV, a corto plazo (7 días), no se detectaron diferencias en la transmisión viral
entre tratamientos (Figura 4), aunque el número de ninfas de A. gossypii en presencia de
parasitoides fue significativamente menor. A largo plazo (14 días), la momificación de los
pulgones pudo haber reducido su vida activa como vectores, limitando con ello la incidencia
viral de CABYV en presencia del parasitoide respecto al tratamiento control (Figura 4). Además,
en ausencia del enemigo natural, el virus se distribuyó ampliamente por toda la superficie
experimental, mientras que en su presencia el movimiento de virus estuvo restringido a un gran
foco central (Figura 5).
xii
Figura 3. Mapas de distribución espacial de la población de Aphis gossypii y la infección viral de CMV a corto plazo (2 días), así como la asociación entre los dos agentes, virus y vector. Cada punto indica una planta receptora individual. Los puntos pequeños rellenos representan a índices de agrupación con valores de 0 a ±0.99, los círculos huecos a valores de ±1 a ±1.49 y los puntos grandes rellenos a valores >1.5 o <1.5. Las líneas rojas encierran focos de población con valor v=1.5 y las líneas azules a huecos sin población con valor v=-1.5. Las líneas negras muestran contornos de valor v=0, es decir, regiones intermedias entre zonas con focos y huecos. El índice de agregación, Ia, el índice positivo de agrupación en focos, vi, el índice negativo de agrupación en huecos, vj, y el índice de asociación espacial, X, se muestran encerrados por una línea naranja si son estadísticamente significativos. La letra N y la flecha se refieren al norte geográfico.
Foco Hueco 0 1 -1 -1.5 1.5 V Asociación
Disociación 0 0.025 0.05 0.95 0.975 p
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Resumen
xiii
Figura 4. Tasa de transmisión viral de los virus CMV y CABYV (%) en plantas de pepino dentro de jaulones control sin parasitoides y con parasitoides. Los asteriscos se refieren a diferencias estadísticamente significativas de acuerdo al test Chi-cuadrado (p≤0.05).
Figura 5. Mapas de distribución espacial de la población de Aphis gossypii y la infección viral de CABYV a largo plazo (14 días), así como la asociación entre los dos agentes, virus y vector. Los símbolos y contornos son los mismos que para la Figura 3.
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xiv
Los resultados del segundo objetivo mostraron diferencias en el comportamiento de vuelo entre
las distintas especies de insectos y los materiales que recubrían los jaulones donde se realizaron
las sueltas. El film de plástico absorbente PL afectó a los pulgones y las moscas blancas y las
condiciones pobres en radiación UV no permitieron iniciar el vuelo de estos insectos desde la
plataforma de liberación, mientras que los materiales fotoselectivos no tuvieron efecto en la
polilla del tomate, parasitoides y sírfidos. El film absorbente PL también redujo la capacidad de
M. persicae para localizar las trampas amarillas dentro de los jaulones. Así, una menor
proporción de pulgones alcanzó las trampas más alejadas de estos jaulones, mientras que los
insectos pudieron volar hasta el final de los jaulones revestidos con materiales transparentes,
como las mallas P y T (Figura 6.c y d). En cuanto a B. tabaci, el primer año de ensayos se
obtuvieron más capturas en las dianas cercanas a la plataforma de liberación independientemente
del material utilizado en los jaulones, y fueron disminuyendo con la distancia al punto de suelta.
Por el contrario, durante el segundo año, las capturas de mosca blanca sólo fueron
significativamente menores en las trampas más alejadas (T2 y T3) bajo los materiales más
absorbentes de radiación UV, el film PL y la malla O (Figura 6.a y b). Durante el primer año de
ensayos, la orientación de la polilla del tomate T. absoluta no estuvo afectada por la ausencia de
radiación UV. Sin embargo, cuando la jaula se amplió de tamaño en el segundo año, la
oviposición en la planta de tomate más cercana al punto de liberación fue significativamente
inferior bajo el film PL. En relación a los enemigos naturales, durante el primer año de ensayos
la tasa de parasitismo de A. colemani fue muy baja y significativamente menor en jaulones con
film PL frente a las mallas G y A. Tras mejorar el diseño experimental en el segundo año, la
absorción de radiación UV no afectó negativamente al parasitoide y no se encontraron
diferencias entre los tratamientos. De manera semejante, la tasa de oviposición del sírfido S.
rueppellii fue similar dentro de todos los jaulones.
Resumen
xv
Figura 6. Capturas de Myzus persicae y Bemisia tabaci (%) en trampas amarillas situadas a 1 (T1), 1.4 (T2) y 1.8 (T3) metros de distancia de la plataforma de liberación en jaulones de 2 metros cubiertos por diferentes materiales fotoselectivos: film PL (a), malla O (b), malla P (c) y malla T (d). Los asteriscos se refieren a diferencias estadísticamente significativas de acuerdo al test ANOVA (p≤0.05).
Los resultados del tercer objetivo evidenciaron que la radiación UV-A regula de manera directa
la fisiología vegetal con implicaciones para algunos insectos fitófagos. Ambas especies
vegetales, pimiento y berenjena, respondieron al estrés lumínico con una reducción del tallo, de
manera significativa en pimiento. En cambio, no se encontraron diferencias en el área foliar entre
tratamientos. Las plantas de pimiento sometidas a radiación UV-A tuvieron mayor contenido de
compuestos fenólicos, azúcares, aminoácidos libres y proteínas. El descenso del contenido en
aminoácidos en ausencia de radiación UV pudo modificar de manera indirecta la eficacia
biológica del pulgón M. persicae, ya que se observó un retraso en las tasas de crecimiento y un
descenso de la fecundidad cuando el pulgón se desarrolló en plantas crecidas sin radiación UV-A
independientemente del régimen lumínico al que se expuso a la plaga con posterioridad
(tratamientos UVA-/UVA- y UVA-/UVA+) (Figura 7). No existió un efecto directo de la luz
sobre el crecimiento del pulgón. Por el contrario, los niveles de clorofilas y carotenoides de las
hojas de berenjena disminuyeron con la radiación UV-A, y no se encontraron diferencias en el
resto de compuestos analizados. Respecto a B. tabaci, se observó un efecto directo negativo de la
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xvi
radiación UV-A que se tradujo en mayor duración del desarrollo larvario y menor fertilidad de
los huevos de mosca blanca (tratamientos UVA+/UVA+ y UVA-/UVA+). No existió un efecto
indirecto de las condiciones lumínicas en las que la planta fue crecida previamente a la
infestación del insecto (Figura 7).
Figura 7. Fecundidad de Myzus persicae en plantas de pimiento y Bemisia tabaci en plantas de berenjena sometidas a cuatro regímenes lumínicos de radiación UV-A. Las letras se refieren a diferencias estadísticamente significativas de acuerdo al test ANOVA (p≤0.05).
En el cuarto objetivo, se comprobó que todas las mallas impregnadas en deltametrina y bifentrín
evitaban el paso de M. persicae y A. gossypii en condiciones de laboratorio (<16% pulgones en
la hoja diana), teniendo las dos mejores mallas impregnadas en bifentrín un poro de 0.71 y 0.44
mm2. La persistencia de bifentrín fue buena tras un mes de exposición en condiciones de campo
durante el otoño, sin embargo su eficacia disminuyó tras dos meses expuestas en el exterior. En
cuanto al control de B. tabaci, fue necesario disminuir el tamaño del poro hasta 0.29 mm2 para
evitar su paso a través de la malla (6.77±2.46%). Asimismo, se produjo una disminución de la
eficacia de las mallas desde el primer mes de exposición a la luz solar.
En cuanto a los ensayos de campo, los resultados de 2011 indicaron que la malla con insecticida
de 0.46 mm2 redujo de manera significativa la densidad de población y la tasa de ocupación de
plantas con pulgones dentro de los módulos (Figura 8.a), si bien no fue eficaz en el control de la
mosca blanca, a pesar de que dicha malla fue efectiva en condiciones de laboratorio. La
incidencia de las virosis CMV y CABYV, así como el número de infecciones mixtas, fue
significativamente inferior dentro los módulos con malla insecticida (Figura 8.b). En el año
2013, los resultados observados siguieron la misma tendencia, con una menor tasa de ocupación
de pulgones y menor incidencia viral bajo la malla tratada de 0.29 mm2. El estudio espacial con
SADIE mostró que los pulgones colonizaron todo el módulo interior en el tratamiento control,
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Resumen
xvii
mientras que estuvieron limitados a los bordes con malla impregnada. La infección de CMV
tuvo una distribución regular en el tratamiento control y una distribución agregada bajo malla
impregnada. Además, se observó asociación significativa de ambos agentes en un módulo
control. La distribución de CABYV fue agregada en las líneas de plantas cercanas a las fuentes
de virus exteriores en los módulos control. Por el contrario, la infección en el tratamiento con
malla impregnada fue muy escasa e incluso inexistente en uno de los módulos, por lo que el uso
de estas barreras se perfila como una alternativa prometedora a los insecticidas de aplicación
foliar. La tasa de pulgones parasitados por A. colemani fue similar en módulos control y tratados
a lo largo del ciclo de cultivo, por lo que la malla impregnada en bifentrín fue compatible con la
actividad del parasitoide.
Figura 8. Densidad de Aphis gossipii alados (a) medidos con escala 0-5: 0 (0 pulgones), 1 (1-4 pulgones), 2 (5-19 pulgones), 3 (20-49 pulgones), 4 (50-149 pulgones), 5 (>150 pulgones), y tasa de transmisión viral de los virus CMV y CABYV (b) en plantas de pepino dentro de módulos control y módulos con malla impregnada en bifentrín. Los asteriscos se refieren a diferencias estadísticamente significativas de acuerdo al test de Student (a) y test Chi-cuadrado (b) (p≤0.05).
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xviii
4. DISCUSIÓN
La aplicación de estrategias alternativas al control químico, como el uso de enemigos naturales y
barreras físicas, ha permitido avanzar en el control de insectos vectores de hortícolas de una
forma más respetuosa con el medio ambiente (Jacas y Urbaneja, 2008; Robledo et al., 2009;
Colomer et al., 2011).
Los resultados obtenidos en la primera sección de este trabajo muestran cómo la presencia de A.
colemani ocasionó una rápida dispersión de A. gossypii desde la planta fuente, probablemente
debido a la emisión de feromonas de alarma (Losey y Denno, 1998; Day et al., 2006; Jeger et al.,
2011). Este escape se tradujo en un incremento de la transmisión de CMV en las plantas
colindantes (Roitberg y Myers, 1978; Weber et al., 1996; Belliure et al., 2011; Hodge et al.,
2011). Este resultado se explica debido al modo de transmisión no persistente de CMV, durante
breves periodos de adquisición e inoculación y sin periodo de latencia (Fereres y Moreno, 2009).
La incidencia de CMV fue similar a corto y largo plazo debido a que los pulgones perdieron
movilidad conforme fueron parasitados, mientras que se incrementó en las jaulas control por la
propia acción colonizante del vector. Como se comprobó en el análisis espacial mediante
SADIE, el pulgón mostró la distribución típica de un vector colonizante (Jeger et al., 2011). A
corto plazo, la infeccion por CMV se limitó a plantas aisladas en el tratamiento control, mientras
que la distribución fue agregada en las jaulas con parasitoides debido al movimiento del vector
desde la planta fuente a las plantas colindantes como consecuencia de la presión de A. colemani
(Jeger et al., 2011).
Estudios previos han demostrado que los enemigos naturales también pueden promover la
dispersión de virus persistentes, aunque la respuesta del vector está muy influenciada por los
hábitos del enemigo natural (Bailey et al., 1995; Smyrnioudis et al., 2001; Hodge y Powell,
2008a). No se encontraron diferencias entre tratamientos en la tasa de transmisión de CABYV a
corto plazo, sin embargo la infección fue menor en las jaulas con parasitoides a largo plazo
(Smyrnioudis et al., 2001), debido a la menor esperanza de vida y movilidad de los pulgones
virulíferos tras ser parasitados (Calvo y Fereres, 2011). Dentro de las jaulas se observaron
momias y parasitoides adultos tras dos semanas desde su liberación, lo que demuestra que A.
colemani fue capaz de establecerse en el medio (Zamani et al., 2007). Por lo tanto, la reducción
de la población de pulgones virulíferos limitó la dispersión de CABYV, tal y como se observó en
el patrón espacial de la enfermedad, corroborando el efecto beneficioso de los enemigos
naturales en el control de vectores de virus transmitidos de modo persistente.
Resumen
xix
El uso de revestimientos plásticos fotoselectivos se ha implementado de manera satisfactoria en
el control de plagas y enfermedades de cultivos protegidos (Antignus et al., 1998; Chyzik et al.,
2003; Díaz et al., 2006; Weintraub, 2009; Ben-Yakir et al., 2012; Legarrea et al., 2012a, b). Los
resultados obtenidos con anterioridad sugieren que el modo de acción de estas barreras es doble.
Por un lado, se bloquea el acceso de los insectos al interior de los invernaderos porque estos
exhiben una preferencia positiva por el ambiente exterior rico en radiación UV, y por otro lado
las condiciones creadas dentro del invernadero alteran el comportamiento de las plagas y limitan
su capacidad de dispersión una vez establecidas dentro del cultivo (Raviv y Antignus, 2004).
En los estudios del segundo objetivo se observó cómo el plástico absorbente PL impidió la salida
de pulgones y mosca blanca desde los tubos de liberación, lo que sugiere que el movimiento de
dichas plagas podría limitarse dentro de los invernaderos (Chyzik et al., 2003; Raviv y Antignus,
2004; Döring y Chittka, 2007), mientras que se observó un efecto neutral en los enemigos
naturales (Chyzik et al., 2003; Chiel et al., 2006; Doukas y Payne, 2007a, b).
Respecto a la capacidad de orientación de los insectos dentro de las jaulas, el plástico absorbente
PL interfirió con el vuelo de M. persicae, ya que redujo su capacidad de localizar las trampas
amarillas (Chyzik et al., 2003; Díaz et al., 2006; Ben-Yakir et al., 2012; Legarrea et al., 2012a).
Bajo ambientes con radiación UV, una mayor cantidad de pulgones recorrieron todo el jaulón
llegando hasta la última trampa de manera significativa y evidenciando un efecto rebote al caer
en la cara trasera de esta (Kring, 1972; Döring y Chittka, 2007). La actividad de vuelo fue muy
distinta entre pulgones y moscas blancas. En B. tabaci, las diferencias más acusadas se
obtuvieron en los plásticos más absorbentes en radiación UV (film PL y malla O), estando las
moscas restringidas a la trampa más cercana al punto de liberación, corroborando el menor
movimiento de B. tabaci en ambientes deficientes en luz UV (Costa y Robb, 1999; Antignus et
al., 2001; Mutwiwa et al., 2005; Legarrea et al., 2012c).
La ausencia de luz UV bajo el plástico absorbente PL afectó negativamente a la puesta de huevos
de T. absoluta, aunque no se observaron diferencias entre plantas colocadas a diferentes
distancias, probablemente porque este insecto localiza la planta por medio de estímulos olfativos
y no visuales (Proffit et al., 2011). En cuanto a los insectos beneficiosos, los revestimientos
absorbentes de radiación UV fueron compatibles con el vuelo de parasitoides y sírfidos y no
mermaron su actividad, posiblemente debido a que dichos insectos también se orientan hacia el
complejo planta-plaga por señales complementarias a las visuales, como las pistas olfativas (Du
et al., 1996; Storeck et al., 2000; Chiel et al., 2006; Boivin et al., 2012).
xx
Como ya se ha mencionado con anterioridad, la radiación UV también afecta a los insectos de
manera indirecta por los cambios que se producen en las plantas como respuesta a dicha
exposición (Vänninen et al., 2010; Johansen et al., 2011). Para intentar dilucidar el papel de los
efectos directo e indirecto en la eficacia biológica de las plagas, se estudiaron dos complejos
planta-insecto: el pulgón M. persicae en planta de pimiento y la mosca B. tabaci en planta de
berenjena.
Se constató un efecto negativo de la ausencia de radiación UV-A en el crecimiento poblacional
del pulgón M. persicae, tal y como se ha visto previamente en otras especies de pulgones
(Antignus et al., 1996; Chyzik et al., 2003; Díaz et al., 2006; Kuhlmann and Müller, 2009a; Paul
et al., 2011; Legarrea et al., 2012a). Este efecto fue indirecto mediado por el menor contenido en
azúcares y aminoácidos de las hojas de pimiento (Roberts y Paul, 2006; Comont et al., 2012),
compuestos involucrados en la nutrición de los pulgones (Dadd y Krieger, 1968; Mittler et al.,
1970; Srivastava y Auclair, 1971, 1975; Weibull, 1987). Por el contrario, la exposición de las
moscas blancas a la radiación UV-A no tuvo un efecto indirecto negativo en su eficacia
biológica. Además, la fisiología y crecimiento de la berenjena no se vio alterada en respuesta a
los distintos régimenes lumínicos aplicados, mostrando cierta tolerancia a la radiación UV-A
(González et al., 2009; Kulhmann y Müller, 2009a, 2010), por lo que presumiblemente el efecto
observado en B. tabaci fue directo y no estuvo mediado por la planta.
La fisiología y crecimiento de la planta de pimiento estuvo condicionada por la exposición a
UV-A. La altura de los pimientos fue menor como consecuencia del estrés lumínico, respuesta
encontrada en otras especies vegetales (Kuhlmann y Müller, 2010; Comont et al., 2012).
También se indujo la síntesis rápida de compuestos fenólicos (Gaberšcik et al., 2002, Izaguirre et
al., 2007; Mahdavian et al., 2008; Kulhmann y Müller, 2009a, 2009b, 2010), que actúan como
fotoprotectores frente a la radiación UV (Middleton y Teramura, 1993; Harborne y Williams,
2000).
Los resultados obtenidos en el cuarto objetivo sugieren que las mallas impregnadas con bifentrín
son una alternativa prometedora para evitar el paso de pulgones al interior de los cultivos
protegidos mientras se mantiene una buena aireación de los invernaderos (Bethke y Paine, 1991;
Muñóz et al., 1999). Todas las mallas impregnadas ensayadas frenaron el paso de M. persicae y
A. gossypii en condiciones de laboratorio frente a las mallas control, por lo que se puso de
manifiesto su beneficio adicional a las propiedades físicas de estos materiales (Martin et al.,
2006, 2007, 2010; Díaz et al., 2004). Con respecto a B. tabaci, fue necesario disminuir el tamaño
Resumen
xxi
del poro hasta 0.29 mm2 para conseguir una exclusión efectiva en condiciones de laboratorio. En
una segunda fase de la investigación se ensayaron dos mallas impregnadas en bifentrín de 0.46 y
0.29 mm2 en sendos ensayos de campo, liberando los insectos en plantas fuente de virus CMV y
CABYV colocadas en el exterior del cultivo protegido, para evaluar su asentamiento y
transmisión viral.
Aunque en condiciones de laboratorio la eficacia de estas mallas decreció con la exposición
solar, la cantidad remanente fue suficiente para el control eficaz de pulgones en los ensayos de
campo. De hecho, el insecticida bifentrín posee un alto efecto de “knockdown” o muerte rápida
por contacto, y mejor estabilidad química que otros piretroides (FAO, 2010). Así, la densidad
poblacional de pulgones y la incidencia de los virus CMV y CABYV fue significativamente
inferior en los módulos con malla tratada en ambos ensayos de campo (Martin et al., 2006;
Licciardi et al., 2008). Esto fue debido a la menor densidad de vectores durante las primeras
semanas del ensayo, ya que cuando intentaron atravesar la malla se impregnaron con bifentrín y
murieron antes de alcanzar el cultivo protegido.
El estudio con la metodología SADIE mostró diferencias espaciales en la distribución de los
virus dependiendo de su naturaleza no persistente o persistente. El virus no persistente CMV se
distribuyó de manera regular o aleatoria en los modulos control, mientras que estuvo agregado
bajo malla tratada. Por el contrario, se observó una agregación significativa del virus persistente
CABYV en los bordes de los modulos control, lo que sugiere que el foco inicial se originó cerca
de las plantas fuentes y se extendió a plantas colindantes. Además, la población de pulgones
estaba asociada a las plantas infectadas con CABYV en los módulos tratados, propio de virus
transmitidos de manera persistente (Irwin y Thersh, 1990).
Ninguna de las dos mallas fue eficaz en el control de la mosca blanca, posiblemente debido al
tamaño del insecto y su resistencia a insecticidas piretroides (Byrne y Bellows, 1991; Whalon et
al., 2008). Estos resultados contrastan con los obtenidos con mallas impregnadas en alfa-
cipermetrina, donde se consiguió controlar la mosca blanca con una malla de 0.9 mm de
diámetro (Martin et al., 2014). Por último, la malla impregnada en bifentrín ensayada en campo
fue compatible con la actividad del parasitoide A. colemani, comprobando que dichas mallas
pueden ser implementadas junto con el control biológico.
En este trabajo se ha puesto de manifiesto el importante papel de los enemigos naturales en la
distribución espacio-temporal de los insectos vectores, factor a tener en cuenta en la dispersión
de las virosis vegetales. Asimismo, tanto las mallas fotoselectivas de luz UV como las
xxii
impregnadas con insecticidas presentaron características beneficiosas adicionales al control
físico de insectos vectores, y constituyen herramientas que deben ser consideradas en los
programas de Manejo Integrado de Plagas en cultivos hortícolas de invernadero como
alternativas al uso de insecticidas.
xxiii
SUMMARY
Insect vectors of plant viruses are the main agents causing major economic losses in vegetable
crops grown under protected environments. This Thesis focuses on the implementation of new
alternatives to chemical control of insect vectors under Integrated Pest Management programs.
In Spain, biological control is the main pest control strategy used in a large part of greenhouses
where horticultural crops are grown. The first study aimed to increase our knowledge on how the
presence of natural enemies such as Aphidius colemani Viereck may alter the dispersal of the
aphid vector Aphis gossypii Glover (Chapter 4). In addition, it was investigated if the presence of
this parasitoid affected the spread of aphid-transmitted viruses Cucumber mosaic virus (CMV,
Cucumovirus) and Cucurbit aphid-borne yellows virus (CABYV, Polerovirus) infecting
cucumber (Cucumis sativus L). SADIE methodology was used to study the distribution patterns
of both the virus and its vector, and their degree of association. Results suggested that parasitoids
promoted aphid dispersal in the short term, which enhanced CMV spread, though consequences
of parasitism suggested potential benefits for disease control in the long term. Furthermore, A.
colemani significantly limited the spread and incidence of the persistent virus CABYV in the
long term.
The flight activity of pests Myzus persicae (Sulzer), Bemisia tabaci (Gennadius) and Tuta
absoluta (Meyrick), and natural enemies A. colemani and Sphaerophoria rueppellii
(Weidemann) under UV-deficient environments was studied under field conditions (Chapter 5).
One-chamber tunnels were covered with cladding materials with different UV transmittance
properties. Inside each tunnel, insects were released from tubes placed in a platform suspended
from the ceiling. Specific targets were located at different distances from the platform. The
ability of aphids and whiteflies to reach their targets was diminished under UV-absorbing
barriers, suggesting a reduction of vector activity under this type of nets. Fewer aphids reached
distant traps under UV-absorbing nets, and significantly more aphids could fly to the end of the
tunnels covered with non-UV blocking materials. Unlike aphids, differences in B. tabaci
captures were mainly found in the closest targets. The oviposition of lepidopteran T. absoluta
was also negatively affected by a UV-absorbing cover. The photoselective barriers were
compatible with parasitism and oviposition of biocontrol agents.
xxiv
Apart from the direct response of insects to UV radiation, plant-mediated effects influencing
insect performance were investigated (Chapter 6). The impact of UV-A radiation on the
performance of aphid M. persicae and whitefly B. tabaci, and growth and leaf physiology of host
plants pepper and eggplant was studied under glasshouse conditions. Plants were grown inside
cages covered by transparent and UV-A-opaque plastic films. Plant growth and insect fitness
were monitored. Leaves were harvested for chemical analysis. Pepper plants responded directly
to UV-A by producing shorter stems whilst UV-A did not affect the leaf area of either species.
UV-A-treated peppers had higher content of secondary metabolites, soluble carbohydrates, free
amino acids and proteins. Such changes in tissue chemistry indirectly promoted aphid
performance. For eggplants, chlorophyll and carotenoid levels decreased with supplemental UV-
A but phenolics were not affected. Exposure to supplemental UV-A had a detrimental effect on
whitefly development, fecundity and fertility presumably not mediated by plant cues, as
compounds implied in pest nutrition were unaltered.
Lastly, the efficacy of a wide range of Long Lasting Insecticide Treated Nets (LLITNs) was
studied under laboratory and field conditions. This strategy aimed to prevent aphids and
whiteflies to enter the greenhouse by determining the optimum mesh size (Chapter 7). This new
approach is based on slow release deltamethrin- and bifenthrin-treated nets with large hole sizes
that allow improved ventilation of greenhouses. All LLITNs produced high mortality of M.
persicae and A. gossypii although their efficacy decreased over time with sun exposure. It was
necessary a net with hole size of 0.29 mm2 to exclude B. tabaci under laboratory conditions. The
feasibility of two selected nets was studied in the field under a high insect infestation pressure in
the presence of CMV- and CABYV-infected cucumber plants. Besides, the compatibility of
parasitoid A. colemani with bifenthrin-treated nets was studied in parallel field experiments.
Both nets effectively blocked the invasion of aphids and reduced the incidence of both viruses,
however they failed to exclude whiteflies. We found that our LLITNs were compatible with
parasitoid A. colemani.
As shown, the role of natural enemies has to be taken into account regarding the dispersal of
insect vectors and subsequent spread of plant viruses. The additional benefits of novel physico-
chemical barriers, such as photoselective and insecticide-impregnated nets, need to be
considered in Integrated Pest Management programs of vegetable crops grown under protected
environments.
1
CHAPTER 1. INTRODUCTION
1.1. VEGETABLE PRODUCTION IN PROTECTED ENVIRONMENTS
Vegetable production plays a major role in Spanish agriculture, with 9,941,200 t and ranks first
in Europe in greenhouse production area (65,055 ha) (EUROSTAT, 2009; MAGRAMA, 2014).
Andalucía holds approximately 70% of the Spanish greenhouse area with 47,367 ha
(MAGRAMA, 2014). Spanish cucumber, pepper and eggplant productions are in the top 10
worldwide ranking (FAOSTAT, 2012) (Table 1.1).
Table 1.1. Crop production in the world, year 2012.
Rank Cucumber and gherkin Chilli and pepper Eggplant
Country Yield (t) Country Yield (t) Country Yield (t)
1 China 48,000,000 China 16,000,000 China 28,800,000
2 Turkey 1,741,878 Mexico 2,379,736 India 12,200,000
3 Iran 1,600,000 Turkey 2,072,132 Iran 1,300,000
4 Russia 1,281,788 Indonesia 1,656,615 Egypt 1,193,854
5 Ukraine 1,020,600 USA 1,064,800 Turkey 799,285
6 USA 901,060 Spain 1,023,700 Indonesia 518,827
7 Spain 713,200 Egypt 650,054 Iraq 460,000
8 Mexico 640,508 Nigeria 500,000 Japan 327,400
9 Egypt 613,880 Algeria 426,566 Spain 246,600
10 Japan 586,500 Ethiopia 402,109 Italy 217,690
Cucumber (Cucumis sativum L.) is an annual warm-season cucurbit native to Southern Asia with
a rough, tender vine, trailing stems and hairy leaves. Cucumber is one of the main vegetables
grown in Southern Spain and comprises 8,902 ha yielding 753,941 t (MAGRAMA, 2013). It is
mainly grown inside greenhouses (90%) and under biological control, being the cotton aphid
Aphis gossypii Glover (Hemiptera: Aphididae) the main key pest (Robledo et al., 2009;
MAGRAMA, 2013).
Sweet pepper (Capsicum annuum L.) is an herbaceous solanaceae native to Central and South
America and is of high agricultural importance (Arranz-Arranz, 2008). The crop cycle starts
2
from summer to autumn in warm and dry climates (optimal conditions: 20-25 ºC during the day
and 16-18 ºC at night), although it can be also cultivated in temperate climates or off-season
inside protected environments (Arranz-Arranz, 2008). In the Mediterranean area, pepper is one
of the main vegetable crops produced under covered structures (65%), with 18,108 ha yielding
1,016,811 t (MAGRAMA, 2013).
Eggplant (Solanum melongena L.) is a tropical perennial solanaceae that is mainly cultivated as
an annual in temperate climates. It is native to Indian subcontinent and was domesticated in
Bangladesh and India. It has spiny stems, large and coarse leaves, and an egg-shaped purple
fruit. Eggplant requires higher temperature and irradiance than tomato or pepper (23-25 ºC
during the day and 16-18 ºC at night. The Spanish production is 206,333 t in 3,665 ha
(MAGRAMA, 2013).
1.2. INSECT PESTS
Arthropods are probably the most successful and diverse animal phylum. Insecta is the largest
class of this phylum, and it is the ecological guild that has most species-richness members.
Within this class, the order Hemiptera comprises small sap-sucking insects with a needle-like
stylet bundle consisting of two mandibular and two maxillary stylets, such as aphids (Hemiptera:
Aphididae) and whiteflies (Hemiptera: Aleyrodidae). In the order Thysanoptera, thrips, or cell-
content feeders, have mouthparts composed of two maxillary stylets and one mandibular stylet
(Hogenhout et al., 2008).
About 450 of the 4700 species of the family Aphididae in the world have been recorded from
crop plants, being 100 of them of major economic importance that feed on herbaceous plants
(Blackman & Eastop, 2000). Most agriculturally important species are in the subfamily
Aphidinae, with life cycles tied to temperate seasonality and the phenology of their hosts
(Blackman & Eastop, 2007). Aphids are considered one of the most important pests worldwide
not only because of the direct damage they cause, but also because their alimentary habits
involve indirect damage. They excrete honeydew and the development of sooty molds eventually
reduces the quality of production. Most importantly, they are the major group of vectors of plant
viruses. The family Aphididae includes the greater proportion of insect vectors. They are thought
to transmit almost half of the plant viruses and be the most efficient vectors of approximately
275 virus species within 19 virus genera (Nault, 1997; Hull, 2014; Ng & Perry, 2004).
Introduction
3
The life cycle of most aphids alternates one sexual generation in the autumn to produce
overwintering eggs -with short photoperiod inducing sexual morphs-, and a succession of female
telescopic parthenogenetic generations during spring and summer, reducing the generation time
and enabling the exploitation of periods of rapid plant growth (holocyclic life cycle) (Dixon,
1973). Thus, they are potentially harmful insects as their populations increase in a very short
period of time by means of telescopic generations (Blackman & Eastop, 2000). They share some
morphological characteristics such as the secretory organ known as siphunculi, two-segmented
tarsi, five- or six-segmented antennae and the cauda. Moreover, aphids exhibit dimorphism with
apterae and winged morphs. Crowding, poor nutrition or low temperature may produce alate
individuals, responsible for long distance dispersal (Blackman & Eastop, 2007).
The cotton aphid A. gossypii and the green peach aphid Myzus persicae (Sulzer) are among the
14 aphid species of most agricultural importance (Blackman & Eastop, 2007). Aphis gossypii is a
highly polyphagous cosmopolitan pest that colonizes more than 100 crop plants, widely
distributed in crops such as cotton, zucchini, melon, cucumber or citrus, belonging to the
families Cucurbitaceae, Malvaceae and Rutaceae (Blackman & Eastop, 2000). During
unfavorable environmental periods, A. gossypii can complete the sexual cycle in species of
genuses Catalpa or Hibiscus acting as primary hosts. However, with optimal climate conditions,
aphids may produce quick offspring with about fifty generations a year. Besides, A. gossypii is
able to efficiently transmit more than 50 plant viruses (Blackman & Eastop, 2000) (Figure 1.1).
Myzus persicae is another cosmopolitan, very polyphagous pest and highly efficient as a virus
vector of more then 100 plant viruses (Blackman & Eastop, 2007). Its sexual phase occurs in
Prunus persica Batsch or Prunus nigra Aiton, where spring populations may become very dense
and curl the leaves. In contrast, the secondary hosts belong to more than 40 plant families, some
of them economically important crops such as pepper or turnip (Blackman & Eastop, 2007)
(Figure 1.2).
4
Figure 1.1. The cotton aphid, Aphis gossypii: nymph (left), apterae adult (center), alate adult (right).
Figure 1.2. The green peach aphid, Myzus persicae: nymph (left), apterae adult (center), alate adult (right).
Around 1200 whitefly species are known within the family Aleyrodidae. Whiteflies are small
sized-tropical pests of equal importance as aphids, in the sense that its feeding involves sooty
mold fungi development and virus transmission (Byrne & Bellows, 1991). Many whiteflies that
feed on woody angiosperms are usually monophagous or oligophagous but polyphagy is found
on whiteflies of herbaceous plants (Mound & Halsey, 1978). Their life cycle consists of four
nymphal instars, the first having functional walking legs (“crawler”). The fourth nymphal stage
is commonly referred as pupa, and is flattened and translucent in the early phase. As the pupa
develops, the eyes and body of the adult become visible. At this point, apolysis is complete and
the adult emerges, showing typical dimorphism with males smaller than females. Then, mating
and oviposition takes place. They produce wax in all life stages except the egg (Byrne &
Bellows, 1991).
One of the most important whitefly pests in agriculture is Bemisia tabaci (Gennadius) (Gerling et
al., 2001). Its body is 0.80-0.95 mm long and 0.5 mm wide. Its wings are held tent-like above the
body and slightly apart (measurement with wing expanse ranges between 1.75 and 2.19 mm).
Bemisia tabaci has a wide range of host plants including crops, vegetables and ornamental plants
in tropics and subtropics such as cotton, soybean, melon, tomato, eggplants or poinsettia (Byrne
Introduction
5
& Bellows, 1991; Ellsworth & Martínez-Carrillo, 2001; Berlinger et al., 2002). It is a major
vector of more than 110 plant viruses, some of them in a persistent manner and as devastating as
Tomato yellow leaf curl virus (TYLCV, Begomovirus) (Berlinger et al., 2002; Glick et al., 2009)
(Figure 1.3). Nowadays, B. tabaci is considered a complex of more than morphologically
indistiguishable 28 cryptic species that differ in host range and virus transmission ability among
other biological properties (Barro et al., 2011).
Figure 1.3. The silverleaf whitefly, Bemisia tabaci: nymph and larvae (left), adult (right).
1.3. PLANT VIRUSES
Plant viruses cause devastating diseases in a wide host range, some of which are of enonomic
importance. Viruses reduce production and quality producing significant economic losses in
vegetable production worldwide (Hull, 2014). It has been estimated that viruses are responsible
for the loss of 10% of global food production, behind losses produced by phytopathogenic fungi
(Strange & Scott, 2005). Transmission from plant to plant by vectors is one of the most useful
virus strategies of dispersal, as they are obliged parasites (Hull, 2014). Virus transmission
entitles a direct interaction between host, virus and vector.
Four phases can be distinguished in this process:
• Acquisition phase: time in which the vector feeds on an infected plant so that it acquires
the viral particles.
6
• Latent period: time between the virus acquisition by the vector and its ability to transmit
the virus.
• Retention period: time in which the vector remains competent for virus transmission
subsequent to acquisition.
• Inoculation phase: process in which the virus is inoculated by the vector to the plant.
Viruses can be classified into two categories differing by the time that the vector is able to
transmit the disease: non-circulative or cuticula-borne viruses (with two different categories:
non-persistent and semipersistent) and circulative or foregut-borne viruses (Nault, 1997; Hull,
2014; Ng & Perry, 2004; Fereres & Moreno, 2009; Bragard et al., 2013; Blanc et al., 2014).
