MULTITROPHIC INTERACTIONS in a POTATO-APHID SYSTEM ASAD ALI B.Sc. (Hons), M.Sc. (Hons) a Thesis Submitted for the Degree of Doct

MULTITROPHIC INTERACTIONS IN A POTATO-APHID SYSTEM

ASAD ALI B.Sc. (Hons), M.Sc. (Hons)

A thesis submitted for the Degree of Doctor of Philosophy of Imperial College London and the Diploma of Imperial College London

Division of Biology

Silwood Park Campus

Imperial College London

November 2010 Dedication



This research work is dedicated to my worthy parents,

My loving wife Gul Naz & Sweet son Asjad Ali

Declaration

The work presented in this thesis is entirely my own and has not been submitted anywhere else.

Signed...... Asad Ali

Certified...... Professor Denis J. Wright

(PhD Supervisor)

Acknowledgements

All praises and appreciations are for ALMIGHTY ALLAH, the most merciful and compassionate, the greatest source of knowledge and wisdom, who bestowed upon me skill and intellect to conduct and accomplish the assigned project in the most humble manner.

The deepest love and inspirations are due to Holy Prophet MUHAMMAD (Peace be Upon

Him), who is forever a torch of guidance and knowledge for humanity.

I wish to express my sincere gratitude to my worthy supervisor Prof. Denis J. Wright for his substantial guidance, continuous inspiration and invaluable assistance in bringing this dissertation to its present form. Without his encouragement and constant guidance, I could not have finished this dissertation. I am also greatly thankful to members of my PRP,

Prof. Jim Hardie and Dr. Glen Powell for their valuable suggestions, constructive criticism and encouragement throughout my research endeavour.

I wish to thank Syngenta, Jealott‟s Hill, UK for supplying me with the culture of

Meloidogyne javanica for this research. I am also thankful to my colleagues; Dr. Khalid

Farooq, Mr. Malik Muhammad Masud and Mr. Shahid Riaz at National Potato Programme,

NARC, Islamabad, Pakistan, for their help and assistance in establishing and data collection of potato field trials at Pakistan.

This study was made possible through the financial support of Higher Education

Commission (HEC) of Pakistan. I am also grateful to the PARC Islamabad, Pakistan for granting study leave to complete this task.

I don‟t have the words to acknowledge the fascinating love, encouragement, support both moral and financial of my beloved parents. Thanks BABA and MOOR for giving me life, for educating me with aspects from both arts and sciences, for unconditional support and encouragement to pursue my interests, even when the interests went beyond boundaries of language, field and geography. Their prayers are the root of my success.

I am grateful to my wife GUL NAZ for her sincerity which was continuous push for me and the joy she gave me in the form of Asjad Ali, my beloved son, my asset, and much more. Thank you both for your time and patience.

My thanks are also due to the kindness and love of my family members, especially my Bibi Gulaney, Masher baba, Daji, Kaji and family, brothers, Yousaf Ali, Ashraf Ali,

Waqas Ali and sister, Nazia Farooq. I am also grareful to my In-laws who added charm to my life, especially Haji saib and Sheena aunty, Amjad Islam, Fazal Karim, Jaini, Bushi, Lado and Uzmi the great, for her amusing chat.

Friends are co-sharer of my struggle and my work. I am also grateful to all my friends during my stay at Imperial College especially Asim Gulzar, Muhammad Tariq, Anjum

Aqueel, Rahim Shah, Muhammad Adrees, Farida and Sofia, Khalid Zafar, Ziab Khan at

Leicester, Dr. Syed Meher Ali Shah and Maqsood Shah at Pakistan and Asim Khan at

Bradford. Also thanks to the folks at the Silwood Park Campus for interesting discussions and being fun to be with. Thanks, Silwoodians!

I am also Thankful to all of my teachers in life who put me on the track for success especially Prof. Muhammad Naeem, Prof. Mian Inayatullah, Dr. Sajjad Ahmad, Dr. Imtiaz Ali

Khan and Mr. Salim Jan for getting me interested in Entomology and for their continuous encouragements during the course of my studies.

All errors and limitations remaining in this thesis are mine alone.

Abstract

In interactions between plants, insect herbivores and natural enemies, resistant plant varieties and soil abiotic/biotic factors can affect natural enemies through changes in the plant or host insect. Interactions can be negative, neutral or positive in relation to herbivore populations. The majority of studies have used above-ground plant–insect–parasitoid systems and relatively few studies have included below- ground effects on above-ground tritrophic interactions. The aim of the present project was to understand how soil-based stress factors (nutrient availability and root pathogens) influence multitrophic interactions in a potato-aphid (Myzus persicae)-parasitoid (Aphidius colemani) system. Specific objectives were: to assess aphid performance on different potato cultivars under laboratory and field conditions; use aphid resistant and susceptible cultivars to assess the effects of soil nutrition and a plant pathogen (root-knot nematode) on aphid performance; conduct multifactorial ditrophic (aphid performance) and tritrophic (parasitism success) experiments with selected combinations of soil treatments; use olfactometry to determine whether plant volatiles are involved where significant tritrophic interactions occur between treatments. Potato cv. Anya, a cv derived from Desiree and Pink Fir Apple, was found to be consistently resistant to M. persicae compared with the three other cultivars tested (Desiree, Santé and Pink Fir Apple) in both laboratory experiments (UK) and field trials (Pakistan and UK). For Anya (resistant) and Desiree and Pink Fir Apple (susceptible), high levels of soil nutrients enhanced aphid performance, their parasitism by A. colemani, and adult parasitoid emergence. Root-knot nematodes had a negative effect on aphid performance at both low and high soil nutrition but had a positive effect on parasitism at low soil nutrition. Olfactometry showed Desiree to be more attractive to M. persicae compared with Anya. The work is discussed in relation to the development of pest management strategies for the control of M. persicae on potato.

TABLE OF CONTENTS

Dedication...... 2

Declaration...... 3

Acknowledgements...... 4

Abstract...... 6

Table of Contents...... 7

CHAPTER 1: Introduction

1.1 Background……………………………………………………………….....13

1.2 Aims and objectives…………………………………………………….…..16

1.3 Hypotheses…………………………………………………………….....…17

CHAPTER 2: Literature Review

2.1 Multitrophic interactions……………………………………………..……..18

2.2 Soil Nutrition and insect-plant interactions…………………….…...…….20

2.2.1 Effect of soil nutrition on plants………………………………..…..21

2.2.2 Indirect effects of soil nutrition on insect herbivores……….....…22

2.2.3 Indirect effects of soil nutrition on parasitoids…………………....24

2.3 Potato………………………………………………………………………...25

2.4 Aphids…………………………………………………………………….….26

2.4.1 Myzus persicae……………………………………………….…..…30

2.4.2 Host plant resistance……………………………………….……….31

2.5 Parasitoids…………………………………………………………...……...32

2.6 Plant parasitic nematodes………………………………………….……...34

2.6.1 Meloidogyne spp…………………………………….….…………..34

CHAPTER 3: General Materials and Methods

3.1 Plant production……………………………………………………………..37

3.1.1 Potato cultivars……………………………………………….…...... 37

3.1.2 John Innes compost…………………………………………..…....38

3.2 Insect culture……………………………………………………….…….….39

3.3 Parasitoids…………………………………………..……………………….39

3.4 Plant parasitic nematodes………………………………………………….40

3.5 Data analysis……………………………………………..……………....…40

CHAPTER 4: The performance of Myzus persicae on potato cultivars at different soil nutritional levels

4.1 Introduction……………………………………………………………….….41

4.2 Materials and Methods……………………………………………………..42

4.2.1 Plant production…………………………………………………..…42

4.2.2 Insect culture…………………………………………………....…..43

4.2.3 Performance experiments……………………………………..…..43

4.2.4 Data analysis………………………………………………………..45

4.3 Results…………………………………………………………………….…45

4.3.1 Developmental time……………………………………………..….45

4.3.2 Fecundity and intrinsic rate of increase…………………………..46

4.4 Discussion…………………………………………………………………...49

CHAPTER 5: Field performance of Myzus persicae on potato cultivars in Pakistan and the UK

5.1 Introduction…………………………………………………………………..52

5.2 Materials and Methods…………………………………………………..…53

5.2.1 Field sites and farming practices………………………………….53

5.2.2 Aphid survey……………………………………………...... 55

5.2.3 Data analysis…………………………………………...... 56

5.3 Results………………………………………………………………………..56

5.3.1 Meteorological data for Silwood Park……………………...... 56

5.3.2 Meteorological data for Pakistan…………………………………..57

5.3.3 Field performance of M. persicae on potato cultivars: Silwood Park

2008…...... 59

5.3.4 Field performance of M. persicae on potato cultivars: Pakistan

2009………...... 60

5.3.5 Performance of M. persicae on potato cultivars in pots (semi-field):

Silwood Park 2009 ……………………………………………...... 64

5.3.6 Field performance of M. persicae on potato cultivars: Pakistan

2010……...... 66

5.4. Discussion ...... 70

CHAPTER 6: Performance of Myzus persicae on upper, middle and lower leaves of three potato cultivars at low and high soil nutrition

6.1 Introduction...... 74

6.2 Materials and Methods...... 75

6.2.1 Plant production...... 75

6.2.2 Insect culture...... 75

6.2.3 Performance Experiments...... 76

6.2.4 Data Analysis...... 77

6.3 Results...... 77

6.3.1 Developmental time...... 77

6.3.2 Fecundity and Intrinsic rate of increase...... 79

6.4 Discussion...... 82

CHAPTER 7: The preference of Myzus persicae on potato cultivars at different soil nutrition levels

7.1 Introduction...... 85

7.2 Materials and Methods...... 86

7.2.1 Plant production...... 86

7.2.2 Insect culture...... 86

7.2.3 Preference (settling) bioassay with winged aphids...... 86

7.2.4 Olfactometer experiment...... 87

7.2.5 Trichome density on potato cultivars...... 89

7.2.6 Data analysis...... 89

7.3 Results...... 90

7.3.1 Preference (settling) bioassay with winged aphids...... 90

7.3.2 Olfactometer experiment...... 93

7.3.3 Trichome density on potato cultivars...... 94

7.4 Discussion...... 96

CHAPTER 8: Performance of Aphidius colemani on Myzus persicae reared on three potato cultivars at different soil nutrition levels

8.1 Introduction...... 100

8.2 Materials and Methods...... 101

8.2.1 Plant production...... 101

8.2.2 Insect culture...... 101

8.2.3 Parasitoids...... 101

8.2.4 Experimental Procedure...... 102

8.2.5 Data analysis...... 103

8.3 Results...... 104

8.3.1 Percentage parasitism...... 104

8.3.2 Percentage emergence...... 105

8.3.3 Developmental time...... 106

8.3.4 Sex ratio...... 108

8.3.5 Adult size...... 108

8.4 Discussion...... 109

CHAPTER 9: Multitrophic interactions in a potato-aphid- parasitoid system: influence of root herbivory and soil nutrition

9.1 Introduction...... 113

9.2 Materials and Methods...... 115

9.2.1 Plant production...... 115

9.2.2 Insect culture...... 115

9.2.3 Parasitoids...... 115

9.2.4 Root knot nematodes (RKN)...... 115

9.2.5 Experimental treatments...... 116

9.2.6 Foliar herbivore performance...... 116

9.2.7 Parasitoid performance...... 117

9.2.8 Plant performance...... 118

9.2.9 Data analysis...... 118

9.3 Results...... 119

9.3.1 Plant performance...... 119

9.3.2 Foliar herbivore performance...... 121

9.3.3 Parasitoid performance...... 124

9.4 Discussion...... 128

CHAPTER 10: Summary and General Discussion

10.1 Summary of the experimental findings...... 133

10.2 General Discussion ...... 135

10.2.1 Soil nutrition...... 135

10.2.2 Host plant resistance...... 136

10.2.3 Root herbivory...... 138

10.2.4 Conclusion...... 139

10.3 Future work...... 139

References……………………………………………………………..…….141

Appendices………………………………………………………………...... 192

CHAPTER 1

INTRODUCTION

1.1 Background

All organisms in an ecosystem are linked chemically and their relationship is apparent in food chains and food webs (van Veen et al. 2006; Olff et al. 2009). If linear food chains in a generalized food web are considered, these contain at least three trophic levels

(Price et al. 1980; Schmitz et al. 2000). In tritrophic interactions, organisms at lower trophic levels are forced to evolve to reduce feeding by organisms at higher trophic levels; whereas organisms at higher trophic levels evolve to increase consumption (Price et al. 1980; Hare

2002).

To understand tritrophic interactions, it is necessary to study the chemical factors

(e.g. plant secondary compounds including plant volatiles, insect pheromones and soil nutrients) that mediate them. For example, when using natural enemies to control herbivorous pests it is essential to understand the interactions between the plants, herbivores and natural enemies that are involved. Manipulation of plant attributes that promote the success of the natural enemies may enhance pest control (Verkerk et al.

1998a; Cortesero et al. 2000; Symondson et al. 2002; Powell and Pickett 2003; Poppy and

Sutherland 2004; Wilkinson and Landis 2005; Fiedler et al. 2008; Greenstone et al. 2010).

Price et al. (1980) highlighted the importance of also considering the third trophic level in insect–plant interactions. Thus, natural enemies of herbivores can help plants by reducing herbivore damage, and plants can help natural enemies by making herbivores more vulnerable to them (Nordlund 1981).

Numerous studies on tritrophic interactions have contributed to our knowledge of how plants, herbivores, and parasitoids interact between different trophic levels and what consequences this can have for their respective populations (Poppy and Sutherland 2004;

Turlings and Wäckers 2004; Ode 2006; Stout et al. 2006; Zehnder et al. 2007; Poelman et al. 2008; Schädler et al. 2010). Tritrophic interactions include various aggressive and defensive interactions both between and within trophic levels, including morphological, behavioural, and physiological relationships (de Moraes et al. 2000; Bowers 2003; Bezemer et al. 2005; Vencl et al. 2005; Griffin and Thaler 2006).

Plants possess a variety of biochemical and physical defence mechanisms that can deter, poison, or starve herbivores feeding on them (Hartley and Jones 1997; Sabelis et al.

1999; Kessler and Baldwin 2002; Dicke and van Poecke 2002; Gershenson 2002; Strauss and Murch 2004; Arimura et al. 2005; Schilmiller and Howe 2005; Stout 2007; Bruinsma et al. 2008; Agrawal and Konno 2009). Interactions between plants and parasitoids may have evolved in a race between plants and herbivores. For instance, where there has been a competitive advantage for plants that produce more efficient signals when attacked, for parasitoids that utilize these signals, and for herbivores that minimise plant responses to herbivory (de Moraes et al. 2000; Bezemer et al. 2003; Nyman et al. 2007; Bailey et al.

2009).

Many plant secondary compounds (metabolites) have toxic effects on herbivores and pathogens, while other plant defences appear to have indirect effects upon pests and pathogens, such as attracting predators and inhibiting insect oviposition (Elbanna 2007).

One trophic level can affect diversity maintenance in other trophic levels (Chesson and

Kuang 2008). In these interactions, herbivore-induced volatiles of plants attract the third trophic level, e.g. natural enemies. It has been shown that such induced volatiles play an important role against insect herbivores and pathogens (Mauricio et al. 1997; Stotz et al.

2000; Scutareanu et al. 2003; Snoeren et al. 2007; Liu et al. 2009; Snoeren et al. 2010;

Dicke and Baldwin 2010).

Potato

Potatoes, Solanum tuberosum L. (Solanales: Solanaceae) were first domesticated in

Peru and Chile and introduced to the British Isles around 1600 AD. Over the next century

14 their popularity grew to such an extent that they became the staple diet of a large part of the population. Potatoes are a valuable crop in the United Kingdom and worldwide, including

Pakistan (Section 2.3).

Potatoes are attacked by various insect pests, including borers and sap feeders

(Section 2.3). Among these insect pests, aphids can cause considerable economic loss through direct and indirect damage to the crop worldwide (Raman 1984; Hooker 1986; Shah

1988; Blackman and Eastop 2000; Kuroli and Lantos 2006; van Emden and Harrington

2007). Because of their role as virus vectors (over 13 viruses are transmitted by aphids in potato), aphids are probably of greater economic importance as potato pests worldwide than are insect defoliators or tuber pests (Hanafi 2006). Apart from insect pests, plant parasitic nematodes, particularly cyst and root-knot species, are also a major threat and play an important role in reducing potato production all over the world (Hanafi 2006; Scurrah et al. 2005; Mugniéry 2007; Hodda and Cook 2009).

Aphids

Aphids (Hemiptera: Aphididae) are phytophagous insects that feed on plant phloem, producing serious damage to crops (Section 2.4; Dixon 1998). The peach-potato aphid

Myzus persicae Sulzer (Hemiptera: Aphididae) is a generalist aphid species, attacking many host plants including potatoes (van Emden et al. 1969; Blackman and Eastop 2000).

Although chemical pest management has been implemented with relative success against aphid pests of agriculture (Parker et al. 2002), their capacity to develop insecticide resistance has become an important problem (Field et al. 1988; Field and Devonshire 1997;

Devonshire et al. 1998; Foster et al. 2000; Denholm and Devine 2001; Field and Foster

2002; Srigiriraju et al. 2009). Unlike many lepidopteran and some coleopteran pests, there has been no commercialization of transgenic crops expressing toxins for aphid control

(Sharma et al. 2000; Romeis et al. 2006; Burgio et al. 2007). Cultural and biological methods for aphid control have become increasingly important in protected crops and more widely as part of integrated crop management programmes (Albajes et al. 1999; Gullino et

15 al. 1999; Heinz et al. 2004; Yano 2006; van Emden and Harrington 2007; Bailey et al.

2009).

The role of natural enemies in the regulation of insect populations has been the subject of considerable debate (Stiling 1988; Walde and Murdoch 1988; Murdoch 1990;

Hawkins 1992; Kos et al. 2008). Among the natural enemies of aphids, parasitoid wasps can play an important role in controlling the aphids including pest of potato crops (Tomanovi and Brajkovi 2001; Herzog et al. 2007). Among parasitoids, the genus Aphidius

(Hymenoptera: Braconidae) contains numerous species which attack and provide biological control of aphids (Section 2.5). Aphidius colemani Viereck (Hymenoptera: Braconidae) is the most commonly used parasitoid species for controlling aphid species in protected crops

(Kim and Kim 2003; Vásquez et al. 2006; Van Driesche et al. 2008).

1.2 Aims and objectives

The main aim of the project was to understand how two soil-based factors (soil fertilizer levels, plant pathogens) and host plant resistance influence multitrophic interactions in a potato - aphid - parasitoid system.

Objectives:

1) To assess aphid (M. persicae) performance on a range of potato cultivars under controlled environmental conditions to determine which were the most susceptible and resistant cultivars.

2) Using the most aphid resistant and aphid susceptible cultivars (1), to assess the separate effects of soil fertilizer levels, plant cv and pathogen (plant parasitic nematode) on aphid performance.

3) To conduct multifactorial ditrophic (aphid performance) and tritrophic (parasitism success) experiments with selected combinations of soil fertilizer or pathogen treatments.

4) Where significant differences occur between treatments in multifactorial experiments, to use whole plant olfactometry on aphids to determine whether plant volatiles are involved in specific trophic interactions.

5) To conduct field trials (UK and Pakistan) with potato cultivars to help determine the extent that the effects of cultivars on aphid performance observed in the laboratory are manifested under field conditions.

1.4 Hypotheses

The above objectives are linked to the following hypothesis:

1) That soil based factors will significantly influence aphid performance on foliage through direct effects on plant quality and indirect effects (e.g. plant volatiles) on parasitism.

[Objectives 2, 3, 4 and 5].

2) That host plant resistance will be a major compounding factor with soil-based factors in influencing aphid performance [Objectives 1, 2, 3 and 4].

CHAPTER 2

LITERATURE REVIEW

2.1 Multitrophic interactions

In the last three decades, the study of trophic interactions between organisms has evolved from work on plant-herbivore or prey-predator interactions to a more complex, multitrophic approach involving three or more trophic levels that addresses the complexity of food webs much more realistically (Tscharntke and Hawkins 2002; van der Putten et al.

2004; Green et al. 2005; Hopkins et al. 2009; Megίas and Müller 2010).

Research on multitrophic interactions aims to identify the forces that regulate populations, including bottom-up forces controlled by resources and top-down forces controlled by predators and antagonists. Multitrophic interactions are however more complex than linear bottom-up / top-down interactions. For instance, food webs can be characterised in two dimensions: vertically, in which they form a food chain of rarely more than four or five trophic levels, or horizontally, i.e. within one trophic level (Hassell and

Waage 1984; Murray et al. 2006; Wardle 2006; Huntley and Kowalewski 2007; Schneider and Maraun 2009; Harvey et al. 2010). Ecological interactions between two species are also often indirectly mediated by a third species of the same or another trophic level (Bronstein and Barbosa 2002; Salminen et al. 2010). This is found in intra-guild interactions, where two species that share a host or prey also engage in a trophic interaction with each other

(Rosenheim et al. 1995; Rosenheim 1998; Moore et al. 2003; Putten et al. 2009), and in apparent competition, where two species, that do not come into direct contact, interact because they share a natural enemy (Holt and Lawton 1993; Denno et al. 1995; Chaneton and Bonsal 2000; Brassil and Abrams 2004; van Veen et al. 2006; Diaz et al. 2010).

The present study focused on bottom-up interactions and especially the effects of soil nutrition and soil pathogens on herbivores and their parasitoids (Fig. 2.1).

Fig. 2.1 Conceptual model of interactions between three trophic levels; solid lines show bottom-up interactions, dotted lines show top-down interactions (from Jansson 2003).

