INTEGRATION OF PHYSICAL, CHEMICAL AND BIOLOGICAL …oa.upm.es/35073/1/BEATRIZ_DADER_ALONSO.pdf ·...

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


5.2. OBJECTIVE 51




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


6.2. OBJECTIVE 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


6.4. 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


7.2. OBJECTIVE 93


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


7.3.6. 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

ii

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

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

vi

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

viii

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

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53

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T1

T2

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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.

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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.

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.


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).


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.


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.


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).

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.


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).


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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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


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


37

Figu

re 4

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patia

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stri

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n nu

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(to

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38

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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


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).


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


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

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is g

ossy

pii

and

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ulat

ive

num

ber

of C

AB

YV

-inf

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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

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f th

e as

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atio

n be

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n C

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tor,

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for

Fig

ure

4.5.

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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


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.


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.

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.



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


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).


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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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).


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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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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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

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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


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


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.

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.



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


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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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)

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34

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104

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UVA- (pepper: n=56, eggplant: n=50)





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


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).


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


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 %


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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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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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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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).


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


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


7.3.1. 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”


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,


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


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


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).


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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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).


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

!"#$%&'()"%

#

Ia =

1.3

1

P

a =

0.04

V

i = 1

.35

P

vi =

0.0

3 V

j = -

1.32

P

vj =

0.0

4

Ia =

1.5

7

P

a =

0.00

V

i = 1

.55

P

vi =

0.0

0 V

j = -

1.57

P

vj =

0.0

0

Ia =

0.8

9

P

a =

0.74

V

i = 0

.89

P

vi =

0.7

6 V

j = -

0.89

P

vj =

0.7

3

Ia =

1.0

9

P

a =

0.28

V

i = 1

.00

Pvi

= 0

.36

Vj =

-1.

09

Pvj

= 0

.28

Ia =

0.9

5

P

a =

0.56

V

i = 0

.93

P

vi =

0.6

1 V

j = -

0.95

P

vj =

0.5

6

X =

0.0

1

P

= 0

.49

X =

-0.

34

P

= 0

.98

X =

0.1

7

P

= 0

.15

X =

0.3

6

P

= 0

.02

X =

0.3

3

P =

0.0

3

N

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


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

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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


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.

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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.

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

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