Non-persistent viruses are efficiently acquired and inoculated during very brief (5-10 seconds)
intracellular probes on the epidermal plant tissues without a latent period. Non-persistent viruses
have very short retention times up to 12 hours (Ng & Falk, 2006) and they are unable to cross the
insect’s gut, thus viruses can be retained in the cuticula at the tip of the stylets (Fereres &
Moreno, 2009). For semipersistent viruses, the vector remains viruliferous from hours to days
(Bragard et al., 2013).
Depending on the capacity of the virus to replicate in vector cells, circulative viruses can be
classified as propagative or non-propagative. Most circulative viruses require aphids to feed
from the phloem of an infected plant during a sustained and/or long period of time (phloem-
restricted virus). Circulative viruses exhibit a delay called latent period, which is defined as the
time elapsed beween the moment in which the vector feeds on the infected plant and its ability to
transmit the virus. The latent period ranges from days to weeks. The virus circulates through the
vector body and the vector is viruliferous even after molting (Hogenhout et al., 2008). These
viruses have a very specific relationship with their vectors. Viral particles must encounter a
series of selective barriers to finally reach the accessory salivary glands from which they will be
inoculated together with watery saliva (Moreno et al., 2011; Gray et al., 2014).
Aphids are major vectors of plant viruses. Two good examples are Cucumber mosaic virus
(CMV) and Cucumber aphid-borne yellows virus (CABYV), both transmitted by A. gossypii that
cause severe yield loss in cucurbits. Both viruses have different transmission modes. CMV is one
of the most important plant viruses, with a very broad host range, infecting more than 1200 plant
species in 100 families, including ornamentals, woody plants and important crops such as
pepper, lettuce, beans or cucurbits (Scholthof et al., 2011). CMV is the type member of the
Introduction
7
genus Cucumovirus in the family Bromoviridae. It is an isometric virus with a tripartite genome
of messenger-sense, single stranded RNA. CMV is transmitted in a cuticula-borne, non-
persistent manner by more than 80 aphid species in 33 genera. Besides, CMV may be
transmitted by mechanical contact, seed and pollen (Palukaitis & García-Arenal, 2003). CMV-
infected plants show yellowish patches, green and yellow mottling on leaves, and reduced plant
growth (Figure 1.4).
CABYV is a circulative, non-propagative virus that belongs to the Polerovirus genus in the
Luteoviridae family, and was first described by Lecoq et al. (1992). CABYV causes serious
losses on field-grown cucurbits in Spain (Juárez et al., 2013; Kassem et al., 2013). It is a 2.5 nm
in diameter, isometric virus with a single stranded positive-sense RNA 5.7 kb in length (Guilley
et al., 1994). Typical symptoms of CABYV-infected plants are interveinal yellowing of the basal
leaves and stunting that later turn into a general yellowing and necrosis at the end of the cycle
(Figure 1.4).
Figure 1.4. Symptoms of Cucumber mosaic virus (left) and Cucumber aphid-borne yellows virus (right) in cucumber leaves.
1.4. INTEGRATED PEST MANAGEMENT
Integrated Pest Management (IPM) is defined as “a decision support system for the selection and
use of pest control tactics, singly or harmoniously coordinated into a management strategy,
based on cost/benefit analyses that take into account the interests of and impacts on producers,
society and the environment” (Kogan, 1998). IPM programs are designed around four basic
components: action thresholds, pest monitoring, preventive cultural practices and control. They
were implemented to minimize insecticide hazards to crops, humans and environment, and to
adopt non-chemical measures. Nowadays, existing European and Spanish regulations aim to
8
reduce the dependence on chemical products used in agriculture to lower the maximum residue
levels for each product (BOE, 2004; OJEC, 2005, 2009). Andalucía holds 62.5% Spanish
production area dedicated to IPM with 520,324 ha of horticultural crops and fruit trees
(MAGRAMA, 2013).
1.4.1. BIOLOGICAL CONTROL
Biological control is the major component of IPM programs in Spain since 2004, especially in
the Mediterranean area, which concentrates 85% of Spanish vegetable production (Colomer et
al., 2011). Following Spanish regulations, biological control is the main pest control strategy for
sweet pepper, tomato and cucurbit production (BOE, 2002, 2004; Jacobson, 2004; Ramakers,
2004; Beltrán et al., 2010; Van der Blom et al., 2010). Among these crops, sweet pepper is
probably the best adapted with virtually all the production grown under biological control (Van
der Blom et al., 2009).
Predators and parasitoids comprise the third trophic level. Predation is defined as the
consumption of prey resulting in the host’s death. Predators feed on more than one prey during
their lifetime. On the contrary, parasitoids feed on just one prey slowly until the host develops
into a mummy, eventually is consumed and dies (Van den Bosch & Messenger, 1973).
Beneficial insects can be purchased from commercial suppliers, which are expanding in numbers
and replacing the use of insecticides in protected crops in many parts of the world (Van Lenteren
& Bueno, 2014). As an example, the polyphagous solitary endoparasitoid Aphidius colemani
Viereck (Hymenoptera: Braconidae) is a major natural enemy of A. gossypii that is widely used
in biological control (Boivin et al., 2012) (Figure 1.5). The addition of natural enemies to the
plant-virus-vector system might involve more complicated interactions between the agents, as
natural enemies not only reduce the levels of herbivore pressure and vector numbers, but also
may greatly modify the incidence of disease within the plant population.
Introduction
9
Figure 1.5. The parasitoid Aphidius colemani attacking an aphid (left) and developed mummies (right).
1.4.2. PHYSICAL CONTROL
Mechanical and physical controls involve the use of barriers, traps or physical removal, making
the environment unsuitable for pests to survive. More specifically, physical control includes
mulches, steam sterilization of the soil, traps, as well as barriers and screens (Weintraub &
Berlinger, 2004; Antignus, 2014).
1.4.2.1. INSECTICIDE-TREATED NETS
Insecticide-treated nets were developed long ago as bednets in public health to give protection
against malaria (Hougard et al., 2002). This strategy was approved for use with pyrethroids,
compounds that exhibit a rapid knockdown effect and high insecticidal potency at low dosage
without mutagenic or teratogenic effects (Zaim et al., 2000). The insecticide may be applied to
the net surface by immersion or spraying, but also by incorporation in the process while making
the yarns in the factory. In the latter case, the nets are called Long Lasting Insecticide-Treated
Nets (LLITN), and the insecticide may persist more than three years under field conditions
(Martin et al., 2007).
Field experiments using LLITNs have demonstrated promising outcomes against agricultural
pests such as mites in crops such as African eggplant, resulting in higher yields (Martin et al.,
2010), and brassica crops (Díaz et al., 2004; Martin et al., 2006; Licciardi et al., 2008). These
nets have proven to be cost-effective in cabbage production. LLITNs serve as an effective barrier
to control a wide range of Lepidopteran pests, including the diamondback moth Plutella
xylostella L. (Lepidoptera: Plutellidae) and cabbage loopers, or the aphid Lipaphis erysimi
10
Kaltenbach (Hemiptera: Aphididae), but not against the cabbage whitefly Aleyrodes proletella L.
(Hemiptera: Aleyrodidae) (Díaz et al., 2004; Martin et al., 2006; Licciardi et al., 2008).
1.4.2.2. UV-ABSORBING PLASTIC COVERS
Ultraviolet (UV) radiation belongs to the non-ionizing part of the electromagnetic spectrum and
ranges between 100 and 400 nm; 100 nm has been chosen arbitrarily as the boundary between
non-ionizing and ionizing radiation. The International Commission on Illumination
conventionally categorizes the official UV ranges into 3 regions, although there is variation in
usage:
UV-A: 315-400 nm UV-B: 280-315 nm UV-C: 100-280 nm
These photoselective covers act as filters that do not transmit the majority of UV light. The lack
of UV radiation has major consequences on insect pests, since it might greatly modify their
orientation toward potential hosts, flight activity, alighting, arrestment, feeding behaviour, and
interaction between sexes (Raviv & Antignus, 2004). There are two mechanisms reported for the
anti-insect activity of these materials. First, the number of insects that invade the enclosed
greenhouse is lower due to the higher UV reflectance emitted by the sky or reflected from these
covers. Second, the light environment created inside alters the normal behaviour of insects, thus
resulting in reduced flight activity (Raviv & Antignus, 2004; Antignus, 2012).
At the same time, not only does UV radiation directly influence insects but also indirectly via
plant’s physical and biochemical traits (Vänninen et al., 2010; Johansen et al., 2011). UV
cladding materials lead to changes on crop growth, epicuticular waxes, leaf thickness and
trichome density (Hunt & McNeil, 1999; Kittas et al., 2006; Kuhlmann & Müller, 2009a, 2010;
Paul et al., 2011). Also, there is evidence that UV transmitting environments could produce food
plants with improved features such as increased anthocyanins, flavonoids or phenolics involved
in strawberry and grapefruit ripening processes (Tsormpatsidis et al., 2011; Carbonell-Bejerano
et al., 2014).
1.5. EFFECTS OF UV RADIATION ON PLANTS
Knowledge on the effects of UV-B on plant growth and nutritional characteristics relevant to
insects has developed due to past concerns about ozone depletion (Ballaré et al., 1996; Hunt &
McNeil, 1999; Mackerness, 2000; Jansen, 2002; Comont et al., 2012; Mewis et al., 2012). Our
knowledge on the effects of UV-B suggests that typical plant responses would include the
Introduction
11
accumulation of UV-screening metabolites, increased leaf thickness and trichome density or
reduction in cell elongation (Smith et al., 2000; Paul & Gwynn-Jones, 2003; Liu et al., 2005;
González et al., 2009; Kulhmann & Müller, 2009a, 2010).
Only a few authors have considered UV-A impacts on plant growth (Tezuka et al., 1994;
Jayakumar et al., 2003, 2004; Verdaguer et al., 2012). Verdaguer et al. (2012) showed that
radiation in the UV-A range produces alterations in leaf morphology and anatomy, with the most
characteristic response mainly observed in the adaxial epidermal cells, which were thicker and
longer than those grown without UV-A. Understanding of the indirect effects of UV-A on
insects via plants remains limited to what we know about current practices in horticulture.
However, evidence suggests that supplemental UV-A may improve plant growth, yield and
quality of soybean (Middleton & Teramura, 1993) and black lentil (Jayakumar et al., 2003).
1.6. EFFECTS OF UV RADIATION ON PESTS, VIRUSES AND BENEFICIALS
Many insects, including aphids and pollinators, have a trichromatic system with an ultraviolet
receptor peaking at 320-330 nm, a second one with the peak in the blue region at 440-480 nm
and a third green receptor with a maximum sensitivity around 530 nm (Briscoe & Chittka, 2001;
Kirchner et al., 2005; Skorupski et al., 2007). Two ranges of the spectrum have been identified
in whiteflies, with UV radiation correlated to migratory behaviour and yellow wavelengths with
settlement (Mound, 1962; Coombe, 1982). Thrips (Thysanoptera: Thripidae) have two peaks of
efficiency, one sensitive to UV wavelengths at 365 nm and another in the green region at 540
nm, although there is no physiological evidence for a third photoreceptor in the blue region
(Matteson et al., 1992; Mazza et al., 2010).
Aphids drastically reduce their flight activity under UV-deficient ambients (Chyzik et al., 2003;
Díaz & Fereres, 2007; Döring & Chittka, 2007; Legarrea et al., 2012a). Moreover, a decrease in
fecundity and population density has been also demonstrated (Antignus et al., 1996; Chyzik et
al., 2003; Díaz et al., 2006; Kuhlmann & Müller, 2009a; Paul et al., 2011; Legarrea et al.,
2012b). Lower densities of several whitefly species have been found under UV-deficient screens
in greenhouse and field studies (Costa & Robb, 1999; Antignus et al., 2001; Mutwiwa et al.,
2005; Legarrea et al., 2012c). Research on spider mites concluded that Tetranychus urticae Koch
(Acari: Tetranychidae) exploits UV-A information to avoid ambient UV-B radiation (Sakai &
Osakabe, 2010). At the same time Panonychus citri McGregor (Acari: Tetranychidae) eggs were
tolerant to UV-B radiation and females successfully oviposited on the upper side of leaves
exposed to UV-B (Fukaya et al., 2013).
12
The management of aphid and whitefly-borne viruses by optical barriers suggests that the
blockage of UV light may decrease virus incidence and spread because of the reduced dispersal
of vector populations (Antignus et al., 1996, 1998; Kumar & Poehling, 2006; Díaz et al., 2006;
Díaz & Fereres, 2007; Weintraub, 2009; Ben-Yakir et al., 2012; Legarrea et al., 2012a).
The spectral efficiency of the parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae)
has been previously described, with a primary peak at 520 nm and a secondary peak in the UV
region (340-400 nm) (Mellor et al., 1997). It has been reported that aphid and whitefly
parasitoids are attracted to high UV radiation but they can perform well in a UV-filtered
environment, occasionally having a better distribution and dispersal (Chyzik et al., 2003; Chiel
et al., 2006; Doukas & Payne, 2007a, b). Conversely, there is a scarcity of data on predators and
further knowledge is needed in this area (Reitz et al., 2003; Legarrea et al., 2012c). Finally, a
large body of material on UV effects on plant-pollinator interactions has been published
(Stephanou et al., 2000; Petropoulou et al., 2001; Dyer & Chittka, 2004).
13
CHAPTER 2. OBJECTIVES
The general objective of this Thesis focuses on the integration of different tactics and the
implementation of new alternatives to pesticides for the control of insect vectors of plant viruses
under protected agriculture. Among these, novel physico-chemical barriers, such as UV-
photoselective covers and insecticide-impregnated nets, were tested with pests and beneficials.
In addition, the effect of biological control on aphid dispersal and virus spread was also studied.
Specific objectives were covered:
1. To investigate the role of parasitoid Aphidius colemani Viereck in the dispersal of the
cotton aphid Aphis gossypii Glover and the spread of plant viruses Cucumber mosaic
virus (CMV, Cucumovirus) and Cucumber aphid-borne yellows virus (CABYV,
Polerovirus) in a cucumber crop under glasshouse conditions.
2. To evaluate the direct effect of UV-absorbing cladding materials with different light
transmittances on the flight activity and orientation of pests Myzus persicae (Sulzer),
Bemisia tabaci (Gennadius) and Tuta absoluta (Meyrick), and natural enemies A.
colemani and Sphaerophoria rueppellii (Weidemann) under field conditions.
3. To study the direct impact of UV-A radiation on the performance of the green peach
aphid M. persicae and the silverleaf whitefly B. tabaci, and on the physiology and
biochemistry of their host plants, pepper and eggplant, as well as the indirect impact of
UV-A radiation on pests mediated by host plant growth under glasshouse conditions.
4. To evaluate the efficacy of a wide range of Long Lasting Insecticide Treated Nets
(LLITNs) against M. persicae, A. gossypii and B. tabaci under laboratory conditions, and
to test the feasibility of selected nets under a high insect infestation pressure to assess
vector colonization and the spread of aphid-transmitted viruses CMV and CABYV in a
cucumber crop under field conditions. To test the compatibility of LLITNs with aphid
parasitoids.
14
15
CHAPTER 3. MATERIALS AND METHODS
3.1. EXPERIMENTAL SITES
Experiments were conducted at the Institute of Agricultural Sciences (ICA) of the Spanish
National Research Council (CSIC) (Madrid, Spain) (40° 26’ 23’’ N, 3° 41’ 14’’ W), at “La
Poveda Experimental Farm” (Madrid, Spain) (40°18' 58"N, 3°29' 05"W) and in the experimental
field at the College of Agricultural Engineering (ETSIA) of the Polytechnic University of
Madrid (UPM) (Spain) (40º 26’ 35’’ N, 3º 44’ 19’’ W). Some experiments were performed in a
short term research placement in collaboration with Dr. Dylan Gwynn-Jones at the Institute of
Biological, Environmental and Rural Sciences (IBERS) of the Aberystwyth University (United
Kingdom) (52°24' 59"N, 4°03' 56"W).
Plants and insects used in the experiments were grown and reared in controlled conditions inside
climate chambers or greenhouse facilities acording to protocols previously established in the
Insect Vectors of Plant Pathogens laboratory at ICA-CSIC.
3.2. PLANT DEVELOPMENT
Several plant species were grown for the experiments at ICA-CSIC, “La Poveda Experimental
Farm” and ETSIA-UPM. Seeds of cucumber (Cucumis sativum L. cv. ‘Marumba’ (Enza Zaden
España S.L., Almería, Spain) and cv. ‘Ashley’ (Rocalba S.A., Gerona, Spain)), pepper
(Capsicum annuum L. cv. ‘California Wonder’) (Ramiro Arnedo S.A., La Rioja, Spain),
eggplant (Solanum melongena L. cv. ‘Black beauty’) (Batlle, S.A., Barcelona, Spain), tomato
(Solanum lycopersicum L. cv. ‘Marmande’) (Semillas Fitó S.A., Barcelona, Spain) and melon
(Cucumis melo L. cv. ‘Primal’) (Syngenta Seeds B.V., Enkhuizen, The Netherlands) were
germinated in 10.5 cm diameter pots filled with a 50:50 mixture of vermiculite (No. 3, Asfaltex
S.A., Barcelona, Spain) and soil substrate (Kekkilä Iberia, Quart de Poblet, Spain). Plants were
watered three times a week and fertilized using 20-20-20 (N:P:K) Nutrichem 60 fertilizer (Miller
Chemical & Fertilizer Corp., Pennsylvania, USA) at a 0.25 g L-1 dosage. Plants were grown in a
walk-in insect-proof chamber (Ibercex S.A., Arganda del Rey, Spain) at 23:20 °C temperature
(day:night), a photoperiod of 16L:8D, 60-80% RH, 200 µmol m-2 s-1 PAR, 0.62 W m-2 UV-A
and 0.038 W m-2 UV-B at canopy level (31 cm from light source) (Figure 3.1).
16
Figure 3.1. Insect-proof climatic chamber for plant development.
3.3. INSECT REARING
Six insect species were used in the experiments, pests Myzus persicae (Sulzer) (Hemiptera:
Aphididae), Aphis gossypii Glover (Hemiptera: Aphididae), Bemisia tabaci (Gennadius)
(Hemiptera: Aleyrodidae), and Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), and natural
enemies Aphidius colemani Viereck (Hymenoptera: Braconidae) and Sphaerophoria rueppellii
(Weidemann) (Diptera: Syrphidae). Geographical origin, host and rearing climatic conditions are
summarized in Table 3.1.
Plants with insect colonies were placed inside small boxes or cages sealed with a fine cloth to
allow ventilation inside a walk-in chamber (Ibercex S.A., Arganda del Rey, Spain). Insects were
maintained continuously for several generations prior to the experiments. Aphids, A. colemani,
S. rueppellii and T. absoluta were reared in environmental-controlled chambers with 100 µmol
m-2 s-1 PAR, 0.31 W m-2 UV-A and 0.013 W m-2 UV-B at canopy level (31 cm from light
source) (Figure 3.2, Table 3.1). Whiteflies were reared in glasshouse facilities.
Materials and methods
17
Figure 3.2. Climatic chamber for insect rearing.
Table 3.1. Geographical origin, host and rearing climatic conditions of the six insect species used in the experiments.
Insect Place Year Host/Prey Climatic conditions
Myzus persicae El Encín (Madrid, Spain) 1989 Pepper 23:18 °C 60-80% RH 16L:8D
Aphis gossypii Almería (Spain) 1998 Melon 23:18 °C 60-80% RH 16L:8D
Bemisia tabaci Q biotype
Almería (Spain) 2007 Melon Eggplant
23:20 °C 70-80% RH 16L:8D
Tuta absoluta Cartagena (Murcia, Spain) 2009 Tomato 25 °C 60-80% RH 16L:8D
Aphidius colemani
Almería (Spain) 2011 Cucumber/ Aphis gossypii
24:20°C 60-80% RH 16L:8D
Sphaerophoria rueppellii
San Vicente del Raspeig (Alicante, Spain)
2011 Pepper/ Myzus persicae
23:18 °C 60-80% RH 16L:8D
Aphid colonies were first started from a single viviparous female collected in the field. When no
experiments were conducted, both aphid colonies were maintained at low density placing five
18
healthy apterae adults on four-week old plants. Plants and aphids were grown for two weeks, and
then the protocol was repeated to maintain the populations in high quality host plants. Alate
morphs were induced by overcrowding aphid colonies in insect cages placed in the same climatic
chambers. All M. persicae generated in a two-week old colony were maintained for extra time.
Aphis gossypii alates were produced by placing ten apterae adults per plant and developing the
colony for 3 weeks.
Bemisia tabaci Q biotype was supplied by Dr. Enrique Moriones Alonso in 2007 (La Mayora
Experimental Station, Málaga, Spain). Four six-week old melon or eggplants and 500 recently
emerged whiteflies were placed inside a cage (60 x 60 x 100 cm) (Figure 3.3). Eggs laid on the
plants took five weeks to complete their cycle and produce newly emerged adults. When the
population reached several thousands of individuals, the same procedure was started again to
maintain the colony. Identity of biotype status of the population in rearing was periodically
confirmed by determining the sequence of cyto-chrome oxidase I mitochondrial gene (Frolich et
al., 1999).
Figure 3.3. Cages where whitefly was reared at glasshouse facilities.
Leaves with eggs laid by T. absoluta adults were collected. When larvae emerged, they were fed
with new tomato leaves until pupae development. Then those pupae were transferred to a new
cage with tomato plants for oviposition.
Materials and methods
19
Sphaerophoria rueppellii was supplied by Dr. María Ángeles Marcos García (University of
Alicante, Spain). Aphidius colemani adults, originally supplied by Koppert España S.L.
(Almería, Spain), were mantained on A. gossypii colonies as host according to the methodology
described by Calvo & Fereres (2011). Insects were synchronised prior to assays to ensure that
individuals were the same age.
3.4. GLASSHOUSE FACILITIES
Glasshouse facilities with cooling, heating and illumination systems were located at ICA-CSIC
(Figure 3.4). Temperature, relative humidity and specific photoperiod when required were
remotely controlled through a central computer. Climatic conditions were registered by Tinytag
TGU-4500 data loggers (Gemini Ltd., Chichester, United Kingdom).
Figure 3.4. Glasshouse facilities at ICA-CSIC.
3.5. VIRUS INOCULATION AND DETECTION
Cucumber plants cv. ‘Marumba’ were inoculated with Cucumber mosaic virus (CMV,
Cucumovirus) and Cucumber aphid-borne yellows virus (CABYV, Polerovirus) 13 days after
sowing at a 1-true leaf stage and used 4 weeks post-inoculation as viral sources. Plants were
mechanically inoculated with CMV isolate M6 (subgroup IA), obtained from a melon crop in
1996 in Tarragona (Spain) and provided by Dr. Enrique Moriones Alonso (Díaz et al., 2003).
20
Carborundum was used to facilitate the inoculation (Figure 3.5). CMV-infected plants were
maintained at ICA-CSIC in an insect-proof chamber at 25:20 °C temperature (day:night), a
photoperiod of 16L:8D and 60–80% RH.
Figure 3.5. Mechanical inoculation of CMV (left) and comparison between a healthy and a CMV-infected plant four weeks post-inoculation (right).
The CABYV isolate, provided by Dr. Hervé Lecoq (INRA, Montfavet, France), was obtained
from zucchini squash in 2003 in Montfavet (France) and mantained by aphid serial transmission.
Aphis gossypii adults were allowed to feed for 48 hours on previously CABYV-infected plants
and nymphs produced during this period fed on these plants for one extra day, reaching an
acquisition access period (AAP) of three days. After the AAP, 20 nymphs were transferred to
each healthy receptor plant for an inoculation access period (IAP) of 3 days, and then they were
removed (Figure 3.6). CABYV-inoculated plants were maintained in a chamber at 25:20 °C
temperature (day:night), a photoperiod of 16L:8D and 60-80% RH until experiments took place.
Figure 3.6. Inoculation of CABYV by transferring viruliferous aphids from a previously infected source plant (left) to receptor healthy plants (right).
Materials and methods
21
Viral infection was confirmed by Double Antibody Sandwich Enzyme-Linked ImmunoSorbent
Assay (DAS-ELISA) (Clark & Adams, 1977) using specific commercial antibodies against
CMV (Agdia Inc., Indiana, USA) and CABYV (Sediag, France).
3.6. PHOTOSELECTIVE COVERS
Nets studied in this work were provided by Ginegar Plastic Products Ltd. (Kibbutz Ginegar,
Israel): net Optinet, reference 32050502505; G-Anti Insect, reference 2223200428; A-Anti
Insect, reference 2223200481; P-Anti Insect, reference 32025502501; and T-Anti Insect,
reference 39125502501. The UV-opaque plastic film (PL), used as a UV-blocking control, was
provided by Solplast S.A. (Murcia, Spain). Net threads and film were made out of high-density
polyethylene (HDPE). All nets were 10x20 threads/cm (mesh 50), which resulted in holes
smaller than 0.5 mm2. Insects could not escape as all species tested had a bigger size. The UV-
opaque film was 200 µm thick. Color was white for A-Anti Insect and Optinet, and transparent
for T-Anti Insect, P-Anti Insect, G-Anti Insect and UV-opaque film. Optical properties were
analysed at the Instituto de Óptica Daza de Valdés (IO-CSIC, Madrid, Spain). Total
transmittance from 250 to 1500 nm and diffuse reflectance from 380 to 1500 nm were evaluated
in steps of 2 nm with a double monochromator Lambda 900 UV/Visible/NIR spectrophotometer
(PerkinElmer Life & Analytical Sciences Ltd., Connecticut, USA). The properties of all covers
are detailed in Table 3.2, including their physical and UV absorbing properties.
Table 3.2. Characteristics of the UV-absorbing materials used in the experiments.
Type Name Code Companya Colour Meshb Hole size (mm)
Yarn diameter (µm)
Thickness (µm)
UV absorption
Net
T-Anti Insect T 1 - 50 0.80x0.27 240 480 No
P-Anti Insect P 1 - 50 0.80x0.27 240 480 Yes
A-Anti Insect A 1 White 50 0.80x0.27 240 480 Yes
G-Anti Insect G 1 - 50 0.80x0.27 240 480 Yes
Optinet O 1 White 50 0.80x0.27 240 480 Yes
Film Antivirus PL 2 - - - - 200 Yes a 1: Ginegar Plastic Products Ltd., 2: Solplast S.A. b Mesh refers to the number of threads per cm in either direction of the woven net
22
3.7. LONG LASTING INSECTICIDE-TREATED NETS (LLITNs)
Nets were made of polyethylene yarns knitted in different patterns and provided by the
companies Intelligent Insect Control SAS (Castelnau Le Lez, France) and Ginegar Plastic
Products Ltd. (Kibbutz Ginegar, Israel) (Figure 3.7). The net yarns were pre-treated with
insecticides during the manufacturing process to produce LLITNs. A total of 23 insecticide-
treated nets and 10 untreated controls classified according to the following criteria were tested: a
hole size ranging from 0.12 to 3.42 mm2, and various insecticide compounds and dosages (Table
3.3).
Figure 3.7. Long lasting bifenthin-treated net used in the experiments.
Materials and methods
23
Table 3.3. Characteristics of nets used in experiments.
Net code Colour Hole (mm2) UV-adda Deltamethrin (g kg net-1) 151 Green 2.41 No - 210 White 1.93 No - C25warp White 0.73 No - 149 White 3.42 No 2.0 412 White 3.38 No 2.0 404 Light blue 2.78 No 1.4 406 Green 2.77 Yes 1.4 150 Blue 2.65 No 2.0 190 Dark blue 2.62 No 2.0 147 Yellow 2.59 No 4.0 405 White 2.47 Yes 1.4 191 Light blue 2.07 No 2.0 206 Yellow 2.06 No 4.0 148 White 2.00 No 4.0 207 White 1.82 No 4.0 25 White 0.66 No 1.2 25-30 White 0.35 No 2.8 Net code Colour UV-add Bifenthrin (g kg net-1) 1.4 Yellow 0.77 No - C7x11 Yellow 0.70 No - 2.4 Yellow 0.56 No - 3.4 Yellow 0.45 No - C10x11 Yellow 0.41 No - 64/11/07 Yellow 0.29 No - 42 Yellow 0.12 No - 1 Yellow 0.83 Yes 4.0 TR11-291 Yellow 0.71 Yes 4.0 2 Yellow 0.60 Yes 4.0 TR11-290 Yellow 0.46 Yes 3.8 3 Yellow 0.44 Yes 5.0 64/11/08 Yellow 0.29 Yes 2.1 40 Yellow 0.12 Yes 3.4 Net code Colour UV-add Chemical compound 196 Violet 2.92 No Deltamethrin+PBOb 195 Pink 2.67 No PBO
a Addition of UV-blockers, b Piperonyl butoxide
3.8. SPATIAL ANALYSIS
The Spatial Analysis by Distance IndicEs (SADIE) methodology has proved itself to be one of
the most powerful tools for studying distribution patterns, which was introduced to replace
traditional abstract mathematical approaches to more understandable biology-based measures
(Perry, 1995). Based on the transportation algorithm, the principle of SADIE is the measurement
24
of the minimum value, in terms of distance travelled, which individuals would have to move
from unit to unit so that all they are spaced as uniformly as possible, known as distance to
regularity, D. Also, the method provides the distance to crowding, C, as the minimum value of
the total distance that individuals must move to be as aggregated as possible (Perry, 1998).
The spatial pattern of a population is described by the Index of aggregation, Ia, which by
convention is an aggregated sample if Ia>1, a spatially-random sample if Ia=1 and a regular
sample if Ia<1. This index is calculated dividing the observed distance to regularity by the
averaged distance individuals moved when their distribution was randomized (Perry et al.,
1999).
SADIE also quantifies the degree to which each count contributes towards the overall degree of
clustering of the entire population, providing the positive Index of clustering in patches, vi, and
the negative Index of clustering in gaps, vj (Perry et al., 1999) for each sample data. For units
within patches of relatively large counts close to another, the Index of clustering in patches
would be large. Conversely, Index of clustering in gaps tends to be large in units within gaps of
small counts close to another. By convention, values <1.5 stand for patches and values <-1.5
indicate a gap, representing significant clustering half as large as that expected by chance alone.
Clustering indices may be plotted on a map as they are correlated on a continuous scale. Both
indices visually indicate the location and extent of clusters in the data so these values can be
contoured with Surfer 9.0 software (Golden Software, 2009), which allows the graphical
representation of patches and gaps.
Furthermore, by correlating the Indices of clustering of each count of two data sets (in this case,
between aphid and virus populations), a local measure of association was given for each sample
unit (Perry & Nixon, 2002). In case there is either a patch or a gap cluster in the same spatial
point of both populations, they are positively associated; if there is the opposite cluster, they are
negatively dissociated. The overall spatial association between aphid population and virus
incidence was calculated with the Index of spatial association, X (Perry & Nixon, 2002), and
contoured as well with Surfer 9.0 software (Golden Software, 2009). Association is significant
when p-values <0.025 and dissociation if p-values >0.975.
Materials and methods
25
3.9. GENERAL STATISTICS
Count data was transformed with either √(x+0.5), x2 or Ln(x+1) in order to decrease
heteroscedasticity and achieve normal distribution. If data was expressed as a percentage, the
angular transformation 2*(arcsin√x) was used. Then the parameters were analysed using IBM
Statistics SPSS 21.0 software (SPSS, 2013).
Parametric procedures were used whenever variables followed a normal distribution with a
Student t-test (p≤0.05) or a one-way ANOVA pairwise comparison followed by LSD (least
significant differences) (p≤0.05) to test differences between more than two treatments. If data did
not follow a normal distribution after transformations, a non-parametric Mann-Whitney U-test or
Kruskal-Wallis H-test (p≤0.05) was applied.
When comparing proportions, a Chi-square goodness of fit test (p≤0.05) using Statview 4.01
software (Abacus Concepts, 1992) was performed to check if the observed frequency
distribution was related to the expected frequency distribution.
Experiments that included repeated measurements over time such as crop height and leaf area
were assessed with ANOVA univariate repeated measures analysis (p≤0.05) using
SuperANOVA v. 1.11 software for Macintosh (Abacus Concepts, 1989).
26
27
CHAPTER 4. SPATIO-TEMPORAL DYNAMICS OF VIRUSES ARE
DIFFERENTIALLY AFFECTED BY PARASITOIDS DEPENDING ON
THE MODE OF TRANSMISSION1
ABSTRACT
Relationships between agents in multitrophic systems are complex and very specific. Insect-
transmitted plant viruses are completely dependent on the behaviour and distribution patterns of
their vectors. The presence of natural enemies may directly affect aphid behaviour and spread of
plant viruses, as the escape response of aphids might cause a potential risk for virus
transmission. The spatio-temporal dynamics of Cucumber mosaic virus (CMV, Cucumovirus)
and Cucurbit aphid-borne yellows virus (CABYV, Polerovirus), transmitted by Aphis gossypii
Glover in a non-persistent and persistent manner, respectively, were evaluated in the short and
long term in the presence and absence of the aphid parasitoid, Aphidius colemani Viereck.
SADIE methodology was used to study the distribution patterns of both the virus and its vector,
and their degree of association. Results suggested that parasitoids promoted aphid dispersal in
the short term, which enhanced CMV spread, though consequences of parasitism suggest
potential benefits for disease control in the long term. Furthermore, A. colemani significantly
limited the spread and incidence of the persistent virus CABYV in the long term. The impact of
aphid parasitoids on the dispersal of plant viruses with different transmission modes is discussed.
4.1. INTRODUCTION
Aphids (Hemiptera: Aphididae) are the primary vectors of plant viruses transmitting almost half
of the known plant viruses, approximately 275 virus species within 19 different virus genera
(Nault, 1997; Hull, 2014; Ng & Perry, 2004). Long distance movements of winged aphids could
eventually lead to virus spread. Transient vectors that land and probe on a plant without
colonising the crop are often the main vectors of non-persistent viruses, while colonising vectors
are involved in transmission of persistent viruses (Fereres & Moreno, 2009). Host preference
first involves phototactic responses to visual cues that may be modified by the emission of plant
volatiles (Döring & Chittka, 2007; Mauck et al., 2010). The interaction between plant pathogens
1 Published in: Dáder B., Moreno A., Viñuela E., Fereres A. 2012. Spatio-temporal dynamics of viruses are differentially affected by parasitoids depending on the mode of transmission. Viruses, 4: 3069-3089.
28
and vectors has been widely discussed but there is no general consensus on whether the parasite-
induced changes in host phenotype favours vector’s responses such as settlement, behaviour,
performance and overall fitness (Hodge & Powell, 2008b; Mauck et al., 2010; Bosque-Pérez &
Eigenbrode, 2011; Calvo & Fereres, 2011). Non-circulative viruses exhibit different effects that
mainly enhance vector attraction to infected hosts but some authors have documented neutral
reproductive performance of vectors and their parasitoids reared on plants infected by circulative
viruses (Hodge & Powell, 2008b; Bosque-Pérez & Eigenbrode, 2011; Calvo & Fereres, 2011).
Biological control is a major component of Integrated Pest Management (IPM) programs.
Although natural enemies reduce the levels of herbivore pressure, their addition to a plant-virus-
vector system may involve complicated interactions between the agents, as they may greatly
modify disease incidence within the plant population (Dicke & van Loon, 2000). Therefore,
there is a need to balance biological control consequences, as the benefits in reducing vector
numbers may be offset by an increase in the spread of the virus. Several authors first studied the
effect of natural enemies on virus spread by aphids throughout the plant population (Roitberg &
Myers, 1978; Bailey et al., 1995; Weber et al., 1996). Roitberg & Myers (1978) discussed the
role of Coccinella californica Mannerheim (Coleoptera: Coccinellidae) in the spread of Bean
yellow mosaic virus (BYMV, Potyvirus). Bailey et al. (1995) described how the predator activity
of Coleomegilla maculata De Geer (Coleoptera: Coccinellidae) resulted in an increase in Barley
yellow dwarf virus (BYDV, Luteovirus) incidence in oats. Similarly, Weber et al. (1996)
observed the increased ability of parasitised Aphis fabae Scopoli (Hemiptera: Aphididae) to
transmit Beet yellows virus (BYV, Closterovirus). The same trend has been confirmed in recent
studies showing greater spread of Pea enation mosaic virus (PEMV, symbiotic mutualism
between an Enamovirus and an Umbravirus) and BYMV by Acyrthosiphon pisum (Harris)
(Hemiptera: Aphididae) in the presence of the aphid parasitioid Aphidius ervi (Haliday)
(Hymenoptera: Braconidae) in Vicia faba L. (Hodge & Powell, 2008a; Hodge et al., 2011).