In insects, interactions between plants, herbivores and their natural enemies are among the most studied multitrophic interactions (e.g. Turlings et al. 1990; Vet and Dicke

1992; Vet et al. 1995; Lewis et al. 1997; Verkerk et al. 1998a; Dicke 1999; Singer and

Stireman 2003; Whitfield 2003; Novotny et al. 2006; Dyer et al. 2007; Inbar and Gerling

2008; Connahs et al. 2009; Harvey et al. 2010). In this type of food chain, the first trophic level, represented by the plant, influences the herbivore (the second trophic level) by its quality and quantity as a food-source. At the third trophic level, natural enemies limit herbivore populations by mortality. As indicated above, interactions between herbivores and their host plants and between herbivores and their natural enemies can only be understood when they are considered together within a multitrophic context (Fig 2.1; Price et al. 1980;

Faeth 1992; van der Meijden and Klinkhamer 2000; Hare 2002; Jansson 2003; Inbar and

Gerling 2008; Desneux and Ramirez-Romero 2009). For instance, the first trophic level

(plant) can also influence the efficiency of the third trophic level (natural enemy) by providing shelter; mediating host/prey accessibility and availability; providing host/prey

19 finding cues; influencing host/prey suitability; and providing supplemental food sources for natural enemies (Cortesero et al. 2000). Natural enemies may „help‟ the plants, using the benefits named above, thus limiting the herbivore population more than would be otherwise possible (Godfray 1994; Bottrell and Barbosa 1998; Van Rijn et al. 2002; Brewer and Elliott

2004; Almohamad et al. 2009).

The influence of a host plant can act directly on the third trophic level for instance by plant volatiles attracting parasitoids (Dicke 1999; Shiojiri et al. 2001; Hilker and Meiners

2002; Steidle and van Loon 2003; Fatouros et al. 2005; Liu 2009; Zhang and Schlyter

2010), plant structures that hinder parasitoids searching for hosts, or plant structures that can provide parasitoid shelter or food for parasitoids (Lovinger et al. 2000; Olson and

Andow 2003; Mulatu et al. 2006; Carrillo 2008; Wise et al. 2010). The influence of a host plant can also act indirectly, for instance by influencing the nutritional quality of the parasitoid´s host (Harvey and Gols 1998; Jansson 2003; Urrutia et al. 2007; Sarfaraz et al.

2009a,b; Sarfaraz et al. 2010; Blake et al. 2010). A parasitoid is also influenced by the host itself, for instance by its preference for a particular developmental stage or stages (Hågvar and Hofsvang 1991; Liu et al. 2004; Li et al. 2006; Martinou and Wright 2007; Mironidis and

Savopoulou-Soultani 2009; Amarasekare et al. 2010).

2.2 Soil nutrition and insect-plant interactions

Soil nutrition means nurturing the soil to the point where it is able to grow nutrient- dense plants and not just maintain fertility but improve fertility with each crop grown (Cook

2010). Soil nutrition plays an important role in multitrophic interactions among plants, phytophagous insect and their parasitoids through its effects on plant quality (Van der

Putten 2001; Harvey et al. 2003; Ohghushi 2005; Blake et al. 2010; Staley et al. 2010).

2.2.1 Effect of soil nutrition on plants

Ingestad (1987) suggested that the relative addition rate of each nutrient, rather than the nutrient concentration in fertiliser or soil is important for plant growth, and that any nutrient shortage can slow down the plant relative growth rate. Some nutrients, e.g. potassium (K) and phosphorous (P) are known to be taken up in excess by plants when available (Mengel and Kirkby 1987; Mengel et al. 2001; Jansson 2003). Excess of nutrients can disturb the uptake of other nutrients (Mengel and Kirkby 1987; Prasad and Power 1997;

Mengel et al. 2001). In general, increased supply of one cation species can lead to lower uptake of other cations. For example, K competes strongly with the uptake of other cations

(Prasad and Power 1997). An increase in soil nitrogen (N) fertilisation can raise the content of both nitrate (N03) and soluble amino acids in the plant, while N deficiency can lead to hydrolysis of proteins and redistribution of amino acids from older leaves to younger organs.

In both cases, the N content in the phloem is increased (Mengel and Kirkby 1987; Mengel et al. 2001; Barker and Pilbeam 2006).

In the phloem, assimilates are transported from actively photosynthetic leaves to growing tissues and breakdown products are transported from senescing leaves to growing tissues (Sengbusch 2003). The phloem content is not only influenced by the plant‟s uptake of nutrients but also by the growth of the plant and shifts between growth and senescence of plant tissues. Potassium is vital for the water balance in the plant and influences the uptake of NO3 and the assimilation of CO2; K deficiency can lead to decreased protein synthesis and accumulation of amino acids (Mengel and Kirkby 1987; Mengel et al. 2001;

Chapagain and Wiesman 2004; Barker and Pilbeam 2006), probably due to inadequate energy (ATP) supply. Phosphorous is essential for energy transfer in plants and deficiency can affect various processes, such as protein synthesis and active ion uptake and thus lead to retarded plant growth (Schachtman et al. 1998; Hermans et al. 2006; Zekri and Obreza

2009).

2.2.2 Indirect effects of soil nutrition on insect herbivores

Soil nutrient availability can alter the nutritional quality of plants and this can influence herbivore growth and reproduction (Larsson 1989; Waring and Cobb 1992 Altieri and Nicholls 2003; Facknath and Lalljee 2005; Hwang et al. 2008; Vandegehuchte et al.

2010b). Nitrogen is one of the plant nutrients that plays an important role for herbivore performance (Mattsson 1980; Scriber 1984; Scriber and Slansky 1981; Bruyn 2002; Altieri and Nicholls 2003; Pennings and Simpson 2008; Sauge et al. 2010). This is especially true in the case of aphids, because the N content is low in the phloem. Phloem sap contains

0.004-0.6% w/v N while most plant tissues contain at least 1% N of dry weight (Mattson

1980).

Nitrogen fertilisation of plants has, in many cases, been shown to have a positive effect on aphids and other phloem feeding insects (van Emden 1966; Waring and Cobb

1992; Nevo and Coll 2001; Throop and Lerdau 2004; Sauge et al. 2010). In 115 studies, crop damage by insect pests increased when the N content of the host plant was increased

(Schoonhoven et al. 2005). In a review, Waring and Cobb (1992) reported that in approximately 50% of studies the response of phloem feeding insects was positive to N fertilisation and in 25% there was no response. While Van Emden (1969) found that in about

40% of the studies he reviewed aphids responded positively to N fertilisation, in 35% they responded negatively, and in about 25% there was no response. One reason for the varying results may be that different plants and different aphid species react differently. For instance, the nutritional demands are not identical for M. persicae and the potato aphid,

Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae), Myzus persicae colonize on the lower senescing leaves while M. euphorbiae mostly found on the upper young leaves

(Aldamen and Gerowitt 2009). Many experiments have shown that M. persicae is positively influenced by N fertilisation (Harrewijn 1983; Jansson and Smilowitz 1986; Nevo and Coll

2001; Jansson and Ekbom 2002; Zehnder and Hunt 2008; Sauge et al. 2010).

Low soil K content has been shown to be positive for M. persicae (Jansson 2003) and Aphis glycines Matsumura (Hemiptera: Aphididae) (Myers and Gratton 2006), High levels of K have mostly been shown to influence reproduction negatively or not at all for aphids and other phloem feeding insects (Myers et al. 2005; Myers and Gratton 2006).

However, some studies have shown a positive effect of increased K on the performance of insects (Woolridge and Harrisson 1968; Perrenoud 1990). Waring and Cobb (1992) concluded that fertilisation with P does not generally influence phloem feeding insects or influences them positively. Skinner and Cohen (1994) reported that P deficiency reduced whitefly ovipositon on true leaves by 40% in the growth chamber and by 38% in the youngest expended leaves in the greenhouse. Bishop et al. (2010) studied the relative abundance of eight arthropod orders and five families in plots that received N, P, or no nutrients for 3–5 years and concluded that in Year 3 orthopteran abundance was associated with a P-mediated increase in plant cover, whereas in Year 5 orthopteran density was not related to cover, diversity or plant %N but rather to unmeasured effects of P.

The ratios between soil nutrients have also been shown to influence herbivores

(Busch and Phelan 1999; Jansson and Ekbom 2002; Huberty and Denno 2006). Van

Emden and Bashford (1971) found that a high N:K ratio was favourable for M. persicae on

Brussels sprout. Harrewijn (1983) found a positive effect of high N:K ratio in the fertilisation of potatoes on M. euphorbiae. Apple et al. (2009) conducted a study on the performance of gelechiid larvae on greenhouse-grown lupins with factorial manipulation of competitors and soil N and P. In the presence of competition, larval mass was highest at intermediate N:P and high % P. Huberty and Denno (2006) also highlighted the importance of interfacing herbivore life-history strategies with ecological stoichiometry (elemental ratios that affect ecological processes at scales from the cellular to the global) of N and P in order to interpret the consequences of N and P limitation on herbivore performance and population dynamics.

Soil nutrition may also alter the attractiveness of plants to insects by enhancing plant vigour, morphology and phenology (Rahier 1978; Stadler 1992; Jansson 2003; Schonhof et

23 al. 2007; Facknath and Lalljee 2005; Sarfraz et al. 2005; Shennan 2008; Hsu et al. 2009;

Sarfraz et al. 2010).

2.2.3 Indirect effects of soil nutrition on parasitoids

Insufficient nutrients in the host can affect development and survival, sex ratio, longevity, fecundity, and the size of parasitoid wasps (Vinson and Iwantsch 1980; Godfray

1994; Hunter 2003; Karimzadeh and Wright 2008; Sarfraz et al. 2009b). Askew and Shaw

(1986) divided parasitoids into two groups based on their life style: koinobiont and idiobiont, which are mostly ectoparasites (e.g. Mastrus ridibundus Gravenhorst (Hymenoptera:

Ichneumonidae) (Section 2.5). For koinobiont parasitoids, such as Aphidius spp., host quality is determined both by host resources at parasitization and the resources the host acquires during parasitoid development (Mackauer et al. 1997; Mackauer and Chau 2001;

Hunter 2003; Jervis et al. 2008; Boivin 2010). The quality of an herbivore as a host for a parasitoid is affected by the quality of the herbivore‟s nutrient intake (Vinson and Barbosa

1987; Fox et al. 1990; Awmack and Leather 2002; Sarfraz et al. 2009a; Gharratt et al. 2010;

Sarfraz et al. 2010).

Soil nutrition has been suggested as one way of manipulating plant attributes for improved biological control (Verkerk et al. 1998a; Cortesero et al. 2000). In a study by Bentz et al. (1996), parasitization of the whitefly Bemisia argentifolii Bellows and Perring

(Hemiptera: Aleyrodidae) by Encarsia formosa Gahan (Hymenoptera: Aphelinidae) was greater on plants treated with calcium nitrate than on unfertilised plants or plants treated with ammonium nitrate.

In field and laboratory studies, the parasitoid Diadegma insulare Cresson

(Hymenoptera: Ichneumonidae) preferred host larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), reared on plants fertilised with NPK and micro nutrients compared with hosts on unfertilised plants (Fox et al. 1996). In a study by Stadler and Mackauer (1996), the negative effects of unfertilised plants on A. pisum (lower fecundity, small body size and prolonged developmental time) were reflected in negative

24 effects on the aphid parasitoid, Ephedrus californicus Baker (Hymenoptera: Aphidiidae) in the form of a more male biased sex ratio and high parasitoid mortality. Sarfraz et al.

(2009b) evaluated the bottom-up effects of five soil fertility regimes on nutritional quality of canola (Brassica napus L.) and on several fitness correlates of female and male D. insulare

(% parasitism, survival, developmental time and longivity) as mediated through P. xylostella.

Variation in soil fertility influenced the nutritional quality of host plants and this in turn affected the performance of D. insulare; with D. insulare performing best on plants grown with 3.0 g fertilizer pot−1. These plants had 2.06-, 3.77-, and 1.02-fold more NPK respectively than ones grown without any added fertilizer.

2.3 Potato

The largest producers of potatoes worldwide are: China, Russia, India, United states and Germany (World Potato Atlas 2010). The United Kingdom ranks No. 11 among world potato producing countries, recording a 2009 harvest of 6 million tonnes and average yields exceeding 45 tonnes per hectare; with an annual per capita potato consumption of 102 kg

(FAOSTAT 2009). Pakistan is the seventh largest potato producing country in the world and potato has emerged as an important cash crop with over 134,000 ha grown p.a. Potato production in Pakistan has risen significantly in recent years and reached 2,941,000 tonnes in 2009 (Agricultural Statistics of Pakistan 2009).

Potatoes are attacked by a large number of insect pests, including borers, aphids, whiteflies, jassids and beetles (Visser 2005; Grafius and Douches 2008). Insect pests of potato can be categorized into three different general types (Hanafi 2007). The most apparent ones are those that damage leaves or stems such as the Colorado potato beetle,

Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) and flea beetles, e.g. Epitrix tuberis Gentner (Coleoptera: Chrysomelidae). Less obvious are those that damage tubers or roots, such as potato tuber moth, Phthorimaea operculella Zeller (Lepidoptera:

Gelechiidae), cutworms, e.g. Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae), wireworms

(Agriotes spp. (Coleoptera: Elaterlidae)), and white grubs or chafer grubs (Phyllophaga spp.

(Coleoptera: Scarabaeidae)) (Hanafi 2007).

Aphids such M. euphorbiae, M. persicae, Aphis nasturti Kaltenbach (Hemiptera:

Aphididae) and Aulacorthum solani Kaltenbach (Hemiptera: Aphididae) and leafhoppers, e.g. Empoasca fabae Harris (Hemiptera: Cicadellidae) cause damage by feeding on the plant sap, and some are vectors of important potato viruses (Piron 1986; Radcliffe et al.

1993; Radcliffe and Ragsdale 2002; Andret-link and Fuchs 2005; Pelletier et al. 2008;

Verbeek et al. 2010).

Potato occupies a prominent position among vegetable crops consumed worldwide due to its high productivity and good nutritional and calorific value (Burton 1989; Gould

1999; Romans 2005). High nutritional and water content however make potatoes vulnerable to attack by pests and diseases, both in the field and in storage (Radcliffe 1982;

Malik 1995; Sharma and Ortiz 2000; Strand 2006; Johnson 2007; Kapsa 2008; Boiteau

2010).

2.4 Aphids

Aphids are considered by many to be the most important insect pests in temperate zone agriculture (Hill 1987; Blackman and Eastop 2000; Sorensen 2009; Hulle et al. 2010).

They primarily cause damage either by direct feeding and subsequent nutrient loss from the plant or by the transmission of plant viruses (Pickett et al. 1992; Ng and Perry 2004; Ng et al. 2005; Alvarez et al. 2007; Brault et al. 2010). Aphids can also adversely affect crops through toxic compounds in their saliva as well as reducing photosynthetic capacity indirectly in leaves through excretion products and saprophytic fungi (Schepers 1989).

Aphids feeding can also induce galling, tissue senescing and leaf curling (Miles 1999).

Aphids range in size from 1.5 to 3.5 mm (Borror et al. 1976). They are pear-shaped, globose, ovate, and spindle-shaped or elongate in shape and vary greatly in their body markings and colour (black, grey, red, orange, yellow, green, brown, blue-green, white-

26 marked, wax-covered, etc.). Single species show polymorphism, with several colour forms and shapes and may be winged (alate) or wingless (apterous) (Blackman and Eastop

2000). Winged forms are usually triggered by environmental changes, i.e. decreasing photoperiod (special sort of alates in host alternating species) or temperature, deterioration of the host plant or overcrowding (Kennedy and Stroyan 1959; Dixon 1987; Dixon 1998;

Flint 2000; Blackman and Eastop 2000; Van Emden and Harrington 2007; Sorensen 2009).

The life cycle of aphids can be complex (Kundu and Dixon 1995; Drees 1997). Most aphids reproduce sexually and develop through incomplete or hemimetabolous metamorphosis but also through parthenogenesis (Blackman and Eastop 2000). Incomplete metamorphosis is the mode of insect development that includes three distinct stages: the egg, nymph, and the adult stage, or imago. These groups go through gradual changes; there is no pupal stage (Minks and Harrewijn 1987). In greenhouse conditions A. gossypi may complete up to 50 generations a year parthenogenetically, with each adult producing about 85 young (Little 1972; Dress 1997). Adult maturity for A. gossypi is reached in four to ten days and the reproductive period is about three weeks (Little 1972). Similarly M. persicae in moderate climates can produce up to 20 generations in one year (Blackman and

Eastop, 2000).

Most aphids are relatively host specific due to their specific dietary requirements, as the phloem sap which they feed on has a strictly defined composition that differs between different plant species (Dixon 1987; Cole 1994; Blackman and Eastop 2000; Peccoud et al.

2010)., Some aphids are however generalist feeders, such as M. persicae, and can have wide host ranges (Kennedy 1958; Theunissen 1989; Margaritopoulos et al. 2005; Peccoud et al. 2010).

Aphids can be controlled by manipulating environmental conditions such as temperature, humidity and moisture (Awmack et al. 1997; Hallman and Denlinger 1998; Mcvean and

Dixon 2001), by cultural control (Hill 1989; Vincent et al. 2003) by chemical control, and by biological control (van Emden and Harrington 2007). Chemical control is the most frequently used method to suppress insect pests (Parker et al. 2002). Chemical control is easy to

27 apply and relatively inexpensive (Chiras 2009). It is estimated that without pesticides, crop losses to pests would increase by 15 - 20 % (Paoletti and Pimentel 2000). There is, however, an increasing problem with insecticide resistance (Foster et al. 2005; Foster et al.

2007; Fenton et al. 2010). The efficacy of insecticides used to control M. persicae are heavily influenced by the level of resistance of the pest towards these chemicals (Foster and Devonshire 1999) and the problem has worsened in glasshouse crops since more individuals carry different combinations of the resistance mechanisms (Foster et al. 2003;

Ghadamyari et al. 2008; Fenton et al. 2010). Widespread use of insecticides carries drawbacks not only for the environment but also for aphid control because important species like M. persicae have developed resistance to most major insecticide classes including organochlorines, organophosphates, carbamates and pyrethroids (Devonshire and

Field 1991; Robert et al. 2000; ffrench-Constant et al. 2004; Anstead et al. 2007; Fenton et al. 2010). Therefore, alternative approaches for its control are needed. To date, genetically modified (GM) crops expressing insecticidal Bacillus thuringiensis crystal (Cry) toxins, which were first introduced commercially in 1996 (Fernandez-Cornejo and McBride 2002;

Toenniessen et al. 2003; Brookes and Barfoot 2005; Ali et al. 2006; James 2007; Lemaux

2008; USDA 2008; Lemaux 2009), have proved very successful in controlling lepidopteran and coleopteran pests of cotton and maize in particular. Commercial GM crops expressing traits conferring resistance to sucking insect pests have however yet to be developed

(Castle et al. 2006; Gatehouse 2008).

Another possibility to control aphids is the use of the non-GM resistant crop cultivars.

Integrating host plant resistance into a pest management system is an economical way to control aphids. Successful examples of the use of host plant resistance to control aphids include resistance to Russian wheat aphid, Diuraphis noxia Mordvilko (Hemiptera:

Aphididae), on wheat (Triticum aestivum L) (Basky 2003; Lynch et al. 2003; Randolph et al.

2003) and on barley (Hordeum vulgare L.) (Bregitzer et al. 2003). More than 60 wild

Solanum species have been reported to possess genotypes with resistance to M. persicae

(Gibson and Pickett, 1983; Flanders et al. 1999; Flanders et al. 1992; Novy et al. 2002; Le

Roux et at. 2008, Le Roux et al. 2010) and these species represent an important alternative source of aphid resistance, which can be exploited to enhance aphid‟s resistance in cultivated potato (Le Roux et at. 2008, Le Roux et al. 2010). But so far, no commercial potatoes have been specifically developed for insect resistance (Flanders et al. 1999;

Jauhar 2006).

Another method for control of insect pests is the use of natural enemies (Hajek et al.

2004). Populations of all living organisms are subject to some extent to reduction by natural enemies, a process known as natural or biological control, and it has been suggested that around 99% of all potential pests are controlled by their natural enemies (Hajek 2004).

When pests are controlled with human intervention by using their predators, parasites, antagonists and diseases, it is known as „biological control‟ (Hajek 2004). In non- subsistence agriculture, where chemical pesticides have often been overused, biological control has increasingly been considered/adopted when pesticides do not work as expected due to resistance or resurgence (where pesticides reduce natural control) and/or where negative environmental and health side-effects of chemical pesticides become unacceptable (Zadoks 1991; Stefan et al. 1995; World Bank 2005; Séralini et al. 2009).

The reliance on chemical control methods over the past 50 years has thus led to various problems for pest management, shifting pest management towards a more sustainable and balanced approach that reflects cultural, biological and biorational standards (Maredia 2003; Koul and Cuperus 2007). As a consequence integrated pest management (IPM) first promoted over 40 years ago is gaining increasing acceptance

(Peshin and Dhawan 2009). IPM, which can include chemical control, combines a range of methods described above and prioritizes a good understanding of the pest biology and ecology, its interaction with the ecosystem, the importance in society and economics (Norris et al. 2003). To illustrate the importance of IPM, in 2003 5% of protected crops worldwide were under IPM, and this is projected to increase to 20% in the next 3 years (van Lenteren

2003). Both conventional and organic growers indicate an increasing interest in the use of

IPM strategies for the control of insect‟s pest both in greenhouse and out door (Cook et al.

2007; Pimentel 2007; Bale 2008; Lester 2009; Pilkington et al. 2010).

2.4.1 Myzus persicae

Myzus persicae has a worldwide distribution (Blackman and Eastop 2000; van

Emden and Harrington 2007; Peccoud et al. 2010) and is the primary aphid pest species on potato crops (Kuroli and Lantos, 2006). Apart from its primary host, Prunus spp., hundreds of plant species have been identified as secondary hosts in more than 40 plant families

(Blackman and Eastop 2000; Van Emden and Harrington 2007). Myzus persicae causes damage principally by its ability to transmit plant viruses (Salazar 1996; Ragsdale et al.

2001; Radcliffe and Ragsdale 2002).