Virus spread might be correlated with the foraging habit that natural enemies exhibit within the
system (Smyrnioudis et al., 2001; Belliure et al., 2011). Interestingly, the presence of the
predator Coccinella septempunctata L. (Coleoptera: Coccinellidae) resulted in more BYDV-
infected wheat seedlings, but conversely reduced virus incidence in the presence of the parasitoid
Aphidius rhopalosiphi de Stefani Perez (Hymenoptera: Braconidae), probably because
coccinellids have a more energetic searching behaviour (Smyrnioudis et al., 2001). Furthermore,
certain species of aphids employ the ‘drop and move’ escape behaviour when they feel disturbed
by foliar-foraging enemies (Losey & Denno, 1998; Day et al., 2006), potentially increasing the
Dynamics of viruses are differentially affected by parasitoids
29
risk of vector dispersal. Alarm pheromones play a crucial role in aphid dispersal and there have
even been several attempts to mathematically model plant-virus-vector-natural enemy
interactions by integrating this alarm signal, enhancing virus spread due to the presence of aphid
parasitoids (Jeger et al., 2011, 2012).
The distribution patterns of aphids and their natural enemies have highlighted the underlying
dynamic relationships between guilds and their implications in biological control (Díaz et al.,
2010), as well as they have provided successful information about interplant movement of
different aphid morphs (Díaz et al., 2012). Previous studies of the spatial spread of major viral
diseases affecting valuable outdoors crops have been studied using the SADIE methodology
(Moreno et al., 2007; Jones et al., 2008).
4.2. OBJECTIVE
The present study aimed to investigate the tritrophic interactions within a system that included
the host plant Cucumis sativus L., the cotton aphid Aphis gossypii Glover (Hemiptera:
Aphididae), a cosmopolitan pest species that colonises more than 600 host plants, and the widely
used parasitoid wasp Aphidius colemani Viereck (Hymenoptera: Braconidae). Furthermore,
parasitoid-mediated effects on the dissemination of two major plant viruses infecting cucurbits,
Cucumber mosaic virus (CMV) and Cucurbit aphid-borne yellows virus (CABYV), both
efficiently transmitted by A. gossypii in non-circulative and circulative manner, respectively,
were assessed. Additionally, the spatial distribution of both viruses and the degree of association
between the two viruses and its vector was analysed under two different time frame scenarios.
4.3. MATERIALS AND METHODS
4.3.1. EXPERIMENTAL DESIGN
Four different assays were carried out in glasshouse facilities at ICA-CSIC to evaluate the
impact of the parasitoid A. colemani on the spread of CMV and CABYV and its vector A.
gossypii in the short and long term. A set of six cages (1×1×1 m) (three replicates, two
treatments) as described by Díaz et al. (2012) was used in each experiment. A potted CMV- or
CABYV-infected plant was placed in the centre of each cage (Figure 4.1). Four trays with holes
on the bottom to allow percolation containing 48 test cucumber cv. ‘Marumba’ plants at a one-
true leaf stage with a planting distance of 12.5 cm within and between rows were placed
surrounding the virus source. The surface was covered with a uniform layer of soil to create a
continuous surface.
30
Figure 4.1. Spatial disposition of an experimental arena, displaying the central virus source surrounded by the 48 test plants at a one-true leaf stage.
Alate aphids from the colony were collected with an aspirator and grouped in falcon tubes the
same morning when the experiment was conducted. One hundred winged aphids were released
in each virus-infected source plant using clip-cages to allow an acquisition access period of 15
minutes. After this period, clip-cages were removed and a falcon tube with five young female
parasitoids was introduced in the experimental arena. A vial with diluted honey (1:1 by volume)
was placed inside the cages as a feeding source for parasitoids (Figure 4.2). Control cages were
similarly implemented with virus-infected sources and test plants without the introduction of
parasitoids. All cages were rotated 180° daily to allow aphid distribution as uniform as possible
and avoid orientation bias.
Dynamics of viruses are differentially affected by parasitoids
31
Figure 4.2. Introduction of aphids (left) and parasitoids (right) in the experimental arena.
Number, stage and position of aphids were recorded in test plants at different periods of time
(short and long term) depending on both the mode of transmission of the virus studied and the
life history of the parasitoid (Zamani et al., 2007). As a first approach, assays with a CMV and
CABYV virus source were evaluated after 2 and 7 days (short term), respectively. CMV is
acquired and transmitted during brief periods of time and without a latent period so both
processes could occur in that short time periods (few days). Conversely, acquisition and
transmission of circulative viruses require vectors to feed during a much longer period of time.
Moreover, circulative viruses exhibit a delay in hours to days between the moment in which the
vector feeds on the infected plant and their ability to transmit the virus, which ranges from
several days to weeks. In the second set of bioassays, the virus-vector-parasitoid complex was
allowed to evolve 7 days in the case of CMV and 14 days for CABYV to evaluate long-term
virus and vector dispersal. Additionally, the number and position of mummies and parasitoid
adults were recorded in the long-term assays as a new cycle of parasitism was established and
mummies were easily recognizable. The virus source plant was removed after each specific time
period and the number and stage of aphids were recorded. Then, all test plants were sprayed with
Confidor® 20 LS (Bayer CropScience S.L., Paterna, Spain) to avoid further virus transmission.
32
Four weeks after the experiment was completed, virus infection in each of the receptor plants
was tested by DAS-ELISA.
4.3.2. STATISTICAL METHODS
Density, stage and morph of aphids (winged or wingless adults, or nymphs) in both central and
receptor test plants were analysed. All the parameters were compared between the control cages
and those containing parasitoids through a Student t-test (p≤0.05). The proportion of plants
infested by one or more aphids (occupancy rate) and the virus incidence in test plants were
compared between the two treatments (with and without parasitoids) using a Chi-square
goodness of fit test (p≤0.05).
4.3.3. SPATIAL ANALYSIS
The spatial distribution of aphid and virus count data was studied using the Spatial Analysis by
Distance IndicEs (SADIE) methodology explained in section 3.8 (Perry, 1995). In this study, the
assessment of aphid density was determined by the average of the three cages of each treatment
in every position. Virus incidence was represented as the cumulative number of infected plants
in the three cages of each treatment in every position. A value of 1 and 0 was assigned to the
infected plants and healthy plants, respectively. The spatial pattern of the entire population was
described by the Index of aggregation, Ia (Aggregated: Ia>1; Random: Ia=1; Regular: Ia<1), the
positive patch cluster index, vi, and the negative gap cluster index, vj (Perry et al., 1999). By
convention, values <1.5 stand for patches and values <-1.5 indicate a gap. Clustering indices
were plotted on a map and their values were contoured with Surfer 9.0 software (Golden
Software, 2009), which allowed the graphical representation of patches and gaps of aphids and
viruses in the experimental arena. The association between aphid and virus populations was
determined with the Index of spatial association, X, and contoured as well (Perry & Nixon,
2002). Moreover, centroids, C, or the average position of either aphids or viruses in the
experimental cage, were used to calculate δ, the displacement of the entire populations between
centroids and the place where both aphids and virus-infected plants were originally located (the
central virus source).
Dynamics of viruses are differentially affected by parasitoids
33
4.4. RESULTS
4.4.1. EFFECT OF Aphidius colemani ON APHID DISPERSAL AND THE SPREAD OF
Cucumber mosaic virus
The population density of adult morphs and nymphs in the CMV-infected source plant located in
the middle of the cage where aphids were released was frequently higher in the control cages
than in those containing the parasitoid A. colemani, although no significant differences were
found (Table 4.1). Parasitoids successfully located a variable number of aphids in the virus-
infected source plant and mummies could be observed (2.3±1.5) 7 days after the release of
parasitoids, whereas they could not be detected after 2 days, as mummies were not yet
developed. There were fewer aphids on the peripheral test plants in the arenas (=cages) without
A. colemani after 2 days (Figure 4.3.a), but this trend was not repeated after 7 days, with
significantly more apterae adults (t=6.775; df=4; p=0.002) and nymphs (t=11.864; df=4;
p<0.001) in the test plants of control arenas (Figure 4.3.b). The number of nymphs increased
considerably after 7 days (Figure 4.3.b). Besides, recognizable mummies could be detected in
peripheral plants in CMV at 7 days (2.0±2.0) but not at 2 days.
Table 4.1. Mean±S.E. density (number of individuals/plant) of adult morphs and nymphs in the CMV-infected virus source plant after 2 and 7 days in cages with and without (control) Aphidius colemani, followed by statistics according to a Student t-test (p≤0.05).
2 days 7 days
Control A. colemani t p Control A. colemani t p
Alate 24.7±5.5 18.0±5.3 0.873 0.947 11.0±3.2 9.0±2.5 0.524 0.628
Apterae - - - - 10.7±4.7 6.3±3.0 0.882 0.428
Nymphs 176.3±55.1 128.7±26.6 0.745 0.498 170.0±50.0 99.7±33.3 1.227 0.287
34
Figure 4.3. Mean±S.E. values of total number of aphids on test plants in cages with (grey bars) and without (control cages, white bars) Aphidius colemani. a) CMV-infected source plant assay at 2 days. b) CMV-infected source plant assay at 7 days. c) CABYV-infected source plant assay at 7 days. d) CABYV-infected source plant assay at 14 days. Bars with asterisks are significantly different according to a Student t-test (p≤0.05).
The occupancy rates were calculated as the percentage of test plants with one or more aphids in
their different morphs. These rates were consistent with aphid density in the peripheral test
plants (Figure 4.3.a and b), as there was significantly fewer plants occupied by aphids in the
control arenas than in the arenas with parasitoids after 2 days, but larger occupancy rate in the
control after 7 days (Table 4.2).
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Dynamics of viruses are differentially affected by parasitoids
35
Table 4.2. Mean±S.E. percentage of test plants occupied by one or more alate, apterae or nymphs (occupancy rate) in CMV-infected source plant assays after 2 and 7 days in cages with and without (control) Aphidius colemani, followed by statistics according to a Chi-square goodness of fit test (p≤0.05).
2 days 7 days
Control A. colemani χ 2 p Control A. colemani χ 2 p
Alate 38.9±2.5 54.2±7.9 9.930 0.002 42.4±3.5 33.3±8.7 2.495 0.117
Apterae - - - - 38.2±2.8 6.3±3.2 42.509 <0.001
Nymphs 38.2±4.2 50.7±8.2 4.556 0.044 50.7±2.5 38.2±7.8 4.556 0.034 The incidence of CMV at short time (after 2 days) was significantly higher in arenas containing
A. colemani compared to that observed in the control arenas (χ2=5.497; p=0.020). When the
same assay was assessed after 7 days, no significant differences in the incidence of CMV
between treatments were detected (Figure 4.4).
Figure 4.4. Mean±S.E. values of virus transmission (%) in the arenas with parasitoids (grey bars) and in those without them (control, white bars). Bars with asterisks indicate significant differences according to a Chi-square goodness of fit test (p≤0.05).
No differences could be found for the mean displacement (δ) of both aphids and CMV (Table
4.3). The spatial analysis of CMV-infected source plant experiments showed a significant
aggregated distribution of A. gossypii in treatments with and whitout parasitoids after 2 and 7
days (Figures 4.5 and 4.6). At 2 days, aphids were restricted to the lower right corner of the
experimental arena (Figure 4.5) but colonised the entire lower area of the arena after 7 days, with
plants remaining unnoccupied in the northern side (Figure 4.6). In the short term, the spread of
CMV followed a regular distribution in the control cages where few isolated plants became
infected, although CMV distribution was significantly aggregated in the presence of parasitoids
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36
(Figure 4.5). When the spatial distribution of aphids and CMV was studied in the long term (7
days), opposite results were obtained (Figure 4.6). The combination of aphid infestation and
virus infection showed a significant association in the control arenas, that was statistically
significant at 7 days (Figure 4.6), and a dissociation in the presence of parasitoids after 2 days
(Figure 4.5).
Table 4.3. Mean±S.E. values of the displacement (δ) of aphids and CMV after 2 and 7 days in arenas with and without (control) parasitoids, followed by statistics according to Student t-test (p≤0.05).
Aphids Virus
Control A. colemani t p Control A. colemani t p
2 days 1.9±0.2 1.6±0.2 0.758 0.491 0.9±0.1 1.4±0.6 –0.690 0.528
7 days 1.3±0.1 1.5±0.4 –0.539 0.619 1.2±0.4 1.0±0.4 0.215 0.840
Dynamics of viruses are differentially affected by parasitoids
37
Figu
re 4
.5.
Cla
ssed
pos
t m
aps
of t
he s
patia
l di
stri
butio
n of
mea
n nu
mbe
r of
Aph
is g
ossy
pii
and
cum
ulat
ive
num
ber
of C
MV
-inf
ecte
d pl
ants
(to
tal
num
ber
of i
nfec
ted
plan
ts p
er t
reat
men
t) a
t 2
days
, an
d co
ntou
red
map
of
the
asso
ciat
ion
betw
een
CM
V-i
nfec
ted
plan
ts
and
its v
ecto
r, A
. gos
sypi
i. Sp
ots
indi
cate
ind
ivid
ual
test
pla
nts.
Sm
all
fille
d sp
ots
repr
esen
t cl
uste
ring
ind
ices
of
0 to
±0.
99 (
clus
teri
ng
belo
w e
xpec
tatio
n), u
nfill
ed s
pots
±1
to ±
1.49
(cl
uste
ring
slig
htly
exc
eeds
exp
ecta
tion)
and
lar
ge f
illed
spo
ts >
1.5
or <
1.5
(mor
e th
an
half
as
muc
h as
exp
ecta
tion)
. R
ed l
ines
enc
losi
ng p
atch
clu
ster
s ar
e co
ntou
rs o
f v=
1.5
and
blue
lin
es a
re o
f v=
–1.5
. B
lack
lin
es a
re
zero
-val
ue c
onto
urs,
rep
rese
ntin
g bo
unda
ries
bet
wee
n pa
tch
and
gap
regi
ons.
The
ind
ex o
f ag
greg
atio
n, I
a, t
he p
ositi
ve p
atch
clu
ster
in
dex,
vi,
the
nega
tive
gap
clus
ter
inde
x, v
j, an
d th
e in
dex
of s
patia
l as
soci
atio
n, X
, en
clos
ed b
y an
ora
nge
line
are
stat
istic
ally
si
gnif
ican
t. L
ette
r N
and
arr
ow in
dica
te n
orth
ori
enta
tion.
Pat
ch
Gap
0
1 -1
-1.5
1.5
V
Ass
oci
atio
n
Dis
soci
atio
n
0 0.
025 0
.05
0.95 0.9
75
p As
soci
atio
n CM
V A.
gos
sypi
i
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Control A. colemani
!"#!"#$--,
"$""!"($(((&"
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N
38
Figu
re 4
.6. C
lass
ed p
ost m
aps
of th
e sp
atia
l dis
trib
utio
n of
mea
n nu
mbe
r of
Aph
is g
ossy
pii a
nd c
umul
ativ
e nu
mbe
r of
CM
V-i
nfec
ted
plan
ts
(tot
al n
umbe
r of
inf
ecte
d pl
ants
per
tre
atm
ent)
at
7 da
ys,
and
cont
oure
d m
ap o
f th
e as
soci
atio
n be
twee
n C
MV
-inf
ecte
d pl
ants
and
its
ve
ctor
, A. g
ossy
pii.
Sym
bols
and
con
tour
s ar
e as
for
Fig
ure
4.5.
CMV
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%("!"+#
$#)-
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Control A. colemani
A. g
ossy
pii
Asso
ciat
ion
Pat
ch
Gap
0
1 -1
-1.5
1.5
V
Ass
oci
atio
n
Dis
soci
atio
n
0 0.
025 0
.05
0.95 0.9
75
p
N
Dynamics of viruses are differentially affected by parasitoids
39
4.4.2. EFFECT OF Aphidius colemani ON APHID DISPERSAL AND THE SPREAD OF
Cucumber aphid-borne yellows virus
The short and long term experiments with CABYV was extended to 7 and 14 days, respectively,
because its circulative mode of transmission requires longer acquisition and inoculation access
periods than the non-circulative CMV. The central CABYV-infected plant contained more
aphids in the control arenas than in those with A. colemani, and significant differences in the
number of apterae adults and nymphs were detected at 7 days, but not at 14 days (Table 4.4).
Moreover, it was possible to detect mummies in the virus source plant 14 days (8.3±2.0) but not
7 days after parasitoid release. In general terms, there were more peripheral test plants occupied
by a greater number of aphids in the control arenas than in those containing A. colemani, with
significant differences in the case of nymphs at 7 days (t=3.152; df=4; p=0.034) (Figure 4.3.c).
However these significant differences were not observed at 14 days (Table 4.5, Figure 4.3.d). As
mentioned before for the CMV experiments, the number of offspring was much higher in the
long term (Figure 4.3.d). Mummies (56.0±21.6) and even parasitoid adults (3.3±1.8) were found
in peripheral test plants at 14 days again but not at 7 days. The mean incidence of CABYV when
evaluated at short time (7 days) was higher in arenas containing A. colemani compared to that
observed in the control ones, although no significant differences were found. In the long term,
significantly fewer CABYV-infected plants were detected in arenas where A. colemani were
introduced (χ2=8.963; p=0.004) (Figure 4.4).
The overall mean transmission rate of both viruses (CMV and CABYV) in the presence and
absence of A. colemani was not significantly different after 7 days. However, the presence of A.
colemani increased the rate the spread of CMV in the short term (2 days) but reduced the spread
of CABYV in the long term (14 days) (Figure 4.4). Moreover, the transmission rate of CABYV
in control cages significantly increased at 14 days compared to 7 days from 11.1±9.0 to
32.6±8.9% (χ2=19.525; p<0.001) (Figure 4.4).
40
Table 4.4. Mean±S.E. density (number of individuals/plant) of adult morphs and nymphs in the CABYV-infected source plant at 7 and 14 days in cages with and without (control) Aphidius colemani, followed by statistics according to a Student t-test (p≤0.05).
7 days 14 days
Control A. colemani t p Control A. colemani t p
Alate 36.0±1.7 24.3±7.8 1.443 0.222 20.0±6.7 10.0±1.2 1.359 0.246
Apterae 60.3±9.9 17.0±1.5 6.524 0.003 179.7±54.3 185.0±8.2 0.168 0.874
Nymphs 388.0±39.1 160.0±10.4 7.026 0.020 1624.7±297.8 1212.0±55.6 1.465 0.217 Table 4.5. Mean±S.E. percentage of test plants occupied by one or more alate, apterae or nymphs (occupancy rate) in CABYV assays at 7 and 14 days in cages with and without (control) Aphidius colemani, followed by statistics according to a Chi-square goodness of fit test (p≤0.05).
7 days 14 days
Control A. colemani χ 2 p Control A. colemani χ 2 p
Alate 18.8±1.2 24.3±4.2 1.315 0.256 37.5±9.8 30.6±9.1 1.547 0.263
Apterae 20.9±5.2 16.0±3.0 1.133 0.293 63.9±15.2 60.4±1.2 0.369 0.627
Nymphs 27.8±2.8 29.8±4.6 0.152 0.670 75.0±11.0 72.2±0.7 0.286 0.596
The same as in CMV experiments, no differences could be found for the mean displacement δ of
both aphids and CABYV-infected plants under the two treatments (Table 4.6). Aphids also
showed an aggregated distribution in the CABYV assays, being significantly aggregated at 14
days (Figure 4.8) as cucumber is an excellent host plant for A. gossypii and large colonies are
produced in a short period of time (Blackman & Eastop, 2000). Aphid spatial distribution
showed a very similar pattern in the CABYV experiments when parasitoids were present, with
population moving to the southern area of the cages and increasing the number of plants
occupied as time progressed (Figures 4.7 and 4.8). In the control arenas, aphid distribution was
limited to the south and center of the experimental cage at short times (Figure 4.7) but reached
almost all edges when aphids were allowed to stay 14 days, in parallel to what happened with the
spread of CABYV (Figure 4.8). Conversely, virus-infected plants were located in the northern
area in arenas with parasitoids at 7 days (Figure 4.7), and continued being restricted to the same
area with a significantly aggregated distribution at 14 days (Figure 4.8). When the spatial
distribution of the virus and the vector were combined, a dissociation at 7 days and association at
14 days between aphid location and the position of CABYV-infected plants were recorded in
both treatments (Figures 4.7 and 4.8).
Dynamics of viruses are differentially affected by parasitoids
41
Table 4.6. Mean±S.E. values of the displacement (δ) of aphids and CABYV after 7 and 14 days in arenas with and without (control) Aphidius colemani, followed by statistics according to a Student t-test (p≤0.05).
Aphids Virus
Control A. colemani t p Control A. colemani t p
7 days 0.8±0.1 1.2±0.2 –1.496 0.209 2.2±1.0 1.0±0.3 1.154 0.313
14 days 0.8±0.3 0.8±0.1 –0.112 0.916 1.4±0.4 1.0±0.0 1.117 0.326
42
Figu
re 4
.7.
Cla
ssed
pos
t m
aps
of t
he s
patia
l di
stri
butio
n of
mea
n nu
mbe
r of
Aph
is g
ossy
pii
and
cum
ulat
ive
num
ber
of C
AB
YV
-inf
ecte
d pl
ants
(to
tal n
umbe
r of
infe
cted
pla
nts
per
trea
tmen
t) a
t 7 d
ays,
and
con
tour
ed m
ap o
f th
e as
soci
atio
n be
twee
n C
AB
YV
-inf
ecte
d pl
ants
and
its
vec
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Dynamics of viruses are differentially affected by parasitoids
43
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44
4.5. DISCUSSION
The findings suggest that A. colemani promoted early movement of A. gossypii from the virus-
infected source towards the peripheral plants. Parasitoids significantly increased the colonization
of adjacent plants by both adults and nymphs. Consistently, the spread of CMV also increased
when parasitoids interacted with aphids for 2 days. However, no differences in virus incidence
could be found after 7 days between both treatments, and aphids dispersed around and
transmitted CMV equally well under the two treatments, in the presence and absence of A.
colemani, which suggest potential benefits in the long term.
These results may be explained by the mode of transmission of non-persistent viruses and the
particular behaviour of A. gossypii. Transmission of non-persistent viruses is highly favoured if
aphids acquire the viral particles during short periods, decreasing its efficiency with a longer
acquisition access time (Fereres & Moreno, 2009). It is likely that soon after release, parasitoids
caused a disturbance and their presence forced aphids to quickly disseminate, which led to an
increase in aphid density on peripheral test plants and subsequent transmission of CMV.
Aphidius colemani forced them to escape and, as non-persistent viruses do not require a latent
period, aphids were able to inoculate CMV to peripheral plants readily after leaving the central
plant. The incidence of CMV remained the same 2 and 7 days after the experiment started in
arenas with A. colemani, probably because parasitised aphids had reduced the distance they
moved after few days and parasitoids were not in the attack mood any longer. The fact that
mummies were observed in both central and test plants at 7 days seems to corroborate this
hypothesis. Conversely, CMV transmission in control arenas increased with time as aphids
continued colonizing test plants because their mobility was not jeopardised by parasitism.
Similar alterations resulting in an increase in non-persistently and semipersistenly transmitted
virus dispersal have been previously reported at short times (1–3 days) (Roitberg & Myers,
1978; Weber et al., 1996; Belliure et al., 2011; Hodge et al., 2011), but no information on the
long term effects of parasitism on virus spread has been reported so far.
Natural enemies orientate towards plant-host complexes (PHC) by responding to host herbivore-
induced plant volatiles and visual cues (Michaud & Mackauer, 1995; Du et al., 1996; Guerrieri
et al., 1997; Yang et al., 2009). The emission of alarm signals by aphids causes conspecifics to
disperse through different strategies such as ‘drop off’ (Losey & Denno, 1998; Day et al., 2006).
That escaping behaviour may even modify virus spread and has been reported with the aphid A.
pisum and its parasitoid A. ervi (Jeger et al., 2011). Furthermore, damaged plants emit the “cry
Dynamics of viruses are differentially affected by parasitoids
45
for help” signal that may indirectly benefit hosts under herbivore attack (Jeger et al., 2012).
Therefore, it is evident that there is a need to integrate all these multitrophic reactions between
agents resulting in epidemiological consequences (Jeger et al., 2011, 2012).
In the experiments, A. gossypii showed the typical pattern of a colonizing aphid vector species
because of its strong preference for cucumber as a host (Blackman & Eastop, 2000). However,
the spread and incidence of the virus progressed differently at short and long times of evaluation.
The contoured maps of CMV after 2 days revealed the typical pattern of a non-persistent virus in
the absence of parasitoids (control cages). However, the spatial distribution was modified in the
presence of A. colemani and the clear consequence of the immediate disturbance of aphids,
which promoted the distribution of CMV around the entire arena and its aggregation in several
patches, in contrast with the few red spots indicating isolated infections under the control arenas.
At 7 days, the infection in control arenas showed how the initial localised foci had merged in a
larger patch, whereas a regular distribution (Ia<1) was found in the presence of parasitoids. The
A. gossypii-infested and CMV-infected plants were significantly associated in the control arenas,
whilst A. colemani induced dissociation between both agents, highlighting again the strong effect
of natural enemies in the early dispersal of aphids as previously reported (Roitberg & Myers,
1978; Hodge et al., 2011).
Previous studies have described enhanced spread of persistent viruses in the presence of natural
enemies (Bailey et al., 1995; Hodge & Powell, 2008a), although the vector response might be
influenced by the natural enemy’s searching habits (Smyrnioudis et al., 2001). At 7 days, there
were no differences in virus incidence between the two treatments, however significantly fewer
CABYV-infected plants were observed at 14 days in the presence of A. colemani, the same as
seen by Smyrnioudis et al. (2001) for both predators and parasitoids. Despite the fact that some
studies show that virus transmission rate is not reduced by parasitoids (Christiansen-Weniger et
al., 1998), Calvo & Fereres (2011) reported a reduction in the rate of spread of a circulative
Polerovirus due to a decrease in the life span of viruliferous aphids in the presence of
parasitoids. In the study, it was found that mummification might have positively diminished the
duration of aphids as active vectors after 14 days, so CABYV incidence was significantly
reduced in comparison to that in the control cages. The recording of mummies and parasitoid
adults provided evidence that A. colemani was able to establish itself well in the experimental
arenas. It has been reported an average of 10 days at a constant temperature of 25 °C for A.
colemani to complete its development (Zamani et al., 2007). With the mean temperatures in
which the experiments were performed, a new generation of parasitoids could have started to
46
emerge after 14 days so that the reduction in vector populations may have possibly limited
further virus spread. The study shows that the reduction of herbivore damage in the long term
may offset the initial risk of potential virus spread when natural enemies first encounter their
hosts/preys.
In the CABYV assays, there were major clustered areas of either patches or gaps of infected
plants at 7 days in both treatments. Patch clusters seemed to be wider than gap ones in the A.
colemani treatment. Clusters of infected plants are frequently observed in viruses transmitted in a
persistent circulative manner (Irwin & Thresh, 1990). Moreover, just two weeks were enough for
aphids to expand CABYV to all the edges of the experimental arena in the absence of A.
colemani, whilst parasitoids limited the incidence of CABYV to specific patches. These results,
together with the reduction in the rate of transmission of CABYV in the presence of A. colemani
after 14 days, prove the beneficial role of natural enemies in the long term, specially when
dealing with viruses transmitted in a persistent circulative manner.
The evaluation periods were selected depending on the type of transmission of each virus-vector
combination and the life history of A. colemani. These periods cannot be comparable to the
crop’s growth cycle that approximately lasts three to four months in commercial greenhouses.
Making a prediction in a hypothetical scenario where the experiments had been run for a longer
period is adventurous, but all plants would have possibly become infected by the persistent virus
CABYV if parasitoids had not been released in the arena. Data suggests that the spread of
CABYV would be strongly constrained in the presence of parasitoids under a real field situation
in the long term because the mobility and population growth of aphid vectors would be
jeopardised. However, it would be more difficult to predict the long-term consequences of
parasitoid release in the case of the non-persistent virus CMV. Further research would be helpful
to assess the effect of parasitism on aphid dispersal and virus transmission over time.
This study refers to a specific virus-vector-host-natural enemy complex, but natural enemy
diversity occurring in cucurbit crops could influence the spread of both viruses in either
direction. As a first approach, it will be desirable to carry out further studies with different
natural enemies to study how this beneficial guild could modify viral dynamics and to
investigate differences between their behaviour. As a second step, different virus-vector
combinations could be evaluated to gain a better understanding of multitrophic interactions in
pathosystems where whiteflies or thrips are present.
Dynamics of viruses are differentially affected by parasitoids
47
It is clear that the outcome in the system might be also influenced by the vector preference for
healthy or diseased plants, the plant response to pest damage caused by vectors in attracting
parasitoids (“cry for help” signal) and the vector response to the presence of parasitoids (alarm
signal) in the short as well as in the long term (Jeger et al., 2011). The infected-host
attractiveness mediating settlement and arrestment behaviours constitute a key point in this
process because both of them are correlated to the time required to positively acquire the viral
particles (Hodge & Powell, 2008a, b; Mauck et al., 2010; Bosque-Pérez & Eigenbrode, 2011;
Carmo-Sousa et al., 2014). It is known that both persistent and non-persistent viruses would tend
to enhance vector attraction to infected plants, increasing alighting and arrestment of their
vectors. However, it is known that both type of viruses have contrasting effects on vector settling
and feeding preferences, with persistent viruses tending to improve host quality for vectors and
promote long-term feeding while non-persistent viruses tend to reduce plant quality and promote
rapid disersal (Hodge & Powell, 2008a; Mauck et al., 2012). Moreover, in the experiments
aphids released on the virus-infected source plant were non-viruliferous. It has been shown that
aphids infected with circulative Luteovirus BYDV prefer to settle on virus-infected than on non-
infected plants (Ingwell et al., 2012). All together, it seems that both non-persistent and
persistent viruses have coevolved and adapted to exploit specific behavioral traits of their vectors
to enhance their own spread. Thus the spatial and temporal pattern of virus spread will depend
on the mode of transmission (persistent or non-persistent) as well as on the “infection” status of
the virus vector. Overall, these observations suggest the importance of taking into account the
degree of activity of natural enemies when implementing IPM programs for controlling vectors
of plant diseases.
48
49
CHAPTER 5. FLIGHT BEHAVIOUR OF VEGETABLE PESTS AND
THEIR NATURAL ENEMIES UNDER DIFFERENT UV-BLOCKING
ENCLOSURES2
ABSTRACT
Ultraviolet radiation (UV) is the fraction of the solar spectrum that regulates almost every aspect
of insect behaviour, including orientation toward hosts, alighting, arrestment and feeding
behaviour. To study the role of UV on the flight activity of five insect species of agricultural
importance (pests Myzus persicae (Sulzer), Bemisia tabaci (Gennadius) and Tuta absoluta
(Meyrick), and natural enemies Aphidius colemani Viereck and Sphaerophoria rueppellii
(Weidemann)), one-chamber tunnels were covered with six cladding materials with different
light transmittance properties ranging from 2-83% UV and 54-85% photosynthetically active
radiation (PAR). Inside each tunnel, insects were released from tubes placed in a platform
suspended from the ceiling. Specific targets varying with insect species were placed at different
distances from the platform. Evaluation parameters were designed for each insect and tested
separately. The ability of insects to leave the platform was assessed, as well as the number of
captures, eggs or mummies in each target, either sticky traps or plants. The results suggest
differences in flight activity among insect species and UV-blocking nets. The UV-opaque film
drastically prevented aphids and whiteflies from flying outside the tubes whereas T. absoluta,
syrphids and parasitoids were not affected. Aphid flight behaviour was affected by the UV-
opaque film compared to the other nets, especially in the furthest target of the tunnel. Fewer
aphids reached distant traps under UV-absorbing nets, and significantly more aphids could fly to
the end of tunnels covered with non-UV blocking materials. Bemisia tabaci and T. absoluta
orientation was also negatively affected by the UV-opaque film although in a different trend.
Unlike aphids, differences in B. tabaci captures were mainly found in the closest targets.
Ultraviolet transmittance did not have any effects on parasitoids and S. rueppellii, implying cues
other than visual for these insects under our experimental conditions. Further effects of
photoselective enclosures on greenhouse pests and their natural enemies are discussed.
2 Published in: Dáder B., Plaza M., Fereres A., Moreno A. 2015. Flight behaviour of vegetable pests and their natural enemies under different UV-blocking enclosures. Annals of Applied Biology, accepted.
50
5.1. INTRODUCTION
Solar ultraviolet radiation, particularly in the UV-A+B range (280-400 nm), is an abiotic factor
that has major consequences for insect pests, since it might greatly modify their orientation
toward potential hosts, flight activity, alighting, arrestment, feeding behaviour and interaction
between sexes (Raviv & Antignus, 2004). Many insects, including aphids and pollinators, have a
trichromatic system in the compound eye with an ultraviolet receptor peaking at 320-330 nm, a
second one with the peak in the blue region at 440-480 nm and a third green receptor with a
maximum sensitivity around 530 nm (Briscoe & Chittka, 2001; Kirchner et al., 2005; Skorupski
et al., 2007). Aphids (Hemiptera: Aphididae), one of the most important pests of crops
worldwide, have been reported to drastically reduce their flight activity under UV-deficient
ambients (Chyzik et al., 2003; Döring & Chittka, 2007; Legarrea et al., 2012a). In whiteflies
(Hemiptera: Aleyrodidae), two ranges of the spectrum have been identified, with UV radiation
correlated to migratory behaviour and yellow wavelengths with settlement (Mound, 1962;
Coombe, 1982). Other pests such as thrips (Thysanoptera: Thripidae) have two peaks of
efficiency, one sensitive to UV wavelengths at 365 nm and another in the green region at 540
nm, although similar to whiteflies there is no physiological evidence for a third photoreceptor in
the blue region (Matteson et al., 1992; Mazza et al., 2010). On the other hand, little attention has
been given to the effect of UV light on lepidopteran insects (Meyer-Rochow et al., 2002), and no
information about pest moths appears to be known.
Among new Integrated Pest Management strategies, UV-absorbing photoselective nets have
been shown to satisfactorily perform under field situations and reduce the impact of insect
vectors and plant pathogens in protected crops (Antignus et al., 1998; Chyzik et al., 2003; Díaz
et al., 2006; Weintraub, 2009; Ben-Yakir et al., 2012; Legarrea et al., 2012a, b). These covers
act as filters that do not transmit the majority of UV light. The management of aphid and
whitefly-borne viruses by optical barriers suggests that the blockage of UV light may interfere
with insect vision and their ability to orientate within the crop (Antignus et al., 1996; Kumar &
Poehling, 2006; Díaz et al., 2006; Díaz & Fereres, 2007; Legarrea et al., 2012a). At the same
time, UV radiation influences insects not only directly but also indirectly via the plant’s physical
and biochemical traits (Vänninen et al., 2010; Johansen et al., 2011).