At least 54 viruses are known to infect potatoes (Brunt et al. 1996) and aphids are their most important vectors (Peters 1987; Raman and Radcliffe 1992; Nault 1997; Jeffries

1998; Blackman and Eastop 2000; Basky 2002; Radcliffe and Ragsdale 2002; Qi et al.

2004; Kuroli and Lantos 2006; Anstead et al. 2008). In potato crops, the percentage of plants infected with Potato leaf roll virus (PLRV) strongly depends on the number of aphids that have previously visited PLRV-infected plants (Beekman 1987; van den Heuvel et al.

1993; Thomas et al. 1997; Radcliffe and Ragsdale 2002). Myzus persicae is the most efficient, cosmopolitan, and commonly abundant vector of PLRV (Woodford et al. 1995;

Robert and Bourdin 2000; Robert et al. 2000) and Potato virus Y (Piron 1986; Ragsdale et al. 2001 Radcliffe and Ragsdale 2002). Kanavaki et al. (2006) evaluated the transmission efficiency of Potato virus YN from and to tobacco plants by M. persicae nicotianae

Blackman, a tobacco specialist, and M. persicae a generalist, and found that M. persicae had a significantly higher propensity to transmit PV YN (31.9 versus 15.3%).

Due to its importance as a virus vector, M. persicae is considered of great economic importance in all potato growing areas (van Emden et al. 1969; Radcliffe 1982; Radcliffe et al. 1999; Radcliffe and Ragsdale 2002; Kuroli and Lantos, 2006). Castle and Berger (1993) and Castle et al. (1998) reported that M. persicae grows faster, has higher fecundity and

30 preferentially settles on cultivated potato infected by the potato leaf-roll virus (PLRV) than on uninfected potato plants. Eigenbrode et al. (2002) and Srinivasan et al. (2006) have observed that M. persicae prefers PLRV-infected potato plants to non-infected plants, PVY- infected plants or PVX-infected plants.

2.4.2 Host plant resistance

Painter (1951) defined resistance of plants to insect attack as the relative amount of heritable qualities possessed by the plant that influence the ultimate degree of damage done by the insect. Plant resistance is, however, relative and can be measured only by comparison with other genotypes, and it can be modified by physical, chemical and biological factors (Auclair 1989; van Emden 2007; Pedigo and Rice 2009). The multiplication of aphids and the development of an infestation on plants are highly dependent on either genetically inherent or environmentally induced plant characteristics

(van Emden et al. 1969; Wellings and Ward 1994; van Emden 2007).

For phytophagous insects, host plants represent heterogeneous resources due to temporal and spatial variability in host quality and availability (Fox et al. 1995; Olivares-

Donoso et al. 2007). Plant cultivars can vary with respect to various morphological and physiological traits, including trichome number, tissue toughness, leaf shape, leaf structures, wax contents, secondary plant substances, primary metabolite content and the number of genes involved in these traits (Khan et al. 1986; Sandström and Pettersson 1994;

Panda and Khush 1995; Castro et al. 1996; Legrand and Barbosa 2000; Gniwotta et al.

2005; Goggin et al. 2007; Campitelli et al. 2008; Kang et al. 2010). All these factors may have direct or indirect effects on herbivore performance (van Emden 2007; Khan and Port

2008; Raupp et al. 2010).

Host plant resistance can be classified as either antibiosis (changes in insect biology and demographic parameters) or antixenosis (changes in insect behavior leading to low or non-acceptance of the host plant) (Flanders et al. 1992; Panda and Khush 1995; Le Roux et al. 2007, 2008). Antixenosis-based resistance may be conferred by glandular trichomes

(Yencho and Tingey 1994; Alvarez et al. 2006; Pelletier and Dutheil 2006) and volatile organic compounds (VOC) of plants (Holopainen 2008; Dewhirst and Pickett 2010).

Antibiosis-based resistance may be due to protease inhibitors (Goggin 2007), the accumulation of alkaloids (Cardoza et al. 2006), cardenolides (Zehnder and Hunter 2007), glucosinolates (Broekgaarden et al. 2008) and a diverse range of phenolic compounds

(Ranger et al. 2007).

Variation in host-plant characteristics may influence the population dynamics of herbivores (Hunter and Price 1992; Panda and Khush 1995; van Emden 2007). Several studies have demonstrated that plant genotypes vary in their suitability for insect growth, survival and reproduction (Panda and Khush 1995; Leimu and Koricheva 2006; Wise 2007;

Leimu et al. 2008). This variation in plant genotypes may affect the life history parameters such as fecundity, longevity and survival of insects (Cronin and Abrahamson 1999;

Underwood and Rausher 2000; Nikolakakis et al. 2003; Fritz and Hochwender 2005, Davis et al. 2007; Johnson and Agrawal 2007; Johnson 2008 ; Schädler et al. 2010).

2.5 Parasitoids

A parasitoid is „an organism which develops on or in another single ("host") organism, extracts nourishment from it, and kills it as a direct or indirect result of that development' (Eggleton and Gaston 1990, following Kuris 1974). It is estimated that around

25% of all insects are „parasitoids‟ (Godfray 1994). While there are around 70,000 described species of parasitoids, it is believed that there could be more than 1.5 - 2 million still undescribed (Godfray 1994; Quicke, 1997). About 75% of the species belong to the orders Hymenoptera (Feener and Brown 1997; Belshaw and Quicke 2002) but the orders

Diptera, Coleoptera, Lepidoptera, Trichoptera, Neuroptera and Strepsiptera also have representatives (Godfray 1994; Quicke 1997).

The main characteristic shared by parasitoids is the need for a second insect species to complete their life cycle. While adult parasitoids are free-living, their larvae use a

32 second insect species as a food source (Godfray 1994; Quicke 1997). Among parasitoids, the genus Aphidius (Hymenoptera: Braconidae) is a large group containing numerous species which attack and provide biological control of aphids in horticulture, field crops and urban landscapes (Kos et al. 2008). Female Aphidius spp. lay eggs singly in aphid nymphs.

The wasp larvae then hatch and consume the aphids from inside.

Parasitoids can be divided into two different groups, depending on host growth: koinobionts, which usually allow the host to continue its development after oviposition, and idiobionts, that do not (Askew and Shaw 1986). Idiobionts inject venom into their host during oviposition, either causing permanent paralysis or arrested development (Gauld and Bolton

1988). While koinobionts do not paralyse the host, or do so temporarily, and the host can continue to grow while the parasitoid larvae feed on its hemolymph or non vital organs

(Shaw 1997; Völkl et al. 2007).

Parasitic wasps (parasitoids) are important biological control agents as discussed earlier and are used extensively in biological control and IPM in many parts of the world

(Purcell 1998; Gahukar 2006). Some parasitoids are specific to aphids and several species of these are produced commercially for use against aphid pests, principally on protected crops (Section 2.2.3; Messing and Rabasse 1995; van Lenteren 2003).

Aphid parasitoids have considerable potential as biological control agents but their efficiency is dependent upon their presence in the right place at the right time; their appearance in crops should be synchronized with colonising pest populations early in the season (Powell et al. 1998). Biological control strategies by parasitoids are being increasingly applied for aphid management in greenhouse crops (van Lenteren and Woets

1988, Parrella et al. 1999; Hajek 2004; Lopes et al. 2009; Henry et al. 2010). Aphidiine wasps are all parasitoids of aphids (Mackauer and Starý 1967; Quicke 1997; Stary 2007;

Völkl et al. 2007; Latham and Mills 2010). Many aphidiine species have been successfully used in biological control programmes against pest species in the field and glasshouses

(Ramakers 1989; van Steenis and El-Khawass 1995; Yano 2006).

Aphidius colemani is one of the most important parasitoids worldwide for the control of A. gossypii and other aphid species, including M. persicae (van Steenis 1993). Aphidius colemani is believed to be indigenous to India but has been found in many other regions of the world (Starý 1975; Goh et al. 2001; Starý, 2002; Langhöf et al. 2005; Starý et al. 2007;

Frank 2010) and it is produced commercially for biological control of M. persicae and A. gossypii in protected cropping systems such as greenhouses (van Schelt et al. 1990;

Messing and Rabasse 1995; Grasswitz 1998; Elenberg et al. 2000; Bolckmans and Tetteroo

2002; van Lenteren 2003; Yano 2006).

2.6 Plant parasitic nematodes

Another group of pests which severely affect potato production are plant parasitic nematodes (Brodie et al. 1993; Minnis et al. 2002; Scurrah et al. 2005; Mugniéry 2007;

Hodda and Cook 2009). Plant nematodes generally damage plant roots rather than the aerial parts of plants and reduce yield, even when infestations produce no obvious symptoms in the above-ground plant stem and leaves. Severely infested plants are stunted, often chlorotic and typically occur in patches within fields (Brodie et al. 1993; Scurrah et al.

2005; Mugniéry 2007). Rhizoctonia and other fungal diseases associated with nematode feeding may also contribute to the yield loss (Hugot et al. 2001; Bhattarai et al. 2009). In potato crops, the major nematode species are potato cyst nematodes, Globodera spp., in temperate regions (Winslow and Willis 1972; Trudgill 1985; Den Nijs 2007), and root-knot nematodes, Meloidogyne spp., in warmer climates (Stirling and Wachtel 1985; Vovlas et al.

2005; Mugniéry 2007).

2.6.1 Meloidogyne spp.

Meloidogyne spp. are obligate endoparasites and the most important species can infect a very wide range large number of crop plants and cause severe losses in yield

(Sasser 1980; Mai 1985; Williamson and Hussey 1996; van der Putten et al. 2006). The

34 disease is characterized by the presence of galls or „root-knots‟ on infected plants.

Symptoms include poor fruit yield, stunted growth, wilting and susceptibility to other pathogens (Brodie et al. 1993; Scurrah et al. 2005; Mugniéry 2007). Second-stage juveniles

(J2) of Meloidogyne spp. penetrate the host, generally near root tips; migrate intercellularly toward the meristem then up the developing vascular cylinder to the region of cell differentiation where they lose their vermiform shape and develop to form sedentary swollen adults (Potenza et al. 1996; Abad et al. 2009).

Meloidogyne spp. can cause significant losses in potato in both warm and cool climates depending upon the nematode species (Chitwood, 1949; Mitchell et al. 1971;

Dabaj and Khan 1981; Nakasono et al. 1990; Brodie et al. 1993; Al-Hazmi et al. 1993;

Cinarli and Eterkin 1996; Charchar 1997; Chaves and Torres, 2001; Vovlas 2005).

Besides direct yield losses, root-knot nematodes may cause indirect damage in the form of blisters on tubers, as well as brown spots in tuber flesh where the maturing egg mass is located directly under the epidermis. These deformations and blemishes make tubers unmarketable and, more importantly, facilitate dissemination of the pathogen in infected seed tubers to new areas. Although many species of Meloidogyne are known to infect potato, only six are considered to be of global importance: Meloidogyne chitwoodi, M. fallax, M. hapla, M. arenaria, M. incognita and M. javanica (Jatala and Bridge 1990; Brodie et al. 1993; Molendijk and Mulder 1996). The first three of those six species are found in cool temperate regions, whereas the others are more important in warm temperate, tropical and subtropical regions of the world (Brodie et al. 1993).

In response to signals from the nematode, phenotypic changes occur in root cells in the vascular cylinder adjacent to the head of the nematode enlarge forming „giant cells‟, large multinucleate, metabolically active cells that serve as a source of nutrients for the developing, endoparasitic form of the nematode (Jones 1978; Dangl and Jones 2001; Davis et al. 2004; Abad and Williamson 2010). Concurrent swelling and division of cortical cells around the nematode lead to the formation of the galls or „root knots‟ characteristic of

Meloidogyne spp. infection (Hussey 1985). Root-knot nematodes obtain nutrients from the

35 phloem via the giant cells (Dorhout 1993) and cause a strong nutrient sink effect on the plant (McClure 1977). After female development, which usually takes about 3 weeks, eggs are released on the root surface in a protective, gelatinous matrix (Hussey 1985).

The complex morphological and physiological changes that occur during the establishment of feeding sites are reflected by altered gene expression in the host (Niebel et al. 1994; Sijmons et al. 1994; Williamson and Hussy 1996; Abad et al. 2003; Sukno et al.

2006; Caillaud 2008). Molecular responses include those to wounding or stress caused by nematode infection as well as perturbations directed toward the initiation and maintenance of feeding sites (Foyer and Noctor 2005).

CHAPTER 3

General Materials and Methods

3.1 Plant production

Potato, Solanum tuberosum, cv. Anya (Suttons Ltd, Paignton, UK), and cvs Desiree,

Pink Fir Apple and Santé (Marshalls and Co, Cambridge, UK) were sprouted at 20 ºC for 2 to 3 weeks before cultivation. The sprouted seed potatoes were then planted in plastic pots

(15 cm diameter) in John Innes Seed, No.1, No. 2 or No. 3 compost (Fargro Ltd,

Littlehampton, UK) in a glasshouse at Silwood Park. The minimum temperature in the glasshouse was kept at 20 ºC + 2 ºC. Additional lighting (16 : 8 L : D cycle) was provided

(mercury halide and sodium) to ensure a minimum light intensity during day time. Potatoes emerged after 2 weeks and when the plants were 4 to 5 week-old they were transferred to a controlled environment room at 20 ºC+ 1 ºC, 65-70% RH with a 16 : 8 L : D cycle for experiments.

Chinese cabbage, Brassica chinensis L. var pekinensis cv. Wonk Bok (Kings Seeds,

Colchester, UK) were grown in pots (10 cm diameter) in John Innes No. 2 compost (Fargo

Ltd., Littlehampton, UK) in a glasshouse as above. Tomato, Lycopersicon esculentum cv.

Moneymaker (Tozer Seeds, Cobham, UK), a cultivar susceptible to root-knot nematodes and widely used for their culture (Khan et al. 2000; Darban et al. 2003) were grown in pots

(15 cm diameter) in John Innes No.2 compost in a glasshouse as above.

3.1.1. Potato cultivars

Morphological descriptions of the potato cultivars used in the experiment are given in

Table 3.1. Cultivar Desiree has thick stems and broad, dark green foliage (Appendix 1).

Cultivar Anya has thin delicate, hairy stema, with narrow, light green foliage (Appendix 2).

Cultivar Pink Fir Apple has tall thin violet stems, with narrow to broad dark green foliage.

The leaves are broader than that of cv. Anya (Appendix 3). Cultivar Santé has thick stems, with broad, dark green foliage (Appendix 4).

Table 3.1 Morphological characteristics of potato cultivars used in experiments.

Desiree Anya Pink Fir Santé Apple Pedigree Urgenta x Desiree x Bred from SVP Y 66 13 Depesche Pink Fir Apple Darbre 636 x SVP AM 66 42 Country of origin Netherlands United France Netherlands Kingdom Foliage cover Moderate Poor Moderate Good Growth habit Semi erect Spreading to Semi erect Spreading to to erect semi erect semi erect Maturity Late Intermediate Very late Intermediate to late Resistant to potato Very low to Medium to Very Low to Medium to leaf roll virus low high low high Resistance to other Medium to Very low to Low Medium to viruses high low high Resistance to potato Low Low Low Medium to cyst nematodes high

Source: The European cultivated potato data base. (www.europotato.org/display_discription.php?variety_name)

3.1.2 John Innes compost

The John Innes range of loam-based composts was used for experiments. John

Innes composts are a blend of loam, peat, course sand or grit and fertilizers. Compound fertilizer in John Innes composts provide a wide spectrum of plant nutrients needed for balanced growth, including nitrogen, phosphorus and potassium (NPK) (John Innes

Manufacturers‟ Association 2010)

John Innes seed compost has no added N and K (Table 3.1). John Innes No.1 compost has added base fertilizer (Harvey and Baker 2002) to the standard mixture of steam-sterilized loam, peat and grit (Lawrence 1950; Bunt 1963). The concentration of N, P

38 and K in the compost can be increased by doubling (John Innes No.2) or tripling (John Innes

No.3) the amount of base fertiliser (John Innes No.1).

Table 3.2 Composition (% w/w) of the composts used in the experiments.

Compost Loam:Peat:Sand N P K NPK (g/l)

John Innes Seed 2 : 1 : 1 0 3.2% 0 0

John Innes No.1 7 : 3 : 2 5.1% 3.2% 8.1% 3

John Innes No.2 7 : 3 : 2 10.2% 6.4% 16.2% 6

John Innes No.3 7 : 3 : 2 15.3% 9.6% 24.3% 9

Source: www.gardeningdata.co.uk/soil/john_innes/john_innes.php

3.2 Insect culture

Myzus persicae was obtained from the MpSIL colony established at Silwood Park in

March, 1996 from a sub-population of a clone that had been maintained for over 10 years at

Rothamsted Research, Hertfordshire, UK (Verkerk et al. 1998b; Kalule and Wright 2005;

Kati 2009). In order to avoid a behavioural bias toward susceptible cultivars of potato, aphids were reared on 6 week-old Chinese cabbage in all experiments except those involving parasitoids. The aphids were reared in a cage (45 x 45 x 35 cm) in a controlled environment room (Section 3.1). All experiments were conducted under the same environmental conditions.

3.3 Parasitoids

Aphidius colemani (Syngenta-Bioline, Little Clacton, UK) were reared on M. persicae on Potato cv. Santé in a controlled environment room (Section 3.2) for six generations. Sixth generation mummified aphids were collected from the plant and placed in plastic containers

39 provided with a 30% w/v honey solution. Female and male parasitoids that emerged were recorded and transferred to Eppendorf tubes (1.5 ml), with one female plus one male per tube with honey solution (30 %) and left to mate for 24 h. The mated females were then used for experiments.

3.4 Plant parasitic nematodes

Meloidogyne javanica were obtained from Syngenta (Jealott‟s Hill, UK) and cultured on tomato plants (Section 3.1). Each tomato plant was inoculated with approximately 3000

M. javanica second stage infective juveniles (J2) and incubated in a greenhouse at 25 ± 2°C and 70% R.H with 16 : 8 (L:D) cycle. Two months after inoculation, when egg masses were well formed in the tomato roots, the inoculated plants were uprooted, their roots gently washed free of soil. Egg masses were removed from the roots and put in distilled water in a covered dish at 25 ± 1°C to allow J2 to hatch. Freshly hatched J2 (< 1 week) were used in experiments in combination with two levels of soil nutrients: John Innes No.1 (low nutrient) and John Innes No.3 (high nutrient).

3.5 Data analysis

All statistical analyses were performed with R, version 2.10.1 and 2.11.1 (R development Core Team, 2009; 2010). Where possible data were analysed using parametric statistics (ANOVA) after log, square root or arcsine transformation as necessary.

Models were simplified by removing variables that did not improve the explanatory statistical power, as described by Crawley (2005, 2007). Posthoc Tukey HSD tests were performed for the determination of significant differences between treatment levels (Crawley 2005, 2007).

CHAPTER 4

The performance of Myzus persicae on potato cultivars at different soil nutritional levels

4.1 Introduction

Myzus persicae is a polyphagous pest widely distributed in the world. It damages a large variety of crops (Blackman and Eastop 2000), including potatoes (Radcliffe and

Ragsdale 2002; Qi et al. 2004; Anstead et al. 2008), principally because it is a vector for a large number of plant viruses (Nault 1997; Blackman and Eastop 2000) (Section 2.4).

Studies on insect performance are important because the degree of resistance exhibited by a plant against a particular herbivore can be measured by the variation in insect performance on that host (Section 2.4.2; Bethke et al. 1998; Johnson 2008; Rowntree et al.

2010; Schädler et al. 2010). The measures of performance commonly used to assess the level of plant resistance to aphids are the effective fecundity and intrinsic rate of increase

(Castle and Berger 1993; Ruggle and Guitierrez 1995; Blackman and Eastop 2000; Davis et al. 2007; Obopile and Ositile 2010; Pelletier et al. 2010).

A number of plant factors can influence aphid-plant interactions (Awmack and

Leather 2002; Will and Van-Bel 2006; Peccoud et al. 2010). These factors could be plant cultivars (Davis et al. 2007), plant physiological age (Taylor 1962), differences in plants growth patterns (Taylor 1962; Nderitu and Mueke 1989) and soil fertilization (Painter 1951,

1958; Jansson 2003). Potato cultivars can differ in plant quality, defensive compounds and physical properties such as leaf hairiness, leaf texture, colour and shape that can affect the performance of herbivores including M. persicae (Awmack and Leather 2002; Section

2.4.2).

Soil nutrition and plant genotype/cultivar have also been shown to affect herbivore performance on various crops (Osier and Lindroth 2001; Glynn et al. 2007; Krauss et al.

2007; Zehnder et al. 2009). A positive correlation has been shown between reproduction of

M. persicae and the amount of soluble N in plants in various studies (Van Emden 1966;

Harrewijn 1970; Scriber 1984; Slansky 1990; Jansson 2003; Sauge et al. 210). Increasing levels of K in soil have been shown to have positive (Woolridge and Harrisson 1968;

Perrenoud 1990; Jansson 2003), negative or no effects (Jansson 2003; Myers and Gratton

2006) on the performance of aphids and other phloem feeding insects. Jansson (2003) found that both N and K increased the intrinsic rate of increase and fresh weight of M. persicae but had the opposite effect on M. euphorbiae. Fertilisation with P has been reported to either have no effect on phloem feeding insects or influence them positively

(Waring and Cobb 1992; Skinner and cohen 1994; Jansson 2003). Jansson (2003) found no effect of high soil P levels on either M. persicae or M. euphorbiae on pepper.

The objective of the present study was to understand the combined effects of host cultivar and soil nutrition on the developmental time, fecundity and intrinsic rate of increase of M. persicae.

4.2 Materials and Methods

4.2.1 Plant production

Potato cvs Anya, Santé, Desire and Pink Fir Apple (Section 3.1.1) were grown in a glasshouse in plastic pots (15 cm diameter x 14 cm depth) with John Innes® seed, No. 1,

No. 2 or No. 3 compost (Table 3.1). The plants were watered every 2 days with 200-250 ml per plant. When plants were 4-5 weeks old, at the growth stage II that is vegetative growth, last for 30-70 days (Appendix 5; Johnson 2008) they were transferred to a controlled environment room (Section 3.1).

4.2.2 Insect culture

Myzus persicae were maintained on 6 week-old (growth stage IV) (Andaloro et al.

1983) Chinese cabbage cv. Wonk Bok for several generations prior to experiments (Section

3.1). All aphids used in the experiments came from a single virginoparous apterous individual from a colony at Silwood Park (Section 3.2).

4.2.3 Performance experiments

Experiments were carried out on 4-5 week-old (pre-tuber-filling) potato plants. Four plants of each cultivar in the four soil composts (Section 4.2.1) were used in a randomised block design in a controlled environment room. Clip cages (2.5 cm diameter x 1.5 cm with mesh on both sides) were used to confine aphids (Adam and van Emden 1972). Clip cages can shade and damage the part of the leaf they cover, and consequently they were moved every second day (MacGillivray and Anderson 1957; Adams and van Emden 1972). A clip cage was fitted onto each of four mature leaves (2nd leaf from the base up to 5th leaf) per plant (Jansson and Smilowitz 1985a; Storer and van Emden 1995) (Fig. 4.1). The experiment was arranged in four blocks based on the four cultivars and each block was sub- divided into four based on the four soil composts (16 plants per block).

To assess aphid performance, three apterous adults of M. persicae were placed onto the lower leaf surface within each clip cage. They were then left for 24 h after which the adults and all but one nymph were removed per cage. Any nymphs that died or escaped the cage were replaced by a nymph of the same age until adulthood. The nymphs in the clip cages were used to assess performance. Measurements were taken of the time it took for each nymph to reproduce (developmental time or pre-reproductive period) and the nymphs produced were recorded daily and removed from each cage after counting.

Fig 4.1 Potato cv. Pink Fir Apple, showing clip cages on four mature leaves from the bottom (2nd to 5th leaf).

The following were calculated:

a. the effective fecundity, by allowing the aphids to reproduce during a period

equivalent to the developmental time (the time taken by nymph from birth

till it start reproduction);

b. the intrinsic rate of increase (rm).

The intrinsic rate of increase (rm) for apterous aphids on different cultivars was estimated using the equation:

rm = 0.738 (lnMd)/d (Wyatt and White 1977),

Where d is the developmental time from birth to onset of reproduction, Md is the

reproductive output per original female during a period equal to d, and 0.738 is a

correction factor.

4.2.4 Data analysis

Data for effective fecundity and intrinsic rate of increase were analysed in R

(Section 2.11.1; R Developmental Core Team 2010) using two-way ANOVA to test the effects of different soil composts and potato cultivars on aphid performance. The development and fecundity data were not distributed normally and were log and square root transformed, respectively, to give a normal distribution before analysis. Post-hoc Tukey

HSD test was used to determine which factor level differed significantly (Crawley 2007).

4.3 Results

4.3.1 Developmental time

The developmental time of M. persicae differed significantly (P <0.05) among the potato cultivars and composts tested (Table. 4.1). A significantly longer developmental time was observed on Anya with John Innes seed compost, followed by Pink Fir Apple and Santé

(Fig 4.1). Significantly faster development was recorded for Desiree on John Innes No. 3 compost (Fig. 4.2)

Table 4.1: ANOVA of developmental time of M. persicae on four potato cultivars sown in four different composts (***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Cultivars 3 0.48 10.11 846.41 *** Composts 3 0.10 7.59 174.05 *** Cultivars* Composts 9 0.00 0.13 2.06

Myzus persicae took significantly longer to develop on Anya grown in low nutrient compost (John Innes seed compost) compared with Desiree grown in high nutrient based compost (John Innes No. 3) (Fig. 4.2).

Fig. 4.2: Developmental time (mean + SEM) of M. persicae on four potato cultivars grown in four different composts. Treatment with different letters are significantly different (P<0.05).

4.3.2 Fecundity and intrinsic rate of increase

Myzus persicae differed significantly (P <0.05) in fecundity and intrinsic rate of increase among potato cultivars sown in different soil composts (Tables 4.2, 4.3). 46

Significantly greater fecundity of offspring was observed on cv. Desiree grown in John Innes

No. 3 Compost followed by cv. Santé. Significantly lower fecundity was found on cv. Anya

(Fig. 4.3).

Table 4.2: ANOVA of effective fecundity of offspring of M. persicae on four potato cultivars sown in four different composts(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Cultivars 3 17.15 5.72 303.16*** Composts 3 9.76 3.25 172.58*** Cultivars* Composts 9 0.97 0.11 5.70***

Fig. 4.3: Fecundity (mean + SEM) of offspring of M. persicae on four potato cultivars grown in four different composts. Mean with different letters are significantly different (P<0.05)

For all cultivars the fecundity of M. persicae was significantly greater on John Innes

No.3 compost compared with the John Innes Seed compost (Fig. 4.3). There was a trend for aphids on all the potato cultivars to have greater fecundity on John Innes No. 3 compost followed by John Innes No. 2, John Innes No. 1 John Innes seed. These differences were significant for all the cultivars (Table 4.2: Fig. 4.3).

Table 4.3 ANOVA of intrinsic rate of increase of offspring of M. persicae on four potato cultivars sown in four different composts(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Cultivars 3 0.17 0.06 883.99*** Composts 3 0.07 0.03 384.54*** Cultivars* Composts 9 0.00 0.00 6.66***

The intrinsic rate of increase (rm) of M. persicae differed significantly (P < 0.05) among the compost used as well as among the potato cultivars (Table 4.3). Significantly greater intrinsic rate of increase of aphids was observed on potato cv. Desiree grown in

John Innes No. 3 compost followed by John Innes No. 2 while significantly lower intrinsic rate of increase was observed on potato cv. Anya grown in John Innes Seed compost (Fig.

4.4).

Fig. 4.4: Intrinsic rate of increase of offspring of M. persicae on four potato cultivars grown in four different composts (mean + SEM). Mean with different letters are significantly different (P<0.05)

4.4 Discussion

Potato cultivar as well as the nutrient level of the compost used for plant production was shown to greatly influence the performance of M. persicae. The indirect effects of soil nutritional levels act through changes in the nutrient composition of the plant, which influence M. persicae performance. Among the nutritional factors that influence the level of arthropod damage in a crop, total N has been considered critical for both plants and their consumers (Mattson 1980; Scriber 1984; Slansky and Rodriguez 1987; Throop and Lerdau

2004; Sauge et al. 2010).

49 persicae on Anya appeared to be due to antibiosis. That is, a form of plant resistance that affects the biology of an insect so pest abundance and subsequent damage is reduced compared with a susceptible crop (Smith 2005; Teetes 2007). Antibiosis resistance often results in increased mortality or reduced longevity and reproduction of insects (Smith 2005).

The present study showed that Desiree was consistently the most susceptible to M. persicae on all soil types while Anya showed considerable resistance, with the lowest performace indicators compared with the other cultivars. Desiree has also been reported to be susceptible to M. persicae in previous studies (e.g. Bintcliffe and Wratten 1982; Saljoqi et al. 2003; Fréchette et al. 2010). The present work is the first report of aphid resistance in

Anya.

The intrinsic rate of increase of M. persicae was lowest (0.115) on Anya grown in

John Innes seed compost and highest (0.349) on Desiree grown in John Innes No.3 compost. These results are similar to those of Davis et al. (2007) who tested 49 potato cultivars for resistance to M. persicae and found that in greenhouse life table studies, the intrinsic rate of increase of M. persicae was lowest (0.167) on cv. Russet Norkotah and highest (0.350) on cv. Red La Soda. Effects of soil nutrition on pest resistance/susceptibility can be mediated through changes in nutritional content of crops (Dixon 1987). Among the nutritional factors that influence the level of arthropod damage in a crop, total N has been considered critical for both plants and their consumers (Mattson, 1980; Scriber, 1984;

Slansky and Rodriguez, 1987; Douglas 1993).

The present results showed that fecundity of M. persicae was greater on composts with higher nutrient levels compared with composts with low nutrients. There was a trend for the aphid fecundity to be greater on potato plants receiving the highest level of nutrition

(John Innes No. 3). A similar trend was found for the intrinsic rate of increase for aphids on

Desiree, Santé and Pink Fir Apple but no trend was observed for Anya.

Nevo and Coll (2001) studied the effect of four N fertilization levels on A. gossypii colour and size, fertility, and intrinsic rate of increase and found that adult and nymph densities, as well as intrinsic rate of increase were positively correlated with N fertilization.

Rosenheim et al. (1994) also found a significant positive effect of N fertilization on fecundity of cotton aphids. van Emden (1966) studied the effect on N fertilization on fecundity and intrinsic rate of increase of M. persicae on Brussels sprouts and found that the fecundity of the offspring increased with increasing level of N or decreased levels of K individually but if the nutrients is applied in combination as high N : K increased the fecundity of M. persicae and B. brassicae.

Vijverberg (1965) reported an increase in infestation of M. persicae on potatoes and of giant willow aphid Tuberolachnus salignus Gmelin (Hemiptera: Aphididae) on willow when the aphids were reared on plants receiving high N/K ratios. While Jansson et al.

(1986) observed that M. persicae population growth on potato was positively correlated with amino acid concentration, which is directly related to N levels in the phloem, an observation compatible with the results reported here. An increase in the nutrient content of a plant can be argued to increase its acceptability as a food source to pest populations (Pimentel and

Warneke 1989; Rowntree et al. 2010).

In conclusion, the present results showed that there was a very substantial difference in the intrinsic rate of increase between M. persicae populations on plants sown in high nutrient compost with the susceptible potato cv. Desiree compared with plants grown in low nutrient compost with cv. Anya. Management of soil nutrients and the potato cultivars grown may therefore significantly reduce the number of aphids and thus damage to the crop.

CHAPTER 5

Field performance of Myzus persicae on potato cultivars in Pakistan and the UK

5.1 Introduction

Potatoes are an important crop throughout the world and attacked by a wide range of insect pests, including aphids, jassids, leaf hoppers, whitefly, beetles, wireworms, cutworms and potato tuber moth (Zanoni 1991; Section 2.3). Among aphid species, M. persicae is an important pest affecting potato production causing direct damage due to feeding and indirect damage as a virus vector (Raman 1984; Hooker 1986; Sections 2.3 and 2.4). Both the nymphs and the adults of M. persicae transmit plant viruses but since the adults are more mobile they play by far the most important role in the field (Flanders et al.

1991).

As a consequence of management failures, notably due to the development of resistance to insecticides, alternative and sustainable methods have to be developed for aphid management. One approach has been to look for natural sources of plant resistance to find genes amenable to breeding programmes and several potato cultivars (e.g. Epicure,

Great Scot, Record, Roslin Tana and Russet Norkotah) with low M. persicae infestation rates have been observed in the field (Landis et al. 1972; Bintcliffe and Wratten 1982;

Mndolwa et al. 1984; Nderitu and Mueke 1989; Davis et al. 2007).

The objective of the present study was to compare M. persicae infestations on potato cultivars in field trials in two countries (Pakistan and UK) and under varying climatic conditions in one country (Pakistan) to determine whether the relative resistance and susceptibility to M. persicae found in these cultivars in the laboratory (Chapter 4) was maintained under field conditions.

5.2 Materials and Methods

Potato cv. Desiree, Santé, Anya and Pink Fir Apple (Section 3.1) were used in field experiments. In Pakistan, planting was in mid to late January in Peshawar and Islamabad, and in the last week of January in Abbottabad (2009, 2010). In the UK (Silwood Park), planting was in mid-May in 2008 and 2009.

5.2.1 Field sites and farming practice

Silwood Park: Field sites were established at Silwood Park, Ascot, Berkshire, UK

(51.4086N, 0.6495S) in 2008. The soils found throughout Silwood Park are free-draining, acidic, sandy soils of the Bagshot Series with pH range 5.1 to 6.2 (Crawley 2005). Field sites were rotovated prior to potato seed planting (cvs Anya, Desiree, Pink Fir Apple and

Santé) on 17/5/2008.

In 2008, each potato cultivar was planted in four separate experimental blocks of 16 x 9 m2, with four replications. The experimental plot size was 4 x 3 m, with in total sixteen experimental plots (Fig 5.1). Potato tubers were planted at an inter-row distance of 75 cm and an intra-row distance of 30 cm. The seed rate ranged from 2.0 to 2.5 tonnes/ha depending on the seed grade. Fertilizer was applied with the recommended dose of 140 :

280 : 250 (N : P : K) kg/ha for potato crops (Potato Council 2008). Irrigation, weeding and earthing-up were done by hand as required.

Weeding was a consistent problem in 2008, and it was very labour intensive to eliminate and keep the crop clean from weeds. In 2009, potato cv. Anya, Desiree and Pink

Fir Apple were therefore planted (13/05/2009) in 25 l buckets filled with field soil and put in the field in four blocks; each block containing 18 plants (6 each of cultivar) Due to the similar performance of Santé and Pink Fir Apple in 2008, only the latter cultivar was used in 2009.

Block I Block II

8m 3x4 m2

CV2 CV1 6m

CV3 CV4

Block III Block IV

Fig. 5.1 General layout (fully randomized block design) for the field experiments in UK (2008) and Pakistan (2009). Field trials in subsequent years did not include Santé.

Pakistan: Three field sites with different soil/climatic characteristics were established in Pakistan in 2009 and 2010.

1. Islamabad: the field trial was conducted at the experimental farm of the National

Agricultural Research Centre, Islamabad (33o 42‟N latitude, 73o 15‟E longitude; altitude 507 m above sea level). The soil type is Shujabad loam belonging to the Missa soil series and is described as fine-silty, belonging to the hyperthermic family of Typic Ustochrept (Farooq

2005). The soil is low in organic matter and has a pH of 7.8 to 8.1. The field site was rotovated prior to potato seed planting on 21/01/2009 (Anya, Desiree, Pink Fir Apple and

Santé) and 19/01/2010 (Anya, Desiree and Pink Fir Apple).

2. Peshawar: the field trial was conducted at the experimental farm of the Agricultural

Research Institute, Peshawar (34o 02‟N latitude, 71o 37‟E longitude; altitude 359 m above sea level). The soil of Peshawar has developed in Piedmont alluvium and is a silty-clay- loam (Jan and Noor 2007). The soil is low in organic matter and has a pH of 8.1 to 8.4. The field site was rotovated prior to potato seed planting on 17/01/2009 (Anya, Desiree, Pink Fir

Apple and Santé) and 15/01/2010 (Anya, Desiree and Pink Fir Apple).

3. Abbottabad: the field trial was conducted at the experimental farm of the Hazara

Agricultural Research Station, Abbottabad (34o 10‟N latitude, 73o 15‟E longitude; altitude

1260 m above sea level). The soil of Abbottabad is loam and clay, mainly non-calcareous; inceptisols and chromudertic soils being the most common soil types found in the district

(Pastakia 2004). The soil is low in organic matter and has a pH of 6.1 to 6.7. The field site was rotovated prior to potato seed planting on 25/01/2009 (Anya, Desiree, Pink Fir Apple and Santé) and 27/01/2010 (Anya, Desiree and Pink Fir Apple).

Each potato cultivar was planted in four separate experimental blocks as described for the Silwood Park 2008 field trial above. The seed rate ranged from 2.0 to 2.5 tonnes/ha depending on the seed grade. Fertilizer was applied at the recommended dose rate of 120 :

120 : 180 (N : P : K) kg/ha in Abbottabad; while in Peshawar and Islamabad the recommended fertilizer dose rate used was 250 :125 :125 (N : P : K) kg/ha. Irrigation, weeding and earthing-up were done manually.

5.2.2 Aphid survey

Naturally occurring aphid infestations were studied in all the experimental locations.

Aphid counts were carried out weekly throughout the potato growing season (c. 10 weeks), starting one week after potato emergence and finishing just prior to harvest. In each replicated plot, the number of aphids (adult and nymph) was counted on six randomly selected plants from each plot. In the field trials in Pakistan, the number of M. persicae on

55 three randomly selected leaves from the upper, middle and lower parts of each plant was taken as single sample. In the field trials at Silwood Park the number of aphids was counted on the whole plant due to low populations of aphids.

5.2.3 Data analysis

Aphid performance was analysed in R (2.10.1), R Developmental Core Team 2009) using ANOVA to test the effects of different potato cultivars on aphid performance at three locations in Pakistan and Silwood Park, UK. Where data were not normally distributed, it was square root or log transformed prior to ANOVA. Posthoc Tukey HSD test were used to determine which factor level differed significantly (Crawley 2007).

5.3 Results

5.3.1 Meteorological data for Silwood Park

In 2008, the mean maximum summer temperature was 21.5 °C and the mean minimum temperature was 10.9 °C (Table 5.1). Rainfall varied from 120 mm in June to 56 mm in August 2008. The average RH varied from 66% in June to 69% in August 2008.

In 2009, the mean maximum summer temperature recorded was 22.7 °C and the mean minimum temperature was 11.3 °C (Table 5.1). Rainfall varied from 35 mm in June to

36 mm in August. The average RH varied from 65% in June to 73% in August.

Table 5.1 Meteorological data during field trials: Silwood Park (2008-2009) and Pakistan (2009-2010).

Month Max (°C) Min (°C) Total Rainfall Mean RH (%) (mm)

Silwood Park, UK (Summer)

(2008) (2009) (2008) (2009) (2008) (2009) (2008) (2009)

May 19.8 18.9 6.2 5.2 68 29 66 67 June 20.4 22.4 7.9 9.7 120 34 61 65 July 22.9 22.8 11.2 12.4 76 69 69 65 August 21.5 23.8 13 13.1 56 36 69 74

Peshawar (spring)

(2009) (2010) (2009) (2010) (2009) (2010) (2009) (2010)

January 18.8 21 7.3 5.3 51 21 70 63 February 20.5 18.9 8.6 8.3 75 118 63 73 March 23.3 29 12.4 15 85 17 64 65 April 28.7 33.4 16.2 14.3 180 32 61 54

Abbottabad (spring)

February 14.9 13.1 3.1 2.1 57 215 68 73 March 19.2 23.9 6.4 8.2 196 53 70 58 April 24.3 27.1 10.1 11.8 122 49 64 56 May 29.8 30.1 13.3 15.5 25 19 47 50

Islamabad (spring)

January 18.8 20.8 5.3 3.7 51 12.1 69 55 February 20.4 19.8 7.6 7.6 80 103 63 65 March 25.6 29.1 11.3 13.8 50 82 56 53 April 29.5 33.9 15.6 18.4 110 38 56 41

5.3.2 Meteorological data for Pakistan

Peshawar is not in a monsoon region, unlike other parts of Pakistan. Rainfall occurs in both winter and summer. The climate of Peshawar is semi-arid and subtropical.

Spring in Peshawar occurs from mid-February to the end of April. Summer months are May to September. In 2009, the mean maximum temperature in spring was 24.4 °C and the mean minimum temperature was 12.0 °C (Table 5.1). The spring rainfall varied from 75 mm in February to 180 mm in April-May. The RH varied from 63% in February to 61% in April

2009. In 2010, the mean maximum temperature in spring was 25.7 °C and the mean minimum temperature was 13.2 °C. The rainfall varied from 119 mm in February to 52 mm in April-May. The RH varied from 73% in February to 54% in April (Table 5.1).

Islamabad has an atypical version of a humid subtropical climate, with long, very hot summers accompanied by a monsoon season followed by short, mild winters. Spring in

Islamabad occurs from mid-February to the end of April. Summer months are May to

September. In 2009, the mean maximum temperature in spring was 23.3 °C and the mean minimum temperature was 9.6 °C (Table 5.1). Rainfall varied from 80 mm in February to

110 mm in April 2009. The RH varied from 63% in February to 56% in April 2009. In 2010, the mean maximum temperature in spring was 25.9°C and the mean minimum temperature was 10.2 °C. The rainfall varied from 103 mm in February to 38 mm in April-May. The RH varied from 65% in February to 41% in April (Table 5.1).

Abbottabad is located at the base of the Himalayas, which gives it a temperate climate throughout most of the year. Abbottabad falls in the active region of monsoon.

Spring usually begins in March and continues untill May; summers are mild and short. The winter is severe with snowfall on surrounding hills. In 2009, the mean maximum temperature in spring was 21.3 °C and the mean minimum temperature was 7.9 °C (Table 5.1). Rainfall varied from 57 mm in February to 25 mm in May. The RH varied from 68% in March to 47% in May (Table 5.1). In 2010, the mean maximum spring temperature was 20.6°C and the mean minimum temperature was 8.2 °C (Table 5.1).The rainfall varied from 215 mm in

February to 46 mm in April-May. The RH varied from 73% in February to 50% in April (Table

5.1).

5.3.3 Field performance of M. persicae on potato cultivars: Silwood Park 2008

Myzus persicae differed significantly (P < 0.05) in performance on the four potato cultivars tested (Table 5.2). A significantly higher number of aphids were observed on

Desiree; a significantly lower number of aphids were observed on Anya (Fig. 5.2).

Table 5.2 ANOVA of number of Myzus persicae on potato cultivars: Silwood Park 2008 and 2009(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

UK (2008)

Cultivars 3 20.98 6.99 16.13***

UK (2009)

Cultivars 2 21.05 10.52 42.79***

Fig. 5.2: Mean seasonal number (+ SEM) of Myzus persicae recorded per plant on four potato cultivars: Silwood Park 2008. Treatments with different letters are significantly different (P < 0.05).

In summer 2008, M. persicae appeared from 2nd week of June and continued until the 3rd week of August. Initially the aphid‟s numbers were low on all potato cultivars due to the wet start to the season (Table 5.1). In the 3rd week of July the aphid population started building with a peak in the last week of July. The aphid‟s numbers then declined due to crop maturation and increased rainfall (Fig. 5.3). Aphids colonized Desiree significantly to the greatest degree, followed by Santé and Pink Fir Apple, while significantly poor colonization was observed on Anya (Fig. 5.2). As Anya is an early maturing cultivar M. persicae peaked early and declined early (Fig. 5.3).