Successful IPM systems use a wide variety of control means. Any optical barrier that is
implemented in greenhouses should not interfere with biological agents in the existing
Flight behaviour of pests and beneficials under UV-blocking enclosures
51
system. The spectral efficiency of the hymenopteran parasitoid Encarsia formosa Gahan
(Hymenoptera: Aphelinidae) has been previously described, with a primary peak at 520 nm and
a secondary peak in the UV region (Mellor et al., 1997). It has been reported that aphid and
whitefly parasitoids are attracted to high UV radiation but they can perform well in a UV-filtered
environment (Chyzik et al., 2003; Chiel et al., 2006; Doukas & Payne, 2007a, b). Conversely,
there is a scarcity of data on predators and further knowledge is needed in this area (Reitz et al.,
2003; Legarrea et al., 2012c). In this work, a common hoverfly species of Mediterranean outdoor
and greenhouse crops was studied, whose visual capacity has not been tested yet (Amorós-
Jiménez et al., 2012).
5.2. OBJECTIVE
The aim of this research was to evaluate the orientation and flight activity of a wide range of
insects of agricultural importance under six new cladding materials with different optical
properties. The goal was to find an optical barrier effective against key pests but compatible with
natural enemies commonly used under greenhouse conditions in IPM programs. For this study,
we included pest species Myzus persicae (Sulzer) (Hemiptera: Aphididae), Bemisia tabaci
(Gennadius) (Hemiptera: Aleyrodidae) and Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae),
as well as two natural enemies, the parasitoid, Aphidius colemani Viereck (Hymenoptera:
Braconidae), that is commonly released in protected crops, and the predator Sphaerophoria
rueppellii (Weidemann) (Diptera: Syrphidae), an aphidophagous hoverfly that is being tested as
a biocontrol agent in Southeastern Spain.
5.3. MATERIALS AND METHODS
5.3.1. EXPERIMENTAL DESIGN
Two experimental designs were carried out under field conditions in two experimental stations
located at the Polytechnic University of Madrid and “La Poveda Experimental Farm” during the
spring of two consecutive years, 2011 and 2012. In 2011, film PL and nets G, A, P and T were
tested in a design consisting of three plots (replications) in which five one-chamber tunnels
(experimental unit, 1 m long x 0.6 m wide x 0.6 m height) were covered with each type of
cladding material (treatments). Ends of the tunnels were covered with the cladding materials as
well. Tunnels were placed standing on bare soil in a north-south orientation, with the release
platform directed toward the north. The position of each cladding material within plots was
52
randomized. Distances between tunnels and plots were 0.6 m and 2 m, respectively (Figure
5.1.a).
A 15 x 15 cm release platform was placed in each tunnel, and suspended from the ceiling at a
height of 0.3 m and 0.3 m away from one side of the tunnel. Release platforms consisted of black
cardboards attached to glass tubes filled with insects. Four targets (T1, T2, T3 and T4) were
placed at 0.6, 0.7, 0.8 and 0.9 m from the entrance of the tunnel (Figure 5.1.b). A specific target
was designed after conducting preliminary tests in the laboratory for assessing the attraction and
flight capacity of each insect species under each type of cladding material: a) a 10 x 10 cm
yellow sticky trap for testing the number of captures of M. persicae and B. tabaci; b) three-week
tomato plants for assessing T. absoluta oviposition; c) four-week-old cucumber plants infested
with twenty-five 2nd-instar A. gossypii nymphs for evaluating the number of mummies
parasitized by A. colemani; d) four-week-old pepper plants infested with 20-49 M. persicae for
evaluating oviposition by S. rueppellii.
Upon placing targets in each tunnel, the glass tubes from the release platform were opened and
insects were allowed to fly across the tunnel. All trials were conducted starting at solar noon for
three hours except for T. absoluta for 24 hours because the oviposition peak occurs during the
first hours of the morning. Two S. rueppellii, three female A. colemani, 25 T. absoluta, 50 M.
persicae and 100 B. tabaci were released into each tunnel. Each trial run was repeated several
times on different dates but always within a four-week interval for each insect species. The
experiment was run once for T. absoluta (n=3), twice for M. persicae (n=6) and S. rueppellii
(n=6), three times for A. colemani (n=9) and four times for B. tabaci (n=12), depending on
ongoing results, insect availability and climatic conditions. Insects trapped on front and back
sides of each sticky trap were counted separately using a Nikon SMZ1500 lamp microscope
(Nikon Inc., Melville, USA). Cucumber plants with potentially parasitised aphids were kept in a
greenhouse (23:20 °C, 70-80% RH and 16L:8D) for 7 days to assess the number of mummies.
The number of T. absoluta and S. rueppellii eggs were counted immediately after the assays. All
insects unable to exit the glass tubes were counted. Average, minimum and maximum relative
humidity, and temperature were recorded inside the tunnels with Tinytag TGU-4500 data loggers
(Gemini Ltd., Chichester, United Kingdom).
In 2012, several changes were made to improve the experimental design, based on results
obtained during 2011. This time, two plots with four tunnels each were tested (Figure 5.1.c).
Nets A and G, which gave no extra information and did not reduce vector dispersal compared to
Flight behaviour of pests and beneficials under UV-blocking enclosures
53
UV-transparent nets in 2011, were excluded. UV-absorbing net O, a new material with potential
differences compared to the control treatment, was included in the trials of the second year of
study. In addition, flight tunnels were expanded to 2 m long x 0.6 m wide x 0.6 m height
(experimental unit) to force insects to fly longer distances using three targets (T1, T2 and T3)
placed at 1, 1.4 and 1.8 m (Figure 5.1.d) from the entrance of the tunnel. Due to availability, the
number of insects released from the flying platform also varied; 12 T. absoluta, five female A.
colemani, 50 M. persicae, and 100 B. tabaci were introduced into each tunnel. Each trial run was
repeated several times on different dates but always within an eight-week interval for each insect
species. The experiment was repeated twice for T. absoluta (n=4) and M. persicae (n=4), and
three times for B. tabaci (n=6) and A. colemani (n=6), depending on ongoing results, insect
availability and climatic conditions. The rest of the experimental conditions were the same as in
2011.
Figure 5.1. Experimental set-ups, displaying plots and overhead view of tunnels with the position of the release platform and targets (numbers 1 to 4) in years 2011 (a, b) and 2012 (c, d).
5.3.2. STATISTICAL METHODS
The proportion of insects able to exit the release platform was compared by type of net and
insect species using a Chi-square goodness of fit test (p≤0.05). Percentages of M. persicae and B.
tabaci relative captures referred to the number of insects able to leave the platform, and A.
colemani mummies referred to the number of nymphs available per plant (100 the first year and
75 the second year) were transformed by 2*arcsin√x. Number of T. absoluta and S. rueppellii
eggs was transformed with either √(x+0.5), x2 or Ln(x+1). Differences among photoselective
enclosures and distances to platform were tested with one-way ANOVA pairwise comparison
(p≤0.05). Student t-tests were used to assess differences between both sides of the traps (p≤0.05).
54
When data did not follow a normal distribution, a non-parametric Mann-Whitney U-test or
Kruskal-Wallis H-test was performed (p≤0.05).
5.4. RESULTS
5.4.1. PHOTOSELECTIVE COVERS
As shown in Figure 5.2, net T was selected as a UV-transparent control because it transmitted
most of the UV radiation (83%) when compared to the UV-opaque film PL, the negative control
that blocked 98% of UV radiation. Nets O, G and A were able to transmit UV from 38 up to
44%, and net P up to 65%. Among these nets, O was able to transmit only 54% PAR compared
to 74-87% transmitted by the rest of the nets.
Figure 5.2. Total transmittance from 250 to 750 nm of the six cladding materials studied (film PL, and nets O, G, A, P and T).
5.4.2. ABILITY TO LEAVE THE RELEASE PLATFORM
The proportion of insects able to leave the platform differed among species and enclosures. In
2011, the UV-opaque film PL, which transmitted only 2% UV radiation, reduced the ability of
M. persicae (21.7±5.2%) and B. tabaci (88.2±3.6%) to exit the release tubes significantly
compared to the rest of the nets (p<0.001) (Figure 5.3.a). The same situation applied to A.
colemani (p<0.001), with slightly lower proportions of parasitoids flying out of the platform
under all types of cladding materials (Figure 5.3.a). In general, insects were able to fly out of the
glass tubes under all types of nets without significant differences among treatments. No
differences among treatments were found in T. absoluta and S. rueppellii (p>0.05), and the
majority of insects (>70%) were able to leave the platform under all kinds of cladding materials
!"
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Flight behaviour of pests and beneficials under UV-blocking enclosures
55
(Figure 5.3.a). In 2012, PL significantly reduced the ability of aphids (74.5±4.3%) and whiteflies
(66.3±14.3%) to leave the tubes compared to all nets (p<0.001), where percentages ranged from
91.8±1.7% to 96.0±1.1% of insects leaving the tubes (Figure 5.3.b). Unlike the previous year,
the UV-opaque film PL did not affect the ability of A. colemani to leave the platform compared
to the UV-transparent net T, which had 83% UV transmittance (χ2=0.577; p=0.480), although
significant differences were found between PL and nets O and P, with a 38% and 65% UV
transmittance respectively (Both nets, χ2=5.455; p=0.026). For T. absoluta, differences were
found between nets O and T (38% and 83% UV transmittance, respectively) (χ2=5.790; p=0.030)
(Figure 5.3.b). In general, a lower percentage of T. absoluta was found outside the tubes under
all type of nets when compared to the other insect species tested, and that tendency was
significant under nets P and T (65% and 83% UV transmittance, respectively) (p<0.05).
Figure 5.3. Percentage of insects that left the platform tubes and flew through the tunnels during 2011 (a) and 2012 (b). Different letters in bars (mean±S.E.) indicate statistically significant differences within each insect species among the type of material (film PL, and nets O, G, A, P and T) according to Chi-square goodness of fit test (p≤0.05). UV transmittance of all materials is also listed.
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56
5.4.3. SHORT TUNNELS – DISPERSAL OF PESTS
M. persicae orientation was perturbed under the UV-opaque film, which transmitted only 2%
UV radiation, and its ability to reach the end of the tunnel was somehow affected when
compared to the net enclosures (Table 5.1). Significantly fewer aphids were trapped in T3 (Front
side: H=10.081; df=4; p=0.039), T4 (Front and back sides: H=12.601; df=4; p=0.013. Back side:
H=10.324; df=4; p=0.035) and on the front side of the four traps (H=12.601; df=4; p=0.013)
under the UV-opaque film than under the rest of the cladding materials tested (data not shown).
When the influence of trap distance was studied, we observed a significant increase in captures
in distant traps under net T (83% UV transmittance), ranging from 5.42±1.34% (T4) to
6.94±1.85% (T3), as opposed to aphids trapped in closer traps T1 (2.44±1.39%) and T2
(1.92±0.67%) (F=3.929; df=20(3); p=0.023). No differences were found between both sides of
traps for M. persicae captures (p>0.05).
Flight behaviour of pests and beneficials under UV-blocking enclosures
57
58
Conversely, B. tabaci had a distinct preference for closer targets no matter the cladding material,
and the percentage of whitefly captures decreased with distance to the release site under all
treatments (PL: F=4.064; df=44(3); p=0.012. G: F=6.067; df=44(3); p=0.002. A: F=12.228;
df=44(3); p<0.001. P: F=6.568; df=44(3); p=0.001. T: F=11.476; df=44(3); p<0.001) (Figure
5.4). Whitefly captures increased for T3 and then decreased for T4 under net G (40% UV
transmittance (Figure 5.4). No apparent differences among covers for the same target or side of
trap were found (p>0.05).
Differences in UV transmittance inside tunnels did not have an effect on T. absoluta oviposition
and a similar number of eggs were laid on the same target of each tunnel (p>0.05) and under all
enclosures, regardless the target distance to platform (p>0.05) (Table 5.1).
Figure 5.4. Mean percentage of Bemisia tabaci captures±S.E. in yellow sticky traps placed at 0.6 (T1), 0.7 (T2), 0.8 (T3) and 0.9 (T4) m distance from the release platform inside one-meter tunnels covered with different cladding materials (film PL, and nets G, A, P and T). UV transmittance of all materials is also listed. Different letters stand for statistical differences according to one-way ANOVA or Kruskal-Wallis H-test (p≤0.05).
5.4.4. SHORT TUNNELS – DISPERSAL OF NATURAL ENEMIES
In general, parasitism by A. colemani was very low under all treatments and reached a peak of
18.67±6.62% under net G, which transmitted 40% UV radiation (Table 5.1). Aphidius colemani
behaved in a similar way under all enclosures in targets T1, T2, and T4 (p>0.05), but UV-opaque
film reduced the rate of parasitism in T3 (H=16.166; df=4; p=0.003) compared to nets G and P.
Total parasitism was also significantly lower under PL compared to nets G and A (H=10.443;
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Flight behaviour of pests and beneficials under UV-blocking enclosures
59
df=4; p=0.034) (Table 5.1). When comparing the distance of cucumber plants to the release
platform, no differences could be found among targets (p>0.05).
The results with S. rueppellii were not as clear-cut as with other insects. Indeed, females showed
a very erratic behaviour and few eggs were found on M. persicae-infested pepper plants (Table
5.1). Surprisingly, when counting total oviposition per tunnel we observed that females laid more
eggs under the UV-opaque film than under the rest of nets, although oviposition was statistically
similar under all enclosures and the distance of pepper plants to the release point had no
influence on oviposition (p>0.05) (Table 5.1).
5.4.5. LONG TUNNELS – DISPERSAL OF PESTS
M. persicae flight behaviour was mostly affected by the UV-opaque film compared to nets.
These differences mainly occurred in the furthest target of the tunnel (Front side: F=5.654;
df=12(3); p=0.012. Back side: F=5.962; df=12(3); p=0.010), in the back side of the three traps
(F=4.526; df=12(3); p=0.024) and on the total number of captures within tunnels (F=3.673;
df=12(3); p=0.044). Fewer aphids reached distant targets under UV-absorbing nets and
significantly more aphids could fly up to the end of the tunnels covered with net P (F=26.268;
df=9(2); p<0.001) (Figure 5.5.c). Furthermore, when front and back sides of yellow sticky traps
were compared, significantly more aphids were trapped on the back side under net P (65% UV
transmittance) (Front side: 10.74±1.68, back side: 18.13±0.86. t=-3.916; df=6; p=0.008),
whereas this trend was reversed under UV-absorbing materials, such as film PL (Front side:
3.52±2.16, back side: 1.39±0.81) and net O (Front side: 12.92±5.16, back side: 9.94±3.80),
which had 2% and 38% UV transmittance, respectively. However, differences were not
significant in the latter materials (p>0.05).
60
Figure 5.5. Comparison between Myzus persicae and Bemisia tabaci flights, displaying the percentage of captures±S.E. in yellow sticky traps placed at 1 (T1), 1.4 (T2) and 1.8 (T3) m distance from the release platform inside two-meter tunnels covered with different cladding materials: film PL (a), net O (b), net P (c) and net T (d). UV transmittance of all materials is also listed. Asterisks stand for statistical differences among targets according to one-way ANOVA (p≤0.05).
B. tabaci flight activity was also negatively affected by the UV-opaque film, although in a
different trend as M. persicae. Unlike aphids, differences in whitefly captures were mainly found
in the closest targets: T1 (Front side: F=4.065; df=20(3); p=0.017. Back side: F=5.909;
df=20(3); p=0.005) and T2 (Front side: F=3.138; df=20(3); p=0.048. Back side: F=9.009;
df=20(3); p=0.001) (data not shown). Total whitefly captures under UV-opaque film were
significantly lower on the front (F=4.300; df=20(3); p=0.017) and back side of the three traps
(H=11.803; df=3; p=0.008), and per tunnel (F=6.925; df=20(3); p=0.002) (data not shown). As
distance from platform to target increased, fewer whiteflies were trapped under all enclosures,
being significant for two UV-absorbing materials: film PL (F=4.285; df=15(2); p=0.034) and net
O (F=19.746; df=15(2); p<0.001) (Figure 5.5.a and b). Under the latter, there was an abrupt
difference among targets placed at 1 m versus 1.4 and 1.8 m, suggesting that B. tabaci flight was
restricted to the first trap when UV radiation was low (Figure 5.5.b). However, the slope
smoothed as UV transmittance increased under P and T treatments (65% and 83% UV
transmittance, respectively) and no differences in captures among targets were recorded (Figure
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Flight behaviour of pests and beneficials under UV-blocking enclosures
61
5.5.c and d). Similarly, in year 2012 no differences could be found between both sides of the
yellow trap (p>0.05).
In contrast to year 2011, where we could not find differences among treatments, UV-opaque film
deterred oviposition by T. absoluta in T1 (F=7.642; df=12(3); p=0.004) and per tunnel (F=5.675;
df=12(3); p=0.012) in 2012 (Table 5.2). Similar to what we observed for short tunnels, no
differences in oviposition were found among the distance of targets to platform for each material
enclosure (p>0.05).
Table 5.2. Mean number of Tuta absoluta eggs and percentage of Aphidius colemani mummies±S.E. in targets placed at 1 (T1), 1.4 (T2) and 1.8 (T3) m distance from the release platform inside two-meter tunnels covered with different cladding materials (film PL, and nets G, A, P and T). UV transmittance of all materials is also listed. Different letters stand for statistical differences among cladding materials in each target and total per tunnel (one-way ANOVA (p≤0.05)).
Insect Material UV tran. (%) T1 T2 T3 Total
T. a
bsol
uta PL 2 0.00±0.00 a 0.50±0.50 a 3.75±3.43 a 4.25±3.92 a
O 38 33.00±20.93 b 16.25±9.12 a 19.25±6.33 a 68.50±29.86 b P 65 15.50±4.66 b 11.25±9.07 a 35.75±23.84 a 62.50±28.47 b T 83 15.50±2.33 b 26.75±17.57 a 10.25±6.09 a 52.50±25.69 b
A. c
olem
ani PL 2 0.00±0.00 a 4.67±4.67 a 5.11±3.75 a 9.77±8.28 a
O 38 8.23±3.68 a 6.89±4.43 a 13.56±4.51 a 28.67±10.81 a P 65 4.67±2.55 a 8.89±3.40 a 6.89±4.39 a 20.44±7.93 a T 83 9.77±3.73 a 3.11±3.11 a 11.77±5.87 a 24.67±5.16 a
5.4.6. LONG TUNNELS – DISPERSAL OF NATURAL ENEMIES
Total parasitism rates increased in 2012 compared to 2011 and ranged from 9.77±8.28% to
28.67±10.81%, depending on the type of enclosure. Although rates under UV-opaque film were
lower, no statistically significant differences were found on the ability of A. colemani to find the
furthest targets under the absorbing film PL in 2012 (p>0.05). Furthermore, UV-absorbing nets
did not affect the ability of A. colemani to locate their hosts and overall parasitism was similar
under UV-absorbing and UV-transparent nets (F=2.182; df=20(3); p=0.122) (Table 5.2). No
differences were found among host distance to platform for each material tested (p>0.05).
5.5. DISCUSSION
Ultraviolet (UV) radiation has a crucial impact on insect vision, and insect flight activity might
be greatly affected under UV-filtered environments. We first tested the ability of insects to exit
the glass tubes and leave the release platform. Our data support the use of UV-opaque films to
62
deter aphids or whiteflies when they first approach a greenhouse. Raviv & Antignus (2004)
reported two mechanisms for the anti-insect activity of these materials. First, the number of
insects that invade enclosed greenhouses is lower due to the higher UV reflectance emission by
the sky or reflected from these covers. Second, the light environment created inside alters the
normal behaviour of insects, thus resulting in reduced flight activity.
In this work, the initial entry of insects into the greenhouse was not studied but the findings
suggest that pests could be restricted to the area they first encounter and might not be able to
search for suitable hosts (Chyzik et al., 2003; Raviv & Antignus, 2004; Döring & Chittka, 2007).
Around 30% of parasitoids were not able to fly outside the tubes in 2011, even under the UV-
transparent materials P and T. Very few parasitoids were reported under UV-opaque film.
Consequently, this conditioned the rate of parasitism found in those assays, which was very low.
Some observations suggest that the behaviour of parasitoids was altered due to heat inside the
tunnels in 2011. Despite that A. colemani has a subtropical to warm climate distribution
including areas of Mediterranean Europe (Stary, 1975), high temperatures were reached during
the spring season of 2011 (30 ºC outdoors and about 39 ºC inside PL tunnels). Literature has
reported neutral effects of the lack of UV on natural enemies (Chyzik et al., 2003; Chiel et al.,
2006; Doukas & Payne, 2007a, b). Indeed, experiments in 2012 provided better results for
parasitoids by expanding the size of tunnels. The milder average temperatures recorded during
the second year (20 ºC) probably allowed this very sensitive insect to exit the tubes and fly along
the tunnel (Zamani et al., 2007). Tuta absoluta and S. rueppellii flight was not affected by
different enclosures, implying that olfactory signals may mediate attraction, landing and
oviposition much more than visual stimuli (Proffit et al., 2011).
Secondly, unravelling the role of UV radiation on insect orientation was considered. Although
insects have similar photoreceptors in their compound eyes, our results suggest a variety of
responses depending on each of the species studied. Moreover, it has to be acknowledged that
some insect species showed slightly different flight behaviour inside the tunnels between the
2011 and 2012 experimental design. Suppression of UV from the light spectrum reduced aphid
captures in both short and long tunnels, agreeing with previous studies that have shown how UV-
absorbing screens contribute to reduce aphid movement and dispersal (Chyzik et al., 2003; Díaz
et al., 2006; Ben-Yakir et al., 2012; Legarrea et al., 2012a). On the contrary, once they reached
the end of the UV-transparent tunnels, their flight dispersal response probably triggered by UV
light was followed by a plant-responsive foraging behaviour that ended up with aphids caught in
Flight behaviour of pests and beneficials under UV-blocking enclosures
63
the furthest target of both experimental designs, especially in the back side of the yellow sticky
traps (Kring, 1972; Döring & Chittka, 2006).
Aphid and whitefly flight activities greatly differed from each other. On one side, aphid captures
increased with distance to platform, especially under the most UV-transparent materials.
Conversely, B. tabaci flew short distances in the absence of UV light and no differences were
found under nets P and T as UV transmittance increased. Besides, whiteflies showed this
gradient in relation to the number of captures no matter what tunnel length was used. Bemisia
tabaci has been reported to be very sensitive to mid-range visible light (550 nm), expressing a
preferred landing reaction on yellow sticky traps than on green leaves (Mound, 1962; Mutwiwa
et al., 2005). Lower densities of several whitefly species have been found under UV-deficient
screens in greenhouse and field studies (Costa & Robb, 1999; Antignus et al., 2001; Mutwiwa et
al., 2005; Legarrea et al., 2012c).
On the contrary, the migratory behaviour controlled by UV sensitivity (Mound, 1962) resulted in
no differences among targets for UV-transparent nets P and T during the second year. In 2011,
we found significantly different whitefly captures under all enclosures, decreasing with target
distance to platform. During the first year, perhaps the distance between platform and T1 was not
long enough to discriminate among treatments and whiteflies saw the yellow trap right after
leaving the tubes under all enclosures. The short distance triggered a strong attraction, regardless
of UV transmittance. This may explain why the majority of whiteflies were significantly
restricted to T1 under materials P and T, as happened under the rest of materials. On the
contrary, during 2012 we only found differences for the PL and O materials. Possibly, whiteflies
flew a long distance before encountering any visual stimuli. Then, the presence of UV radiation
inside P and T tunnels made possible for whiteflies to distribute equally among targets.
Inside the short tunnels, we found no differences either in the number of T. absoluta eggs among
nets. This might be due to the fact that plants were too close to each other for moths to
discriminate between targets. Also, T. absoluta showed an erratic behaviour, choosing random
targets regardless of distance. Olfactory stimuli seem to be involved in host recognition by T.
absoluta (Proffit et al., 2011), although no references about the role of UV radiation on this
insect have been reported. Indeed, when the experimental design forced insects to fly across a
longer distance we observed significantly fewer eggs under the UV-opaque film. Other
environmental factors, especially high humidity and temperature under the UV-opaque film,
64
might have been deleterious for oviposition by T. absoluta, explaining the low number of eggs
collected under our experimental conditions.
With regard to natural enemies, we observed that parasitism by A. colemani under UV-opaque
film was lower inside short tunnels than long tunnels. Moreover inside such short tunnels, we
found significantly fewer mummies under the UV-opaque film. Aphidius colemani orients
towards the plant-host complex following a “cry for help” mechanism in which plants emit
defence volatile signals when they are under herbivore attack. Once parasitoids land on a
suitable plant, they can detect aphids by olfactory cues such as honeydew secretions or alarm
pheromones (Du et al., 1998). In these experiments, nymphs within a plant were closely located
so that if one A. colemani female landed on a plant, it could eventually find every aphid on it as
odours released by individuals would trigger a strong searching behaviour (Storeck et al., 2000).
That might explain why no differences were seen when testing parasitism rate as a function of
the distance to the release platform, since vision appears to act as a secondary cue for these
insects. In assays with short tunnels, we also observed water condensation and an increase of 2.5
ºC inside the UV-opaque film tunnels compared to those covered with nets. Percentages of A.
colemani parasitism increased in all treatments in 2012 but especially under the UV-opaque film,
proving a certain compatibility with UV-filtered environments. Moreover, higher parasitism
rates were obtained under photoselective nets O, G and A compared to the UV-transparent net T,
although no significant differences were found among materials, suggesting that A. colemani
responds to host-plant signals in order to locate potential hosts, particularly those induced by
aphid feeding (Chiel et al., 2006; Boivin et al., 2012). As a whole, results suggest that
parasitoids may perform well under low UV light levels (Chyzik et al., 2003; Chiel et al., 2006;
Doukas & Payne, 2007a, b), but environmental conditions can be a major drawback that should
be taken into account when implementing these technologies into greenhouses (Legarrea et al.,
2012c). Conversely, no final conclusions can be generalised about syrphids because we found
low egg densities under all treatments, but the findings show promise as S. rueppellii females
laid eggs inside tunnels covered with the UV-opaque film.
Several trade-offs are faced in every decision involving IPM, but these studies suggest that pests
can be managed successfully without risking the activity of beneficial insects. Integrated Pest
Management programs for vegetables could benefit by protecting crops with UV-absorbing nets
that permit airflow, exclude pests and provide a good environment for natural enemies. The
studies suggest there are species-specific responses to UV light, therefore the use of UV-
absorbent materials in greenhouse production cannot be generalised. Nevertheless, it would be
Flight behaviour of pests and beneficials under UV-blocking enclosures
65
rewarding to design further studies and test new species, especially natural enemies, to assess
their compatibility with cladding materials under realistic conditions inside commercial
greenhouses.
66
67
CHAPTER 6. IMPACT OF UV-A RADIATION ON THE PERFORMANCE
OF APHIDS AND WHITEFLIES AND ON THE LEAF CHEMISTRY OF
THEIR HOST PLANTS3
ABSTRACT
Ultraviolet radiation directly regulates a multitude of herbivore life processes, in addition to
indirectly affecting insect success via changes in plant chemistry and morphogenesis. Plant and
insect (aphid and whitefly) exposure to supplemental UV-A radiation in the glasshouse
environment and the effects on insect population growth were investigated. Glasshouse grown
peppers and eggplants were grown from seed inside cages covered by novel plastic filters, one
transparent and the other opaque to UV-A radiation. At a 10-true leaf stage for peppers (53 days)
and 4-true leaf stage for eggplants (34 days), plants were harvested for chemical analysis and
infested by Myzus persicae (Sulzer) and Bemisia tabaci (Gennadius), respectively. Clip-cages
were used to introduce and monitor the insect fitness and populations of the pests studied. Insect
pre-reproductive period, fecundity, fertility and intrinsic rate of natural increase were assessed.
Crop growth was monitored weekly for 7 and 12 weeks throughout the crop cycle of peppers and
eggplants, respectively. At the end of the insect fitness experiment, plants were harvested (68
days and 18-true leaf stage for peppers, and 104 days and 12-true leaf stage for eggplants) and
leaves analysed for secondary metabolites, soluble carbohydrates, amino acids, total proteins and
photosynthetic pigments. The results demonstrate for the first time that UV-A modulates plant
chemistry with implications for insect pests. Both plant species responded directly to UV-A by
producing shorter stems but this effect was only significant in pepper whilst UV-A did not affect
the leaf area of either species. Importantly, in pepper, the UV-A treated plants contained higher
contents of secondary metabolites, leaf soluble carbohydrates, free amino acids and total content
of protein. Such changes in tissue chemistry may have indirectly promoted aphid performance.
For eggplants, chlorophylls a and b, and carotenoid levels decreased with supplemental UV-A
over the entire crop cycle but UV-A exposure did not affect leaf secondary metabolites.
However, exposure to supplemental UV-A had a detrimental effect on whitefly development, 3 Published in: Dáder B., Gwynn-Jones D., Moreno A., Winters A., Fereres, A. 2014. Impact of UV-A radiation on the performance of aphids and whiteflies and on the leaf chemistry of their host plants. Journal of Photochemistry and Photobiology B, Biology, 138: 307-316.
68
fecundity and fertility presumably not mediated by plant cues as compounds implied in pest
nutrition -proteins and sugars- were unaltered.
6.1. INTRODUCTION
Aphids and whiteflies are two of the most important pests worldwide, not only because of the
direct damage they cause, but also because their alimentary habits involve transmission of plant
viruses (Hull, 2014). Ultraviolet radiation plays a major role in herbivores, including insect
pests, by modifying their orientation toward potential hosts, flight activity, alighting, arrestment,
feeding behavior and interaction between sexes (Raviv & Antignus, 2004; Johansen et al., 2011).
Aphids (Hemiptera: Aphididae) and whiteflies (Hemiptera: Aleyrodidae) are among the most
studied insects concerning their flight behaviour. Aphids have been reported to reduce their
flight activity and ability to disperse in UV-deficient environments (Díaz & Fereres, 2007;
Döring & Chittka, 2007). Moreover, a decrease in fecundity and population density has been
also demonstrated (Antignus et al., 1996; Chyzik et al., 2003; Díaz et al., 2006; Kuhlmann &
Müller, 2009a; Paul et al., 2011; Legarrea et al., 2012c). Conversely, UV radiation stimulates
whitefly migration (Mound, 1962; Coombe, 1982). Among new Integrated Pest Management
strategies, UV-absorbent photoselective nets have been successfully tested in field situations by
reducing the impact of insect vectors and plant pathogens on protected crops (Díaz & Fereres,
2007; Weintraub, 2009; Legarrea et al., 2012a).
Knowledge on the effects of UV-B on plant growth and chemistry (nutritional characteristics
relevant to insects) has been developed due to past concerns about ozone depletion (Ballaré et
al., 1996; Hunt & McNeil, 1999; Mackerness, 2000; Jansen, 2002; Comont et al., 2012; Mewis
et al., 2012). In contrast, understanding of the effects of the UV-A fraction of the solar spectrum
on plants and insect pests is very limited. Whilst UV-A radiation is unaffected by ozone
depletion, it is a significant component of the solar spectrum affected by latitude, altitude and
cloud cover. It is also often absent from the glasshouse/horticultural environment. New
environmental concerns suggest that understanding UV-A impacts on plants could be important
given that predictions by the United Nations Environment Programme suggest that there will be
a higher incidence of cloud free periods, particularly in southern Europe and the Mediterranean
Basin. This will result in higher exposure of crops to ambient UV-A radiation
Impact of UV-A on the performance of pests and leaf chemistry of hosts
69
(WMO, 2010). Only a few authors have considered UV-A impacts on plant growth (Tezuka et
al., 1994; Jayakumar et al., 2003, 2004; Verdaguer et al., 2012). The latter work shows that
radiation in the UV-A range produces alterations in leaf morphology and anatomy of several
plants, with the most characteristic response mainly observed in the adaxial epidermal cells,
which were thicker and longer than those grown without UV-A.
There are no known studies that have focused on how UV-A influences the relationship between
phytophagous insects and their plant hosts but there is large body of material published on UV-A
plant pollinator interactions (Stephanou et al., 2000; Petropoulou et al., 2001; Dyer & Chittka,
2004). Furthermore, research on spider mites by Sakai & Osakabe (2010) concluded that
Tetranychus urticae Koch (Acari: Tetranychidae) exploits UV-A information to avoid ambient
UV-B radiation. At the same time other work on Panonychus citri McGregor (Acari:
Tetranychidae) suggested that eggs were tolerant to UV-B radiation and females successfully
oviposited on the upper side of leaves exposed to UV-B via artificial lamps (Fukaya et al.,
2013).
The knowledge on the effects of UV-B on plant-insect interactions suggests that typical plant
responses would include the accumulation of UV-screening metabolites, increased leaf thickness
and trichome density or reduction in cell elongation (Smith et al., 2000; Paul & Gwynn-Jones,
2003; Liu et al., 2005; González et al., 2009; Kulhmann & Müller, 2009a). These impacts have
implications for host success because such physical and biochemical traits affect host acceptance
and success of future insect progeny (Vänninen et al., 2010; Paul et al., 2011).
Understanding of the indirect effects of UV-A on insects via plants remains limited to what we
know about current practices in horticulture. On one hand, the horticulture industry traditionally
grows crop species under glass or plastic with opaque or lowered UV radiation environments.
However, evidence suggests that supplemental UV-A may improve plant growth, yield and
quality. For example, a combination of visible radiation and UV-A at a particular ratio may be
highly suitable for enhanced growth of soybean seedlings (Middleton & Teramura, 1993).
Similar findings have been observed on the yield of Phaseolus mungo L., which was improved
with UV-A exposure (Jayakumar et al., 2003). UV cladding materials have been shown to also
have positive effects on crop growth by increasing stem length, leaf toughness or trichome
density (Hunt & McNeil, 1999; Kittas et al., 2006; Kuhlmann & Müller, 2009a, 2010; Paul et
al., 2011). There is also evidence that UV transmitting environments could produce food plants
commercially with increased human health benefits (Tsormpatsidis et al., 2011).
70
6.2. OBJECTIVE
In this study, it was hypothesised that UV-A is central to the trophic relationships between these
two global pests -aphids and whiteflies- and their host plants. The horticultural hosts Capsicum
annuum L. (pepper) and Solanum melongena L. (eggplant) and their respective insect pests, the
green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) and the whitefly Bemisia
tabaci (Gennadius) (Hemiptera: Aleyrodidae) were grown in the presence and absence of UV-A
radiation. It was targeted how UV-A impacts the success of insects via population growth. In
tandem with direct effect of UV-A, how UV-A exposure indirectly affects insects via changes in
plant chemistry was also assessed. Correlations between the different responses found in leaf
chemicals analysed and plant sensitivity to UV-A were considered as well.
6.3. MATERIALS AND METHODS
6.3.1. EXPERIMENTAL DESIGN
Experiments were undertaken in glasshouse facilities at ICA-CSIC at a temperature of 23:20±2
°C (day:night), a photoperiod of 14:10 (light:dark) and 70-80% RH. Capsicum annuum cv.
‘California Wonder’ and S. melongena cv. ‘Black beauty’ seeds were germinated in pots. For
both species, three seeds were placed in each pot and thinned to one post germination. UV-A
radiation was supplied by two Osram Ultra-Vitalux UV lamps (Osram GmbH, Munich,
Germany). Lamps were switched on and off with no gradual transition for a photoperiod of 14
hours every day throughout the entire length of experiments. The lamps emitted no UV-C
radiation and produced radiation levels representative of typical sunny summer day conditions in
the centre of the Iberian Peninsula (Gutiérrez-Marco et al., 2007; Häder et al., 2007). However,
it should be emphasised that the aim was to expose plants and insects to UV-A under glasshouse
conditions rather that simulate UV-A outdoors. The lamps used were heavily weighted for UV-A
emission so throughout the text the treatment will be referred as UVA+ (supplemental UV-A).
A set of two cages (1 x 1 x 1 m) was covered by filters (Figure 6.1). As a positive control that
allowed UV-A radiation transmission but blocked UV-B radiation (Table 6.1, Figure 6.2), the
upper side of one cage was covered with a 200 µm thickness film (Solplast S.A., Murcia, Spain).