25 Anya 20 Desiree 15 Sante PFA Aphids Aphids / Plant 10

0 1 2 3 4 5 6 7 8 9 10 Weeks ( June to August 2008)

Fig. 5.3 Mean weekly number of Myzus persicae per plant on different potato cultivars: Silwood Park 2008.

5.3.4 Field performance of Myzus persicae on potato cultivars: Pakistan 2009

Myzus persicae differed significantly (P < 0.05) in performance on the four potato cultivars tested (Table 5.3). A significantly higher number of aphids were observed on

Desiree at Peshawar followed by Santé, while a significantly lower number of aphids were recorded on Anya in all three locations (Fig. 5.4).

The data in 2009 were not normally distributed and was square root transformed for normal distribution for analysis (Table 5.3). The performance of M. persicae was

60 significantly greater in Peshawar compared with Abbottabad for all potato cultivars (Fig.

5.4). The number of M. persicae was significantly greater on Desiree and significantly lower on Anya in all three locations (Fig. 5.4).

Table 5.3 ANOVA of the number of Myzus persicae on potato cultivars at three different locations in Pakistan: 2009 and 2010(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Pakistan (2009)

Locations 2 1492.38 746.19 126.10*** Cultivars 3 2671.67 890.56 150.50*** Location* Cultivars 6 526.28 87.71 14.82***

Pakistan (2010)

Locations 2 540.52 270.26 29.29*** Cultivars 2 2327.47 1163.73 126.13*** Location* Cultivars 4 102.14 25.53 2.77*

Fig. 5.4: The seasonal mean (+ SEM) number of Myzus persicae recorded per plant on four potato cultivars: Pakistan 2009. Treatments with different letters are significantly different (P < 0.05).

140

120

100 Anya 80 Desiree 60 Sante PFA Aphids/ Plant 40

0 1 2 3 4 5 6 7 8 9 10 Weeks (February to March 2009) a)

180 160 140

120 Anya 100 Desiree 80 Sante

60 PFA Aphids/ plant 40 20 0 1 2 3 2 5 6 7 3 9 10 Weeks ( February to April 2009) b)

100 90 80 70 Anya 60 Desiree 50 Sante 40 PFA Aphids/ plant 30 20 10 0 1 2 3 4 5 6 7 8 9 10 Weeks (March to May 2009) c)

Fig. 5.5 Mean weekly number of Myzus persicae per plant on different potato cultivars in 2009: (a) Islamabad, (b) Peshawar and (c) Abbottabad.

In Islamabad (2009), M. persicae appeared from the 2nd week of February and continued until last week of April. The population started to build rapidly due to optimum temperatures but declined in the last two weeks of March due to heavy showers (Table

5.1). The aphid population started building up again in the first week of April and peaked in the 3rd week of April, then started to decline due to crop maturity and increasing temperature (Table 5.1). Desiree had the highest populations of aphids except at the end of the season while Anya had the lowest populations (Fig. 5.5a).

In Abbottabad (2009), M. persicae appeared from the 2nd week of March and continued until the 3rd week of May. The population of M. persicae was low initially due to cool March temperatures and rain during this month (Table 5.1). The aphid population started building in the 2nd week of April, peaking in the last week of April and then started a gradual decline due to crop maturity and increasing temperature (Table 5.1). Desiree had the highest populations of aphids except at the end of the season while Anya had the lowest populations (Fig. 5.5c).

In Peshawar (2009), M. persicae appeared from the 2nd week of February and continued until the 3rd week of April. Due to favourable climatic conditions (Table 5.1) the aphid population started building in the last week of February and peaked in the last week of March. It then started a gradual decline due to crop maturity and increasing temperature

(Table 5.1). Unexpected heavy showers in the last week of April also contributed to the decline in aphid populations at the end of the growing season (Table 5.1). Desiree had the highest populations of aphids except at the end of the season while Anya had the lowest populations (Fig. 5.5b).

5.3.5 Performance of Myzus persicae on potato cultivars in pots (semi-field): Silwood Park 2009

Myzus persicae differed significantly (P < 0.05) in performance among three potato cultivars (Table 5.2). A significantly higher number of aphids were observed on Desiree; a significantly lower number of aphids were observed on Anya (Fig. 5.7).

20 Anya Desiree 15 PFA Aphids/Plant 10

0 1 2 3 4 5 6 7 8 9 10 Weeks ( June to August 2009)

Fig. 5.6 Mean weekly number of Myzus persicae per plant on different potato cultivars: Silwood Park 2009.

In 2009, M. persicae appeared from the 1st week of June and continued until the 2rd week of August. The aphid density was low on all potato cultivars until the 1st week of July but with the rise in temperature the aphid population started building from the 2nd week of

July, peaking in the last week of July. Aphid populations then started to decline due to crop maturation and low populations were observed in the 2nd week of August (Fig. 5.6). Aphid populations were significantly greater on Desiree followed by Pink Fir Apple, the significantly poorest aphid populations were observed on Anya. As Anya is an early maturing cultivar M. persicae peaked early and the population declined earlier due to early crop maturation (Fig.5.6).

Fig. 5.7: The seasonal mean (+ SEM) number of Mizus persicae recorded per plant on three potato cultivars: Silwood 2009. Treatments with different letters are significantly different (P < 0.05).

The data were not normally distributed in 2009 and were log transformed prior to

ANOVA. Desiree had significantly highest populations of aphids while Anya was significantly the poorest host for M. persicae in both 2008 and 2009 (Figs 5.2, 5.7).

5.3.6 Field performance of Myzus persicae on potato cultivars: Pakistan 2010

Myzus persicae differed significantly (P < 0.05) in performance among the potato cultivars tested (Table 5.3). A significantly higher number of aphids were observed on

Desiree at Peshawar followed by Pink Fir Apple; a significantly lower number of aphids were found on Anya in all the three locations (Fig. 5.8).

The data were not normally distributed in 2010 (Table 5.3) and was square root transformed for normal distribution for analysis. The performance of M. persicae was

66 consistently significantly greater in Peshawar compared with Abbottabad for all potato cultivars (Fig. 5.8). Desiree was significantly the best host cultivar for aphids while Anya was significantly the poorest host cv. in all three locations (Fig. 5.8).

Fig. 5.8: The seasonal mean (+ SEM) number of Myzus persicae recorded per plant on four potato cultivars: Pakistan 2010. Treatments with different letters are significantly different (P < 0.05).

In Islamabad (2010), M. persicae appeared from the 2rd week of February and continued until last week of April. The population started building rapidly due to optimum temperatures but then declined in the last week of March due to heavy showers (Table 5.1).

The aphid population started building again in the 2nd week of April and peaked in the 3rd

67 week of April, then started to decline due to crop maturity and increasing temperature

(Table 5.1). Desiree had consistently the highest populations of aphids while Anya consistently had the lowest populations (Fig. 5.9a).

In Abbottabad (2010), M. persicae appeared from the 1st week of March and continued until the 2rd week of May. The population of M. persicae was low initially due to cool March temperatures and rain during this month (Table 5.1). The aphid population started building in the 3nd week of April, peaking in the last week of April and then started a decline due to crop maturity and increasing temperature (Table 5.1). Desiree had consistently the highest populations of aphids while Anya had the lowest populations except for one week (Fig. 5.9c).

In Peshawar (2010), M. persicae appeared from the 2nd week of February and continued until the 3rd week of April. Due to favourable climatic conditions (Table 5.1) the aphid population started building in the 2nd week of March and peaked in the first week of

April. It then started a gradual decline due to crop maturity and increasing temperature

(Table 5.1). Desiree had consistently the highest populations of aphids while Anya consistently had the lowest populations (Fig. 5.9b).

140

120

100

80 Anya Desiree

60 Pink Fir Apple Aphids/plant

0 1 2 3 4 5 6 7 8 9 10 Weeks (February to April 2010) a)

160

140

120

100 Anya 80 Desiree Pink Fir Apple

Aphids/Plant 60

0 1 2 3 4 5 6 7 8 9 10 Weeks (February to April 2010) b)

40 Anya Desiree

30 Pink Fir Apple Aphids/plant

0 1 2 3 4 5 6 7 8 9 10 Weeks (March to MAy 2010) c)

Fig. 5.9 Mean weekly number of Myzus persicae per plant on different potato cultivars in 2010: (a) Islamabad, (b) Peshawar and (c) Abbottabad.

5.4 Discussion

In all the field experiments (UK and Pakistan), the potato cultivar was found to greatly influence the weekly mean number of M. persicae per plant. There can be a number of factors responsible for aphid population levels (Awmack and Lether 2002; Will and Van-

Bel 2006; Peccoud et al. 2010), including plant cultivar (Davis et al. 2007), plant physiological age (Taylor 1962), differences in plant growth patterns (Taylor 1962; Nderitu and Mueke 1989), soil fertilization (Painter 1951), environmental stress (Aheer et al. 2007) and natural enemies (Leather and Owuor 1996; Landis et al. 2000).

In the present study, the performance of M. persicae on the different potato cultivars tested was consistent under the various field conditions found in the UK and Pakistan. The field results also agreed with laboratory studies on the same potato cultivars (Chapter 4).

The field experiments showed that Desiree was consistently the most susceptible to M.

70 persicae in all localities in 2008, 2009 and 2010, with the greatest number of aphids per plant. While Anya showed considerable resistance, with the lowest weekly aphids count throughout the growing season, compared with the other cultivars.

Desiree has been reported to be susceptible to M. persicae in numerous studies

(e.g. Bintcliffe and Wratten 1982; Saljoqi et al. 2003; Karley et al. 2003; Parker 2005; Ali and

Jan 2006; Jan et al. 2006; Fenton et al. 2010), including the Peshawar, Abbottabad (Saljoqi et al. 2003; Khurshid 2007) and Khaghan Valley (Ali and Jan 2006; Jan et al. 2006) regions of Pakistan. The present work is, however, the first report of aphid resistance in Anya.

Resistance to M. persicae in Anya was shown to have an antibiosis component, with reduced aphid performance (Section 4.3 and present chapter). The earlier colonisation by

M. persicae of Desiree and Santé compared with Anya (Figs 5.3, 5.5, 5.6) also suggests that a difference in preference (antixenosis) may also be involved in resistance in Anya.

Antixenosis resistance affects the behaviour of an insect pest and is usually expressed as non-preference of the insect for a resistant plant compared with a susceptible plant (Teetes

2007). For example, Klingler et al. (2005) used two closely related resistant and susceptible genotypes of Medicago truncatula Gaert to characterize the aphid-resistance phenotype and found that the migratory blue green aphid, Acyrthosiphon kondoi Shinji, was deterred from settling on resistant plants.

The overall level of aphid infestation observed in the UK (Parker et al. 2000; and present work) is less than that in Pakistan. This may be due to the cooler summer and frequent showers in the UK which make the conditions less favourable for aphids. The low and selective use of insecticide sprays in potato crops in the UK may also favour natural enemies that can reduce the aphid population in the field (Cowgill et al. 2004).

At Silwood Park, M. persicae appeared in the 2nd week of June in both 2008 and

2009 and peaked in the last two weeks of July. Similar patterns of infestation were observed by Parker (2005), who carried out a series of field experiments to asses the direct effect of feeding by aphids on potato yield in the UK. Cowgill et al. (2004) also noted that populations of M. persicae peaked in the last two weeks of July in a UK field study.

In the Peshawar field trials, M. persicae appeared in the 2nd week of February in both

2009 and 2010, its population peaking in the last two weeks of March and then starting to decline in the 2nd week of April. Similar results were also obtained by Khan et al. (2003) in field studies in the Peshawar area. The same seasonal pattern for aphid infestations on potato was observed in Islamabad. In Abbottabad, aphid infestations were somewhat delayed compared with other two localities. Aphids started to appear in the 2nd week of

March, almost one month later than at the other localities due the later start of spring in

Abbottabad. The population peaked in 3rd week of April and then started to decline in May.

These results are similar to the survey of Ali and Jan (2006) who studied the population fluctuation of M. persicae in Abbottabad and the Khaghan Valley and by Khan et al. (2004) in the Swat Valley in northern Pakistan.

The high levels of M. persicae infestations in Pakistan (Umar et al. 1995; Niaz and

Ayub 2007; present study) may be due to several factors: (a) a traditional potato mono- cropping pattern, with consequent favourable conditions to the over-wintering of this aphid species; (b) high N applications by farmers, which stimulates vegetative plant growth, thus increasing plant susceptibility to aphids; (c) the mild climate in spring, which favours aphid reproduction; and (d) the frequent and high rates of application of broad-spectrum insecticides that eliminate natural enemies (Khattak et al. 1996; Jansen 1998; Haseeb et al.

2000; Saljoqi and van Emden 2003; Youn et al. 2003; Ali and Jan 2006; Cabral et al. 2008;

Sauge et al. 2010).

Due to high aphid infestation during spring (Umar et al. 1995; Niaz and Ayub 2007), most farmers have switched to autumn crops in the plains of Pakistan (e.g. Islamabad and

Peshawar). In hilly areas (e.g. Abbottabad), farmers grow potato only in spring, and aphids migrate to other vegetables in summer and to its primary host (Prunus spp.) in autumn for overwintering.

Aphid resistance in potato is as desirable today as when Adams (1946) said, "Aphid resistance alone would be a material aid to the industry." The results of the present study shows that the potato cultivars tested differed significantly in their suitability for aphid

72 reproduction. Anya appeared to be a highly resistant cv. to aphids in the field and in combination with biological control and/or selective insecticide use this level of resistance could provide substantial control.

While Anya is a specialist salad potato and unsuitable as a main crop, these results may provide insights to plant breeders who wish to incorporate aphid resistance traits from

Anya or its progenitors into breeding programmes.

CHAPTER 6

Performance of Myzus persicae on upper, middle and lower leaves of three potato cultivars at low and high soil nutrition

6.1 Introduction

The preference and performance of Myzus persicae is associated with the physiological age of potato leaves. For example, on turnip and sugar beet, M. persicae settled consistently on leaves of a specific age and on specific sites on leaves of a given age (Hodgson 1978; Jepson 1983). Various studies have shown that the population parameters of M. persicae are greater on older plants or leaves than on younger plants or leaves (Taylor 1955, 1962; Harrewijn 1970; Jansson and Smilowitz 1985a,b; Lara et al.

1999; Van Emden and Harrington 2007; Aldamen and Gerowitt 2009). Collectively, the evidence suggests that the population dynamics of M. persicae may be associated with phloem concentrations of amino-nitrogen compounds, which are mobilized during leaf senescence (Thomas and Stoddart 1980).

Jansson and Smilowitz (1985b) reported that M. persicae were associated with potato plant phenology. Myzus persicae were more abundant and their populations peaked sooner and declined earlier on potato cultivars with shorter growing seasons compared with late maturing cultivars. Taylor (1955) found more aphids on the lower leaves of potato plants and suggested that aphid performance was related to the physiological age of leaves and various studies have since shown that field populations of M. persicae increase more rapidly on lower leaves than on upper or middle leaves (Jansson and Smilowitz 1985a;

Nderitu and Mueke 1986; Aldamen and Gerowitt 2009).

The literature on the performance of M. persicae on plants fertilized with different nutrient levels and the effect on total soluble N in leaves at different plant position (lower, middle and upper) is confusing. Van Emden and Bashford (1969) showed that fecundity of

M. persicae was positively correlated with concentrations of soluble N within Brussels sprout

74 leaves of the same age, but not between leaves of different ages. However, Harrewijn

(1970) and van Emden and Bashford (1971) however found poor correlations between performance of M. persicae and concentrations of N in leaves. Other studies have shown that population parameters of M. persicae increase with an increase in soluble N or free amino acids in plants fertilized with different levels of fertilizer (van Emden, 1966; Jansson and Smilowitz, 1986).

To address the hypothesis that nutrients content in soil has positive effects on the performance of M. persicae the present study looked at the effect of leaf position on the performance of M. persicae on three potato cultivars grown in two soils with different nutritional levels.

6.2 Materials and methods

6.2.1 Plant production

Potato, cvs Anya, Desiree and Pink Fir Apple (Section 3.1) were grown in plastic pots (15 cm diameter x 14 cm depth) using John Innes® No. 1 and No. 3 compost (Table

3.2). The potted plants were kept in a glasshouse and watered every 2 days with 200-250 ml per plant. When plants were at the growth stage II (section 4.2.1) they were transferred to a controlled environment room (Section 3.1).

6.2.2 Insect culture

Myzus persicae were maintained on 6 week-old Chinese (Section 4.2.2) cabbage cv.

Wonk Bok for several generations prior to the experiments (Section 3.1). All aphids used in the experiments came from a single virginoparous apterous individual from a colony at

Silwood Park (Section 3.2).

6.2.3 Performance experiments

Experiments were carried out using 4-5 week-old (pre-tuber-filling) potato plants.

Five plants of each cultivar per soil compost were randomised on benches in a controlled environment room (Section 4.2.3). The experiment was split into three blocks based on the three cultivars. Each block was then divided into two based on the two soil composts used to give two sub-plots per block; these were replicated five times to give 10 sub-plots per block. Two clip cages (Section 4. 2.3) were attached to two randomly selected leaves per plant position: upper (sixth to eight newly emerged expanded leaves), middle (third to fifth expended compound leaves) and lower (second mature but not visibly senescing leaf)

(Fig. 6.1).

Fig. 6.1 Potato plant showing clip cages on upper, middle and lower leaves. Assessment procedure involved taking three apterous adults from the culture and placing onto the lower leaf surface within each clip cage (Section 4.2.3).

The following parameters were determined:

 pre-reproductive time (time from birth until onset of reproduction);

 the effective fecundity by allowing the aphids to reproduce during a period

equivalent to the developmental time;

 the intrinsic rate of increase (rm).

The intrinsic rate of increase (rm) for apterous aphids on different cultivars was

estimated using the following equation (for details see Section 4.2.3):

rm = 0.738 (lnMd)/d (Wyatt & White 1977)

6.2.4 Data analysis

Data for developmental time, effective fecundity and intrinsic rate of increase were analysed in R (Section 2.11.1; R Developmental Core Team 2010) using ANOVA to test the effects of different soil composts and potato cultivars on aphid performance at different plant positions. Where data were not distributed normally it was log transformed before analysis.

Post-hoc Tukey HSD test was used to determine which factor level differed significantly

(Crawley 2007).

6.3 Results

6.3.1 Developmental Time

The developmental time of M. persicae differed significantly (P < 0.05) between the potato cultivars and composts tested at different plant positions (Table. 6.1). A significantly longer developmental time was observed on Anya in John Innes No.1 compost on top young leaves, followed by Pink Fir Apple. A significantly faster developmental time was recorded for Desiree in John Innes No. 3 compost on lower older leaves (Fig 6.2).

Table 6.1 ANOVA of developmental time of offspring of Myzus persicae on leaves in different positions on three potato cultivars sown in compost with high or low nutrient levels(***P < 0.001; **P < 0.01; *P < 0.05)..

Source of variation d.f. Sum Sq Mean Sq F value

Cultivars 2 0.38 0.19 240.75*** Composts 1 0.02 0.02 28.46*** Position 2 0.03 0.02 21.17*** Cultivars : Composts 2 0.00 0.00 0.21 Cultivars : Position 4 0.01 0.00 1.42 Composts: Position 2 0.00 0.00 0.11 Cultivars : Composts : Position 4 0.00 0.00 0.09

Fig. 6.2: Mean (+ SEM).developmental time of M. persicae on leaves in different positions on three potato cultivars sown in compost with high or low nutrient levels. Treatments sharing the same letter are not significantly different (P >0.05).

The data were not distributed normally and were log transformed to give a normal distribution before analysis. Myzus persicae took significantly longer to develop on young leaves of Anya grown in low nutrient compost (John Innes No.1) compared with older leaves of Desiree grown in high nutrient based compost (John Innes No. 3) (Fig. 6.2).

6.3.2 Fecundity and Intrinsic rate of increase

Myzus persicae differed significantly (P < 0.05) in fecundity and intrinsic rate of increase between potato cultivars sown in different soil composts (Tables 6.2, 6.3).

Significantly greater fecundity was observed on Desiree grown in John Innes No. 3 compost followed by Pink Fir Apple grown in the same media while significantly lower fecundity was found on Anya grown in John Innes No.1 and 3 (Fig. 6.3). Similarly the fecundity and intrinsic rate of increase of M. persicae differed significantly (P < 0.05) between leaf zones

(Tables 6.2, 6.3). A significantly greater fecundity and intrinsic rate of increase occurred on the older leaves (bottom leaves) of Desiree grown in John Innes No. 3, while a significantly lower fecundity and intrinsic rate of increase occurred on the younger leaves (top leaves) of

Anya grown in John Innes No. 1 (Fig. 6.3, 6.4).

Table 6.2 ANOVA of effective fecundity of M. persicae on leaves in different positions on three potato cultivars sown in composts with high or low nutrient levels(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Cultivars 2 4.57 2.29 157.93*** Composts 1 2.23 2.23 153.72***

Position 2 1.18 0.59 40.57*** Cultivars : Composts 2 0.03 0.02 1.05

Cultivars : Position 4 0.03 0.01 0.54 Composts: Position 2 0.07 0.0 2.31

Cultivars : Composts : Position 4 0.03 0.01 0.49

The fecundity of M. persicae was significantly greater in John Innes No.3 compared with John Innes No.1 compost for all three potato cultivars (Fig. 6.3). There was a trend for aphids on all the potato cultivars to have greater fecundity on John Innes No. 3 compost and lower leaves. These differences were significant (P < 0.05) for all the cultivars (Table

6.2).

Fig. 6.3: Mean (+ SEM).effective fecundity of M. persicae on leaves in different positions on three potato cultivars sown in compost with high or low nutrient levels. Treatments sharing the same letter are not significantly different (P >0.05).

The intrinsic rate of increase (rm) of M. persicae differed significantly (P < 0.05) with the compost used as well as between potato cultivars and leaf position (Table 6.3). A significantly greater intrinsic rate of increase of aphids was observed on Desiree grown in

John Innes No. 3 compost on lower leaves while a significantly lower intrinsic rate of increase was observed on Anya grown in John Innes No.1 compost (Fig. 6.4). Among

80 different plant positions, the intrinsic rate of increase of M. persicae was significantly greater on lower plant leaves compared with young upper leaves on all cultivars.

Table 6.3 ANOVA of intrinsic rate of increase of offspring of M. persicae on leaves in different positions on three potato cultivars sown in composts with high or low nutrient levels(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Cultivars 2 0.24 0.12 539.54***

Composts 1 0.04 0.04 178.62*** Position 2 0.03 0.17 76.15***

Cultivars : Composts 2 0.00 0.00 9.17

Cultivars : Position 4 0.00 0.00 2.16 Composts: Position 2 0.00 0.00 0.39

Cultivars : Composts : Position 4 0.00 0.00 0.60

Myzus persicae performance was significantly influenced by soil nutrition levels (low and high), by potato cv. (Table 3.1) and by the leaf zone on which the aphids were feeding.

Aphid performance was significantly higher on the lower leaves of Desiree grown in John

Innes No. 3 compost, while performance on Anya was lower, especially on the younger leaves in John Innes No.1 compost.

Figure 6.4: Mean (+ SEM) intrinsic rate of increase of M. persicae on leaves in different positions on three potato cultivars sown in composts with high or low nutrient levels. Treatments sharing the same letter are not significantly different (P >0.05).

6.4 Discussion

A number of factors are known to influence aphid-plant interactions, including plant cultivar. (Davis et al. 2007; Aldamen and Gerowitt 2009), plant physiological age (Taylor

1962), differences in plants growth patterns (Taylor 1962; Nderitu and Mueke 1989) and soil fertilization/nutrition (Painter 1951). Among the nutritional factors that influence the level of arthropod damage in a crop, total N has been considered critical for both plants and their consumers (Mattson 1980; Scriber 1984; Slansky and Rodriguez 1987; Throop and Lerdau

2004; Sauge et al. 2010).

In the present study, the performance of M. persicae on potato cultivars differed consistently between the different soil nutritional levels tested and based on the effects on developmental time, effective fecundity and intrinsic rate of increase. As in previous laboratory (Chapter 4) and field studies (Chapter 5), Desiree was consistently the most susceptible to M. persicae, in the present case both at low and high soil nutritional levels, while Anya showed considerable resistance, with the lowest performance indicators compared with the other cultivars.

Myzus persicae was shown to perform more efficiently on the lower leaves than on the middle or upper leaves of each cv. examined. This finding is consistent with previous studies (Shands et al. 1954; Mack and Smilowitz 1981; Jansson and Smilowitz 1985a;

Nderitu and Mueke 1986) and the preference for lower leaves is likely to be related to leaf senescence (Taylor 1955; 1962). Myzus persicae populations also tend to increase more rapidly on early maturing varieties because they have a greater proportion of senescing leaves (Taylor 1962; Nderitu and Mueke 1989).

As leaves senesce, the composition of phloem sap changes from predominantly carbohydrates to predominantly amino acids (Thomas and Stoddart 1980). Jansson et al.

(1986) reported that the population growth of M. persicae on potato was positively correlated with amino acid concentrations. The fecundity of M. persicae has been shown to be positively correlated with plant nitrogen content, reproducing more rapidly on lower leaves due to higher quantities of amino-nitrogen compounds in the phloem (Wooldridge and Harrison 1968). This supported the contention that the population growth of M. persicae on potato could be correlated with physiological development of leaves (Taylor 1962). The present study also showed that M. persicae had higher fecundity and intrinsic rate of increase on lower senescing leaves than on the young upper leaves.

Bald et al. (1946) and Taylor (1955) compared five potato cultivars for aphid susceptibility. Both authors concluded that the main reason for the observed differences was the different growth patterns of the varieties. Davis et al. (2007) compared 49 commercial potato cultivars, primarily of North American origin, for the intrinsic rate of

83 increase of M. euphorbiae and M. persicae and found that this differed considerably between cultivars. Aldamen and Gerowitt (2009) showed that eight current German potato cultivars also differ significantly in their suitability for aphid reproduction.

In conclusion, the present results showed that there is a substantial difference in the intrinsic rate of increase and fecundity between M. persicae populations on both susceptible and resistant potato cultivars in high and low nutritive compost. Thus manipulation of soil nutrition and the potato cultivars can significantly affect the number of aphids and thus damage to the crop. These results also support the previous laboratory and field results

(Chapter 4 and 5) and suggest that aphid susceptibility and resistance can be included in official descriptions of potato cultivars and become an additional trait in farmer's choice of cultivar. More information on the traits associated with aphid resistance in potato cultivars is required for plant breeding programmes.

CHAPTER 7

The preference of Myzus persicae on potato cultivars at different soil nutritional levels

7.1 Introduction

Host plant recognition and acceptance is a critical period for aphid colonization and population establishment. Final host plant acceptance occurs stepwise through behavioural sequences until continuous feeding commences (Schoonhoven et al. 1998; Powell et al.

2006). At each stage, aphids may either continue with their colonization behaviour or depart the plant (Le Roux et al. 2008).

Plant factors, including plant species (Castle et al. 1998; Thomas 2002; Alvarez and

Srinivasan 2005; Davis et al. 2007; Steenken and Halaweh 2010), resistance (Gibson and

Pickett 1983; Jayasinghe 1990; Jayasinghe et al. 1998; Teetes 2007) morphology, e.g. leaf thickness and toughness, leaf hair density and waxy layers (Tingey et al. 1982; Sanford et al. 1991; Yencho and Tingey 1994; Speight et al. 1999), and nutritional status (Fereres et al.

1990; Karley et al. 2002; Traicevski and Ward 2002) are known to modify host-plant preference of aphids.

Among non-plant factors, reflective mulches, barriers, fallow ground or skip rows, chemical pest control practices and environmental factors also modify the host selection behaviour of aphids (Eckel and Lampert 1996; Ragsdale et al. 2001). These modified host- plant preferences may influence aphid population dynamics (Schoonhoven et al. 1998;

Awmack and Leather 2002; Powell et al. 2006).

Host specific chemical cues also influence host preference and host acceptance by aphids (Caillaud and Via 2000; Del Campo et al. 2003) and studies have shown that host selection and feeding behaviour of aphids are affected by plant volatile organic compounds

(Visser 1986; Nottingham et al. 1991; Visser et al. 1996; Pickett et al. 1992, Hildebrand et al. 1993; Bruce et al. 2005; Webster et al. 2008). 85

The present studies investigated the preference of M. persicae for potato cultivars with the following objectives: (1) to determine the response of winged aphids toward potato cultivars grown in compost with different nutrient levels; (2) if a preference exists, to assess whether volatile cues are involved using olfactometry; and (3) to determine the trichome density on leaves of the each potato cv.

7.2 Materials and Methods

7.2.1 Plant production

Potato, cvs Anya, Desiree, Pink Fir Apple and Santé (Section 3.1) were grown in plastic pots (15 cm diameter x 14 cm depth) in three different John Innes composts (Table

3.2). The potted plants were kept in a glasshouse and watered every 2 days with 200-250 ml per plant. When plants were at the growth stage II (Section 4.2.1), they were transferred to a controlled environment room for preference experiments (Section 3.1).

7.2.2 Insect culture

Myzus persicae were maintained on 6 week-old Chinese at growth stage IV (Section

4.2.1) cabbage cv. Wonk Bok in a controlled environment room for several generations prior to the experiments (Section 3.1). All aphids used in the experiments came from a single virginoparous apterous individual from a colony at Silwood Park (Section 3.2).

7.2.3 Preference (settling) bioassay with winged aphids

Four treatment plants (one each of Anya, Desiree, Santé and Pink Fir Apple) were placed at random in the corners of a wooden cage (90 x 90 x 90 cm) with fine mesh sides.

Pots were spaced so that the leaves of each plant did not touch those of the other plants. A plastic Petri dish (9 cm diameter) with 40 winged M. persicae was placed on top of a centrally located wooden dowel (50 cm long) with a circular top (7 cm diameter). In

86 experiments, winged aphids were used preferentially because this is the main dispersal morph.

To obtain winged individuals, the aphids were raised on six-week-old Chinese cabbage plants that were slightly water stressed and aphids were overcrowded. The four treatment plants were randomly placed in the experimental arena, and the experiment was replicated eight times for Experiment No.1 and four times for Experiment No. 2 under laboratory condition (Sections 3.1 and 3.2). The experimental set-up was left undisturbed in the laboratory for 24 h so the aphids could fly and settle on any of the four plants. After 24 h, the number of winged aphids per plant was recorded. The percentage of aphids that moved and the mean percentage response to each treatment was calculated.

7.2.4 Olfactometer experiment

The host-selection response of M. persicae to potted potato plants was studied using a whole-plant four-way Perspex olfactometer based on the design of Pettersson

(1970) modified by Bruce (2005) and placed in a controlled environment room (Section

3.2.). Connected to the olfactometer were four Perspex cylinders (28.7 cm internal diameter

× 63.5 cm). The top and bottom of each cylinder had removable Perspex lids that could be tightly closed. There was an air inlet 5 cm above the base, and an air outlet 5 cm from the top on the opposite side of the cylinder from the inlet. The outlet from each cylinder was connected via an air flow meter to one of four arms around the central arena of the olfactometer (Fig. 7.1). The olfactometer chambers were enclosed in a box (66 x 56 x 66 cm) fitted with a curtain of black cloth to prevent extraneous light from entering the box. The bottom of the four-arm olfactometer apparatus was lined with filter paper (Whatman No. 1).

Air was pumped (Model:, CAPEX 8C, Charles Austin pumps ltd., Byfleet, Surrey, UK) through an activated-charcoal filter (10–18 mesh size), and regulated by an airflow meter, placed outside the light box and drawn through the four arms towards the centre at 240 ml min−1 (flow rate through each individual arm was 60 ml min−1) (Bruce et al. 2005). Light-

87 induced preference was avoided by providing even illumination through the use of two 12-W strip lights placed equidistant on the ceiling of the olfactometer chamber.

Single alate virginoparae which had been starved for 1 h prior to experiments

(Johansson et al. 1997) were introduced into the central arena and allowed to settle for 5 min before the plant chambers were connected to the olfactometer. The time the aphid spent in each olfactometer arm was recorded using specialist software (OLFA, Udine, Italy) over a 16 min period (Bruce et al. 2005; Webster et al. 2010). Single aphids were used to avoid the intraspecific interactions between aphids during the experiment (Pettersson et al.

1995). Control experiments without plants indicated that there was no positional bias within the apparatus. As an additional measure, the apparatus was rotated a quarter of a turn every 2 min to eliminate any directional bias (Webster et al. 2010).

Olfactometer experiments were carried out between 0900 and 1500 h to reduce any circadian bias (Troncoso et al. 2005). The olfactometer was dismantled after every daily session and cleaned in 50 % ethanol, soaked overnight in 10 % non-perfumed cleaning solution (Lipsol, Bibby Sterilin, Staffordshire, UK), rinsed in tap water then distilled water, and air dried.

Four to five-week old potted potato, cvs Anya and Desiree grown in two different composts (John Innes No.1 and John Innes No.3) were compared in a series of seven paired experiments (below) with two opposing olfactometer arms having the same treatment

(i.e. arm 1+3 vs arm 2+4). Ten replicates for each comparison were made. Insects and plants were tested once and then discarded ito avoid pseudoreplication (Ramirez et al.

2000).

Exp 1; Desiree (John Innes No. 3) vs Empty control

Exp 2; Anya (John Innes No.3) vs Empty control

Exp 3; Desiree (John Innes No. 3) vs Anya (John Innes No.3)

Exp 4; Desiree (John Innes No. 3) vs Desiree (John Innes No. 1)

Exp 5; Desiree (John Innes No. 1) vs Anya (John Innes No. 3)

Exp 6; Desiree (John Innes No. 1) vs Anya (John Innes No. 1)

Exp 7; Anya (John Innes No. 3) vs Anya (John Innes No. 1)

Fig. 7.1 The five regions of the four-arm olfactometer (250 mm diameter).

7.2.5 Trichome density on potato cultivars

Leaves were randomly selected from different plant positions (top, middle and bottom) in each cultivar and three disks (1.5 cm diameter) were removed with a cork borer from each leaf. Trichomes on the under-sides (abaxial) and upper-side (adaxial) of leaf disks (where M. persicae primarily reside and feed) were counted using a dissecting microscope, and the average count for the three disks was used to estimate trichome density. Although potato plants possess both glandular and non-glandular trichomes, total number of the trichomes (i.e., glandular and non-glandular) were analyzed because the two trichome types are highly correlated (Kaplan et al. 2009).

7.2.6 Data analysis

Data were analysed in R (Section 2.11.1; R Developmental Core Team 2010).

Percentage data were converted into proportion data and square root transformed to

89 normalise distributions (Dytham 2003) and subjected to ANOVA. Post-hoc Tukey HSD test was used to determine which factor level differed significantly (Crawley 2007). The effect of potato cv. on trichome density was assessed by ANOVA. Wilcoxon‟s two-tailed rank sum test was used to compare the total time spent by each individual aphid in the two opposing olfactometer arms (arm 1+3 vs arm2+4).

7.3 Results

7.3.1 Preference (settling) bioassay with winged aphids

Significant differences (P <0.05) were detected in the number of M. persicae present on different cultivars 24 h after release when choice was among cultivars (Table 7.1). Anya attracted a significantly lower number of M. persicae compared with Desiree, Pink Fir Apple and Santé while Desiree attracted significantly more aphids (Fig. 7.2 a).

Desiree grown in high nutritive compost attracted significantly (P <0.05) more aphids compared with Desiree in low nutritive compos, while the compost did not affect (P >0.05) the attractiveness of Anya (Fig. 7.2 b). When a choice was offered between two cultivars

(Anya and Desiree) sown in two different composts, Anya was significantly less attractive than Desiree, even in high nutritive compost (John Innes No.3) compared with Desiree grown in low nutritive compost (John Innes No.1) (Table 7.1 and Fig. 7.2 b).

Table 7.1: ANOVA of preference (settling) bioassay for M. persicae on potato cultivars

(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Experiment No.1

Cultivars 3 0.50 0.17 13.66 ***

Experiment No.2

Cultivars 1 0.40 0.40 112.51 *** Composts 1 0.04 0.04 12.05 ** Cultivars* Composts 1 0.00 1.00 1.11

Fig. 7. 2 Mean (± SEM) number of M. persicae settled on potato cultivars after 24h of (a) four potato cultivars (b) on two potato cultivars at low and high soil nutrition. Treatments with the same letters were not significantly different (P >0.05).

7.3.2 Olfactometer bioassay

No significant preference (P = 0.41) was observed between different olfcatometer arms in the absence of plant odours (Fig 7.3).

Fig. 7.3 Mean (± SEM) time M. persicae spent in each arm of the olfactometer in the absence of any odour source (n= 5). Treatments with the same letters were not significantly different (P >0.05).

When alates of M. persicae were exposed to volatiles from Desiree and Anya, significantly (P <0.05) more alates were attracted to Desiree (Fig. 7.4). Similarly when alates of M. persicae were exposed to volatiles from plants grown in high and low nutritive compost, significantly (P <0.05) more alates were attracted to the high nutritive Anya and

Desiree (Fig. 7.4).

Treatment A vs Treatment B p-value

Anya 1 Anya 3 0.007

Anya 3 Desiree 1 <0.001

Anya 1 Desiree 1 0.025

<0.001 Desiree 1 Desiree 3

Anya 3 Desiree 3 <0.001

Control Anya 3 <0.001

Control Desiree 3 <0.001

80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 Time (%) spent / treatmen

Fig. 7.4 Olfactory responses of M. persicae (mean percent time ± SEM) to potato cultivars in different treatment combinations. The level of significant differences was set at P<0.05 (n=10). [Anya 1 = grown in John Innes No.1, Anya 3 = grown in John Innes No.3, Desiree 1 = grown in John Innes No.1, Desiree 3 = grown in John Innes No.3].

7.3.3 Trichome density on potato cultivars

Desiree in general had a higher number of trichomes on both the under and upper side of leaves compared with Anya (Fig 7.5 a,b) but these differences were not statistically significant (Table 7.2). When the trichome density was analysed on different plant positions, the upper leaves had significantly (P <0.05) more trichomes then lower leaves (Table 7.2 and Fig 7.5 a,b) on all cultivars. However the interaction between plant position and cultivars was not significant (Table 7.2).

Table 7.2: ANOVA of trichome density/cm2 on the abaxial and adaxial surface of leaves from the top, middle and bottom of three potato cultivars(***P < 0.001; **P < 0.01; *P < 0.05).

Source of variation d.f. Sum Sq Mean Sq F value

Trichome density (abaxial)

Cultivars 2 68.79 34.39 2.15 Position 2 2589.55 1294.78 80.91*** Cultivars: Position 4 25.13 6.28 0.39

Trichome density (adaxial)

Cultivars 2 5.86 2.930 0.40 Position 2 409.43 204.715 27.87 *** Cultivars: Position 4 20.60 5.149 0.70

(a)

(b)

Fig.7.5 Mean (± SEM) trichome density on leaves (a) abaxial (b) adaxial of three potato cultivars at different plant positions. Treatments with the same letters were not significantly different (P >0.05).

7.4 Discussion

Differences in aphid host plant susceptibility may be attributed to several factors including host plant nutritional quality, morphology and genotype, and also to aphid host

96 finding or preference (Bruyn 2002; Awmack and Leather 2002; Zehnder 2010). Finding a host can be a challenge for aphids because they tend to be host specific and have poor control over their flight path (Dixon 1998, Powell et al. 2006). Even if aphids locate a suitable cultivar or species, the plant may have traits (antixenosis and/or antibiosis mechanisms) that help it resist aphid attack (Painter 1968, Kogan and Ortman 1978, Panda and Khush 1995, Hill et al. 2004).

Antixenosis (non-preference) refers to plant traits that reduce insect establishment by changing insect preference behaviours, where preference is measured by the number of insects landing and settling on plants (Tetees 2007). Host selection by alates is normally the first stage of colonization and plays a major role in determining aphid populations in the field

(Klingauf 1987). Kennedy and Kishaba (1977) compared settling behaviour of alates of A. gossypii on resistant and susceptible lines of melon (Cucumis melo) and found a highly significant preference for the susceptible genotype. Bosland and Ellington (1996) suggested that the resistance of Capsicum pubescens towards M. persicae is due to antixenosis and da Costa et al. (2010) reported evidence for antixenosis in studies on commercially available cultivars of Capsicum spp. In the present study results from the settling bioassays with alates showed that significantly greater numbers of M. persicae preferred to settle on

Desiree compared with Anya irrespective of the level of soil nutrition.

Plant volatiles are important cues because they are the first chemical barrier encountered by the aphids (Kerkut and Gilbert 1985; Nottingham et al. 1991; Pickett et al.,

1992; Kessler and Baldwin 2001). The present olfactometer studies showed that the relative antixenosis observed for Anya compared with Desiree had a volatile component. This is the first report of antixenosis resistance to aphids in a cultivated potato, cv. Anya. This kind of resistance has been previously reported in wild Solanum spp. to M. persicae (Le Roux et al.

2008, 2010).

When the choice offered was between plants grown in high nutritive and low nutritive composts, M. persicae preferred plant with high nutrition for both Desiree and Anya. Le

Roux et al. (2008 and 2010) also reported Desiree the more attractive in olfactometer

97 studies to M. persicae when compared with resistant wild potato, Solanum bukasovii and S. stoloniferum and found antixenosis resistance against M. persicae in all wild Solanum accessions. In contrast to the settling bioassay Anya where preference for Anya was not affected by the high and low nutritive compost, olfactometery results showed Anya in high nutritive soil was the most preferred then Anya on low nutritive compost. This may be due to the fact that high soil nutrient make the plant more vigorous and attractive and change the volatile organic compounds in the plant which affect the attraction of insects. Hoffland et al.

(2000) also reported that differences in nutrient availability lead to variations in plant chemistry, which in turn can influence host selection by herbivores. Facknath et al. (In press) also reported that leafminer, Liriomyza huidobrensis Blanchard (Diptera:

Agromyzidae) adults were more attracted to vigorous plants of Anya compared to the nutrient-limited ones.

Expression of antixenosis through host plant odour has also been reported in olfactometer for many other insect species. For example, Gaum et al. (1994) reported that the western flower thrip Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) showed different responses to rose cultivars, while Storer and van Emden (1995) reported that A. gossypii showed different responses to host plant odours from different cultivars of chrysanthemum.

It is generally believed that pubescent leaves will be more resistant against herbivores than less pubescent ones of the same species due to mechanical restrictions to herbivore activities caused by a high density of trichomes (Levin 1973). The effects of trichomes are however, often variable within the herbivore community (van Dam and Hare

1998; Hare and Elle 2002; Andres and Connor 2003; Gruner et al. 2005; Kaplan et al. 2009;

Dai et al. 2010). To date, only in wild potatoes has the role of glandular trichomes in defence against insects been well-documented (Gibson 1971; 1974; 1976; Tingley et al.

1982; Yencho and Tingey 1994; Alvarez et al. 2006). Glandular trichome-based resistance is however genetically complex and it is strongly associated with poor agronomical characteristics (Kalazich and Plaisted 1991; Bonierbale et al. 1994) and this trait has yet to

98 be used successfully in breeding for aphid resistance. In the present study, the differences in trichome density between Desiree and Anya were not statistically significant, which suggested that the resistance to M. persicae observed in Anya did not involve trichomes.

In conclusion, the results of the present experiments suggest that the resistance of

Anya to M. persicae observed in laboratory (Chapters 4 and 6) and field (Chapter 5) studies have an antixenosis component involving plant volatiles. Further studies are required to determine whether there is a second antixenosis component present on or in the leaf and feeding and probing bioassays are needed.