The four lateral sides were covered to a 50 cm height with a UV-transparent net T 50 mesh
(Ginegar Plastic Products Ltd., Kibbutz Ginegar, Israel) to permit airflow inside the cage. The
remaining upper 50 cm were covered with plastic film. For the suppressed UV-A radiation
treatment, a 200 µm thickness Antivirus UV-blocking film (Solplast S.A., Murcia, Spain) and a
Impact of UV-A on the performance of pests and leaf chemistry of hosts
71
UV-absorbing Optinet 50 mesh (Ginegar Plastic Products Ltd., Kibbutz Ginegar, Israel) were
used. Optical properties (transmitted radiation) of the UV-opaque and UV-transparent films were
analysed at the CSIC Torres Quevedo Institute (Madrid, Spain) using a double monochromator
Lambda 900 UV/Visible/NIR spectrophotometer (PerkinElmer Life and Analytical Sciences
Ltd., Connecticut, USA). The main difference between both filters was that the UV-opaque film
blocked UV-A transmission (315-400 nm) and the UV-transparent film allowed UV-A
transmission, as seen in Figure 6.2.
Figure 6.1. Set of two cages covered by two different plastic films used in the experimental design. The disposition of plants inside the cages is also shown.
Lamps were hung at a distance of 1 m above the plant canopy. Irradiance per second was
measured daily above cage and at canopy level as well as on the abaxial side of the leaves and
through the leaves with clip-cages where insects were monitored with an ALMEMO 25904S
radiometer (Ahlborn GmbH, Holzkirchen, Germany). The radiation received by the plants
(irradiance) under both treatments is shown in Table 6.1. The UV daily doses were 71.67 KJ m-2
day-1 UV-A and 0.55 KJ m-2 day-1 UV-B for treatment UVA+, and 1.76 KJ m-2 day-1 UV-A and
0.10 KJ m-2 day-1 UV-B for treatment UVA-. Daily UV-A radiation inside the cage covered by
the blocking film was very low (1.76 KJ m-2 d-1) hence this treatment was called UVA- (near
zero UV-A). A fourty-fold increase in UV-A transmittance at the plant canopy level inside the
regular cage was measured when compared to the cage covered by the UV-absorbing barrier
(1.422 vs. 0.035 W m-2) (Table 6.1). Low levels of UV-B radiation inside both experimental
treatments were detected although represented less than 1% of the light received by plants (0.011
W m-2 in treatment UVA+ and 0.002 W m-2 in treatment UVA-) (Table 6.1).
It should again be noted that the experimental set up was used to evaluate how supplemental
UV-A affects plant-insect interactions and performance in the glasshouse environment. The
72
focus was on crop production and this study was not designed to simulate outdoor environmental
conditions, hence any extrapolation of findings to field conditions should be done with caution.
Table 6.1. Radiation conditions at canopy level outside and inside the experimental cages (UVA+ and UVA- treatments), on the abaxial side of the leaves and through the leaves with clip-cages where insects were monitored. Transmission percentages represent radiation transmitted inside both cages in relation to the same level outside cages.
Treatment UVA+ Treatment UVA- PARa UV-Ab UV-Bb PAR UV-A UV-B Canopy level outside cage 515.0 (112.8) 11.722 0.561 505.0 (110.6) 11.290 0.575 Canopy level inside cage 441.8 (96.8) 1.422 0.011 334.6 (73.3) 0.035 0.002 Abaxial side w/ clip-cage 25.3 (5.5) 0.083 0.002 21.8 (4.8) 0.003 0.002 Through leaves w/ clip-cage - 0.030 0.002 - 0.000 0.000 Transmission inside cage (%) 85.79 12.13 1.96 66.26 0.31 0.35
a µmol m-2 s-1 (W m-2), b W m-2
Figure 6.2. Total transmittance from 250 to 750 nm of the UV-transparent (UVA+) and UV-opaque (UVA-) plastic films measured by a double monochromator spectrophotometer.
Pots with seeds were placed inside cages and plants were grown from seeds under two different
radiation regimes, either with supplemental (UVA+) or near zero UV-A radiation (UVA-). At a
10-true leaf stage (53 days) for peppers and 4-true leaf stage (34 days) for eggplants, half of the
plants of each cage were moved from the UVA+ to the UVA- treatment and vice versa. Some of
the plants were infested by aphids (n=19) or whiteflies (n=16) to study the performance of
insects. In this way, there were four UV-A treatments: positive control UVA+/UVA+, plants
grown under supplemental UV-A radiation for the entire growth cycle; negative control UVA-
/UVA-, plants grown at near zero UV-A radiation for the entire growth cycle; UVA+/UVA-,
plants grown under supplemental UV-A radiation before insect introduction and at near zero
UV-A after insect introduction; and UVA-/UVA+, plants grown at near zero UV-A radiation
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Impact of UV-A on the performance of pests and leaf chemistry of hosts
73
before insect introduction and under supplemental UV-A after insect introduction. Figure 6.3
represents a timeline diagram of the experimental procedure. Stem height, and leaf length and
width were monitored weekly using a ruler (n=6). The relationship between these measurements
and actual leaf area (cm2) was calculated by scanning ten leaves of different stages of each plant
species and contouring them with Adobe Acrobat software (Pepper: 0.66±0.01. Eggplant:
0.73±0.01). Experiments were repeated twice over one year. Leaf material harvested throughout
the experiment was either snap-frozen and maintained at -80°C or air-dried at 70°C as relevant
for further analyses.
Figure 6.3. Timeline diagram of the experimental design, showing the four different UV-A treatments (T1: UVA+/UVA+, plants grown under supplemental UV-A radiation for the entire growth cycle; T2: UVA+/UVA-, plants grown under supplemental UV-A radiation before insect introduction and under near zero UV-A after insect introduction; T3: UVA-/UVA+, plants grown under near zero UV-A radiation before insect introduction and under supplemental UV-A after insect introduction and T4: UVA-/UVA-, plants grown under near zero UV-A radiation for the entire growth cycle), dates of insect infestation to study the performance of aphids and whiteflies and plant harvests for peppers and eggplants. The arrows refer to the moment when half of the plants of each treatment were moved from treatment UVA+ to UVA- and vice versa.
Both insect species were synchronised prior to assays to ensure that individuals were the same
age. Pepper plants were infested by M. persicae at the 10-true leaf stage (53 days old) (Figure
6.3). One single wingless aphid adult was placed in a clip-cage on the abaxial side of the
youngest fully developed leaf of each pepper plant and allowed to produce nymphs for 24 hours
(Figure 6.4). Surplus nymphs were removed leaving three nymphs per plant, which were
monitored until adulthood stage. When the first nymph reached the adult stage, the other two
were removed. Offspring from the remaining insect was monitored by removing nymphs daily
for an equal number of days to the pre-reproductive period. The parameters pre-reproductive
period (d), effective fecundity (Md), intrinsic rate of natural increase (rm=0.738*(logeMd)/d),
Insect introduction Second harvest (n=6)
Sowing First harvest (n=6)
Pepper
Eggplant 0 Time
(days)
0
53
34
68
104
UVA+ (pepper: n=56, eggplant: n=50)
UVA- (pepper: n=56, eggplant: n=50)
UVA+ (pepper: n=25, eggplant: n=22)
UVA- (pepper: n=25, eggplant: n=22)
UVA+ (pepper: n=25, eggplant: n=22)
UVA- (pepper: n=25, eggplant: n=22)
T1
T2
T3
T4
74
mean relative growth rate (RGR=rm/0.86) and mean generation time (Td=d/0.738) were
calculated (n=19). Eggplants were infested by B. tabaci at the 4-true leaf stage (34 days old)
(Figure 6.3). Ten pairs of adult whiteflies were left to produce eggs inside clipcages on the
abaxial side of the youngest fully developed leaf of each plant for 24 hours and ten eggs were
monitored until adult emergence (Figure 6.4). A newborn female and male were placed on a new
leaf and their offspring monitored for 30 days. Pre-reproductive period, larvae viability, female
fecundity and fertility were studied (n=16).
Figure 6.4. Clipcages for aphid and whitefly monitoring placed on the abaxial side of the leaves.
Plants from the two species were harvested at two different growth stages for determining
biomass and content of chemical compounds (Figure 6.3). Plants were harvested from each of
the treatment cages at the 10-true leaf stage (53 days after sowing) for peppers plants and 4-true
leaf stage (34 days after sowing) for eggplants (n=6). All leaves from each plant were collected
for subsequent chemical analyses. Further plants from the treatments were harvested at 18-true
leaf stage for peppers (68 days after sowing) and at 12-true leaf stage for eggplants (104 days
after sowing). This involved plants from each treatment including those infested with insects and
those not (as above, n=6).
6.3.2. PLANT BIOCHEMICAL ANALYSIS
6.3.2.1. SECONDARY METABOLITES
Frozen samples were subsequently freeze-dried for 48 hours and leaf material homogenised with
a pestle and mortar. Samples were analysed for secondary metabolites by extraction in 70%
methanol of freeze-dried samples (100 mg), as described by Comont et al. (2012). Supernatants
were dried using a Savant SpeedVac SPD121P vacuum centrifuge (Thermo Scientific,
Massachusetts, USA) before re-suspension in 500 µL 70% methanol. The solid-phase extraction
was performed using a Sep-Pak Vac 500 mg C18 column (Waters Ltd., Elstree, UK) before
Impact of UV-A on the performance of pests and leaf chemistry of hosts
75
vacuum centrifugation of the sample to complete dryness. Dried pellets were suspended in 500
µL 100% methanol and analysed via high pressure liquid chromatography (HPLC) with a system
comprising a Waters 515 pump, a Waters 717 plus autosampler, a Waters 996 photodiode array
detector and a Waters C18 Nova-Pak radial compression column (C18 4.0 µm, 8.0 x 100 mm
cartridge) (Waters Ltd., Elstree, UK) with an injection volume of 30 µL and a flow rate of 2 mL
min-1. The mobile phase consisted of 5% acetic acid (solvent A) and 100% methanol (solvent B)
with a linear gradient from 5 to 75%, B in A, over 35 min. Peak integration was performed using
the Empower software. Liquid chromatography-mass spectrometry (LC-MS) was performed to
identify the major compounds. A Thermo Finnigan LC-MS system (Finnigan Surveyor LC pump
plus, PDA plus detector, Finnigan LTQ linear ion trap) (Thermo Scientific, Massachusetts, USA)
and a Waters C18 Nova-Pak column (C18 4.0 µm, 3.9 x 100 mm) were used with an injection
volume of 10 µL and a flow rate of 1 mL min-1. The mobile phase consisted of purified water-
0.1% formic acid (solvent A) and MeOH-0.1% formic acid (solvent B) with a linear gradient
from 5 to 65%, B in A, over 60 min. Phenolics were characterised by UV absorption spectra, MS
fragmentation patterns in negative ion mode and comparison with standards and previously
reported data in the literature (Clifford et al., 2003; Stommel et al., 2003; Marín et al., 2004;
Park et al., 2012).
6.3.2.2. SOLUBLE SUGARS
Air-dried samples (100 mg) were extracted in 3 mL of distilled water at 80 °C three times.
Extracts were centrifuged for 10 min at 10,000 rpm. Supernatants were retained, combined and
frozen until the analysis. Then 50 µL of sample were added to 950 µL of a buffer comprising 5
mM H2SO4 with a 5 mM crotonic acid internal standard. Samples were analysed via HPLC
comprising a Jasco LG-980-02 ternary gradient unit, a Jasco PU-1580 pump, a Jasco AS-1555
sampler and a Jasco RI-2031 detector (Jasco Ltd., Essex, UK). Injection volume was 25 µL.
Sugars were identified by comparison with an internal library of standard compounds (Comont
et al., 2012).
6.3.2.3. FREE AMINO ACID AND PROTEINS
Freeze-dried plant material (100 mg) was extracted in 4 mL of boiling distilled water for 25
minutes. Extracts were allowed to cool and a 1.5 mL aliquot was centrifuged to clarify the
solution, following the methodology described by Winters et al. (2002). Amino acid absorbance
was measured at 570 nm using an Ultrospec 4000 UV/Vis spectrophotometer (GE Healthcare,
Buckinghamshire, England). Histidine was used for the calibration curve as most amino acids
76
have the same response. Total proteins were extracted from 100 mg of freeze-dried sample by
grinding in 1.8 mL Mclivaine buffer pH 7 containing 50 mM ascorbic acid, and 0.2 mL 20%
lithium dodecyl sulphate. Protein content was analysed by the Lowry protein assay (Lowry et al.,
1951) following precipitation of protein in extracts with 20% trichloroacetic acid, 0.4%
phosphotunstic acid and resuspension in 0.1 M NaOH. Absorbance was measured at 700 nm
with a µQuant microtitre plate reader spectrophotometer (Bio-Tek Instruments Inc., Winooski,
USA). Protein contents were determined against a bovine serum albumin calibration curve.
6.3.2.4. PHOTOSYNTHETIC PIGMENTS
Chlorophyll a, chlorophyll b, chlorophylls a+b and carotenoid contents were analysed in freeze-
dried sample extracts. Leaf material (50 mg) was extracted in 80% acetone and supernatants
were diluted 1:15 in 80% acetone with absorbance measured at 470, 646.6, 663.6 and 750 nm
using an Ultrospec 4000 UV/Vis spectrophotometer (GE Healthcare, Buckinghamshire,
England). Pigment contents were determined using equations by Lichtenthaler (1987) and Porra
et al. (1989).
6.3.3. STATISTICAL METHODS
All parameters were analysed with Student t-test (p≤0.05) to assess differences prior to exchange
of plants or one-way ANOVA pairwise comparison (p≤0.05) to test differences after the
exchange of plants. If data did not follow a normal distribution, a non-parametric Mann-Whitney
U-test or Kruskal-Wallis H-test (p≤0.05) was performed. Stem height and leaf area over the crop
cycle (repeated measures over time) were assessed with ANOVA univariate repeated measures
analysis (p≤0.05) using SuperANOVA v. 1.11 software for Macintosh (Abacus Concepts, 1989).
6.4. RESULTS
6.4.1. PLANT GROWTH
Addition of UV-A to pepper plants over the entire plant growth cycle (UVA+/UVA+) caused a
significant reduction in plant height (Treatment: F=15.399; df=3; p<0.001. Time: F=137.122;
df=6; p<0.001. Time x Treatment: F=7.311; df=8; p<0.001). By 68 days, plants grown with
supplemental UV-A were 57% shorter compared to plants grown at near zero UV-A (23.9 cm vs.
37.7 cm) (Figure 6.5.a). Pepper leaf area appeared lower with UV-A but not significantly
different (Treatment: F=2.618; df=3; p=0.068. Time: F=262.928; df=6; p<0.001. Time x
Impact of UV-A on the performance of pests and leaf chemistry of hosts
77
Treatment: F=1.271; df=8; p=0.267) when compared to the near zero UV-A treatment (Figure
6.5.b).
Eggplants exposed to UV-A were shorter from 84 days onwards although not significant
(Treatment: F=0.018; df=3; p=0.997. Time: F=311.450; df=11; p<0.001. Time x Treatment:
F=1.575; df=29; p=0.042). By the end of the experiment, plants exposed to supplemental UV-A
during their entire cycle were 23% shorter than plants that had been grown at near zero UV-A
(50.5 cm vs. 62.2 cm) (Figure 6.5.a). For leaf area no significant effects were observed with UV-
A (Treatment: F=0.191; df=3; p=0.901. Time: F=262.753; df=11; p<0.001. Time x Treatment:
F=1.528; df=29; p=0.054) (Figure 6.5.b). Later addition of UV-A when insects were introduced
to plants (53-68 days for aphids and 34-104 days for whiteflies) did not alter the height or leaf
area responses observed above.
Figure 6.5. Stem height (a) and leaf area (b) of peppers and eggplants grown under four different UV-A radiation regimes for 68 and 104 days, respectively. Bars refer to standard errors. Significant differences (p≤0.05) were observed only for the stem height of pepper plants exposed to supplemental UV-A radiation during the entire growth cycle (UVA+/UVA+) (reduction in height) according to ANOVA univariate repeated measures test.
6.4.2. INSECT RESPONSES
For aphids, the pre-reproductive period (d) from birth to adult stage was similar in all treatments
(H=2.656; df=3; p=0.448) (Table 6.2). However, effective fecundity (Md) was significantly
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78
higher (F=2.888; df=70(3); p=0.042) in early supplemental UV-A treatment scenario compared
to the near zero UV-A treatment (UVA-/UVA-) (Table 6.2 and Figure 6.6). This latter treatment
lowered intrinsic rate of natural increase (rm: F=2.974; df=70(3); p=0.037) as well as mean
relative growth rate (RGR: F=2.974; df=70(3); p=0.037) when compared to pepper plants
exposed to UV-A during early growth (UVA+/UVA-, Table 6.2). UV-A treatment after insect
infestation had no effects on aphid fecundity and development (Figure 6.6).
The response of whiteflies to UV-A exposure was different to that of aphids. The pre-
reproductive period (d) from birth to adult stage was significantly shortened by two days
(H=10.409; df=3; p=0.015) at near zero UV-A during insect development on plants (UVA-
/UVA- and UVA+/UVA-) (Table 6.2). Direct exposure of whiteflies to supplemental UV-A on
plants raised at near zero UV-A (UVA-/UVA+) significantly lowered fecundity -egg numbers-
compared to all other treatments (F=13.256; df=60(3); p<0.001) (Table 6.2 and Figure 6.6).
Moreover, egg numbers were significantly lower in treatments UVA+/UVA+ and UVA-/UVA+,
47% and 123% respectively, when compared to insects maintained on plants raised at near zero
UV-A over the entire experiment (UVA-/UVA-). Supplemental UV-A exposure also lowered
egg fertility (F=6.254; df=60(3); p=0.001) (Table 6.2). This resulted in a significantly lower
(F=14.380; df=60(3); p<0.001) number of larvae in the treatments where insects were exposed to
UV-A, regardless of the previous conditions in which eggplants were raised (treatments
UVA+/UVA+ and UVA-/UVA+, Table 6.2). UV-A treatment after insect infestation had a
negative impact on whitefly fecundity, fertility and development (Figure 6.6).
Table 6.2. Life parameters of Myzus persicae and Bemisia tabaci raised under four different UV-A radiation regimes. Different letters stand for statistical differences (p≤0.05).
Insect Parameters UVA+/UVA+ UVA-/UVA- UVA+/UVA- UVA-/UVA+
M. p
ersi
cae da 8.89±0.15 8.71±0.17 8.63±0.14 8.74±0.15
Mdb 37.53±2.57 ab 29.71±2.41 c 39.32±2.88 a 31.26±3.18 bc Tdc 12.05±0.20 11.80±0.23 11.70±0.19 11.84±0.20 rm
d 0.298±0.006 ab 0.284±0.007 b 0.310±0.006 a 0.283±0.010 b RGRe 0.346±0.007 ab 0.330±0.008 b 0.361±0.007 a 0.329±0.011 b
B. t
abac
i Viabilityf 72.43±10.48 81.38±8.37 77.86±8.78 75.71±6.61 d 26.99±0.89 a 24.40±0.48 b 24.66±0.46 b 26.94±0.84 a No. eggs 78.69±8.12 b 115.69±7.90 a 98.06±8.72 ab 51.88±5.58 c No. larvae 50.69±7.22 b 87.44±8.25 a 73.81±9.54 a 25.94±3.25 c
Fertilityf 60.30±4.91 b 73.48±3.51 a 72.12±4.10 a 50.31±4.23 b a Pre-reproductive period, b Effective fecundity, c Mean generation time, d Intrinsic rate of natural increase, e Mean relative growth rate, f %
Impact of UV-A on the performance of pests and leaf chemistry of hosts
79
Figure 6.6. Comparison between Myzus persicae and Bemisia tabaci fecundity, showing the number of nymphs and eggs per female on peppers and eggplants, respectively, under four different UV-A radiation regimes. Bars refer to standard errors and different letters stand for statistical differences (p≤0.05).
6.4.3. PLANT BIOCHEMICAL RESPONSES
6.4.3.1. SECONDARY METABOLITES
HPLC and LC-MS analysis revealed that there were two hydroxycinnamic acids and four
flavonoids identifiable in pepper leaves. Analysis of eggplants revealed phenolics belonging to
three classes (chlorogenic acid isomers, hydroxycinnamic acid amide conjugates and
isochlorogenic acid isomers), as well as 3-O-feruloylquinic acid, which were determined based
on HPLC elution times, UV spectra and LC-MS fragmentation data (Table 6.3). Two
kaempferol-hexosides with UV absorption maxima at 265 and 349 nm were also identified on
the basis of their MS2, however signals were too low to permit effective quantification of these
compounds.
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Impact of UV-A on the performance of pests and leaf chemistry of hosts
81
Secondary metabolites were increased in peppers by long term UV-A exposure (68 days) but this
depended on time of harvest and whether plants were simultaneously exposed to insects. Total
content was similar under both UV-A regimes at 53 days (t=0.947; df=10; p=0.366) (Figure
6.7.a). However, when plants were harvested at 68 days, the four main flavonoid contents of
pepper plants previously exposed to UV-A and later moved to a near zero UV-A regime
(UVA+/UVA-) were comparable to levels found in those that had been grown entirely without
UV-A radiation (UVA-/UVA-). This implies that phenolic expression declined when UV-A
radiation was withdrawn. Pepper plants grown initially without UV-A and subsequently
transferred to UV-A (UVA-/UVA+) also showed phenolic levels that were significantly higher
than plants continuously grown under supplemental UV-A (UVA+/UVA+) (Compound 2:
F=3.987; df=20(3); p=0.022. Compound 3: F=5.229; df=20(3); p=0.008. Compound 4:
F=11.145; df=20(3); p<0.001. Compound 5: F=20.618; df=20(3); p<0.001. Compound 6:
F=35.214; df=20(3); p<0.001. Total: F=29.945; df=20(3); p<0.001) (Figure 6.7.a). Results for
pepper suggest rapid acclimation to UV-A with aphid introduction and damage influencing
flavonoid profiles, as significantly higher levels were found in plants exposed to supplemental
UV-A early but withdrawn from this treatment (UVA+/UVA-) (Compound 4: F=4.632;
df=20(3); p=0.013. Compound 5: F=7.755; df=20(3); p=0.001. Compound 6: F=7.884;
df=20(3); p=0.001. Total: F=10.546; df=20(3); p<0.001) (Figure 6.7.a). N-caffeoylputrescine
content in both uninfested and infested plants did not differ significantly.
Addition of UV-A radiation did not affect eggplant phenolic expression after the first harvest (34
days) prior to whitefly infestation (t=0.697; df=10; p=0.502) (Figure 6.7.a). In contrast to pepper
plants, eggplant phenolic compounds were unaffected by treatment over the duration of the
experiment (F=0.306; df=20(3); p=0.821) (Figure 6.7.a). Whitefly infestation did not appear to
influence these patterns (F=0.193; df=20(3); p=0.900) (Figure 6.7.a).
82
Figure 6.7. Total phenolic (a) and soluble carbohydrate content (b) of pepper and eggplant leaves grown under four different UV-A radiation and two herbivore regimes, and harvested at two dates. Bars refer to standard errors and different letters stand for statistical differences (p≤0.05).
6.4.3.2. SOLUBLE SUGARS
Data showed different carbohydrate profiles with species and treatments (Figure 6.7.b). Polymer
content was similar under all treatments at any harvest time for both species. Polymer content
was very high in eggplant leaves. Significantly lower levels of total non-structural sugars
(raffinose, sucrose, glucose and fructose) were observed in uninfested pepper plants grown under
treatment UVA+/UVA+ at 68 days (F=3.484; df=20(3); p=0.035). Raffinose and glucose in
particular were significantly higher following treatment UVA-/UVA+ (Raffinose: F=3.440;
df=20(3); p=0.036. Glucose: F=5.365; df=20(3); p=0.007). For infested plants, total non-
structural levels were similar (F=1.205; df=20(3); p=0.334) although sucrose content was
significantly higher in treatments where aphids were grown under supplemental UV-A (F=3.227;
df=20(3); p=0.044). No differences were found at any date in eggplant non-structural sugars.
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Impact of UV-A on the performance of pests and leaf chemistry of hosts
83
When total sugar content was analysed, UVA+/UVA+ level was lowest in uninfested peppers
(F=4.622; df=20(3); p=0.013) but highest in infested plants (F=3.402; df=20(3); p=0.038)
(Figure 6.7.b). Carbohydrate levels under herbivory were lower than those observed in
uninfested peppers possibly due to aphid feeding (Figure 6.7.b). Conversely, no differences were
found among treatments on eggplants samples both uninfested and infested by whiteflies (Figure
6.7.b).
6.4.3.3. FREE AMINO ACID AND PROTEINS
At 53 days, pepper plants exposed to supplemental UV-A had significantly higher levels of free
amino acids (t=2.755; df=10; p=0.020). However, this trend was not significant at 68 days in
uninfested peppers (F=1.871; df=20(3); p=0.167) (Figure 6.8.a). Infested plants had a lower
level compared to uninfested plants possibly due to in situ aphid feeding activity but no
differences could be found between different radiation regimes (F=0.609; df=20(3); p=0.617)
(Figure 6.8.a). A similar pattern was observed for total protein content with a significantly higher
amount in plants continuously grown under supplemental UV-A at 68 days (F=15.062; df=20(3);
p<0.001) (Figure 6.8.b). No differences were observed between treatments in eggplants for free
amino acids (34 days: t=0.291; df=10; p=0.777. 104 days uninfested: F=0.255; df=20(3);
p=0.857. 104 days infested: F=0.217; df=20(3); p=0.883) and total proteins (34 days: t=0.245;
df=10; p=0.812. 104 days uninfested: F=0.783; df=20(3); p=0.517. 104 days infested: F=1.634;
df=20(3); p=0.213) when exposed to UV-A and/or feeding by whiteflies (Figure 6.8.a and b).
84
Figure 6.8. Free amino acids expressed as histidine (a) and total protein (b) content of pepper and eggplant leaves grown under four different UV-A radiation and two herbivore regimes, and harvested at two dates. Bars refer to standard errors and asterisks stand for statistical differences (p≤0.05).
6.4.3.4. PHOTOSYNTHETIC PIGMENTS
There was no significant effect of UV-A exposure on pepper plant photosynthetic pigments
either at any harvest time or under aphid herbivory (Table 6.4). In contrast, eggplant leaves
exposed to supplemental UV-A had lower chlorophyll content radiation at 34 days (Chlorophyll
a: t=-2.531; df=10; p=0.030. Chlorophylls a+b: t=-2.426; df=10; p=0.036) and under whitefly
infestation at 104 days (Chlorophyll a: F=4.613; df=20(3); p=0.013. Chlorophyll b: F=3.887;
df=20(3); p=0.024. Chlorophylls a+b: F=4.994; df=20(3); p=0.010) (Table 6.4). Carotenoids
also showed significant accumulation at near zero UV-A (34 days: t=-2.630; df=10; p=0.025.
104 days uninfested: F=3.803; df=20(3); p=0.026. 104 days infested: F=4.467; df=20(3);
p=0.015). Contents were highest for treatment UVA-/UVA- and mixed treatments where plants
received both radiation regimes had intermediate contents (Table 6.4). Chlorophyll a/b ratio was
statistically equal in all treatments, ranging from 2.3 to 2.5 in peppers and from 2.7 to 2.9 in
eggplants.
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Impact of UV-A on the performance of pests and leaf chemistry of hosts
85
86
6.5. DISCUSSION
In the present work, the effects of UV-A radiation on two key global pests, the aphid M. persicae
and whitefly B. tabaci and their host plants, pepper and eggplant were investigated. The aim was
to determine how UV-A in the glasshouse environment influenced plant growth and chemistry,
and insect performance. This work was undertaken in cages placed in a glasshouse facility where
plants received UV-A radiation via artificial lamp sources. Although the glass of the facility and
filter-covered cages absorbed a considerable amount of radiation, at least some natural UV
reaching the plants cannot be neglected. In particular a higher UV:PAR ratio may have occurred
at the start and end of each day because lamps were already switched on early in the morning
and after sunset. These diurnal changes in the UV:PAR ratio might have influenced plant
chemistry and insect response. However, UV irradiance reaching the plant canopy was
predominantly originating from the lamps (70 %) because sunlight was partially filtered by
greenhouse glass. Most (99%) of the UV radiation received by plants and insects in the UVA+
treatment was UV-A. However, the possibility of a small amount of UV-B irradiance, well
below ambient UV-B levels, present during the experiments has to be acknowledged.
Considering the 14 h photoperiod, plants received 71.67 KJ m-2 d-1 of UV-A while only 0.55 KJ
m-2 d-1 of UV-B, which is 0.76% of the total UV irradiance. Therefore, any changes observed in
plants and insects under the UVA+ treatment were predominantly elicited by UV-A. To our
knowledge, this is the first study that has looked at supplemental UV-A effects on plant-insect
interactions in the glasshouse environment, as opposed to previous research mainly focused on
UV-B impacts (Hunt & McNeil, 1999; Kittas et al., 2006; Kuhlmann & Müller, 2009a, 2010;
Paul et al., 2011).
For both plants species studied, the supplemental UV-A treatment appeared to alter the size and
morphology over the entire crop cycle. Although plants had similar numbers of leaves, pepper
internodes were significantly shorter, similar as previously reported in other plant species
(Kuhlmann & Müller, 2010; Comont et al., 2012). For eggplants, plant height appeared
shortened but there were no significant effects on height or leaf area. This contrasts with
previous work focussing on enhanced UV-B impacts on reduced leaf area (Kittas et al. 2006). In
the current study, chlorophyll and carotenoid contents were lowered in eggplant with UV-A
treatment at both harvest dates and under whitefly infestation, as found on buckwheat or quinoa
with supplemental UV-B (Gaberšcik et al., 2002; González et al., 2009). A reduction in
chlorophyll has been proposed as an indicator of UV sensitivity (Smith et al., 2000).
Impact of UV-A on the performance of pests and leaf chemistry of hosts
87
The relevance of components of leaf chemistry was measured in order to try to interpret the
insect responses observed. Phenolic patterns in peppers changed in response to UV-A and under
herbivory. No secondary metabolite differences were observed during the earlier harvest at 53
days prior to insect introduction but were apparent at 68 days. As expected, 5-O-caffeoylquinic
acid and flavonoid contents were significantly induced with enhanced UV-A (Gaberšcik et al.,
2002, Izaguirre et al., 2007; Mahdavian et al., 2008; Kulhmann & Müller, 2009a, b, 2010). In
the absence of aphids at 68 days, evidence showed how plants grown at near zero UV-A but later
moved to a UV-A regime (treatment UVA-/UVA+) had higher level of leaf secondary
metabolites, which even exceeded the levels found in UV-A treated plants over the entire crop
cycle (UVA+/UVA+). This readiness of peppers to induce ‘sunscreen’ compounds might be
correlated with UV tolerance (Middleton & Teramura, 1993; Harborne & Williams, 2000).
Meanwhile, the flavonoid contents of plants grown with supplemented UV-A but subsequently
moved to near zero UVA- declined rapidly to levels comparable to the control treatment UVA-
/UVA- after stress recovery. Hence the effect of UV-A was not cumulative over time (Comont et
al., 2012). Besides UV-shielding metabolites, elevated contents of phenolics have been proposed
as antifeedants or digestibility reducers (Ballaré et al., 1996; Paul & Gwynn-Jones, 2003).
Flavonoid levels are thought to be an important factor in herbivore nutrition and they may be
partially induced by the same signaling pathway as UV protection, in which the jasmonic acid
plays a key role (Mackerness, 2000; Stratmann, 2003; Demukra et al., 2010; Mewis et al., 2012).
Aphid feeding affected pepper phenolics, as seen previously in tobacco (Izaguirre et al., 2007).
Whether the flavonoids detected acted also as a defense against M. persicae needs further
investigation but results suggest aphid damage influencing their accumulation compared to
uninfested peppers. Indeed one of the flavonoids present in the samples, luteolin-7-O-(2-
apiosyl)glucoside, has been previously proposed as a deterrent compound against the leafminer
fly species Liriomyza trifolii Burgess (Diptera: Agromyzidae) in sweet pepper leaves (Kashiwagi
et al., 2005). Phenolics found in eggplants were mainly hydroxycinnamic acids, with 5-
transcaffeoylquinicacid as the major compound (Stommel et al., 2003). As opposed to peppers,
no significant increases in secondary metabolites were observed with UV-A or whitefly
infestation in eggplants. However, induction of several flavonoids has been stated to protect
tissues from UV damage in this species (Toguri et al., 1993). Past research has shown that
eggplants already have high constitutive defences. Exposure to high UV-B irradiances did not
influence phenolic accumulation, leaf area and chlorophyll a/b ratio (Smith et al., 2000;
González et al., 2009). These results altogether may indicate a high tolerance to UV irradiance in
this species possibly related to its ancestral origin from tropical regions.
88
Total non-structural carbohydrates were lowest in uninfested peppers grown under UV-A during
the complete duration of the experiment (68 days) compared to all other treatments. Comont et
al. (2012) also reported reductions in sucrose, glucose and fructose contents on Arabidopsis
thaliana L. following UV-B treatment although contrasting results have been obtained on maize
leaves (Barsig & Malz, 2000). However when insects were introduced, sucrose content was
significantly higher in treatments where M. persicae was grown under UV-A. This might agree
with previous research done under UV-B stress where higher soluble sugar content, mainly
sucrose, was observed under addition of UV-B (González et al., 2009). Carbohydrate
accumulation may have affected aphid fitness because sucrose is a strong feeding stimulant and
the major component of the phloem sap of plants (Mittler et al., 1970; Srivastava & Auclair,
1971). Indeed when UV-A was withdrawn, adults produced less progeny with lower growth
rates. By contrast, eggplant soluble sugars were unaffected by UV-A and total levels were
similar at every harvest time and under whitefly herbivory, displaying another reliable indicator
to UV tolerance (González et al., 2009).
Amino acids are the major nitrogen source for aphids. In this work, significantly higher free
amino acids in pepper leaves exposed to UV-A radiation were observed, suggesting that insects
could prefer such plants. Amino acids are an essential dietary component for M. persicae growth
(Dadd & Krieger, 1968) that has a mainly nutritive role in aphid feeding (Srivastava & Auclair,
1975; Weibull, 1987). Nitrogen content is thought to act as a feeding stimulant for insects
(Schoonhoven et al., 2006), being higher when high radiation intensities are present in the
environment (Roberts & Paul, 2006). It is likely that phloem quality under supplemented UV-A
conditions had a richer composition that may have triggered a positive plant-mediated effect on
M. persicae development and fecundity. Moreover, free amino acids levels were unsurprisingly
lower under herbivore attack due to aphid feeding. It should be emphasized that the focus was on
the chemical composition of entire pepper leaves and this may not necessary reflect that in the
phloem sap (Kehr, 2006). Further studies should be conducted to find out if the observed
changes in leaf chemistry due to supplemental UV-A radiation are reflective of the chemical
changes in the phloem sap, extracted by stylectomy (Kennedy & Mittler, 1953) or via leaf
incisions (Milburn, 1970).
There were no differences according to UV-A in protein and free amino acid content in
eggplants. Very little is known about the impact of UV radiation on the composition of free
amino acids in phloem sap, but the same trend has been observed in other species of the family
Impact of UV-A on the performance of pests and leaf chemistry of hosts
89
Brassicaceae such as broccoli, where authors reported similar contents except for increased
proline under low UV-B compared to high levels of UV-B (Kulhmann & Müller, 2009a, 2010).