CHAPTER 8

Performance of Aphidius colemani on Myzus persicae reared on three potato cultivars at different soil nutrition levels

8.1 Introduction

Aphids are one of the most problematic insect pests of protected crops worldwide because they can produce high population densities, and resistant strains to insecticides can frequently develop in a short period of time (Goh et al. 2001; Kim and Kim 2003;

Després et al. 2007). Biological control of aphids and other arthropod pests of protected crops have become widespread in many parts of the world, particularly in Northern Europe and North America (van Lenteren 2000; van Driesche et al. 2008; Pilkington et al. 2010).

In IPM a combination of population regulation strategies may be used, including biological and chemical control and host plant resistance (Gray et al. 2009). In interactions between plants, aphids, and their natural enemies, growing resistant varieties can affect natural enemies directly, by modifying their behaviour, or indirectly, by changing the nutritional quality of the prey and/or host (van Emden 1995). The impact of this interaction can be negative, thus demonstrating the incompatibility between the two control methods

(Obrycki and Tauber 1984; Gamarra et al. 1997), or positive, having cumulative effects on the reduction of aphid populations (Farid et al. 1998; Messina and Soureson 2001).

Natural enemies of phytophagous insects function and develop in multitrophic systems (Price et al. 1980). Their behaviour and physiology, which determines their fitness or performance, are influenced by both the plant (first trophic level) and their herbivore host

(second trophic level) (Reed et al. 1991). The effect of the plant on the parasitoid may be manifested directly by a plant‟s physical and chemical characteristics. For example, foliar morphology and plant volatiles may influence natural enemy activity directly (Price 1986;

Hare 1992; Powell et al. 1998), or indirectly via the nutritional properties of the host plant

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(Bergman and Tingey 1979; Orr et al. 1985). Herbivores of poor quality may affect the survival and fitness of the parasitoid by influencing parasitism success, parasitoid size, fecundity and development time (Reed et al. 1991; Godfray 1994).

The present study aimed to determine the extent to which potato cultivars influenced the performance of A. colemani when grown at low and high level of nutrients using John

Innes No.1 and No.3 compost, respectively. Potato, cvs Anya, Pink Fir Apple and Desiree were chosen because of their different levels of susceptibility to M. persicae (Chapters 4 and 5).

8.2 Materials and Methods

8.2.1 Plant Production

Potato, cvs Anya, Pink Fir Apple and Desiree were grown in a glasshouse in John

Innes No. 1 and John Innes No. 3 compost (Table 3.2) as described previously (Section

3.1). When plants reached the growth stage II (Section 4.2.1), they were transferred to a controlled environment room for experiments (Section 3.2).

8.2.2 Insect culture

Myzus persicae were obtained from a colony established at Silwood Park (Section

3.2). For this experiment aphids which were previously cultured on Chinese cabbage were transferred to individual potato cultivars (Anya, Pink Fir Apple and Desiree) and cultured for several generations to acclimatize them to each of the cultivars used in the experiments.

Culture conditions as described previously (Section 3.2).

8.2.3 Parasitoids

Aphidius colemani (Section 3.3) were maintained on M. persicae on potato, cv.

Santé (Section 3.1). By the time the study was initiated, the parasitoid had completed more than six generations on this cultivar. Douloumpaka and van Emden (2003) have reported

101 that parasitoids need to establish on an aphid species for at least two generations before use in the experiments to minimize maternal host plant effects. The colonies were maintained in a controlled environment room 20°C, 65-70% RH and 16 : 8 L : D (Section

3.2) and experiments were conducted under the same conditions.

8.2.4 Experimental procedure

Five apterae of M. persicae (previously cultured on the corresponding cultivar were put in five clip cages per plant attached to the abaxial side of a mature leaves of each cv. – soil compost combination on different leaves of plant (Section 4.2.3). Each treatment was replicated eight times. The clip cages were checked after 24 h and if nymphs had been produced the apterae and all but 10 nymphs per plant, (two from each clip cage) were removed. Following nymph establishment the clip cages were removed from the leaves and each plant was covered with a transparent micro-perforated bag measuring 38 x 90 cm

(Asianet UK, Leicester, UK) to prevent aphid escape. The nymphs were allowed to develop and reproduce on the host plant. As the percentage of parasitism by A. colemani is similar between aphid instars (van Emden and Kifle 2002), after 3 weeks aphid colonies were checked and nymphs were removed at random to create colonies of 60 individuals of mixed instars (as described by Jarošík and Lapchin 2001) on each potato plant. The potato plants were then placed in wooden framed mesh cages (75 x 45 x 35 cm).A single mated female

A. colemani was then placed in each cage. As most Aphidius spp. lay more than 80% of their eggs on the first day of release (Stary 1970; Hofsvang and Hågvar 1975b), the parasitoids were removed from the cages after 24 h. The cages were checked on a daily basis after five days from parasitoid release for the presence of parasitoid mummies. The aphids were allowed to continue their development for 14 days, the maximum time required from parasitisation to mummy formation (van Emden and Kifle 2002).

Mummies were collected daily in labelled Eppendorf tubes (1.5 ml) and checked every 24 h for parasitoid emergence. The emerging adult A. colemani were placed in individual 2.5 x 8 cm glass tubes. The following data were collected for each replicate:

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i. percentage parasitism: number of mummies divided by the original number of

aphids (60);

ii. percentage emergence: number of hatched mummies, divided by the total

number of mummies;

iii. developmental time: from day 0 to day of adult parasitoid emergence;

iv. sex ratio: proportion of adult male parasitoids (the sex of emerged parasitoids

was determined by morphological differences: the shape of the end of the

abdomen (Hofsvang and Hågvar 1975a) and antennal size where males

have 15-1 6 segments and females 13-14 segments (Mecheloff and Rosen

1990);

v. adult size: hind tibiae length (linear dimensions) was used as an indicator of

body size (Hohmann et al. 1988; Van Emden and Kifle 2002; Blande et al.

2004); the length of of the hind tibiae of 30 individual females selected at

random from the pooled replicates per treatment was measured under a

binocular microscope fitted with an eyepiece graticule.

8.2.5 Data analysis

All statistical analyses were performed with R 2.11.1 (R Development Core Team,

2010). Parasitoid performance data were log, square root or arcsine transformation as necessary to stabilise the variance and subjected to ANOVA. Models were simplified by removing variables that didn‟t improve the explanatory statistical power (Crawley, 2005,

2007). Differences between means were analysed using Tukey‟s HSD test. Parasitoid sex ratios were analysed using a generalised linear model with quasibinomial errors due to over dispersion of the bounded proportion. Cultivar and fertiliser treatment factors were included in the model. Significant differences between composts and cv. treatment means were calculated from P-values given by the model.

103

8.3 Results

8.3.1 Percentage parasitism

Potato cultivar did not affect the percentage parasitism observed. Compost had a significant effect on the parasitism but the interaction between cultivar and compost was not significant (Table 8.1). The percentage parasitism by A. colemani was significantly greater on aphids reared on Desiree grown in John Innes No.3. While the percentage parasitism by

A. colemani decreased significantly on aphids reared on Anya grown in John Innes No.1

(Fig. 8.1).

Table 8.1 ANOVA of parasitism success of Aphidius colemani on Myzus persicae reared on Anya, Desiree and Pink Fir Apple in different composts (***P < 0.001; **P < 0.01; *P < 0.05).

F test

Sources of Developmental time variance Parasitism Emergence (%) (%) Male Female Tibia length

Cultivars 2.55 5.58 28.45 21.56 10.05 (A) (**) (***) (***) (***)

Composts 11.44 5.21 6.13 4.34 39.78 (B) (**) (*) (*) (*) (***)

A x B 0.56 2.48 0.90 0.09 1.97

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Fig. 8.1 mean (± SEM) percentage parasitism by Aphidius colemani on Myzus persicae reared on three potato cultivars grown in two composts. Treatments with the same letters were not significantly different (P >0.05).

8.3.2 Percentage emergence

Potato cultivar and compost had significant effects on percentage parasitoid emergence but the interaction between cv. and compost was not significant (Table 8.1). The percentage emergence of A. colemani was significantly greater on aphids reared on Pink Fir

Apple and Desiree grown in John Innes No.3 compared with the percent emergence by A. colemani for Anya in John Innes No.1 (Fig. 8.2).

105

Fig. 8.2 Mean (± SEM) percentage emergence of Aphidius colemani on Myzus persicae reared on three potato cultivars grown in two composts. Treatments with the same letters were not significantly different (P >0.05).

8.3.3 Developmental time

Potato cultivar and composts had significant effects on parasitoid developmental time but the interaction between cultivar and compost was not significant (Table 8.1).

Developmental time for both male and female A. colemani was significantly greater on aphids reared on Anya in John Innes No.1. The developmental time of A. colemani was significantly lower on Desiree and Pink Fir Apple in John Innes No.3 (Figure 8.3). The developmental time of females was slightly longer than males on all cultivars and composts but not statistically significant (Fig. 8.3).

106

Fig. 8.3 Mean (± SEM) developmental time of Aphidius colemani (male and female) on Myzus persicae reared on three potato cultivars in two composts. Treatments with the same letters were not significantly different (P >0.05).

107

8.3.4 Sex ratio

The sex ratioranged from 39 to 58% (Figure 8.4) and was not significantly affected by potato cultivar (F2,177 = 0.4578, P = 0.6334) or compost treatment (F1,176 = 1.7741, P =

0.1846).

Fig. 8.4 Mean (± SEM) sex ratio of Aphidius colemani emerging from Myzus persicae reared on three potato cultivars in two composts. Treatments with the same letters were not significantly different (P >0.05).

8.3.5 Adult size

Adult size, as measured by the length of the female A. colemani hind tibia was significantly influenced by both the potato cultivar and compost (Table 8.1). The hind tibia was significantly longer on Desiree grown in John Innes No.3 compared with Anya on John

Innes No.1. The tibia length of female A. colemani was significantly shorter on Anya grown in John Innes No.1 (Fig. 8.5).

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Fig. 8.5 Mean (± SEM) hind tibia length of female Aphidius colemani emerging from Myzus persicae reared on three potato cultivars grown in two composts. Treatments with the same letters were not significantly different (P >0.05).

8.4 Discussion

Cultivar and soil nutrition can both affect the life history parameters of A. colemani directly by altering the population or the quality of the host aphids (Jiang and Schulthess

2005; Kalule and Wright 2005; Krauss et al. 2007; Garratt et al. 2010) or indirectly by influencing the host plant volatile cues that attract these parasitoids (Powell et al. 1998;

Schworer and Volkl 2001).

In the interactions between plants, aphids and their natural enemies, resistant varieties can affect natural enemies directly, by modifying their behaviour, or indirectly, by changing the nutritional quality of the prey and/or host (van Emden 1995). The impact of this interaction could be either negative, or positive, having cumulative effects on the reduction of aphid populations (Section 8.1). Similarly low nutritional quality or low digestibility of plant tissues are often regarded as defences against herbivores (Williams 1999) by making the

109 herbivore growth rate slower (= long developmental time) thus exposing the herbivores to the natural enemies for long time (Price et al. 1980; Kidd et al. 1994). In contrast, other studies suggest that slow growing herbivores may be at less risk from parasitism than fast- growing ones because parasitoids make a larger investment in each herbivore as each herbivore provides all the nutrients for the parasitoid larval development (Fox et al. 1990,

1996; Williams 1999). Thus herbivores feeding on high plant nutrition will favour natural enemies positively.

In the present study, susceptible (Desiree and Pink Fir Apple) and a partially resistant (Anya) cultivars did not differ significantly in their percentage parasitism by A. colemani. Similar results were obtained by Soglia et al. (2006) for A. colemani with M. persicae on resistant and susceptible chrysanthemum cultivars. Saljoqi and van Emden

(2003) however found in field experiments that M. persicae on the partially resistant potato cultivar Cardinal had a greater rate of parasitism by Aphidius matricariae (Haliday) compared with Desiree.

The present study did show that parasitism of aphids by A. colemani was greater on potato cultivars in high nutritive compost compared with low nutritive compost. Jansson

(2003) found that plant fertilization enhanced parasitism rates in M. euphorbiae but not in M. persicae on pepper and petunia plants. Sarfraz et al. (2005, 2009b) found that parasitism of

P. xylostella by D. insulare varied considerably between the soil fertility regimes on which

Brassica plants were grown. The percentage parasitism observed in the present study (39 -

58%) was similar to previous studies with A. colemani on M. persicae on resistant and susceptible chrysanthemum cultivars (Soliga et al. 2006) but considerably less than reported by Sampaio et al. (2001, 2005) on A. gossypii and M. persicae.

In the present study, parasitoid emergence was significantly affected by soil nutrition and plant cultivar. In general, A. colemani performed best on M. persicae that developed on susceptible plants grown with high nutritional value. Aphids in these treatments were of high quality and faster growing (Chapter 4 and 6), and were thus presumably more suitable for larval development of A. colemani. A similar finding was reported for emergence of D.

110 insulare on P. xylostella on Brassica napus (Sarfraz et al. 2009a,b). Changes in parasitoid development time can be mediated by „„bottom up‟‟ factors associated with the first trophic level (plant) that, in turn, affect the second trophic level (aphid) (Mackauer and

Kambhampati 1984; Stadler and Mackauer 1996). For example, the nutritional sufficiency and quality of the plant can affect parasitoid development (Vinson and Iwantsch, 1980;

Sequeira and Mackauer 1992).

In the present study, the development time of A. colemani was fastest in aphids on a good host (Desiree) grown in high nutritive compost and slowest on hosts on a partially resistant (Anya) grown in low nutritive compost, suggesting a link between parasitoid development and host quality. Similar trends were reported by Reed et al. (1991, 1992) and

Sarfraz et al. (2009b) in studies on Diaeretiella rapae McIntosh and D. insulare, respectively. In contrast, Soglia et al. (2006) reported no differences in the developmental time of A. colemani reared on A. gossypii on resistant and susceptible chrysanthemum cultivars.

Overproduction of males of parasitic Hymenoptera contributes to higher costs for biological control because only females directly kill pests (Chow and Heinz 2005). The role of plants in sex allocation is commonly assumed to be indirect; difference in plant quality first influence the properties of herbivores and parasitoids respond to these properties in their hosts (Godfray 1994). The sex ratio for A. colemani in the present study was not affected significantly by either plant cultivar or the nutritional quality of the plant compost.

These results are in line with Kalule and Wright (2005) who reported no significant interaction between the sex ratio of A. colemani on M. persicae on partially resistant and susceptible cabbage cultivars, and studies with other aphid parasitoids (Farid et al. 1998;

Garratt et al. 2010).

One possible explanation for the present results was that mixed host instars were used in the trial and female A. colemani may have adjusted the sex ratio by preferring to parasitise older host stages in low nutritive conditions and on the resistant Anya. Chow and

Heinz (2005) have reported that using hosts of mixed sizes can reduce a male-biased sex

111 ratio in the parasitoid Diglyphus isaea Walker (Eulophidae: Hymenoptera). Another possibility is that aphid quality in all treatments in the present study was optimal for sex allocation of A. colemani.

Hind tibia length is commonly used to estimate the relative size of parasitoids and is a correlate of parasitoid fitness in terms of longevity and fecundity (Visser 1994; Lauziere et al. 2000). Size, therefore, is a significant indicator of host suitability (Godfray 1994). It has also been shown that female fitness increases with adult size (King 1988), since larger individuals may have a higher potential fecundity (Rosenheim and Rosen 1991; Godfray

1994) and better searching capacities (Visser 1994; Kazmer and Luck 1995).

In the present study, susceptible potato cultivars combined with high soil nutrition significantly increased the size of female A. colemani. Similar findings were reported for the aphid predator, Aphidoletes aphidimyza in a brassica system (Verkerk et al. 1998b) and for

A. ervi on M. persicae reared on pepper plants (Jansson 2003).

In conclusion, most parameters studied in the present work indicated optimal fitness when A. colemani were reared on M. persicae fed on plants grown in high nutritive compost.

Thus, while higher levels of soil fertility can predispose plants to increased infestations of herbivores (Price 1991; Waring and Cobb 1992; Meyer 1996), increased fertility may also enhance the performance of their parasitoids. Vigorous, well nourished plants in combination with plant resistance may thus be better able to compensate for insect attack than plants under nutrient stress and maintaining relatively high levels of soil fertility combined with plant resistance may prove effective components for integrated management of aphids on potatoes.

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CHAPTER 9

Multitrophic interactions in a potato-aphid-parasitoid system: influence of root herbivory and soil nutrition

9.1 Introduction

In multitrophic food webs, plants, herbivores and their natural enemies interact with each other and thus influence community dynamics (Ohgushi 2005; Trotter et al. 2008; Van

Der Putten 2009). In these interactions plants may be simultaneously challenged by above- and below-ground herbivores that can influence plant-mediated interactions between herbivores (Gange and Brown 1989; Masters 1995b; Bezemer et al. 2003).

Root feeders are believed to induce changes in plant physiology by their feeding which results in increased water stress in plants (Smith 1977), and may strongly affect the quality and quantity of nutrients available to foliar feeders (Masters and Brown 1992). These physiological and biochemical changes, driven by below-ground communities, produce a variety of responses within plants that can directly influence foliar herbivores (Masters and

Brown 1992; Masters et al. 1993; Masters 1995a; Bezemer and Van Dam 2005). The impact of root feeders on the performance of foliar herbivores can be positive (Gange and

Brown 1989; Masters and Brown 1992; Masters 1995a; Gange 2001; Wurst et al. 2008;

Kaplan et al. 2009), negative (Bezemer 2003; Soler et al. 2005) or neutral (Moran and

Whitham, 1990; Salt et al. 1996). The majority of these studies have focused on tissue feeding root herbivores.

Root-feeding nematodes are infamous for their devastating effects on crop plants

(Smiley et al. 2005; Fuller et al. 2008). It has been however shown that low amounts of root grazing by nematodes can indirectly enhance plant performance (Denton et al. 1999; Poll et al. 2007). Effects of root-feeding nematodes on above-ground herbivores can be negative due to the induction of systemic defences (Bezemer and Van Dam 2005), or by the lowering

113 of amino-acid contents of leaves (Bezemer et al. 2005). Positive effects can for instance arise as a result of the destruction by root-feeding nematodes of production sites of secondary metabolites in the roots (Kaplan et al. 2008a,b).

The objective of the present study was to investigate interactions in a potato system involving M. persicae, its natural enemy A. colemani, with the root-knot nematode,

Meloidogyne javanica (Treub) Chitwood (Tylenchida: Heteroderidae), as the root herbivore.

Root-knot nematodes form giant cells in plant roots, which act as a metabolic sink that directs nutrients to the site of nematode parasitism (Burgeson 1966; McClure 1977). It was hypothesized that the performance of M. persicae and its parasitoid would respond negatively to changes in the host plant induced by root herbivory by M. javanica due to the induction of systemic defences (Bezemer and Van Dam 2005), or by the lowering of amino- acid contents of leaves (Bezemer et al. 2005) which negatively affect aphids performance and hence parasitoids of the aphids. Soil nutrition was included as an additional factor.

The effects of host plant nutrition on foliar insects including aphids have been shown empirically to be complex (Rowntree et al. 2010). Altering soil nutrition, can force the physiological susceptibility of crop plants to insect pests by either affecting the resistance of individual plant to attack or by altering plant acceptability to certain herbivores (White 1984;

Altieri and Nicholls 2003). In the present study, soil nutrition has been shown to have positive effect on the performance of aphids by increasing its fecundities and intrinsic rate of increase (Chapters 4, 6) or enhancing the attractiveness of the plants to aphids in olfactometer (Chapter 7) and also aphids parasitoids fitness related parameters were influenced positively (Chapter 8).

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9.2 Materials and methods

9.2.1 Plants production

Potato, cv. Desiree was grown in plastic pots in a greenhouse with John Innes No. 1 and No. 3 compost (Section 3.1; Table 3.2). When plants were at the 3 to 4 leaf stage, groth

Stage I (Johnson 2008) they were transferred to a controlled environment room at 25 ± 2°C and 70% R.H with 16 : 8 (L : D) cycle for experiments.

9.2.2 Insect culture

Myzus persicae were obtained from a colony established at Silwood Park (Section

3.2). For this experiment, aphids which were previously cultured on Chinese cabbage were transferred to potato cv. Desiree and cultured for several generations (Section 8.2).

9.2.3 Parasitoids

Aphidius colemani (Section 3.3) were maintained on M. persicae on potato cv.

Santé. By the time the study was initiated, the parasitoid had completed more than six generations on this cultivar. The colonies were maintained in a controlled environment room

(Section 3.2).

9.2.4 Root knot nematodes (RKN)

Meloidogyne javanica were obtained from the culture already established at Silwood

Park (Section 3.4) on tomato cv. Moneymaker. Roots with egg masses (8 to 9-week old plants) were washed free of soil and examined under a stereo microscope at low power (6 x). Egg masses were removed from the roots using fine forceps and put in a covered dish with distilled water and incubated at 28 °C to allow the infective second stage juvenile (J2) to hatch. Only freshly hatched second stage juveniles (24-48 hours old) were used in experiments. The nematode inoculum was adjusted by counting 2 ml aliquots from a thoroughly shaken solution containing J2 in a counting dish under a stereo microscope and

115 then adjusting the volume of the nematode suspension to give the required number of J2 / ml.

9.2.5 Experimental treatments

Five replicate plants of Desiree per treatment were used for each experiment.

Treatments consisted of John Innes No.1 (low nutritive) and John Innes No. 3 (high nutritive) composts with and without root herbivory by RKN. When plants were at the 3–4 leaf stage (groth stage I), they were transferred to a controlled environment room at 25 ±

2°C and 70% RH with 16 : 8 (L : D) cycle. Half the treatment plants were inoculated with approximately 5000 J2 nematodes in 30 ml tap water per pot, adding the inoculum to the soil around the plant. This inoculum level was within the range documented for M. javanica infestations on potato crops (Vovlas et al. 2005; Jada et al. 2007). Treatments without root herbivory were inoculated with 30 ml of tap water. Plants were left for three weeks for RKN to establish in plant roots (Das et al. 2008) prior to the start of experiments. At the end of each experiment nematode-inoculated plants were uprooted and washed carefully and look for galls and egg masses to confirm that the nematodes had become established and all of the experimental plants were found well infested.