The addition of UV-A to the environment had complex effects on aphids. Mainly, an indirect
plant-mediated impact on M. persicae effective fecundity was observed. The effective fecundity
measured was higher in early UV-A treatment scenarios compared to the near zero UV-A
treatment (UVA-/UVA-). This latter treatment also resulted in lowered intrinsic rate of natural
increase and mean relative growth rate when compared to the scenario where plants had only
been exposed to UV-A during early growth (UVA+/UVA-). This may indicate that alterations in
tissue chemistry occurred prior to aphid infestation and contributed to its performance. The
reduction in the population growth without UV-A exposure is in agreement with findings
previously reported for several aphid species (Antignus et al., 1996; Chyzik et al., 2003; Díaz et
al., 2006; Kuhlmann & Müller, 2009a; Paul et al., 2011; Legarrea et al., 2012a). The pre-
reproductive period from birth to adult stage was similar for all treatments. In contrast, results
provided evidence that supplemental UV-A exposure had an impact on the fitness of whiteflies,
this contrasted with aphids. The pre-reproductive period was significantly increased by two days
with supplemental UV-A during insect growth on plants regardless of the radiation regime
before insect introduction (treatments UVA+/UVA+ and UVA-/UVA+). Exposure of whiteflies
to UV-A on plants raised at near zero UV-A (UVA-/UVA+) significantly lowered the number of
eggs compared to near zero UV-A for the entire crop cycle (UVA-/UVA-). There was no
statistically significant difference in the number of eggs between treatments UVA-/UVA- and
UVA+/UVA-, which supports the hypothesis that this effect was not mediated by host cues as it
did not depend on the UV-A regime the plants had been grown under before whitefly infestation.
This resulted in a significantly lower fertility in the treatments where UV-A was supplemented
during insect growth.
When whiteflies were subjected to supplemental UV-A treatments, eggplants received radiation
at the same time although the chemical compounds involved in whitefly nutrition analysed (free
amino acids and sugars) were unaffected by supplemental UV-A. UV-A radiation inside the clip-
cages where insects were monitored was 0.00 W m-2 in the treatment UVA- vs. 0.03 W m-2 in
the treatment UVA+, a difference that may not be sufficient to conclude that UV-A had a direct
impact on whitefly performance. However, the floor of the cages was aluminium and reflected
part of the UV radiation into the clip-cages in the supplemental UV-A treatment. Radiation
transmitted through the leaves could reach the ventral part of the whitefly nymphs and the
radiation reflected by the floor reaching the abaxial side of the leaves could irradiate the dorsum
90
of whiteflies. While results indicate a possible negative effect of UV-A that cannot be explained
by changes in plant chemicals measured, the possibility of an effect triggered by aspects of host
plant chemistry that were not measured cannot be dismissed. Further work to isolate direct from
plant-mediated effects of UV-A radiation on whitefly performance should be conducted in the
future by irradiation of insects under a free-plant environment.
The effect of UV on the life processes of whiteflies has been little studied. Traditionally research
has focused on flight behavior in host choice assays, with more whiteflies being trapped under
environments with UV radiation (Antignus et al., 1996; Costa & Robb, 1999; Kuhlmann &
Müller, 2009a), but to the best of knowledge, for the first time its performance has been tested
under different UV-A regimes. In past studies, it is likely that whiteflies were driven by the
radiation spectrum rather than by the plant chemistry as they tested orientation and alighting
(Kuhlmann & Müller, 2009b), whereas in this work insects were caged and forced to feed on
each plant. Whiteflies showed an explicit tendency to grow slower under the UV-A source after
insect infestation. This might be explained by the mechanism by which UV radiation triggers a
migratory behaviour (Mound, 1962; Coombe, 1982). However, the absence of UV might have
extended the mating period so whiteflies fed and laid eggs over a greater period at near zero UV-
A radiation.
Allocation of UV-A-shielding compounds responsible for physicochemical defense involved
some constrains on peppers, as plant growth decreased under high UV-A conditions. The UV-
induced phenolic pattern in pepper contrasted with lack of changes observed in eggplants. In
addition, this latter species also showed other characteristics present in plants tolerant to high
UV irradiances, such as no changes in leaf area and content of soluble carbohydrates irrespective
of UV-A exposure. These findings might be related to a high tolerance to UV-A. UV-A radiation
altered the chemical composition of pepper plants, with consequences to pest fitness. It is clear
that UV-A enriched pepper nutritional quality for aphids. In contrast for whiteflies, there was a
direct negative effect of UV-A rather than via tissue quality. As a whole, results reported in the
two complexes suggest that UV-mediated changes are highly dependent on the plant and insect
studied. Nevertheless, UV-absorbing nets might be an useful tool against aphids without
detrimental effects on crops. Further knowledge is needed to unravel the complete role of UV-A
radiation in plant-insect interactions, and to elucidate whether these responses present
interactions with effects occurring as a consequence of other fractions of the solar spectrum.
91
CHAPTER 7. CONTROL OF INSECT VECTORS AND PLANT VIRUSES
IN PROTECTED CROPS BY NOVEL PYRETHROID-TREATED NETS4
ABSTRACT
Long Lasting Insecticide-Treated Nets (LLITNs) constitute a novel alternative that combines
physical and chemical tactics to prevent insect access and the spread of insect-transmitted plant
viruses in protected enclosures. This approach is based on a slow release insecticide-treated net
with large hole sizes, which allow improved ventilation of greenhouses. The efficacy of a wide
range of LLITNs was tested under laboratory conditions against Myzus persicae (Sulzer), Aphis
gossypii Glover and Bemisia tabaci (Gennadius). Two nets were selected for field tests under a
high insect infestation pressure in the presence of plants infected with Cucumber mosaic virus
(CMV, Cucumovirus) and Cucurbit aphid-borne yellows virus (CABYV, Polerovirus). The
efficacy of Aphidius colemani Viereck, a parasitoid commonly used for biological control of
aphids was studied in parallel field experiments. LLITNs produced high mortality of aphids.
Certain nets excluded whiteflies under laboratory conditions, however they failed in the field.
Nets effectively blocked the invasion of aphids and reduced the incidence of both viruses in the
field. The parasitoid A. colemani was compatible with LLITNs. For this reason, LLITNs of
appropriate mesh size can become a very valuable tool for additional protection against insect
vectors of plant viruses under IPM programs.
4 Published in: Dáder B., Legarrea S., Moreno A., Plaza M., Carmo-Sousa M., Amor F., Viñuela E., Fereres A. 2014. Control of insect vectors and plant viruses in protected crops by novel pyrethroid-treated nets. Pest Management Science, DOI: 10.1002/ps.3942. Beatriz Dáder performed laboratory experiments with bifenthrin nets and field experiments with pests and plant viruses, in addition to data analysis and writing the paper. Saioa Legarrea performed laboratory experiments with deltamethrin nets. Results presented in her Ph.D. Thesis: Selective physico-chemical barriers against insect vectors of virus diseases in vegetable crops. Universidad Politécnica de Madrid, 2011. Fermín Amor performed the field experiment with A. colemani. Results presented in his Ph.D. Thesis: Compatibilidad de Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) y Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), depredadores importantes en cultivos hortícolas protegidos, con nuevas barreras físicas selectivas y modernos plaguicidas. Universidad Politécnica de Madrid, 2013.
92
7.1. INTRODUCTION
Vegetable crops suffer from economically damaging insect-pests and insect-transmitted virus
pathogens. Integrated Pest Management programs entail an interdisciplinary combination of
chemical and biological measures to manage pest damage (Stern et al., 1959). The control of
these pests generally involves intensive insecticide spraying with undesirable effects on the
environment, growers and public health. Therefore, there is an urgent need to develop
alternatives under the scope of IPM. The use of physical barriers is an excellent method to
reduce pest access to crops and impede virus transmission to plants (Weintraub & Berlinger,
2004). The selection of an appropriate insect screen depends on several factors: the thoracic size
of the insect, the size and geometry of the hole and the way threads are interlaced. Unfortunately,
effective barriers against small insects also reduce the airflow and the ventilation inside
greenhouses that frequently increase fungal problems (Bethke & Paine, 1991; Muñoz et al.,
1999). For this reason, new strategies together with physical barriers need to be developed to
prevent damage due to small insect pests and reduce the incidence of plant pathogens.
Insecticide-treated nets were developed long ago as bednets in public health to give protection
against malaria (Hougard et al., 2002). This strategy was approved for use with pyrethroids,
compounds that exhibit a rapid knockdown effect and high insecticidal potency at low dosage
without mutagenic or teratogenic effects (Zaim et al., 2000). The insecticide may be applied to
the net surface by immersion or spraying, but also by incorporation in the process of making the
yarns in the factory. In the latter case, the nets are called Long Lasting Insecticide-Treated Nets
(LLITN), and the insecticide may persist more than three years under field conditions (Martin et
al., 2007).
Field experiments using LLITNs have demonstrated promising results against agricultural pests
such as mites in African eggplant, resulting in higher yields (Martin et al., 2010), and brassica
crops (Díaz et al., 2004; Martin et al., 2006; Licciardi et al., 2008). These nets are cost-effective
in cabbage production. LLITNs serve as an effective barrier to control a wide range of
Lepidopteran pests, including the diamondback moth Plutella xylostella L. (Lepidoptera:
Plutellidae) and Hellula undalis (Fabricius) (Lepidoptera: Crambidae), or the aphid Lipaphis
erysimi Kaltenbach (Hemiptera: Aphididae), but not against the cabbage whitefly Aleyrodes
proletella L. (Hemiptera: Aleyrodidae) (Díaz et al., 2004; Martin et al., 2006; Licciardi et al.,
2008).
Control of vectors and viruses by pyrethroid-treated nets
93
Nevertheless, more accurate studies under laboratory and field conditions are necessary to fully
understand the mechanism of action of this novel pest control strategy and its compatibility with
natural enemies commonly used in biocontrol under protected enclosures. Up to date, little
attention has been given to this issue and there are no studies concerning the effects of LLITNs
on the behaviour or performance of natural enemies. The bioassays in this study were designed
to select the most appropriate LLITN among nets with different properties (insecticide dosages
and hole size) against two key pests in vegetable crops, aphids and whiteflies. These insects are
polyphagous and cause great concern because of the direct damage by extracting plant fluids, but
more importantly, because of their ability to transmit devastating virus pathogens (Gerling et al.,
2001; Blackman & Eastop, 2007). Standard insecticide applications have lead to the
development of insect resistance to many substances (Whalon et al., 2008) so that the integration
and understanding of control alternatives is necessary (Ellsworth & Martínez-Carrillo, 2001;
Margaritopoulos et al., 2009).
Aphids are the most important vectors of plant viruses (Bragard et al., 2013). Therefore, it is
crucial to interfere with the immigration and dispersal of potentially viruliferous vectors inside
protected crops. In this chapter, two major aphid-transmitted plant viruses affecting cucurbits,
Cucumber mosaic virus (CMV) and Cucumber aphid-borne yellows virus (CABYV) were
studied. Both viruses have different transmission modes. CMV is transmitted in a stylet-borne,
non-persistent manner during brief probes on epidermal cells, whilst CABYV is a circulative,
non-propagative phloem-restricted virus that requires long feeding probes for transmission
(Fereres & Moreno, 2009).
7.2. OBJECTIVE
The objective of our work was to test under laboratory and field conditions a wide range of
LLITNs designed to reduce the incidence of pests Aphis gossypii Glover (Hemiptera: Aphididae)
and Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and viruses CMV and CABYV of
horticultural crops grown under protected enclosures. The new approach is based on a slow
release insecticide-treated net that can provide relatively larger mesh sizes and thereby greater
airflow than standard untreated nets. Moreover, the effect of such nets on the spatial distribution
of aphids and aphid-transmitted viruses was studied with the SADIE methodology. Finally, the
compatibility of LLITN with the aphid parasitoid Aphidius colemani Viereck (Hymenoptera:
Aphidiinae) was tested under field conditions.
94
7.3. MATERIALS AND METHODS
7.3.1. LABORATORY EXPERIMENTS
A novel experimental set-up was designed to force insects to go through the insecticide-treated
net so that the effectiveness of the net killing the insects could be evaluated. All assays were
conducted at ICA-CSIC using an experimental tube composed by two glass cylinders (12 cm
long x 4 cm in diameter) separated by the test net (Figure 7.1). An untreated net of the same
mesh and vials with no net were used as controls in every experiment. Insects were released in
the bottom chamber and a leaf of a preferred aphid host plant was placed in the top chamber as
target. The vial was surrounded with black fabric except at the top, which was covered with a
thin cloth that allowed ventilation and light penetration into the structure (Figure 7.1). Both the
target leaf and light coming from the top were the stimuli to induce insects to climb up and cross
the LLITN. Insects located either feeding on the target leaf, dead or alive in the release chamber
were assessed 6 and 24 hours after aphid and whitefly release, respectively. Mortality values
were corrected (Abbott, 1925). Trials were conducted in the laboratory at room temperature (22-
24 °C).
The influence of deltamethrin concentration on M. persicae mortality was tested with different
insecticide concentrations (n=9), presence of UV-blocking additives (n=3), net colour (white
versus yellow) (n=6) and chemical compounds (deltamethrin, piperonyl butoxide [PBO], and a
combination of both) (n=6), using a turnip leaf as target. The efficacy of deltamethrin-treated
nets against B. tabaci (n=3) was tested using a tomato leaf cv. ‘Marmande’ as target. Ten M.
persicae and 50 B. tabaci were released in the bottom chamber for the deltamethrin trials.
In parallel experiments, net samples were placed in trays and exposed for one month to field
conditions at “La Poveda Experimental Farm” during winter and spring seasons to assess the
persistence of delthametrin (n=4) in the nets. Daily average climatic data was 6.4 °C mean
temperature, 11.7 °C maximum temperature, 1.8 °C minimum temperature and 6.5 MJ m-2 day-1
radiation during winter, and 15.8 °C mean temperature, 23.4 °C maximum temperature, 8.9 °C
minimum temperature and 21.1 MJ m-2 day-1 radiation during spring.
Bifenthrin was also tested because it has been reported to have a longer persistence and stability
than deltamethrin (FAO, 2010). Therefore, a similar experimental procedure was applied to
compare different types of bifenthrin-treated nets (n=9) and the persistence of bifenthrin in nets
exposed two months during the autumn season at the field site “La Poveda Experimental Farm”
Control of vectors and viruses by pyrethroid-treated nets
95
(Madrid, Spain) (n=4) were tested in glass tubes against A. gossypii using a cucumber leaf cv.
‘Ashley’ and B. tabaci with a tomato leaf cv. ‘Marmande’ as targets. Twenty A. gossypii and 20
B. tabaci individuals were tested in each vial for the bifenthrin assays.
Figure 7.1. Laboratory set-up, showing the two-chamber glass tube with the target leaf on top (left) and tubes wrapped with black fabric (right).
7.3.2. DETERMINATION OF THE INSECTICIDE CONCENTRATION OF LLITNs
Insecticide concentration was performed using an adaptation of the CIPAC method
454/LN/M/3.2 for alpha-cypermethrin by Gas Chromatography with Flame Ionisation Detection
(GC-FID) (CIPAC, 2009). Insecticide was determined by extraction in xylene (25 mL) of net
samples (200 mg) in a conical flask. The flask was connected to a condenser and heated to reflux
for 60 minutes until the net sample was completely dissolved. The solution was cooled to room
temperature, filtered through a nylon filter (0.2 µm) and analysed by GC-FID with a system
comprising an Agilent Technologies 7890A gas chromatograph, an Agilent Technologies 7693A
auto sampler and a capillary fused silica column (5% phenyl methyl siloxane 0.25 µm, 30 m x
0.25 mm) (Agilent Technologies Inc., Santa Clara, USA) with an injection volume of 1 µL and a
flow rate of 300 mL min-1. Insecticide content was calculated with an external standard
calibration.
96
7.3.3. EFFICACY OF LLITNs IN FIELD CONDITIONS AGAINST APHIDS AND
WHITEFLIES
Field trials were designed to test the incidence of pest population dynamics and plant viruses in
natural conditions. The two nets that provided the best results in the laboratory trials and could
be produced at a large scale were tested in two field experiments at “La Poveda Experimental
Farm” during autumn of 2011 and 2013. Three identical net-houses “tunnel type” (8 m long x
6.5 m width x 2.6 m high), separated five meters and with the same E-W orientation, soil
properties and irrigation regimes were used. Each net-house was divided in two equal
experimental plots separated with a vertical net following a split-plot design with three replicates
(Figure 7.2). Every plot held 42 cucumber cv. ‘Marumba’ plants, distributed in seven rows. An
experimental net section of 3.5 m long x 4.3 m wide was placed on each side of the tunnels
replacing the standard transparent polyester netting that covered the entire net-house. During
2011, the net-house contained a yellow test net of equal mesh (net TR11-290, 0.46 mm2 hole
size) either treated with 3.8 g bifenthrin kg net-1 or untreated. In 2013, according to the results of
the first field study and after a second set of laboratory trials, the new net 64/11/08 was tested.
The hole size was reduced to 0.29 mm2, either treated with 2.1 g bifenthrin kg net-1 or untreated,
with the aim of decreasing the numbers of living B. tabaci crossing the net. On the outer sides of
the net-houses, an additional row of six cucumber plants (two infected with CMV, two infected
with CABYV and two non-infected) was transplanted to provide inoculum sources at both sides
of the net-house. In 2011, two days after transplant, 240 winged A. gossypii and 500 B. tabaci
per plot were released on the virus-infected cucumber plants that were transplanted on the outer
side of the net-house. Aphids were directly released on the leaf surface to improve settlement
and viral acquisition. Whiteflies were released at the canopy level along the virus-infected source
plants. In 2013, 360 winged A. gossypii and 400 B. tabaci per experimental plot were released.
Aphids and whiteflies crossing the nets were assessed by counting their absence/presence in the
cucumber plants inside the experimental plots. Furthermore, aphid density in 11 marked
cucumber plants inside each experimental plot and in the virus source plants on the outer sides
was monitored weekly by using the following scale previously used in similar studies (Legarrea
et al., 2012a): [0 (0 aphids), 1 (1-4 aphids), 2 (5-19 aphids), 3 (20-49 aphids), 4 (50-149 aphids),
5 (>150 aphids)]. Leaf samples from each cucumber plant under experimental plots were
collected nine weeks after insect release and virus infection was confirmed by Double Antibody
Sandwich Enzyme-Linked ImmunoSorbent Assay (DAS-ELISA) using specific commercial
antibodies against CMV (Agdia Inc., Indiana, USA) and CABYV (Sediag S.A.S., Longvic,
Control of vectors and viruses by pyrethroid-treated nets
97
France) (Clark & Adams, 1977). Net samples were also collected at different time intervals
during each field experiment to measure remaining insecticide concentration after field exposure.
Daily average climatic conditions were 15.2 °C temperature, 61.2 % RH, 12.3 MJ m-2 day-1
radiation and 1.5 mm rainfall during 2011, and 16.1 °C temperature, 69.32 % RH, 13.3 MJ m-2
day-1 radiation and 1.0 mm rainfall during 2013.
Figure 7.2. Field experiment, showing the three net-houses “tunnel type” (left), and the interior of a net-house with the source plants on the outer side of the yellow LLITN (right).
7.3.4. EFFECT OF BIFENTHRIN-TREATED NETS ON THE APHID PARASITOID
Aphidius colemani
The impact of the TR11-290 net on A. colemani was tested in a separate and independent set of
three tunnels with the same dimensions previously described, and located in “La Poveda
Experimental Farm” during the autumn season. We followed a similar split-plot design with
three replicates, being each net-house divided in two equal experimental plots. Each plot
contained a section of either bifenthrin-treated or untreated net (0.46 mm2 hole size) on each side
of the tunnel. Each plot held 42 cucumber plants, distributed in seven rows. At a 2-true leaf
stage, three adults of A. gossypii were released on each of 11 marked plants inside each
experimental plot. As opposed to the field experiments where aphids and whiteflies were
released on the outer side of the net-house, the parasitoids (APHIcontrol® Agrobio, La
Mojonera, Spain) were released inside the net-house and hung on a platform placed in the centre
of each plot at a rate of 10 adults/m2 two weeks after aphid infestation. Insect sampling was
performed weekly for six weeks by scouting their absence/presence in all plants and by counting
their number in the 11 marked plants. Daily average climatic conditions were 15.7 °C
temperature, 70.3 % RH, 12.9 MJ m-2 day-1 radiation and 2.3 mm rainfall.
98
7.3.5. STATISTICAL METHODS
Differences among nets in the percentage of mortality and insects feeding on the target leaf in
the laboratory assays, and insect density in field experiments were assessed by a parametric one-
way ANOVA test followed by pairwise comparison for least significant differences (LSD) or a
Student t-test (p≤0.05). When the data did not follow the ANOVA assumptions, a non-
parametric Kruskal-Wallis H-test or Mann-Whitney U-test (p≤0.05) was performed. Insect
occupancy rate and virus incidence was compared by a Chi-square goodness of fit test (p≤0.05).
7.3.6. SPATIAL ANALYSIS
The spatial distribution of aphids and virus spread was studied using the Spatial Analysis by
Distance IndicEs (SADIE) methodology explained in section 3.8 (Perry, 1995) applied to the
data of year 2011. The spatial pattern of data was described by the index of aggregation, Ia
(Aggregated: Ia>1; Random: Ia=1; Regular: Ia<1), the positive patch cluster index, vi, and the
negative gap cluster index, vj (Perry et al., 1999). By convention, values <1.5 stand for patches
and values <-1.5 indicate a gap. Both indices visually indicate the location and extent of cluster
in the data so their values could be contoured with Surfer 9.0 software (Golden Software, 2009).
Moreover, the degree of local association between aphid presence and virus incidence was
calculated with the index of spatial association, X, and contoured as well (Perry & Nixon, 2002).
In this study, a value of 1 was assigned to plants infested by aphids or infected by virus, and a
value of 0 for uninfested or non-infected plants, for each of the 42 cucumber plants inside every
plot.
7.4. RESULTS
7.4.1. EFFICACY OF LLITNs AGAINST APHIDS IN LABORATORY TRIALS
All LLITNs impregnated with pyrethroids produced high mortality in M. persicae and thereby
reduced the chances that insects would reach the target. Almost half of the aphids reached the
target chamber in the untreated net 151 and differed significantly with deltamethrin tubes, with
fewer insects reaching the target under high concentration (Table 7.1). Addition of UV-blockers
to net 404 did not make a difference on the percentage of aphids that reached the target, although
it was significantly higher in the untreated net 151 compared to LLITNs 404, 405 and 406 (Table
7.1). No synergistic effect of the insecticides deltamethrin and PBO was detected. In contrast,
PBO alone caused significantly higher number of M. persicae on the leaf when compared to
deltamethrin and deltamethrin+PBO nets. The PBO-treated net 195 had lower values than the
Control of vectors and viruses by pyrethroid-treated nets
99
untreated net 151 (Table 7.1). The efficacy of deltamethrin-treated nets decreased over time with
sun exposure in two of the three nets tested, 404 and 412 (Table 7.1). In both cases, the spring-
exposed nets significantly increased the percentage of M. persicae in the target leaf, and even
after winter exposure for net 404. Net 406 was not affected by sun exposure (Table 7.1). Besides,
no significant differences in mortality were found between net colours white and yellow
(p=0.078).
All the bifenthrin LLITNs tested reduced A. gossypii presence on target. The five LLITNs tested
statistically differed in the number of aphids reaching the plant target, and the two most
promising nets had a hole size of 0.71 and 0.44 mm2 (Table 7.1). Numbers of aphids reaching
the leaf significantly differed among periods of field exposure. The unexposed and one-month
field-exposed nets demonstrated relatively good performance, with similar values for aphids
reaching the target. However, when the net was exposed for two months, the percentage of
aphids in the leaf increased up to values similar to those of vials with untreated nets (Table 7.1).
7.4.2. EFFICACY OF LLITNs AGAINST WHITEFLIES IN LABORATORY TRIALS
Over 80% of the whiteflies tested on deltamethrin-treated nets were alive in the target chamber
24 hours after release (Table 7.2). Bemisia tabaci mortality was found to be low in this
experiment, showing a minimum of 0.1±0.4% in net 206, increasing up to 17.1±4.6% in net 25.
However, significant differences in the percentage of whiteflies reaching the target were found
between LLITNs 1, 2 and 3, and untreated nets 1.4, 2.4 and 3.4 (Table 7.2). Among the treated
nets tested during this first laboratory survey, net 3 had the lowest value (Table 7.2). Sun
exposure of the bifenthrin-treated net used in the first field experiment caused a significant
reduction in efficiency against whiteflies under laboratory conditions from one month onwards
(Table 7.2).
100
Control of vectors and viruses by pyrethroid-treated nets
101
102
After these trials and according to the results obtained during the first field experiment, a second
set of laboratory assays were performed with two new LLITNs with a smaller hole size (nets
64/11/08 and 40). Figure 7.3 shows the relation between hole size and B. tabaci reaching the
target in a wide range of nets tested during several years. Pore sizes above 0.44 mm2 were not
sufficient to prevent living whiteflies feeding on the leaf. Net 40 was too dense (0.12 mm2 hole
size) and provided a physical control as no whiteflies were found on the leaf in the untreated
vials with standard net 42. However, the LLITN 64/11/08 with a 0.29 mm2 pore gave promising
results as only 6.77±2.46% whiteflies reached the target, and was therefore selected for the
second field study (Figure 7.3).
Figure 7.3. Mean percentage of Bemisia tabaci feeding on the target after crossing a range of bifenthrin-treated nets with different hole sizes (Net 40, 0.12 mm2 hole size; net 64/11/08, 0.29 mm2 hole size; net 3, 0.44 mm2 hole size; net TR11-290, 0.46 mm2 hole size; net 2, 0.60 mm2 hole size; net 1, 0.83 mm2 hole size).
7.4.3. EFFICACY OF LLITNs IN FIELD CONDITIONS AGAINST APHIDS AND
WHITEFLIES
The nets that gave best results in laboratory conditions every year and could be produced at large
scale were tested in field conditions. The nets used had a hole size of 0.46 and 0.29 mm2 in 2011
and 2013, respectively. In the first field study, the density of alate A. gossypii was significantly
lower in plots protected by the bifenthrin-treated net than in those covered by the untreated nets
from 27 days after aphid release onwards (U=443.0; Z=-2.3; p=0.02) (Figure 7.4.a). Numbers of
apterae and nymphs were also lower in plots protected with the bifenthrin-treated nets from 34
(U=390.5; Z=-2.7; p=0.07) and 20 days (U=448.0; Z=-2.2; p=0.03) onwards, respectively. Plots
protected by bifenthrin-treated nets had a significantly lower aphid occupancy rate from the
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Control of vectors and viruses by pyrethroid-treated nets
103
second sampling date onwards (X2=8.52; p<0.01). Furthermore, the incidence of CMV (X2=8.26;
p<0.01) and CABYV (X2=8.07; p<0.01) was significantly higher in untreated plots than in plots
covered by bifenthrin-treated nets (Figure 7.4.b). Mixed infections with both viruses were also
significantly lower in plots protected with treated-nets (X2=7.14; p<0.01), and just one of the
three plots had plants infected by both viruses whilst incidence of mixed infection in untreated
plots was 13.3±1.4%. However, net mesh was not dense enough to control whiteflies and
dispersal in plots protected with bifenthrin-treated nets was similar to that under the untreated
nets (data not shown). Bifenthrin concentration of the net exposed for two months in field
conditions during autumn decreased from 3.8 to 3.1 g kg net-1 (Table 7.1).
Figure 7.4. Mean±S.E. values of a) Aphis gossypii alate density (scale: 0-5) and b) CMV and CABYV virus transmission (%) inside the plots under bifenthrin-treated and untreated nets during the field experiment in 2011. Asterisks indicate statistical differences according to a) a one-way ANOVA test (p≤0.05) and b) a Chi-square goodness of fit test (p≤0.05).
According to the results of this first field study where whiteflies were not effectively excluded,
we conducted another field study in 2013 using a net with a smaller pore (0.29 mm2), which had
promising results under laboratory conditions (Figure 7.3). Results in 2013 followed the same
trend as in 2011, with a good control of aphid occupancy from 15 days after aphid release
onwards (X2=9.08; p<0.01) but similar whitefly occupancy in plots protected with the treated and
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104
untreated nets (X2=0.51; p=0.51). Aphids readily entered the control plots nine days after insect
release. However, the bifenthrin-treated net prevented aphid entry for three weeks. Virus
incidence was not as high as in 2011 but CABYV infection significantly increased inside the
untreated plots (X2=8.73; p<0.01). Bifenthrin concentration lowered to 1.3 instead of the initial
2.1 g kg net-1 at the beginning of the field experiment.
Due to higher virus transmission, year 2011 data was used to study the spatial distribution of
viruses CMV and CABYV, and its vector A. gossypii in plots with the bifenthrin-treated and
untreated nets. Spatial patterns of aphid presence in untreated plots revealed that aphids
colonized the entire area of the experimental plot in an aggregated distribution although this
aggregation was not significant (Figure 7.5). On the contrary, aphid dispersal was limited to the
borders next to insect release in bifenthrin-treated plots except for the third plot, in which aphid
distribution was more uniform. The spread of CMV followed either a random or a regular
distribution in the control plots, being significantly regular in the second net-house (Ia=0.81;
p=0.97), whereas it was aggregated in the plots protected by bifenthrin-treated nets, with
significant aggregation in the second net-house (Ia=1.78; p=0.00) (Figure 7.5). The combination
of aphid infestation and virus infection showed a significant association between A. gossypii and
CMV in the third untreated plot (X=0.35; p=0.03) (Figure 7.5). For CABYV, the contoured maps
of untreated plots showed virus significant patches restricted to the first two rows of plants that
aphids encountered after crossing the untreated net (Net-house 1: Ia=1.31; p=0.04. Net-house 2:
Ia=1.57; p=0.00) (Figure 7.6). In contrast, only few CABYV spots were found in bifenthrin-
treated plots and infection was not detected in the second plot. A significant dissociation
between the virus and its vector was recorded in untreated net-house 2 (X=-0.34; p=0.98), but a
significant aggregation in the border of the treated areas (Net-house 1: X=0.36; p=0.02. Net-
house 3: X=0.33; p=0.03) (Figure 7.6).
Control of vectors and viruses by pyrethroid-treated nets
105
A. gossypii C
on
tro
l 1
Co
ntr
ol 2
C
on
tro
l 3
Trea
ted
1
Trea
ted
2
Trea
ted
3
Ia =
0.8
6
P
a =
0.83
V
i = 0
.88
Pvi
= 0
.76
Vj =
-0.
83
Pvj
= 0
.90
Ia =
1.1
3
P
a =
0.29
V
i = 1
.14
Pvi
= 0
.20
Vj =
-0.
99
Pvj
=0.
71
Ia =
0.8
3
P
a =
0.88
V
i = 0
.82
Pvi
= 0
.69
Vj =
-0.
91
Pvj
= 0
.87
Ia =
1.0
9
P
a =
0.24
V
i = 1
.10
Pvi
= 0
.21
V
j = -
1.08
P
vj =
0.2
6
Ia =
0.9
6
P
a =
0.53
V
i = 0
.95
Pvi
= 0
.56
Vj =
-0.
96
Pvj
= 0
.54
Ia =
1.0
1
P
a =
0.40
V
i = 1
.01
Pvi
= 0
.39
Vj =
-1.
01
Pvj
= 0
.40
CMV
Ia =
0.9
3
P
a =
0.62
V
i = 0
.94
P
vi =
0.5
8 V
j = -
0.91
P
vj =
0.6
7
Ia =
0.8
1
P
a =
0.97
V
i = 0
.81
P
vi =
0.9
7 V
j = -
0.80
P
vj =
0.9
7
Ia =
0.9
5
P
a =
0.57
V
i = 0
.96
P
vi =
0.5
4 V
j = -
0.95
P
vj =
0.5
7
Ia =
1.2
1
P
a =
0.19
V
i = 1
.05
P
vi =
0.2
9 V
j = -
1.14
P
vj =
0.1
5
Ia =
1.7
8
P
a =
0.00
V
i = 1
.78
P
vi =
0.0
0 V
j = -
1.79
P
vj =
0.0
0
Ia =
1.1
5
P
a =
0.15
V
i = 1
.13
P
vi =
0.1
8 V
j = -
1.15
P
vj =
0.1
5
Association
X =
-0.
11
P
= 0
.68
X =
0.0
3
P
= 0
.44
X =
0.2
6
P
= 0
.09
X =
-0.
04
P
= 0
.57
X =
0.3
5
P
= 0
.03
X =
-0.
13
P
= 0
.79
Pat
ch
Gap
0
1 -1
-1.5
1.5
V
Ass
oci
atio
n
Dis
soci
atio
n
0 0.
025 0.
05
0.95 0.9
75
p
N
Figu
re 7
.5. C
lass
ed p
ost
map
s of
the
spa
tial
dist
ribu
tion
of A
phis
gos
sypi
i an
d C
MV
-inf
ecte
d pl
ants
, and
con
tour
ed m
ap o
f th
e as
soci
atio
n be
twee
n C
MV
-inf
ecte
d pl
ants
and
its
vec
tor,
A. g
ossy
pii d
urin
g th
e fi
eld
expe
rim
ent
in 2
011.
Spo
ts i
ndic
ate
indi
vidu
al t
est
plan
ts.
Smal
l
fille
d sp
ots
repr
esen
t clu
ster
ing
indi
ces
of 0
to ±
0.99
(cl
uste
ring
bel
ow e
xpec
tatio
n), u
nfill
ed s
pots
±1
to ±
1.49
(cl
uste
ring
slig
htly
exc
eeds
ex
pect
atio
n) a
nd la
rge
fille
d sp
ots
>1.
5 or
<1.
5 (m
ore
than
hal
f as
muc
h as
exp
ecta
tion)
. Red
line
s en
clos
ing
patc
h cl
uste
rs a
re c
onto
urs
of
v=1.
5 an
d bl
ue li
nes
are
of v
=–1
.5. B
lack
line
s ar
e ze
ro-v
alue
con
tour
s, r
epre
sent
ing
boun
dari
es b
etw
een
patc
h an
d ga
p re
gion
s. T
he in
dex
of a
ggre
gatio
n, I
a, t
he p
ositi
ve p
atch
clu
ster
ind
ex, v
i, th
e ne
gativ
e ga
p cl
uste
r in
dex,
vj,
and
the
inde
x of
spa
tial
asso
ciat
ion,
X, c
ircl
ed b
y co
lour
ed li
nes
are
stat
istic
ally
sig
nifi
cant
. Let
ter
N a
nd a
rrow
indi
cate
nor
th o
rien
tatio
n.
106
Figu
re 7
.6.
Cla
ssed
pos
t m
aps
of t
he s
patia
l di
stri
butio
n of
Aph
is g
ossy
pii
and
CA
BY
V-i
nfec
ted
plan
ts,
and
cont
oure
d m
ap o
f th
e as
soci
atio
n be
twee
n C
AB
YV
-inf
ecte
d pl
ants
and
its
vec
tor,
A. g
ossy
pii
duri
ng t
he f
ield
exp
erim
ent
in 2
011.
Sym
bols
and
con
tour
s ar
e as
fo
r Fi
gure
7.5
.
Association A. gossypii
Co
ntr
ol 1
C
on
tro
l 2
Co
ntr
ol 3
Tr
eate
d 1
Tr
eate
d 2
Tr
eate
d 3
Ia =
0.8
6
P
a =
0.83
V
i = 0
.88
Pvi
= 0
.76
Vj =
-0.
83
Pvj
= 0
.90
Ia =
1.1
3
P
a =
0.29
V
i = 1
.14
Pvi
= 0
.20
Vj =
-0.
99
Pvj
=0.
71
Ia =
0.8
3
P
a =
0.88
V
i = 0
.82
Pvi
= 0
.69
Vj =
-0.
91
Pvj
= 0
.87
Ia =
1.0
9
P
a =
0.24
V
i = 1
.10
Pvi
= 0
.21
V
j = -
1.08
P
vj =
0.2
6
Ia =
0.9
6
P
a =
0.53
V
i = 0
.95
Pvi
= 0
.56
Vj =
-0.
96
Pvj
= 0
.54
Ia =
1.0
1
P
a =
0.40
V
i = 1
.01
Pvi
= 0
.39
Vj =
-1.
01
Pvj
= 0
.40
CABYV
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Control of vectors and viruses by pyrethroid-treated nets
107
7.4.4. EFFECT OF BIFENTHRIN-TREATED NETS ON THE APHID PARASITOID
Aphidius colemani
Mummies appeared two weeks after parasitoid infestation (week 4) and occupancy rate of plants
remained constant throughout the crop cycle. Parasitism rate expressed as number of mummies
per A. gossypii individuals was the same in the two treatments during the three parasitoid
sampling dates (Week 4: Untreated 0.37±0.03 vs. bifenthrin-treated 0.30±0.14; t=0.43; p=0.69.
Week 5: Untreated 0.91±0.39 vs. bifenthrin-treated 0.65±0.46; t=0.43; p=0.69. Week 6:
Untreated 0.92±0.62 vs. bifenthrin-treated 0.99±0.88; t=0.09; p=0.94). Average numbers of
mummies were statistically similar in untreated (28.33±8.28) and bifenhrin-treated (43.43±8.07)
nets (t=-1.30; p=0.26).
7.5. DISCUSSION
The results obtained in this study suggest that LLITNs can be considered as a promising
approach for reducing aphid immigration into protected crops while allowing suitable airflow in
enclosed environments (Bethke & Paine, 1991; Muñoz et al., 1999). In addition, these nets
produced no harmful effects to A. colemani, an aphid parasitoid frequently used as biocontrol
agent in greenhouse production. To our knowledge, this is the first report of the impact of a
LLITN on a natural enemy. The size of the net hole was big enough to allow proper ventilation,
and at the same time pests that passed through were likely to acquire sufficient pesticide so that
the number of living insect pests entering the greenhouse was strongly reduced. In laboratory
trials, M. persicae and A. gossypii access was reduced below 20% in all of the LLITNs tested
even when a low insecticide dosage (2.0 g kg net-1) and large hole size (0.83 mm2) were used.
No differences were observed among the nets studied when they were treated with different
doses of deltamethrin. Approximately half of the released aphids were able to reach the target
and feed on the leaf when an untreated net of the same mesh as the insecticide-treated net was
used as a barrier. Therefore, the incorporation of insecticide to the yarns acted as a chemical
barrier against aphids and provided additional benefits to the physical exclusion properties of the
net (Díaz et al., 2004; Martin et al., 2006; Licciardi et al., 2008; Martin et al., 2010). The
addition of UV-absorbing additives to the yarn was not an obstacle to the efficacy of the net
because mortality was not different when comparing with a standard LLITN. In contrast, when
PBO alone was used to treat the net, it did not cause significant mortality in aphids and failed to
increase efficacy when combined with deltamethrin. Within the experimental design used in this
study, the insecticide synergist PBO was not shown to enhance efficacy when combined with
108
pyrethroids in LLITNs although further investigations are warranted. Lastly, net colours yellow
and white had no different effect on aphid mortality.
The results also indicate no effect of deltamethrin degradation in one of the nets exposed in the
field. However, the other two LLITNs tested lost efficacy against M. persicae when exposed to
spring conditions, possibly due to higher temperature and radiation than during winter, although
one of them also lost efficacy after winter exposure compared to the unexposed control.
Bifenthrin concentration decreased to 3.1 g kg net-1 instead of the initial 3.8 g kg net-1 after being
exposed for two months in the field, which strongly reduced the efficacy of the nets against A.
gossypii and B. tabaci under laboratory conditions. The persistence of bifenthrin appeared to be
jeopardized from one month onwards, suggesting that nets partially lose efficacy after sun
exposure. However, the amount of bifenthrin left in the nets was enough to reduce aphid
dispersal and virus spread inside treated net-houses during one growing season. This may be due
to the lower insect density during the first weeks of the experiment as the first vectors that
crossed the treated net got impregnated and died before reaching the cucumber crop. Future
work should focus on testing further UV-blocking additives and formulations to maintain the
efficacy of these nets over a period longer than a single crop-growing period.
In the field trials, A. gossypii density was significantly reduced in cucumber plants protected by
completely closed bifenthrin-treated net-houses, so it is likely that these results could be
extended to other aphid species (Díaz et al., 2004; Martin et al., 2006; Licciardi et al., 2008).
Because pyrethroids produce a rapid knockdown effect, the application of LLITNs at field scale
may reduce the spread of plant viruses transmitted by aphids, such as CMV and CABYV. In
particular, bifenthrin has a slower knockdown effect but better chemical stability when compared
to some other pyrethroids, an aspect that needs to be taken into account when developing new
LLITNs (Hougard et al., 2002; CIPAC, 2009). As shown in the spatial analysis of the field
experiment, virus incidence of both viruses and mixed infections significantly decreased under
the insecticide-treated net. Different patterns for CMV and CABYV spread inside control plots
were also found using SADIE. CMV spread had a either regular or random distribution in
untreated plots, a result that matches the typical spread of non-persistent viruses, as opposed to
the aggregation found under plots protected by LLITNs (Fereres & Moreno, 2009). Besides, the
dispersal of A. gossypii was greater inside untreated plots. On the contrary, we found significant
CABYV aggregation in the borders of untreated plots, which suggests an initial focus that led to
infection in adjacent plants (Irwin & Thresh, 1990). CABYV spread and aphid density was very
limited in bifenthrin-treated plots, which may indicate again a low dispersal rate of both agents
Control of vectors and viruses by pyrethroid-treated nets
109
under LLITNs. In addition, aphid population was associated to CABYV infected-plants in
treated plots, as frequently observed in viruses transmitted in a persistent circulative manner
(Irwin & Thresh, 1990).
Protection against aphids would not be enough if untreated nets were placed as a physical barrier
alone in vent openings. A possible solution to this problem would be to reduce the size of the
hole to 0.34-0.40 mm2 (Bethke & Paine, 1991; Weintraub & Berlinger, 2004). One of the
drawbacks of this approach could be the insufficient ventilation inside the enclosed structure
(Muñoz et al., 1999). Results suggest that the use of LLITNs either with deltamethrin or
bifenthrin might allow adequate ventilation of greenhouses. LLITNs placed in the sides of
greenhouses or in window openings would decrease aphid entry, thereby reducing the need for
pesticide treatments and increasing the safety of growers and environment (Hougard et al.,
2002). The use of LLITNs could also help to maintain crop sanitation in regions where vegetable
crops are produced using biological control and IPM programs, as this kind of nets can in some
circumstances at least, be used in a way that is compatible with beneficial insects such as A.
colemani, an important biocontrol agent. This biocontrol production scheme has reduced the
number of insecticide applications when compared to conventional production, but it has also
increased the significance of aphids as pests that cause direct damage to plants in southeastern
Spain (Van der Blom et al., 2009). In this sense, LLITNs could reduce the risk of aphid
infestation. In addition, owing to the increasing importance of biocontrol in greenhouses, further
studies would be necessary to assess the compatibility of both strategies (Willes & Jepson,
1994).
Notwithstanding, the hole size used was sufficiently large to allow the passage of small insects
such as whiteflies. No significant mortality was observed in most nets tested when B. tabaci was
evaluated, although nets treated with bifenthrin appeared to exclude whiteflies better than the
ones treated with deltamethrin. It was necessary to use a 0.60 mm2 hole size and 5.0 g bifenthrin
kg net-1 to reasonably block the access of living whiteflies through the net under laboratory
conditions. Bemisia tabaci is renowned worldwide as an intractable pest that is difficult to
control and one that develops pesticide resistance rapidly (Horowitz et al., 2009). Resistance of
this whitefly to pyrethroids is well known, and this species is registered in the Arthropod
Resistance Pesticide Database (Whalon et al., 2008). The small size of B. tabaci could explain
such unsuccessful results as the body length and width of the whitefly, 0.8-0.95 mm long and 0.5
mm wide, is much smaller than that of aphids (Byrne & Bellows, 1991). It is likely that when it
crossed the LLITNs, B. tabaci was not sufficiently impregnated with the pesticide to suffer from
110
its knockdown effect. The findings of our field experiments also showed that B. tabaci was able
to successfully cross the bifenthrin-treated nets and disperse over the cucumber plots at a similar
rate than when a non-treated net was used as a physical barrier. Results also agree with the
findings on A. proletella survival in cabbage field experiments using deltamethrin-impregnated
nets as a fence (Díaz et al., 2004). Recently, promising results have been obtained with the
pyrethroid alpha-cypermethrin against whiteflies (Martin et al., 2014). The size of the mesh and
the chemical compound appear to be major factors in the production of effective LLITNs, and
dicofol-impregnated nets controlled injurious mites in eggplants even though their size was
smaller than that of whiteflies (Martin et al., 2010). Further experiments should continue to
select the most appropriate mesh size and insecticide to effectively exclude B. tabaci but the
most recent finding suggests that a pore size of 0.29 mm2 might be the best compromise to
control this species.
When properly designed, LLITNs represent a good strategy that combines chemical and physical
control techniques to allow sustainable management and reduce pesticide treatments in crops.
Moreover, LLITNs could be implemented in conjunction with the release of commercially
available natural enemies. In this study, using laboratory and net-house experiments, LLITNs
have reduced aphid populations as well as decreased the spread of plant viruses. Different mesh
sizes and other insecticides should be further assessed for the effective control of B. tabaci. It
would be interesting to test this strategy at field scale again under commercial greenhouses to
develop an alternative tool for IPM programs.
111
CHAPTER 8. GENERAL DISCUSSION
The implementation of new pest control alternatives to chemical tactics, such as the introduction
of natural enemies or physical barriers, has lead to a more sustainable approach towards the
control of insect vectors of plant diseases (Jacobson, 2004; Ramakers, 2004; Van der Blom et
al., 2010). In the present work, the impact of an aphid parasitoid on pest dispersal and virus
spread has been evaluated to understand its role in biocontrol programs of horticultural crops
under greenhouse conditions. Furthermore, two types of new physico-chemical barriers, UV-
absorbing and insecticide-impregnated covers, were studied under laboratory, greenhouse and
field conditions.
The first study aimed to investigate the dispersal of an aphid species, Aphis gossypii Glover, and
the spread of associated viruses Cucumber mosaic virus (CMV, Cucumovirus) and Cucurbit
aphid-borne yellows virus (CABYV, Polerovirus) in a cucumber crop under the presence of the
parasitoid Aphidius colemani Viereck. SADIE methodology was used to study the spatial
distribution of the aphid and the viruses CMV and CABYV, as well as their degree of
association (Chapter 4). Besides, the ability of pests Myzus persicae (Sulzer), Bemisia tabaci
(Gennadius) and Tuta absoluta (Meyrick), and natural enemies A. colemani and Sphaerophoria
rueppellii (Weidemann) to fly under UV-deficient environments and locate targets placed at
different distances was analysed (Chapter 5). Plant-mediated effects influencing insect
performance as a response to UV-A exposure were investigated with the following host-pest
complexes: pepper and M. persicae, and eggplant and B. tabaci. Also, the impact of UV-A on
plant development and leaf chemistry was studied (Chapter 6). Finally, the efficacy of Long
Lasting Insecticide Treated Nets (LLITNs) was tested against A. gossypii and B. tabaci. Two
nets were selected and evaluated in the field under high aphid and whitefly infestation pressure
to assess vector colonization and the spread of aphid-transmitted viruses (CMV and CABYV) in
a cucumber crop. Again, the spatial patterns of both agents were studied with SADIE.
Furthermore, the compatibility of LLITNs with the release of aphid parasitoids in field tunnels
was also studied (Chapter 7).
The aphid parasitoid A. colemani promoted early movement of the vector A. gossypii, and
increased the colonization of plants adjacent to the virus source (Chapter 4). As a consequence,
the spread of CMV also increased in the short term because of its mode of transmission -non-
persistent manner- (Roitberg & Myers, 1978; Weber et al., 1996; Fereres & Moreno, 2009;
112
Belliure et al., 2011; Hodge et al., 2011). Indeed, the emission of alarm signals by aphids causes
conspecifics to disperse, and this escaping behaviour may enhance virus spread (Losey &
Denno, 1998; Day et al., 2006; Jeger et al., 2011). However, no differences were found in the
long term, which suggest potential benefits for disease control (Jeger et al., 2011). Aphis gossypii
showed a spatial ditribution pattern of a colonizing aphid species (Blackman & Eastop, 2000).
Parasitoids promoted the distribution of CMV around the entire arena and its aggregation in
several patches, in contrast with the few aggregated spots indicating isolated infections under the
control arenas. Vector and virus were significantly associated when the parasitoid was absent,
whilst A. colemani induced dissociation between the virus and the vector, highlighting again the
strong effect of natural enemies in the early dispersal of aphids. Whereas previous studies have
described enhanced spread of persistent viruses in the presence of natural enemies (Bailey et al.,
1995; Hodge & Powell, 2008a), this study suggest that A. colemani significantly limited the
incidence and spread of CABYV in the long term, in a similar way as reported earlier for another
Luteoviridae, Barley yellow dwarf virus (BYDV) (Smyrnioudis et al., 2001). One possible
explanation might be that mummification diminished aphid movement and the duration of aphids
as active vectors (Calvo & Fereres, 2011). There were major clustered areas of either patches or
gaps of infected plants in the short term in both treatments, which are frequently observed also in
other persistent viruses such as BYDV (Irwin & Thresh, 1990; Smyrnioudis et al., 2001).
CABYV expanded throughout the entire arena in the absence of A. colemani, whilst parasitoids
significantly limited virus incidence and spread. This study shows that the reduction of herbivore
damage in the long term may offset the initial risk of potential virus spread when natural enemies
first encounter their hosts.
UV-absorbing covers have a double mode of action, first diverting insects away from the
greenhouse walls, and second altering insect behaviour inside the protected environments (Raviv
& Antignus, 2004; Antignus, 2012). The use of these barriers has been satisfactorily
implemented in various crops against pests and virus diseases in the past (Antignus et al., 1998;
Chyzik et al., 2003; Díaz et al., 2006; Weintraub, 2009; Ben-Yakir et al., 2012; Legarrea et al.,
2012a, b; Antignus, 2014). The ability of aphids and whiteflies to reach the targets was
diminished under UV-absorbing barriers, suggesting a reduction of vector activity under this
type of nets (Chyzik et al., 2003; Raviv & Antignus, 2004; Döring & Chittka, 2007). In the
present study (Chapter 5), fewer aphids reached distant traps under UV-absorbing nets, and
significantly more aphids could fly to the end of the tunnels covered with
General discussion
113
non-UV blocking materials. These findings agree with previous studies on how UV-absorbing
screens reduce movement and dispersal of aphid populations (Díaz et al., 2006; Ben-Yakir et al.,
2012; Legarrea et al., 2012a). Whitefly flight activity was different and, unlike aphids,
differences in B. tabaci captures were mainly found in the closest targets. Whiteflies flew shorter
distances in the absence of UV radiation whereas no differences among targets were found under
UV-transparent nets. Lower densities of several whitefly species have been previously found
under UV-deficient screens (Costa & Robb, 1999; Antignus et al., 2001; Mutwiwa et al., 2005;
Legarrea et al., 2012b). The oviposition of lepidopteran T. absoluta was also negatively affected
by the most UV-blocking cover compared to other nets but no differences were found among
targets within cages, implying that olfactory signals may mediate orientation much more than
visual stimuli (Proffit et al., 2011). The photoselective barriers were compatible with A.
colemani parasitism and S. rueppellii oviposition of biocontrol agents (Chyzik et al., 2003; Chiel
et al., 2006; Doukas & Payne, 2007a, b), since natural enemies orient towards the plant-host
complex by olfactory cues such as honeydew secretions, alarm pheromones or plant defence
volatiles (Du et al., 1998; Storeck et al., 2000; Boivin et al., 2012).
As mentioned before, not only does UV radiation directly influence insects but also indirectly
via plant-meediated changes (Vänninen et al., 2010; Johansen et al., 2011). UV-A exposure
caused different responses in the two host-pest complexes studied, pepper and aphid, and
eggplant and whitefly (Chapter 6). Peppers responded directly to UV-A by producing shorter
stems (Kuhlmann & Müller, 2010; Comont et al., 2012). UV-A did not affect the leaf area of
either species. Pepper phenolics accumulated with UV-A exposure (Gaberšcik et al., 2002;
Izaguirre et al., 2007; Mahdavian et al., 2008; Kulhmann & Müller, 2009a, 2009b, 2010).
Results suggested a readiness to induce UV-screening compounds (Middleton & Teramura,
1993; Harborne & Williams, 2000). Sugar, free amino acid and protein levels were also higher in
UV-A-treated peppers (Roberts & Paul, 2006; González et al., 2009; Comont et al., 2012). The
potentially worse quality of leaf tissue without UV-A indirectly diminished aphid performance,
which had lower fecundity and higher growth rates (Antignus et al., 1996; Chyzik et al., 2003;
Díaz et al., 2006; Kuhlmann & Müller, 2009a; Paul et al., 2011; Legarrea et al., 2012a). Indeed,
amino acids and soluble sugars are essential dietary components for M. persicae growth (Dadd
& Krieger, 1968; Mittler et al., 1970; Srivastava & Auclair, 1971; Weibull, 1987). For eggplants,
chlorophyll and carotenoid levels decreased with supplemental UV-A (Smith et al., 2000;
Gaberšcik et al., 2002). Rest of compounds were not affected, which may indicate a high
tolerance to UV irradiance in this species (González et al., 2009). Whiteflies grew slower when
114
exposed to UV-A regardless of the regime where eggplants had been previously grown.
Exposure to supplemental UV-A had a detrimental effect on whitefly development, fecundity
and fertility presumably not mediated by plant cues, as compounds implied in pest nutrition were
unaltered.
Novel impregnated nets with different hole sizes were tested in laboratory conditions against A.
gossypii and B. tabaci (Chapter 7). All LLITNs produced high mortality of aphids, proving
additional benefits to the physical exclusion properties of these nets (Díaz et al., 2004; Martin et
al., 2006, 2007, 2010). The best bifenthrin nets for excluding aphids had a hole size of 0.71 and
0.44 mm2. However, a maximum hole size of 0.29 mm2 was necessary to exclude B. tabaci
under laboratory conditions. Bifenthrin concentration decreased over time with sun exposure,
which strongly reduced the efficacy of the nets under laboratory conditions. However, the
remaining insecticide was enough to effectively block the invasion of aphids during the first
weeks of the field experiments, as vectors that first crossed the treated net got impregnated and
died before reaching the cucumber crop. Furthermore, nets tested in the field (hole sizes 0.46 and
0.29 mm2) reduced the incidence of CMV and CABYV, and it is likely that these results could
be extended to other aphid species (Díaz et al., 2004; Martin et al., 2006; Licciardi et al., 2008).
However, both nets failed for whiteflies, probably due to their small body size and their
resistance to pyrethroids (Byrne & Bellows, 1991; Whalon et al., 2008). Recently, promising
results against whiteflies in laboratory conditions have been obtained with a pyrethroid alpha-
cypermethrin net of 0.9 mm diameter, a pore that is larger than the ones used in the field
experiments (Martin et al., 2014).
CMV spread had a either regular or random distribution in untreated plots, a result that matches
the typical spread of non-persistent viruses (Jones, 2005; Jones et al., 2008), as opposed to the
aggregation found under plots protected by LLITNs. Besides, the dispersal of A. gossypii was
greater inside untreated plots. On the contrary, CABYV aggregation in the borders of untreated
plots suggests an initial focus that led to infection in adjacent plants (Irwin & Thersh, 1990). In
addition, aphid population was associated to CABYV infected-plants in treated plots, as
frequently observed in viruses transmitted in a persistent circulative manner (Irwin & Thersh,
1990). As a whole, LLITNs can be considered as a promising approach for reducing aphid
immigration and virus spread in protected crops while allowing suitable airflow in enclosed
environments (Bethke & Paine, 1991; Muñóz et al., 1999).
General discussion
115
Generally, the best approach to control insect pests should be to integrate different tactics, such
as some of the ones studied in this Thesis. On the other hand, it has to be acknowledged that the
implementation of several strategies at the same time may have unexpected or undesirable
consequences. The interference of these posible effects needs to be investigated to learn if a
combination of several strategies strengthen pest and virus control or not.
This way, and according to the results obtained, now it is posible to say that if biological control
is combined with UV-blocking materials or LLITNs (Chapters 4, 5 and 7), the presumable effect
on biocontrol agents would not be negative, given that their activity is compatible with the
physico-chemical tactics tested. A reduction in CMV and Lettuce Mosaic Virus (LMV,
Potyvirus) spread has been reported under photoselective nets in the field due to to the lower
dispersal of vectors (Legarrea et al., 2012a). This work recommended UV-absorbing nets to
reduce secondary spread of viruses, although not as a measure on its own. That is the reason why
the addition of natural enemies to the complex may help to achieve better results. However, we
need to pay attention to the consequences on virus spread in the long term, especially with non-
persistent viruses, because the mode of transmission plays an important role.
The spatial distribution of CMV primary infection was regular in the short term in the absence of
parasitoids, as seen in Chapter 4. When CMV spreads from primary sources, secondary infection
was concentrated in one patch in the long term. These results are consistent with the polycyclic
nature of non-persistent viruses (Thresh, 1983). These viruses are primarily transmitted by non-
colonizing vectors that acquire the viral particles during brief probes, resulting in localised
spread around initial infection foci (Thresh, 1983; Jones, 2005). Then, the infected plants
provide infection sources for further cycles of acquisition and transmission, so initial scattered
foci expand and coalesce into large clusters (Jones, 2005). Although the aphid studied was a
colonizing vector, this type of vectors has been also described as responsible for compacted
secondary spread of CMV once they settle on the crop (Alonso-Prados et al., 2003).
Jones et al. (2008) observed that the amount of spread increased proportionally with the number
of primary foci present, and that proximity to infection source had a marked effect. In this thesis,
the inoculum sources were placed in two positions: inside and outside the crop (Chapters 4 and
7). The position of the virus source may explain the different spatial patterns obtained. In the
field experiment, where the infected sources were placed outside the crop, a high number of
small foci were present in a regular or random distribution in the control plots (Chapter 7). This
116
distribution has been correlated with the arrival of viruliferous aphids when inoculum is outside
the crop (Alonso-Prados et al., 2003).
CMV spread was promoted in the short term when parasitoids were present, however results
suggest promising outcomes because parasitoids lowered vector populations and there were no
differences in CMV transmission between treatments in the long term, as seen in Chapter 4.
CABYV spread was controlled well just 14 days after parasitoid release. It is likely that if we
had developed the non-persistent system for a few more weeks, the action of natural enemies
would have positively overcome the risk of CMV transmission, in a similar way as observed for
the persistent virus CABYV.
Patterns of CABYV spread were aggregated in the short term no matter parasitoid introduction
(Chapter 4). This trend was repeated in Chapter 7 in control plots, whereas transmission in
treated plots was very low or unexistent, plus associated to vectors. Viruses transmitted in a
circulative manner have a very specific relationship with vectors, which are colonizing pests
(Fereres & Moreno, 2009). Also, CABYV infection spread fast in the absence of biological
control in the long term as shown in Chapter 4, suggesting that both strategies, natural enemies
and LLITNs, are solid options to prevent transmission and spread of persistent viruses.
Another way to integrate different strategies studied in this thesis would be to develop an UV-
blocking net impregnated with pesticides all together (Objectives 5, 6 and 7). The first expected
outcome regarding aphids would be less vector pressure inside the protected crop due to two
factors: the insecticide and the higher UV reflectance reflected from the cover would prevent
aphids to enter the protected environment (Martin et al., 2007, 2010; Antignus, 2012). Besides,
because of the lower UV transmittance, flight behaviour of the aphids that get into the crop
would be disrupted (Ben-Yakir et al., 2012) and fitness would be negatively affected, resulting
in a more powerful solution that will limit aphid spread. With regard to whiteflies, it would be
needed a net with smaller hole size to physically avoid whiteflies or impregnated in other more
effective active ingredients that have been successfully tested recently (Martin et al., 2014).
Once this becomes reality, the UV-blocking properties of the net would reduce the flight activity
of the fewer whiteflies that enter the greenhouse (Antignus et al., 2001; Legarrea et al., 2012b).
In any case, although the combination of tactics may be plausible and it seems the reasonable
way to act, the economic aspect could be the limiting factor. The cost of implementation and the
durability will determine the cost-efficiency of the treatment. Biological control has proven to be
cost-effective in many enclosed horticultural systems tested. Natural enemies settle on the crop
General discussion
117
and reach an ecological balance within the system although periodic releases of new individuals
may be needed (Jacas & Urbaneja, 2008; Van Lenteren & Bueno, 2014). On the contrary, the
production of photoselective materials or LLITNs is still a developing market. Their production
can be expensive and the active ingredients may degrade with time so we would have to replace
them. Another potential problem is the disposal of LLITNs that might requiere additional
legislation as they may be considered as an insecticide residue. One of the first commercial
applications of LLITNs has been in cabbage production, where is cost-effective (Martin et al.,
2006). Also, some companies are selling such type of LLITNs for controlling forest products
against bark beetle pests (Storanet®, BASF SE, Ludwigshafen, Germany).
Overall, observations on the activity of parasitoids have proven the importance of taking
beneficials into account when in the need to control insect vectors and subsequent spread of
plant viruses. Novel physico-chemical barriers, such as photoselective and insecticide-
impregnated nets, may be useful against pests and plant viruses, as well as compatible with
biocontol agents. These strategies need to be further exploited as alternatives to traditional use of
chemicals in Integrated Pest Management programs of vegetable crops grown under protected
environments.
118
119
CONCLUSIONS
Specific conclusions:
1. The aphid parasitoid Aphidius colemani Viereck promoted early dispersal of vector Aphis gossypii Glover and significant spread of Cucumber mosaic virus (CMV, Cucumovirus). The initial risk of non-persistent virus spread was offset by the reduction of herbivore damage in the long term.
2. There was a significant reduction of Cucurbit aphid-borne yellows virus (CABYV, Polerovirus) infection in the presence of A. colemani in the long term, proving the beneficial role of natural enemies against the spread of persistent viruses.
3. The flight activity of the aphid Myzus persicae (Sulzer), the whitefly Bemisia tabaci (Gennadius) and the lepidopteran Tuta absoluta (Meyrick) was diminished under UV-absorbing barriers, suggesting an alteration of their flight behaviour in different ways. These barriers were compatible with the biocontrol agents Aphidius colemani and Sphaerophoria rueppellii (Weidemann).
4. Pepper plants grew shorter under supplemental UV-A radiation. Pepper phenolics, soluble sugars, free amino acid and protein levels increased in UV-A-treated peppers. Eggplant photosynthetic pigments decreased with supplemental UV-A.
5. Myzus persicae fecundity and growth rates were higher in peppers grown under supplemental UV-A radiation. UV-A had a plant-mediated impact on aphid performance as M. persicae benefited from increased amino acid and sucrose content with supplemental UV-A.
6. A detrimental effect of supplemental UV-A radiation on Bemisia tabaci pre-reproductive period, fecundity and fertility was observed. Chemical compounds involved in whitefly nutrition were not altered, which suggests that insect response was not mediated by plant cues.
7. LLITNs avoided the entry and produced high mortality of Myzus persicae and Aphis gossypii under laboratory conditions but a much smaller hole size (0.29 mm2) was needed to exclude Bemisia tabaci. LLITNs tested in the field significantly reduced aphid dispersal, and CMV and CABYV spread, but failed to exclude whiteflies. The aphid parasitoid Aphidius colemani was compatible with LLITNs.
120
General conclusion:
The integration of physico-chemical tactics, such as photoselective materials and long lasting
insecticide-treated nets, with biological control is a plausible solution and the optimal approach
to control insect pests and plant viruses under Integrated Pest Management programs of
vegetable crops with no harmful effects on natural enemies.
121
REFERENCES
Abacus Concepts. 1989. SuperANOVA. Abacus Concepts Inc., Berkeley, CA, USA.
Abacus Concepts. 1992. Statview. Abacus Concepts Inc., Berkeley, CA, USA.
Abbott W.S. 1925. A method of computing the effectiveness of an insecticide. Journal of
Economic Entomology, 18: 265-267.
Alonso-Prados J.L., Luis-Arteaga M., Álvarez J.M., Moriones E., Batlle A., Laviña A., García-
Arenal F., Fraile A. 2003. Epidemics of aphid-transmitted viruses in melon crops in Spain.
European Journal of Plant Pathology, 109: 129-138.
Álvarez A.J., Oliva R.M. 2014. Control de la mosca blanca (Bemisia tabaci) mediante el empleo
de barrera físicas. Phytoma, 264: 24-28.
Amorós-Jiménez R., Pineda A., Fereres A., Marcos-García M.Á. 2012. Prey availability and
abiotic requirements of immature stages of the aphid predator Sphaerophoria rueppellii.
Biological Control, 63: 17-24.
Antignus Y. 2012. Control methods of virus diseases in the Mediterranean basin. Advances in
Virus Research, 84: 533-553.
Antignus Y. 2014. Management of air-borne viruses by “optical barriers” in protected agriculture
and open-field crops. Advances in Virus Research, 90: 1-33.
Antignus Y., Lapidot M., Hadar D., Messika Y., Cohen S. 1998. UV absorbing screens serve as
optical barriers to protect vegetable crops from virus diseases and insect pests. Journal of
Economic Entomology, 91: 1401-1405.
Antignus Y., Mor N., Joseph R.B., Lapidot M., Cohen S. 1996. Ultraviolet-absorbing plastic
sheets protect crops from insect pests and from virus diseases vectored by insects.
Environmental Entomology, 25: 919-924.
Antignus Y., Nestel D., Cohen S., Lapidot M. 2001. Ultraviolet-deficient greenhouse
environment affects whitefly attraction and flight behaviour. Environmental Entomology,
30: 394-399.
122
Arranz-Arranz D. 2008. El pimiento: producción de planta y cultivo. In: MARM (Ed.) Pimiento:
Cultivo y comercialización. Situación actual y perspectivas desde el punto de vista técnico
y comercial. Centro de publicaciones MARM, Madrid, España, pp. 47-62.
Bailey S.M., Irwin M.E., Kampmeier G.E., Eastman C.E., Hewings A.D. 1995. Physical and
biological perturbations: Their effect on the movement of apterous Rhopalosiphum padi
(Homoptera: Aphididae) and localized spread of Barley yellow dwarf virus. Environmental
Entomology, 24: 24-33.
Ballaré C., Scopel A.L., Stapleton A.E., Yanovsky M. 1996. Solar ultraviolet-B radiation affects
seedling emergence, DNA integrity, plant morphology, growth rate, and attractiveness to
herbivore insects in Datura ferox. Plant Physiology, 112: 161-170.
Barro P.J.D., Liu S.S., Boykin L.M., Dinsdale A.B. 2011. Bemisia tabaci: A statement of species
status. Annual Review of Entomology, 56: 1-19.
Barsig M., Malz R. 2000. Fine structure, carbohydrates and photosynthetic pigments of sugar
maize leaves under UV-B radiation. Environmental and Experimental Botany, 43: 121-
130.
Belliure B., Amorós-Jiménez R., Fereres A., Marcos-García M.A. 2011. Antipredator behaviour
of Myzus persicae affects transmission efficiency of Broad bean wilt virus 1. Virus
Research, 159: 206-214.
Beltrán F.D., Parra A., Roldan A., Soler A., Vila E. 2010. Pasado, presente y future del control
integrado de plagas en la provincial de Almería. Cuadernos de Estudios Agroalimentarios,
1: 27-43.
Ben-Yakir D., Antignus Y., Shahak Y. 2012. Colored shading nets impede insect invasion and
decrease the incidences of insect-transmitted viral diseases in vegetable crops.
Entomologia Experimentalis et Applicata, 144: 249-257.
Berlinger M.J., Taylor R.A.J., Lebiush-Mordechi S., Shalhevet S., Spharim I. 2002. Efficiency
of insect exclusion screens for preventing whitefly transmission of Tomato yellow leaf curl
virus of tomatoes in Israel. Bulletin of Entomological Research, 92: 367-373.
Bethke J.A., Paine T.D. 1991. Screen hole size and barriers for exclusion of insect pests of
glasshouse crops. Journal of Entomological Science, 26: 169-177.
123
Blackman R.L., Eastop V.F. 2000. Aphids on the World Crops: An Identification and
Information Guide, 2nd ed. Wiley, New York, USA.
Blackman R.L., Eastop V.F. 2007. Taxonomic issues. In: van Emden H.F., Harrington R. (Eds.)
Aphids as crop pests. Cabi, Wallingford, UK, pp. 1-29.
Blanc S., Drucker M., Uzest M. 2014. Localizing viruses in their insect vectors. Annual Review
of Phytopathology, 52: 403-425.
BOE. 2002. Ley 43/2002, de 20 de noviembre, de Sanidad Vegetal. Boletín Oficial del Estado
(21-11-2002), 279: 40970-40988.
BOE. 2004. Real Decreto 1938/2004, de 27 de septiembre, por el que se establece el Programa
nacional de control de los insectos vectores de los virus de los cultivos hortícolas. Boletín
Oficial del Estado (7-10-2004), 242: 33766-33768.
Boivin G., Hance T., Brodeur J. 2012. Aphid parasitoids in biological control. Canadian Journal
of Plant Science, 92: 1-12.
Bosque-Pérez N.A., Eigenbrode S.D. 2011. The influence of virus-induced changes in plants on
aphid vectors: Insights from luteoviruses pathosystems. Virus Research, 159: 201-205.
Bragard C., Caciagli P., Lemaire O., López-Moya J.J., MacFarlane S., Peters D., Susi P.,
Torrance L. 2013. Status and prospects of plant virus control through interference with
vector transmission. Annual Review of Phytopathology, 51: 177-201.
Briscoe A., Chittka L. 2001. The evolution of color vision in insects. Annual Review of
Entomology, 46: 471-510.
Byrne D.N., Bellows T.S. 1991. Whitefly biology. Annual Review of Entomology, 36: 431-457.
Calvo D., Fereres A. 2011. The performance of an aphid parasitoid is negatively affected by the
presence of a circulative plant virus. BioControl, 56: 747-757.
Carbonell-Bejerano P., Diago M.P., Martínez-Abaigar J., Martínez-Zapater J.M., Núñez-Olivera
E. 2014. Solar ultraviolet radiation is necessary to enhance grapevine fruit ripening
transcriptional and phenolic responses. BMC Plant Biology, 14: 183-198.
124
Carmo-Sousa M., Moreno A., Garzo E., Fereres A. 2014. A non-persistently transmitted-virus
induces a pull-push strategy in its aphid vector to optimize transmission and spread. Virus
Research, 186: 38-46.
Chiel E., Nessika Y., Steinberg S., Antignus Y. 2006. The effect of UV-absorbing plastic sheet
on the attraction and host location ability of three parasitoids: Aphidius colemani,
Diglyphus isaea and Eretmocerus mundus. BioControl, 51: 65-78.
Christiansen-Weniger P., Powell G., Hardie J. 1998. Plant virus and parasitoid interactions in a
shared insect vector/host. Entomologia Experimentalis et Applicata, 86: 205-213.
Chyzik R., Dorinin S., Antignu, Y., 2003. Effect of a UV-deficient environment on the biology
and flight activity of Myzus persicae and its Hymenopterous parasite Aphidius matricariae.
Phytoparasitica, 31: 467-477.
CIPAC. 2009. CIPAC Handbook. M. Marston Book Services Ltd., Oxfordshire, UK, pp. 40.
Clark M.F., Adams A.N. 1977. Characteristics of the microplate method of enzyme-linked
inmunosorbent assay for the detection of plant viruses. Journal of General Virology, 34:
475-483.
Clifford M.N., Johnston K.L., Knight S., Kuhner N. 2003. Hierarchical scheme for LC-MS
identification of chlorogenic acids. Journal of Agricultural and Food Chemistry, 51: 2900-
2911.
Colomer I., Aguado P., Medina P., Heredia R.M., Fereres A., Belda J.E., Viñuela E. 2011. Field
trial measuring the compatibility of methoxyfenozide and flonicamid with Orius laevigatus
Fieber (Hemiptera: Anthocoridae) and Amblyseius swirskii (Athias-Henriot) (Acari:
Phytoseiidae) in a commercial pepper greenhouse. Pest Management Science, 67: 1237-
1244.
Comont D., Winters A., Gwynn-Jones D. 2012. Acclimation and interaction between drought
and elevated UV-B in A. thaliana: differences in response over treatment, recovery and
reproduction. Ecology and Evolution, 2: 2695-2709.
Coombe P.E. 1982. Visual behavior of the greenhouse whitefly, Trialeurodes vaporariorum.
Physiological Entomology, 7: 243-251.
Costa H.S., Robb K.L. 1999. Effects of ultraviolet-absorbing greenhouse plastic films on flight
125
behavior of Bemisia argentifolii (Homoptera: Aleyrodidae) and Frankliniella occidentalis
(Thysanoptera: Thripidae). Journal of Economical Entomology, 92: 557-562.
Dadd R.H., Krieger D.L. 1968. Dietary amino acid requirements of aphid Myzus persicae.
Journal of Insect Physiology, 14: 741-764.
Day K.R., Docherty M., Leather S.R., Kidd N.A.C. 2006. The role of generalist insect predators
and pathogens in suppressing green spruce aphid populations through direct mortality and
mediation of aphid dropping behaviour. Biological Control, 38: 223-246.
Demukra P.V., Abdala G., Baldwin I.T., Ballaré C.L. 2010. Jasmonate-dependent and
independent pathways mediate specific effects of solar ultraviolet B radiation on leaf
phenolics and antiherbivore defense. Plant Physiology, 152: 1084-1095.
Díaz B.M., Barrios L., Fereres, A. 2012. Interplant movement and spatial distribution of alate
and apterous morphs of Nasonovia ribisnigri (Homoptera: Aphididae) on lettuce. Bulletin
of Entomological Research, 31: 1-9.
Díaz B.M., Biurrun R., Moreno A., Nebreda M., Fereres A. 2006. Impact of ultraviolet-blocking
plastic films on insect vectors of virus diseases infesting crisp lettuce. HortScience, 41:
711-716.
Díaz B.M., Fereres A. 2007. Ultraviolet-blocking materials as a physical barrier to control insect
pests and plant pathogens in protected crops. Pest Technology, 1: 85-95.
Díaz B.M., Legarrea S., Marcos-García M.A., Fereres, A. 2010. The spatio-temporal
relationships among aphids, the entomophthoran fungus, Pandora neoaphidis, and
aphidophagous hoverflies in outdoor lettuce. Biological Control, 53: 304–311.
Díaz B.M., Nebreda M., Salas F., Moreno A., García M., Fereres A. 2004. Mallas impregnadas
con insecticidas: un nuevo método para el control de plagas de cultivos hortícolas. Boletín
Sanidad Vegetal Plagas, 30: 623-632.
Díaz J.A., Mallor C., Soria C., Camero R., Garzo E., Fereres A., Álvarez J.M.,
Gómez-Guillamón A.L., Luis-Arteaga M., Moriones, E. 2003. Potential sources of
resistance for melon to nonpersistently aphid-borne viruses. Plant Disease, 87: 960-964.
Dicke M., van Loon J.J.A. 2000. Multitrophic effects of herbivore-induced plant volatiles in an
evolutionary context. Entomologia Experimentalis et Applicata, 97: 237-249.
126
Dixon A.F.G. 1973. Biology of aphids. Edward Arnold Ltd., London, UK.
Döring T.F., Chittka L. 2007. Visual ecology of aphids: A critical review on the role of colours
in host finding. Arthropod-Plant Interactions, 1: 3-16.
Doukas D., Payne C.C. 2007a. The use of ultraviolet-blocking films in insect pest management
in the UK; effects on naturally occurring arthropod pest and natural enemy populations in a
protected cucumber crop. Annals of Applied Biology, 151: 221-231.
Doukas D., Payne C.C. 2007b. Effects of UV-blocking films on the dispersal behavior of
Encarsia formosa (Hymenoptera: Aphelinidae). Journal of Economic Entomology, 100:
110-116.
Du Y.J., Poppy G.M., Powell W. 1996. Relative importance of semiochemicals from first and
second trophic levels in host foraging behavior of Aphidius ervi. Journal of Chemical
Ecology, 22: 1591-1605.
Dyer A.G., Chittka L. 2004. Bumblebee search time without ultraviolet light. Journal of
Experimental Biology, 207: 1683-1688.
Ellsworth P.C., Martínez-Carrillo J.L. 2001. IPM for Bemisia tabaci: a case study from North
America. Crop Protection, 20: 853-869.
EUROSTAT. 2009. URL: <epp.eurostat.ec.europa.eu/portal/page/portal/statistics/
search_database>. European Commission. Visited 31th October 2014.
FAO. 2010. Specifications and evaluations for agricultural pesticides: Bifenthrin. Food and
Agriculture Organization of the United Nations and World Health Organization, Rome,
Italy, pp. 1-33.
FAOSTAT. 2012. URL: <faostat.fao.org/site/339/default.aspx>. Food and Agriculture
Organization of the United Nations and World Health Organization. Visited 31th October
2014.
Fereres A., Moreno A. 2009. Behavioural aspects influencing plant virus transmission by
homopteran insects. Virus Research, 141: 158-168.
127
Frolich D.R., Torres-Jerez I., Bedford I.D., Markham P.G., Brown J.K. 1999. A
phylogeographical analysis of the Bemisia tabaci species complex based in mitochondrial
DNA markers. Molecular Ecology, 8: 1593-1602.
Fukaya M., Uesugi R., Ohashi H., Sakai Y., Sudo M., Kasai A., Kishimoto H., Osakabe M.
2013. Tolerance to solar ultraviolet-B radiation in the citrus red mite, an upper surface user
of host plant leaves. Photochemistry and Photobiology, 89: 424-431.
Gaberšcik A., Voncina M., Trošt T., Germ M., Björn L.O., 2002. Growth and production of
buckwheat (Fagopyrum esculentum) treated with reduced, ambient, and enhanced UV-B
radiation. Journal of Photochemistry and Photobiology B, Biology, 66: 30-36.
Gerling D., Alomar O., Arnó J. 2001. Biological control of Bemisia tabaci using predators and
parasitoids. Crop Protection, 20: 779-799.
Glick E., Levy Y. Gafni Y. 2009. The viral etiology of Tomato yellow leaf curl disease- a
review. Plant Protection Science, 45: 81-97.
Golden Software. 2009. Golden Surfer Software 9.0 version. Colorado, USA.
González J.A., Rosa M., Parrado M.F., Hilal M., Prado F.E. 2009. Morphological and
physiological responses of two varieties of a Highland species (Chenopodium quinoa
Willd.) growing under near-ambient and strongly reduced solar UV-B in a lowland
location. Journal of Photochemistry and Photobiology B, Biology, 96: 144-151.
Gray S., Cilia M., Ghanim M. 2014. Circulative, "Nonpropagative" virus transmission: An
orchestra of virus-, insect-, and plant-derived instruments. Advances in Virus Research, 89:
141-199.
Guerrieri E., Pennacchio F., Tremblay E. 1997. Effect of adult experience on in-flight orientation
to plant and plant-host complex volatiles in Aphidius ervi Haliday (Hymenoptera,
Braconidae). Biological Control, 10: 159–165.
Guilley H., Wipfscheibel C., Richards K., Lecoq H., Jonard G. 1994. Nucleotide-sequence of
cucurbit aphid-borne yellows luteovirus. Virology, 202: 1012-1017.
Gutiérrez-Marco E., Hernández E., Camacho J.L., Labajo A., 2007. Analysis of UVB values in
the centre of the Iberian Peninsula. Atmospheric Research, 84: 345-352.
Häder D.P., Lebert M., Schuster M., del Ciampo L., Helbling E.W., McKenzie R. 2007.
128
ELDONET - A decade of monitoring solar radiation on five continents. Photochemistry
and Photobiology, 83: 1-10.
Harborne J.B., Williams C.A. 2000. Advances in flavonoid research since 1992. Phytochemistry,
55: 481-504.
Hodge S., Hardie J., Powell G. 2011. Parasitoids aid dispersal of a nonpersistently transmitted
plant virus by disturbing the aphid vector. Agricultural and Forest Entomology, 13: 83-88.
Hodge S., Powell G. 2008a. Complex interactions between a plant pathogen and insect
parasitoid via the shared vector-host: Consequences for host plant infection. Oecologia,
157: 387-397.
Hodge S., Powell G. 2008b. Do plant viruses facilitate their aphid vectors by inducing symptoms
that alter behavior and performance? Environmental Entomology, 38: 1573-1581.
Hogenhout S.A., Ammar E., Whitfield A.E., Redinbaugh M.G. 2008. Insect vector interactions
with persistently transmitted viruses. Annual Review of Phytopathology, 46: 327-359.
Holt J., Pavis C., Marquier M., Chancellor T.C.B., Urbino C., Boissot N. 2008. Insect-screened
cultivation to reduce the invasion of tomato crops by Bemisia tabaci: modelling the impact
on virus disease and vector. Agricultural and Forest Entomology, 10: 61-67.
Horowitz A.R., Ellsworth P.C., Ishaaya I. 2009. Biorational pest control – An overview. In:
Ishaaya I., Horowitz A.R. (Eds.) Biorational control of arthropod pests: Aplication and
resistance management. Springer-Verlag New York, USA, pp. 1-20.
Hougard J.M., Duchon S., Zaim M., Guillet R. 2002. Bifenthrin: a useful pyrethoid insecticide
for treatment of mosquito nets. Journal of Medical Entomology, 39: 526-533.
Hull R. 2014. Plant Virology, 5th ed. Academic Press, California, USA.
Hunt J.E., McNeil D.L. 1999. The influence of present-day levels of ultraviolet-B radiation on
seedlings of two Southern Hemisphere temperate tree species. Plant Ecology, 143: 39-50.
Ingwell L.L., Eigenbrode S.D., Bosque-Pérez N.A. 2012. Plant viruses alter insect behavior to
enhance their spread. Scientific Reports, 2: 578.
Irwin M.E., Thresh J.M. 1990. Epidemiology of Barley yellow dwarf: A study in ecological
complexity. Annual Review of Phytopathology, 28: 393-424.
129
Izaguirre M.M., Mazza C.A., Svatoš A., Baldwin I.T., Ballaré C.L. 2007. Solar ultraviolet-B
radiation and insect herbivory trigger partially overlapping phenolic responses in Nicotiana
attenuata and Nicotiana longiflora. Annals of Botany, 99: 103-109.
Jacas J.A., Urbaneja A. 2008. Control biológico de plagas agrícolas. Phytoma España, Valencia,
Spain.
Jacobson R.J. 2004. IPM program for tomato. In: Heinz K.M., van Drieche R.G., Parrella M.P.
(Eds.) Biocontrol in protected culture. Ball Publishing, Batavia, USA, pp. 457-471.
Jansen M.A.K. 2002. Ultraviolet-B radiation effects on plants: induction of morphogenic
responses. Physiologia Plantarum, 116: 423-429.
Jayakumar M., Amudha P., Kulandaivelu G. 2003. Changes in growth and yield of Phaseolus
mungo L., induced by UV-A and UV-B enhanced radiation. Journal of Plant Biology, 46:
59-61.
Jayakumar M., Amudha P., Kulandaivelu G. 2004. Effect of low doses of UV-A and UV-B
radiation on photosynthetic activities in Phaseolus mungo L. Journal of Plant Biology, 47:
105-110.
Jeger M.J., Chen Z., Cunningham E., Martin G., Powell G. 2012. Population biology and
epidemiology of plant virus epidemics: from tripartite to tritrophics interactions. European
Journal of Plant Pathology, 133: 3-23.
Jeger M.J., Chen Z., Powell G., Hodge S., van den Bosch F. 2011. Interactions in a host plant-
virus-vector-parasitoid system: Modelling the consequences for virus transmission and
disease dynamics. Virus Research, 159: 183-193.
Johansen N.S., Vänninen I., Pinto D.M., Nissinen A.I., Shipp L. 2011. In the light of new
greenhouse technologies 2: Direct effects of artificial lighting on arthropods and integrated
pest management in greenhouses crops. Annals of Applied Biology, 159: 1-27.
Jones R.A.C. 2005. Patterns of spread of two non-persistently aphid-borne viruses in lupin
stands under four different infection scenarios. Annals of Applied Biology, 146: 337-350.
Jones R.A.C., Coutts B.A., Latham L.J., McKirdy S.J. 2008. Cucumber mosaic virus infection of
chickpea stands: Temporal and spatial patterns of spread and yield-limiting potential. Plant
Pathology, 57: 842-853.
130
Juárez M., Legua P., Mengual C.M., Kassem M.A., Sempere R.N., Gómez P., Truniger V.,
Aranda M.A. 2013. Relative incidence, spatial distribution and genetic diversity of
cucurbit viruses in eastern Spain. Annals of Applied Biology, 162: 362-370.
Kashiwagi T., Horibata Y., Mekuria D.B., Tebayashi S., Kim C.S. 2005. Ovipositional deterrent
in the sweet pepper, Capsicum annuum, at the mature stage against Liriomyza trifolii
(Burgess). Bioscience, Biotechnology and Biochemistry, 69: 1831-1835.
Kassem M.A., Juárez M., Gómez P., Mengual C.M., Sempere R.N., Plaza M., Elena S.F.,
Moreno A., Fereres A., Aranda M.A. 2013. Genetic fiversity and potential vectors and
reservoirs of Cucurbit aphid-borne yellows virus in Southeastern Spain. Phytopathology,
103: 1188-1197.
Kehr J. 2006. Phloem sap proteins: their identities and potential roles in the interaction between
plants and phloem-feeding insects. Journal of Experimental Botany, 57: 767-774.
Kennedy J., Mittler T. 1953. A method of obtaining phloem sap via the mouthparts of aphids.
Nature, 171: 528.
Kirchner S.M., Döring T., Saucke H. 2005. Evidence for trichromacy in the green peach aphid
Myzus persicae (Sulz.) (Hemiptera: Aphididae). Journal of Insect Physiology, 51: 1255-
1260.
Kittas C., Tchamitchian M., Kasoulas N., Kariskou P., Papaioannou C.H. 2006. Effect of two
UV-absorbing greenhouse-covering films on growth and yield of an eggplant soilless crop.
Scientia Horticulturae, 110: 30-37.
Kogan M. 1998. Integrated pest management: Historical perspectives and contemporary
development. Annual Review of Entomology, 43: 243-270.
Kring J.B. 1972. Flight behaviour of aphids. Annual Review of Entomology, 17: 461-492.
Kuhlmann F., Müller C. 2009a. Development-dependent effects of UV radiation exposure on
broccoli plants and interactions with herbivorous insects. Environmental and Experimental
Botany, 66: 61-68.
Kuhlmann F., Müller C. 2009b. Independent responses to ultraviolet radiation and herbivore
attack in broccoli. Journal of Experimental Botany, 60: 3467-3475.
131
Kuhlmann F., Müller C. 2010. UV-B impact on aphid performance mediated by plant quality
and plant changes induced by aphids. Plant Biology, 12: 676-684.
Kumar P., Poehling H.M. 2006. UV-blocking plastic films and nets influence vectors and virus
transmission on greenhouse tomatoes in the humid tropics. Environmental Entomology, 35:
1069-1082.
Lecoq H., Bourdin D., Wipf-Scheibel C., Bon M., Lot H., Lemaire O., Herrbach E. 1992. A new
yellowing disease of cucurbits caused by a Luteovirus, Cucurbit aphid-borne yellows virus.
Plant Pathology, 41: 749-761.
Legarrea S., Betancourt M., Plaza M., Fraile A., García-Arenal F., Fereres A. 2012a. Dynamics
of nonpersistent aphid-borne viruses in lettuce crops covered with UV-absorbing nets.
Virus Research, 165: 1-8.
Legarrea S., Díaz B.M., Plaza M., Barrios L., Morales I., Viñuela E., Fereres A. 2012b.
Diminished UV radiation reduces the spread and population density of Macrosiphum
euphorbiae (Thomas) [Hemiptera: Aphididae] in lettuce crops. Horticultural Science, 39:
55-60.
Legarrea S., Karnieli A., Fereres A., Weintraub P.G. 2010. Comparison of UV-absorbing nets in
pepper crops: Spectral properties, effects on plants and pest control. Photochemistry and
Photobiology, 86: 324-330.
Legarrea S., Weintraub P.G., Plaza M., Viñuela E., Fereres A. 2012c. Dispersal of aphids,
whiteflies and their natural enemies under photoselective nets. BioControl, 57: 523-532.
Licciardi S., Assogba-Komlan F., Sidick I., Chandre F., Hougard J.M., Martin T. 2008. A
temporary tunnel screen as an eco-friendly method for small-scale farmers to protect
cabbage crops in Benin. International Journal of Tropical Insect Science, 27: 152-158.
Lichtenthaler H.K. 1987. Chlorophylls and carotenoids: pigments of the photosynthetic
biomembranes. Methods in Enzymology, 148: 350-382.
Liu L.X., Xu S.M., Woo K.C. 2005. Solar UV-B radiation on growth, photosynthesis and the
xanthophyll cycle in tropical acacias and eucalyptus. Environmental and Experimental
Botany, 54: 121-130.
132
Losey J.E., Denno R.F. 1998. The escape response of pea aphids to foliar-foraging predators:
Factors affecting dropping behaviour. Ecological Entomology, 23: 53-61.
Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. 1951. Protein measurement with the Folin
phenol reagent. Journal of Biological Chemistry, 193: 265-275.
Mackerness S.A.H. 2000. Plant responses to ultraviolet-B (UV-B: 280-320 nm) stress: what are
the key regulators? Plant Growth Regulation, 32: 27-39.
MAGRAMA. 2013. Superficies y producciones anuales de cultivos. Ministerio de Agricultura,
Alimentación y Medio Ambiente, España.
MAGRAMA. 2014. Encuesta sobre superficies y rendimientos de cultivos. Ministerio de
Agricultura, Alimentación y Medio Ambiente, España.
Mahdavian K., Kalantari K.M., Ghorbanli M., Torzade M. 2008. The effects of salicylic acid on
pigment contents in ultraviolet radiation stressed pepper plants. Biologia Plantarum, 52:
170-172.
Margaritopoulos J.T., Kasprowicz L., Malloch G.L., Fenton B. 2009. Tracking the global
dispersal of a cosmopolitan insect pest, the peach potato aphid. BMC Ecology, 9: 1-13.
Marín A., Ferreres F., Tomás-Barberán F.A., Gil M.I. 2004. Characterization and quantitation of
antioxidant constituents of sweet pepper (Capsicum annuum L.). Journal of Agricultural
and Food Chemistry, 52: 3861-3869.
Martin T., Assogba-Komlan F., Houndete T., Hougard J.M., Chandre F. 2006. Efficacy of
mosquito netting for sustainable small holders’ cabbage production in Africa. Journal of
Economical Entomology, 99: 450-454.
Martin T., Assogba-Komlan F., Sidick I., Ahle V., Chandre, F. 2010. An acaricide-treated net to
control phytophagous mites. Crop Protection, 29: 470-475.
Martin T., Chandre F., Chabi J., Guillet P.F., Akogbeto M., Hougard J.M. 2007. A biological test
to quantify pyrethroid in impregnated nets. Tropical Medicine & International Health, 12:
245-250.
Martin T., Kamal A., Gogo E., Saidi M., Delétré E., Bonafos R., Simon S., Ngouajio M. 2014.
Repellent effect of alphacypermethrin-treated netting against Bemisia tabaci (Hemiptera:
133
Aleyrodidae). Journal of Economical Entomology, 107: 684-690.
Matteson N., Terry I., Ascoli-Christensen A., Gilbert C. 1992. Spectral efficiency of the western
flower thrips, Frankliniella occidentalis. Journal of Insect Physiology, 38: 453-459.
Mauck K., Bosque-Pérez N.A., Eigenbrode S.D., De Moraes C.M., Mescher M.C. 2012.
Transmission mechanisms shape pathogen effects on host-vector interactions: Evidence
from plant viruses. Functional Ecology, 26: 1162-1175.
Mauck K.E., De Moraes C.M., Mescher M.C. 2010. Deceptive chemical signals induced by a
plant virus attract insect vector to inferior hosts. Proceedings of the National Academy of
Science USA, 107: 3600-3605.
Mazza C.A., Izaguirre M.M., Curiale J., Ballaré C.L. 2010. A look into the invisible: ultraviolet-
B sensitivity in an insect (Caliothrips phaseoli) revealed through a behavioural action
spectrum. Proceedings of the Royal Society B: Biological Sciences, 227: 367-373.
Medina P., Adán A., Del Estal P., Budia F., Viñuela E. 2008. Integración del control biológico
con otros métodos de control. In: Jacas J., Urbaneja A. (Eds.) Control biológico de plagas
agrícolas. Phytoma España, Valencia, Spain, pp. 469-476.
Mellor H.E., Bellingham J., Anderson M. 1997. Spectral efficiency of the glasshouse whitefly
Trialeurodes vaporariorum and Encarsia formosa, its hymenopteran parasitoid.
Entomologia Experimentalis et Applicata, 83: 11-20.
Mewis I., Schreiner M., Nguyen C.N., Krumbein A., Ulrichs C., Lohse M., Zrenner R., 2012.
UV-B irradiation changes specifically the secondary metabolite profile in broccoli sprouts:
Induced signaling overlaps with defense response to biotic stressors. Plant Cell
Physiology, 53: 1546-1560.
Meyer-Rochow V.B., Kashiwagi Y., Eguchi E. 2002. Selective photoreceptor damage in four
species of insects induced by experimental exposures to UV-irradiation. Micron, 33: 23-
31.
Michaud J.P., Mackauer M. 1995. The use of visual cues in host evaluation by aphidiid wasps.
Entomologia Experimentalis et Applicata, 74: 267-275.
Middleton E.M., Teramura A.H. 1993. The role of flavonol glycosides and carotenoids in
protecting soybean from ultraviolet-B damage. Plant Physiology, 103: 741-752.
134
Milburn J.A. 1970. Phloem exudation from castor bean: induction bymassage. Planta, 95: 272-
276.
Mittler T., Dadd D, Daniels S.C. 1970. Utilization of different sugars by the aphid Myzus
persicae. Journal of Insect Physiology, 16: 1873-1890.
Moreno A., Garzo E., Fernández-Mata G., Kasse, M., Aranda M.A., Fereres A.
2011. Aphids secrete watery saliva into plant tissues from the onset of stylet penetration.
Entomologia Experimentalis et Applicata, 139: 145-153.
Moreno A., Nebreda M., Díaz B.M., García M., Salas F., Fereres A. 2007. Temporal and spatial
spread of Lettuce mosaic virus in lettuce crops in central Spain: Factors involved in Lettuce
mosaic virus epidemics. Annals of Applied Biology, 150: 351-360.
Mound L.A. 1962. Studies on the olfaction and colour sensitivity of Bemisia tabaci (Genn.)
(Homoptera: Aleyrodidae). Entomologia Experimentalis et Applicata, 5: 99-104.
Mound L.A., Halsey S.H. 1978. Whitef1y of the World. Wiley, New York, USA.
Muñoz P., Montero J.I., Antón A., Giuffrida F. 1999. Effect of insect-proof screens and roof
openings on greenhouse ventilation. Journal of Agricultural Engineering Research, 73:
171-178.
Mutwiwa U.N., Borgemeister C., von Elsner B., Tantau H.J. 2005. Effects of UV-absorbing
plastic films on greenhouse whitefly (Homoptera: Aleyrodidae). Journal of Economic
Entomology, 98: 1221-1228.
Nault L.R. 1997. Arthropod transmission of plant viruses: A new synthesis. Annals of the
Entomological Society of America, 90: 521-541.
Ng J.C.K., Falk B.W. 2006. Virus-vector interactions mediating nonpersistent and semipersistent
transmission of plant viruses. Annual Review of Phytopathology, 44: 183-212.
Ng J.C.K., Perry K.L. 2004. Transmission of plant viruses by aphid vectors. Molecular Plant
Pathology, 5: 505-511.
OJEC. 2005. Regulation EC 396/2005, of 23 February 2005, on maximum residue levels of
pesticide in or on food and feed of plant and animal origin and amending Council Directive
91/414/EEC. Official Journal of the European Communities (16-03-2005), 70: 01-16.
135
OJEC. 2009. Directive 2009/128/EC, of 21 October 2009, establishing a framework for
Community action to achieve the sustainable use of pesticides. Official Journal of the
European Communities (24-11-2009), 309: 71-86.
Palukaitis P., García-Arenal F. 2003. Cucumoviruses. Advances in Virus Research, 62: 241-323.
Park S., Jeong W.Y., Lee J.H., Kim Y.H., Jeong S.W., Kim G.S., Bae D.W., Lim C.S., Jin J.S.,
Lee S.J., Shin S.C. 2012. Determination of polyphenol levels variation in Capsicum
annumm L. cv. Chelsea (yellow bell pepper) using liquid chromatography-tandem mass
spectrometry. Food Chemistry, 130: 981-985.
Paul N.D., Gwynn-Jones D. 2003. Ecological roles of solar UV radiation: towards an integrated
approach. Trends in Ecology and Evolution, 18: 48-55.
Paul N.D., Moore J.P., McPherson M., Lambourne C., Croft P., Heaton J.C., Wargent J.J. 2011.
Ecological responses to UV radiation: interactions between the biological effects of UV on
plants and on associated organisms. Physiologia Plantarum, 145: 565-581.
Perry J.N. 1995. Spatial analysis by distance indices. Journal of Animal Ecology, 64: 303-314.
Perry J.N. 1998. Measures of spatial pattern for counts. Ecology, 79: 1008-1017.
Perry J.N., Nixon P. 2002. A new method to measure spatial association for ecological data.
Ecoscience, 9: 133-141.
Perry J.N., Winder L., Holland J.M., Alston R.D. 1999. Red-blue cages for detecting clusters in
count data. Ecological Letters, 2: 106-113.
Petropoulou Y., Georgiou O., Psaras G.K., Manetas Y. 2001. Improved flower advertisement,
pollinator rewards and seed yield by enhanced UV-B radiation in the Mediterranean annual
Malcolmia maritima. New Phytologist, 152: 85-90.
Porra R.J., Thomson W.A., Kriedemann P.E. 1989. Determination of accurate extinction
coefficients and simultaneous equations for assaying chlorophyll a and b extracted with
four different solvents: verification of the concentration of chlorophyll standards by atomic
absorption spectroscopy. Biochimica et Biophysica Acta, 975: 384-394.
136
Proffit M., Birgersson G., Bendtsson M., Reis R., Witzgall P., Lima E. 2011. Attraction and
oviposition of Tuta absoluta females in response to tomato leaf volatiles. Journal of
Chemical Ecology, 37: 565-574.
Ramakers P.M.J. 2004. IPM program for sweet pepper. In: Heinz K.M., van Drieche R.G.,
Parrella M.P. (Eds.) Biocontrol in protected culture. Ball Publishing, Batavia, USA, pp.
439-455.
Raviv M., Antignus Y. 2004. UV radiation effects on pathogens and insect pests of greenhouse-
grown crops. Photochemistry and Photobiology, 79: 219-226.
Reitz S.R., Yearby E.L., Funderburk J.E., Stavisky J., Momol M.T., Olson S.M. 2003. Integrated
management tactics for Frankliniella thrips (Thysanoptera: Thripidae) in field-grown
pepper. Journal of Economic Entomology, 96: 1201-1214.
Roberts M.R., Paul N.D. 2006. Seduced by the dark side: integrating molecular and ecological
perspectives on the influence of light on plant defence against pests and pathogens. New
Phytologist, 170: 677-699.
Robledo A., Van der Blom J., Sánchez J.A., Torres S. 2009. Control biológico en invernaderos
hortícolas. Coexphal, Almería, Spain.
Roitberg B.D., Myers J.H. 1978. Effect of adult coccinellidae on the spread of a plant virus by
an aphid. Journal of Applied Ecology, 15: 775-779.
Sakai Y., Osakabe M. 2010. Spectrum-specific damage and solar ultraviolet radiation avoidance
in the two-spotted spider mite. Photochemistry and Photobiology, 86: 925- 932.
Scholthof K.B.G., Adkins S., Czosnek H., Palukaitis P., Jacquot E., Hohn T., Hohn B., Saunders
K., Candresse T., Ahlquist P., Hemenway C., Foster G.D. 2011. Top 10 plant viruses in
molecular plant pathology. Molecular Plant Pathology, 12, 938-954.
Schoonhoven L.M., van Loon J.J.A., Dicke M. 2006. Insect-Plant Biology, 2nd ed. Oxford
University Press, Oxford, UK.
Skorupski P., Döring T., Chittka L. 2007. Photoreceptor spectral sensitivity in island and
mainland populations of the bumblebee, Bombus terrestris. Journal of Comparative
Physiology A - Neuroethology Sensory Neural and Behavioral Physiology, 193: 485-494.
137
Smith J.L., Burritt, D.J., Bannister P. 2000. Shoot dry weight, chlorophyll and UV-B absorbing
compounds as indicators of a plant’s sensitivity to UV-B radiation. Annals of Botany, 86:
1057-1066.
Smyrnioudis I.N., Harrington R., Clark S.J., Katis N. 2001. The effect of natural enemies on the
spread of Barley yellow dwarf virus (BYDV) by Rhopalosiphum padi (Hemiptera:
Aphididae). Bulletin of Entomological Research, 91: 301-306.
SPSS. 2013. SPSS statistical package, 21.0 version. SPSS Inc., Chicago, USA.
Srivastava P.N., Auclair J.L. 1971. Influence of sucrose concentration on diet uptake and
performance by the pea aphid, Acyrthosiphon pisum. Annals of the Entomological Society
of America, 64: 739-743.
Srivastava P.N., Auclair J.L. 1975. Role of single amino acids in phagostimulation, growth and
survival of Acyrthosiphon pisum. Journal of Insect Physiology, 21: 1865-1871.
Stary P. 1975. Aphidius colemani Viereck: its taxonomy, distribution and host range
(Hymenoptera, Aphidiidae). Acta Entomologica Bohemoslovaca, 72: 156-163.
Stephanou M., Petropoulou Y., Georgiou O., Manetas Y. 2000. Enhanced UV-B radiation,
flower attributes and pollinator behaviour in Cistus creticus: a Mediterranean field study.
Plant Ecology, 147: 165-171.
Stern V.M., Smith R.F., van den Bosch R., Hagen K.S. 1959. The integrated control concept.
Hilgardia, 29: 81-101.
Stommel J.R., Whitaker B.D. 2003. Phenolic acid content and composition of eggplant fruit in a
germplasm core subset. Journal of the American Society of Horticultural Science, 128:
704-710.
Storeck A., Poppy G.M., van Emden H.F., Powell W. 2000. The role of plant chemical cues in
determining host preference in the generalist aphid parasitoid Aphidius colemani.
Entomologia Experimentalis et Applicata, 97: 41-46.
Strange R.N., Scott P.R. 2005. Plant disease: A threat to global food security. Annual Review of
Phytopathology, 43: 83-116.
Stratmann J. 2003. Ultraviolet-B radiation co-opts defense signaling pathways. Trends in Plant
138
Science, 8: 526-533.
Tezuka T., Yamaguchi F., Ando Y. 1994. Physiological activation in radish plants by UV-A
radiation. Journal of Photochemistry and Photobiology B, Biology, 24: 33-40.
Thresh J.M. 1983. Progress curves of plant virus disease. Advanced in Applied Biology, 8: 1-85.
Toguri T., Umemoto N., Kobayashi O., Ohtani T. 1993. Activation of anthocyanin synthesis
genes by white light in eggplant hypocotyl tissues, and identification of an inducible P-450
cDNA. Plant Molecular Biology, 23: 933-946.
Tsormpatsidis E., Ordidge M., Henbest R.G.C., Wagstaffe A., Battey N.H., Hadley P. 2011.
Harvesting fruit of equivalent chronological age and fruit position shows individual effects
of UV radiation on aspects of the strawberry ripening process. Environmental and
Experimental Botany, 74: 178-185.
Van den Bosch R., Messenger P.S. 1973. Biological control. Intext Educational Publishers, New
York, USA.
Van der Blom J., Robledo A., Torres S., Sánchez J.S. 2009. Consequences of the wide scale
implementation of biological control in greenhouse horticulture in Almería, Spain.
IOBC/WPRS Bulletin, 49: 9-13.
Van der Blom J., Robledo A., Torres S., Sanchez J.A. 2010. Control biológico en horticultura en
Almería: Un cambio radical, pero racional y rentable. Cuadernos de Estudios
Agroalimentarios, 1: 45-60.
Van Lenteren J., Bueno V.H.P. 2014. Pasado, presente y futuro del control biológico. Phytoma,
262: 8.
Vänninen I., Pinto D.M., Nissinen A.I., Johansen N.S., Shipp L. 2010. In the light of new
greenhouse technologies 1: Plant-mediated effects of artificial lighting on arthropods and
tritrophic interactions. Annals of Applied Biology, 157: 393-414.
Verdaguer D., Llorens L., Bernal M., Badosa J. 2012. Photomorphogenic effects of UVB and
UVA radiation on leaves of six Mediterranean sclerophyllous woody species subjected to
two different watering regimes at the seedling stage. Environmental and Experimental
Botany, 79: 66-75.
139
Weber C.A., Godfrey L.D., Mauk P.A. 1996. Effects of parasitism by Lysiphlebus testaceipes
(Hymenoptera: Aphidiidae) on transmission of Beet yellows closterovirus by bean aphid
(Homoptera: Aphididae). Journal of Economical Entomology, 89: 1431-1437.
Weibull J. 1987. Seasonal changes in the free amino acids of oat and barley phloem sap in
relation to plant growth stage and growth of Rhopalosiphum padi. Annals of Applied
Biology, 111: 729-737.
Weintraub P.G. 2009. Physical control: an important tool in pest management. In: Ishaaya I.,
Horowitz A.R. (Eds.) Biorational control of arthropod pests. Springer Science, Berlin,
Germany, pp. 317-324.
Weintraub P.G., Berlinger M.J. 2004. Physical control in greenhouses and field crops. In:
Horowitz A.R., Ishaaya I. (Eds.) Insect pest management: field and protected crops.
Springer, Berlin, Germany, pp. 301-318.
Whalon M.E., Mota-Sánchez D., Hollingworth R.M. 2008. The arthropod pesticide resistance
database. Michigan State University, USA.
Willes J.A., Jepson P.C. 1994. Sub-lethal effects of deltamethrin residues on the within-crop
behaviour and distribution of Coccinella septempunctata. Entomologia Experimentalis et
Applicata, 72: 33-45.
Winters A., Lloyd J.D., Jones R., Merry R.J. 2002. Evaluation of a rapid method for estimating
free amino acids in silages. Animal Feed Science and Technology, 99: 177-187.
WMO. 2010. Scientific assessment of ozone depletion: 2010. World Meteorological
Organization, Global Ozone Research and Monitoring Project, Executive summary.
Geneva, Switzerland.
Yang S., Xu R., Yang S.Y., Kuang R.P. 2009. Olfactory responses of Aphidius gifuensis to odors
of host plants and aphid-plant complexes. Insect Science, 16: 503-510.
Zaim M., Aitio A., Nakashima N. 2000. Safety of pyrethroid-treated mosquito nets. Medical and
Veterinary Entomology, 14: 1-5.
Zamani A.A., Talebi A., Fathipour Y., Baniameri V. 2007. Effect of temperature on life history
of Aphidius colemani and Aphidius matricariae (Hymenoptera: Braconidae), two
140
parasitoids of Aphis gossypii and Myzus persicae (Homoptera: Aphididae). Environmental
Entomology, 36: 263-271.