9.2.6 Foliar herbivore performance

Three weeks after the addition of RKN, five plants for each treatment were used for a

M. persicae performance experiment. Plants were randomised on plastic trays in the controlled environment room. Three clip cages were fitted onto three mature leaves and adult apterae added (Section 4.2.3). The experiment was arranged in two blocks based on the two composts and each block was sub-divided for nematode treatments (with and without RKN), making four sub-plots (total of 20 potted plants). After 24h aphids were removed leaving one nymph per leaf and the pre-reproductive time (time from birth to first reproduction) and the offspring produced (Md) were recorded daily and then removed from each cage. Data was obtained to determine:

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1) the effective fecundity, by allowing the aphids to reproduce during a period equivalent to the pre-reproductive time;

2) the intrinsic rate of increase rm = 0.738 (lnMd) / d (Wyatt and White 1977). where Md = the effective fecundity, d = the pre-reproductive time.

9.2.7 Parasitoid performance

A parallel group of 20 plants were planted under identical treatments as for foliar performance experiments (Section 9.2.6). Three weeks after the addition of nematodes, five adult apterae of M. persicae were put in five clip cages and attached to the abaxial side of different leaves on plant for each treatment. The experimental set up with A. colemani was as described in Chapter 8 (Section 8.2.4). Parasitoids were removed after 24 h of their release and the remaining aphids were allowed to continue their development for 10-14 days, the time required from parasitisation to mummy formation for A. colemani (van Emden and Kifle 2002). The cages were maintained at 25°C, 65-70% RH and 16 : 8 L : D and checked on a daily basis from 5 days after parasitoid release for the presence of parasitoid mummies.

Mummies were collected daily in individual labelled Eppendorf tubes (1.5 ml) and then checked every 24 h for parasitoid emergence. The adult A. colemani were placed singly in labelled 2.5 x 8 cm glass tubes. The following data were collected:

iv. percentage parasitism: calculated as the number of mummies divided by the

starting number of aphids;

v. percentage emergence: calculated as the percentage of the number of

hatched mummies, divided by the total number of mummies;

vi. sex ratio: expressed as the proportion of adults that were males (Section

8.2.4);

iv. adult size using hind tibiae length (linear dimensions) as an indicator

(Hohmann et al. 1988; Van Emden and Kifle 2002; Blande et al. 2004); 30

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individual female A. colemani were randomly selected from the combined

replicates per treatment and the hind tibia measured under a binocular

microscope fitted with an eyepiece graticule.

9.2.8 Plant performance

A second parallel group of 20 plants was grown under identical treatments for plant performance assessments made at a time equivalent to the aphid experiments (8-9 weeks).

Plants were cut at soil level and their fresh weight recorded. Foliar and root biomass and total plant biomass (Vandegehuchte 2010a) were measured after drying in an oven at 80°C for 72 h

9.2.9 Data analysis

All statistical analysis was performed with R 2.11.1 (R Development Core Team,

2010). Foliar herbivore performance, parasitism success and plant performance data were analysed with ANOVA. Log or square root transformation was used if necessary to stabilise the variance. Models were simplified by removing variables that did not improve the explanatory statistical power and Post-hoc Tukey honestly significant difference tests were performed to determine the factor levels that differed significantly (Crawley 2005, 2007).

Parasitoid sex ratios were analysed using a generalised linear model with quasibinomial errors due to over dispersion of the bounded proportion. Cultivars and fertiliser treatment factors were included in the model. Significant differences between treatment means were calculated from P-values given by the model.

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9.3 Results

9.3.1 Plant performance

Soil nutrition significantly affected the shoot fresh weight (Table 9.1) Shoot fresh weight was significantly higher with high nutritive John Innes No.3 compared to low nutritive

John Innes No. 1 ( Fig. 9.1a) but total plant biomass and root and shoot biomass (dry wt) separately didn not change with soil nutrition (Table 9.1; Figs 9.1a and 9.2).

Table 9.1 ANOVA of plant fresh weight, and total biomass, shoot and root biomass (dry wt) of potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition, with and without root herbivory by M. javanica (***P < 0.001; **P < 0.01; *P < 0.05).

F value Source of variation d.f. Plant fresh Total Shoot Root weight biomass biomass biomass

Soil Nutrition (A) 1 27.57*** 0.54 0.67 0.21

Root herbivore (B) 1 57.20*** 26.37*** 27.39*** 4.92*

A x B 1 3.59 0.94 1.30 0.00

Plant fresh weight, total biomass and root and shoot biomass were significantly influenced by root herbivory (Table 9.1). Potatoes planted in low nutritive compost with the addition of M. javanica had significantly reduced plant fresh weight, total plant biomass and root and shoot biomass compared with plants without nematodes in both low and high nutritive composts (Figs 9.1a,b and 9.2).

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Fig. 9.1 Shoot fresh weight (a) and total plant biomass (b) of potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

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Fig. 9.2 Shoot fresh weight (a) and total plant biomass (b) of potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

9.3.2 Foliar herbivore performance

Both soil nutrition level and root herbivory significantly affected the foliar herbivore

(M. persicae) performance (Table 9.2). The development time of M. persicae was significantly greater in the presence of root herbivory in low nutritive compost and was the fastest on the plants without nematodes in high nutritive compost (Fig. 9.3). The interaction between soil nutrition and root hebivory was however not significant (Table 9.2).

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Table 9.2 ANOVA of developmental time, effective fecundity and intrinsic rate of increase of M. persicae on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with and without root herbivory by M. javanica (***P < 0.001; **P < 0.01; *P < 0.05).

F value Source of variation d.f. Developmental Effective Intrinsic rate of time fecundity increase

Soil Nutrition (A) 1 6.37* 5.63* 65.28***

Root herbivore (B) 1 23.21*** 19.39*** 223.76***

A x B 1 1.32 0.13 7.39*

Fig. 9.3 Mean (± SEM) developmental time of M. persicae on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

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The effective fecundity of M. persicae was significantly reduced in the presence of root herbivory and soil nutrition but their interaction was not significant (Table 9.2).

Significantly lower fecundity of M. persicae was recorded on plants with M. javanica in low nutritive compost compared with plants without nematodes in high nutritive compost (Fig.

9.4).

Fig. 9.4 Mean (± SEM) effective fecundity of M. persicae on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

The intrinsic rate of increase of M. persicae was significantly lower in the presence of root herbivory and by low soil nutrition and the interaction between soil nutrition and root herbivory was significant (Table 9.2). A significantly lower intrinsic rate of increase of M. persicae was recorded on plants with M. javanica in low nutritive compost compared with plants without nematodes in high nutritive compost (Fig. 9.5).

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Fig. 9.5 Mean (± SEM) intrinsic rate of increase of M. persicae on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

9.3.3 Parasitoid performance

Soil nutrition had a significant effect on parasitism of M. persicae by A. colemani

(Table 9.3). Root herbivory by M. javanica did not affect parasitism but the interaction between soil nutrition and root herbivory was significant (Table 9.3). Parasitism of M. persicae by A. colemani was significantly greater on aphids reared on plants without nematodes in high nutritive compost compared with low nutritive compost (Fig. 9.6). The presence of nematodes did not affect the percentage parasitism by A. colemani in high nutritive compost but parasitism was significantly lower on plants without nematodes in low nutritive compost (Fig. 9.6).

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Table 9.3 ANOVA of percentage parasitism, parasitoid emergence and hind tibia length of A. colemani grown on M. persicae reared on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with and without root herbivory (***P < 0.001; **P < 0.01; *P < 0.05).

F value Source of variation d.f. Parasitism Emergence Tibia length (%) (%) (mm)

Soil Nutrition (A) 1 12.12** 10.11** 19.91***

Root herbivore (B) 1 0.94 18.94*** 15.39***

A x B 1 4.82* 3.60 0.01

Fig. 9.6 Mean (± SEM) percentage parasitism of A. colemani on M. persicae reared on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

Soil nutrition and root herbivory by M. javanica had significant effects on parasitoid emergence, although the interaction between soil nutrition and root herbivory was not significant (Table 9.3). Nematodes negatively affected the percentage parasitism by A.

125 colemani reared on aphids on plants in low and high nutritive composts (Fig. 9.7).

Percentage parasitism by A. colemani was significantly greater on aphids reared on plants without nematodes in high nutritive compost compared with low nutritive compost with nematodes (Fig. 9.7).

Fig. 9.7 Mean (± SEM) percentage emergence of A. colemani on M. persicae reared on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

The sex ratio of emerging parasitoids ranged from 35 to 70% (Fig. 9.8). The sex ratio was significantly affected by root herbivory (F1, 197 = 9.8358, P = 0.0019) and soil nutrient treatment (F1, 198 = 3.8823, P = 0.050). Parasitoids emerging from the aphids reared on the plants with nematodes in low nutritive compost were mostly male (Fig. 9.8).

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Fig. 9.8 Mean (± SEM) sex ratio (proportion of male) of A. colemani on M. persicae reared on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

Soil nutrition and root herbivory by M. javanica had significant effects on the length of female hind tibia but the interaction between soil nutrition and root herbivory was not significant (Table 9.3). Nematodes negatively affected the hind tibia length of A. colemani reared on aphids on plants in low and high nutritive composts (Fig. 9.9). Hind tibia length was significantly greater for parasitoids on aphids reared on plants without nematodes in high nutritive compost compared with plants on low nutritive compost with nematodes (Fig.

9.9).

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Fig. 9.9 Mean (± SEM) tibia length (mm) of female A. colemanireared on M. persicae reared on potato cv. Desiree planted in low (John Innes No.1) and high (John Innes No.3) soil nutrition with (+N) and without (-N) root herbivory by M. javanica. Treatments with the same letter are not significantly different (P >0.05).

9.4 Discussion

The amounts of nutrient a plant can offer to an herbivore depend on inherent features of that plant, such as root and shoot biomass, and plant age (Vandegehuchte et al.

2010b). In the present study plant fresh weight, total plant biomass and root and shoot biomass were significantly reduced in the presence of root herbivory by M. javanica.

Meloidogyne javanica infestations impair nutrient and water uptake by roots with severe implications for plant growth and development (Melakeberhan et al. 1987; Melakeberhan et al. 1990; Williamson and Gleason 2003; Vovlas et al; 2005). Many studies have reported the adverse effects of root herbivory by M. javanica on potato crops with a reduction in

128 above-ground biomass (e.g. Jatala and Bridge 1990; Mooney et al. 1991; Hussey and

Williamson 1997; Di Vito et al. 2003; Vovlas et al. 2005) but there is some evidence that increasing soil nutrition may enhance tolerance to nematode infections (Melakeberhan et al.

1987; Sikander et al. 2009).

Meloidogyne spp. act as a metabolic sink that directs nutrients to the site of nematode parasitism in the roots (Burgeson 1966; McClure 1977). The presence of nematodes thus hinders nutrient supply to the above-ground plant parts, which in turn can have negative effects on the performance of foliar herbivors (Bezemer et al. 2005; Bezemer and Van Dam 2005; Kaplan et al. 2009). The results of the present study clearly demonstrated that M. javanica infesting potatoes can negatively affect the performance of foliar feeding M. persicae. Aphids had higher effective fecundity and intrinsic rate of increase and shorter developmental times in the absence of root herbivory by M. javanica at both low and high soil nutrition. The results of aphid performance without root herbivory are similar to the aphid performance studies in different nutritional composts reported in

Chapters 4 and 6.

The present results correspond with the lower M. persicae performance on M. incognita inoculated tobacco plants reported in greenhouse trials (Kaplan 2007) and in field cage studies with different genera of nematodes infesting tobacco (Kaplan et al. 2009).

Another study documented lower M. persicae fecundity on Plantago lanceolata attacked by the nematode Pratylenchus penetrans Cobb (Tylenchida: Pratylenchidae) (Wurst and van der Putten 2007). While Vandegehuchte (2010b) reported negative effects of the cyst nematode, Heterodera arenaria Cooper (Tylenchida: Heteroderidae), feeding on the root of

Ammophila arenaria on the foliar feeder Schizaphis rufula Walker (Hemiptera: Aphididae) in the laboratory.

Bezemer et al. (2005) also found that nematode inoculation of roots in the grasses

Agrostis capillaris and Anthoxanthum odoratum reduced the performance of Rhopalosiphum padi L. (Hemiptera: Aphididae) on the leaves and found that effects could be attributed to a

129 reduction of foliar nitrogen and amino acid concentrations. Since root herbivory drastically reduced shoot biomass as shown in the present study, this might explain the negative effect of the nematodes on the density of foliar feeding aphids. As these aphids are sap feeders on the above-ground parts of plants leaves, it is possible that lowering amino acid levels in leaves by root-feeders can negatively affect shoot feeders (Bezemer et al. 2005). The success of aphid herbivory is highly dependent on the nutritional quality of their food plant

(Chapters 4, 6 and 7), and M. javanica may therefore negatively affect sap-feeders by attracting plant resources towards roots, thereby reducing above-ground resource allocation.

In addition to influencing foliar herbivore performance mediated through the host plant, below-ground herbivory by root feeding nematodes and insects can also influence the performance of the herbivore‟s natural enemies (Masters et al. 2001; Bezemer et al. 2005;

Soler et al. 2007). These effects may be direct, by changing the quality or quantity of the shared host plant (Bezemer and Van Dam 2005) or indirect, by producing volatile compounds in response to root herbivory (Soler et al. 2007). In the present study, the percentage parasitism by A. colemani was not influenced by the presence of root herbivory by M. javanica alone. However, the interaction of soil nutrition and root herbivory influenced the percentage parasitism. More parasitoids were found in aphids feeding on plants in high nutrient soils compared with control plants without root herbivory.

The above findings are similar to the percentage parasitism by A. colemani observed in aphids feeding on plants in high nutritive compost (Chapter 8). However, the effect of root herbivory on parasitism was much more pronounced in low nutrient compost, where plants with root herbivory produced more parasitoids compared with control plants. Masters et al.

(2001) also found that root-feeding insects can increase the parasitism rate of seed feeding insects in European swamp thistle, Cirsium palustre. Guerrieri et al. (2004) showed that aphid parasitoids were strongly attracted to mycorrhizal plants compared with control plants without mycorrhiza.

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Bezemer et al. (2005) found positive effects of the root herbivory by an undefined community of plant parasitic nematodes on the A. colemani obtained from R. padi reared on grass species: A. odoratum and A. capillaries. In the present study, increased parasitism in the presence of root herbivory in low nutritive soil may be due to the fact that the parasitoids had no choice in plant selection. Thus, due to the poor plant architecture and prolonged developmental time of aphids in the above treatment, the aphids would be more exposed to parasitism compared with faster developing aphids on more vigorous plants (Cloyd and

Sadof 2000). A second explanatory factor could be differences in plant volatiles in response to root feeding nematodes, which may also vary between cultivars. Therefore it is possible that the chemistry of the leaves on nematode-feeding plants may be altered to such an extent as to affect the attractiveness of plants to foraging parasitoids.

In contrast, parasitoid fitness related parameters such as percentage emergence; adult size and sex ratio were negatively influenced in the presence of root herbivory by M. javanica and positively influenced by high nutritive composts. Fitness related parameters were also positively affected in high nutritive compost in earlier experiments (Chapter 8). In the present experiment, more parasitoids emerged from plants without nematodes, particularly in high nutritive compost. Significantly larger adult A. colemani were collected from aphids feeding on potato plants without nematodes and in high nutritive compost.

Parasitoids collected from plants with nematodes were more male biased than those collected from aphids reared on plants without nematodes in high nutritive compost. These results are in agreement with Hunter‟s (2003) proposed plant quality mechanisms for parasitoid success, where low quality plants are associated with an increase in the production of males and a decline in the size, number and longevity of parasitoids. Hunter

(2003) correlated the performance of parasitoids with host plant quality. Since below-ground root herbivory and low soil nutrition directly affected plant quality, parasitoid success would be influenced by root herbivory (Soler et al. 2005) and nutritional stress (Chapter 8). In the present experiment, the direct link between plant quality and parasitoid performance was

131 associated with the increased production of males, a reduction in hind tibia length and adult emergence under both root herbivory and low nutritive compost.

In conclusion, the present experiment suggests that below-ground herbivory by nematodes is able to cause significant changes in the host plant biomass and consequently affect the behaviour and population dynamics of foliar feeding aphids and their parasitoids.

In the majority of cases decisions on pest management are restricted to above-ground herbivores, and the possible effects of plant parasitic nematodes on the above-ground tritrophic systems are ignored. This study highlights the potential importance of root herbivory by nematodes when considering integrated pest management programmes.

However, these studies need to be repeated under diverse field conditions to see whether the above laboratory observations are translated into the field.

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CHAPTER 10

Summary and General Discussion

10.1 Summary of the experimental findings

CHAPTER 4: The performance of Myzus persicae on potato cultivars at different soil nutritional levels

 In laboratory experiments, potato cv. Pink Fir Apple and in particular cv. Desiree was

more susceptible to M. persicae compared with cv. Anya; aphid performance was

particularly poor on Anya suggesting antibiosis resistance.

 High nutritive compost increased fecundity and intrinsic rate of increase of M.

persicae on all cultivars.

 Low nutritive compost reduced fecundity and increased the developmental time for

the M. persicae on all cultivars.

CHAPTER 5: Field performance of Myzus persicae on potato cultivars in Pakistan and the UK

 The same pattern of susceptibility to M. persicae found in the laboratory (Chapter 4)

was observed in field trials with natural infestations of aphids at three locations in

Pakistan (2009, 2010) and one location in the UK (2008, 2009).

 The field trials also showed that aphids were less attracted to Anya in addition to

perfoming poorly on this cultivar suggesting that antixenosis resistance as well as

antibiosis resistance was present.

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CHAPTER 6: Performance of Myzus persicae on upper, middle and lower leaves of three potato cultivars at low and high soil nutrition

 The performance of M. persicae was better on the lower mature leaves compared

with the upper younger leaves on all the cultivars and soil nutrition levels.

CHAPTER 7: The preference of Myzus persicae on potato cultivars at different soil nutritional levels

 In choice experiments, M. persicae preferred to colonize the susceptible cv. Desiree

compared with cv. Anya.

 Myzus persicae also preferred potato plants planted in high nutritive compost

compared with low nutritive compost.

 In olfactometer bioassays, significantly more M. persicae were attracted to the odour

of cv. Desiree compared with cv. Anya; M. persicae were also significantly more

attracted to potato plants planted in high nutritive compost compared with low

nutritive compost.

 No significant differences in leaf pubescence were found between cvs Desiree, Pink

Fir Apple or Anya.

CHAPTER 8: The performance of Aphidius colemani on Myzus persicae reared on three potato cultivars at different soil nutritional levels

 Percent parasitism by A. colemani was not influenced by potato cultivar.

 Percent parasitism by A. colemani increased with increasing soil nutrition.

 Parasitoid development time, emergence and adult female size (hing tibia length)

were significantly affected by potato cultivar and soil nutrition; A. colemani took

longest to develop on Anya in low nutritive compost; parasitoid emergence and

female hind tibia length was greatest on Desiree and Pink Fir Apple in high nutritive

compost.

134

 Neither soil nutrition nor potato cultivar affected the parasitoid sex ratio.

CHAPTER 9: Multitrophic interaction in potato-aphid-parasitoid system: influence of root herbivory and soil nutrition

 Plant biomass was significantly reduced by root herbivory (M. javanica) and by low

nutritive compost.

 The performance of M. persicae was reduced by root herbivory in low nutritive

compost.

 Root herbivory had a positive effect on percent parasitism by A. colemani at low soil

nutrition.

 Parasitoid emergence, sex ratio and female size (hind tibia length) were significantly

affected by root herbivory and low soil nutrition; fewer parasitoids emerged, and

were smaller in size and male biased on plants with low soil nutrition and root

herbivory.

10.2 General Discussion

In integrated pest management programmes, a combination of population regulation strategies, such as management of soil fertility, host plant resistance and biological control, can maintain insect pests below the economic threshold for damage with little impact on the environment. The present study addressed such factors in combination with soil nutrition and also looked at the impact of root herbivory on multitrophic interactions above ground.

10.2.1 Soil nutition

Various studies have shown that insect performance on plants is linked to the physical, chemical and biological properties of soil (Altieri and Nicholls 2003; Zehnder et al.

2007). Healthy and vigorous plants that grow quickly are better able to withstand pest

135 damage but over-fertilizing crops can actually increase pest problems (Zehnder et al. 2007).

In the present work, nutrient rich composts increased the susceptibility of potatoes to infestation of M. persicae (Chapters 4 and 6). Myzus persicae performed well in terms of greater fecundity and intrinsic rate of population increase (rm) on nutrient rich compared with low nutritive compost and this trend was consistent both for susceptible and resistant potato cultivars. Similarly, aphids showed greater preference for vigorous plants, planted in high nutritive compost (Chapter 7). These results are in line with observations that soil fertility practices can affect the physiological susceptibility of crops to insect pests by either affecting the resistance of plants to attack or by altering plant acceptability (Altieri and

Nicholls 2003; Sarfraz et al. 2009b; Sarfraz et al. 2010). Aphidius colemani also performed well on the aphids reared on the vigorous, well fertilized plants (Chapter 8). These results are in agreement with proposed plant quality mechanisms for parasitoid success, where low quality plants are associated with decreased parasitoid performance (Reed et al. 1991;

Godfray 1994; Hunter 2003). The above work illustrates the importance of attaining an optimal balance of soil fertilization for crop growth and pest population regulation.

10.2.2 Host plant resistance

Plants can defend themselves against herbivores with direct and indirect defence mechanisms (Groot and Dicke 2001). Direct plant defences affect herbivores through physical or chemical means, such as thorns, toxins and digestibility reducers. Indirect defence includes the attraction of natural enemies of herbivores. Natural enemies can be influenced by plant characteristics independently of the herbivore or mediated through herbivore activities. The relevant plant characteristics include: i) plant tissues and products that are used as a source of nutrition by natural enemies; ii) plant morphology; iii) visual and vibrational cues; iv) secondary plant chemicals; v) plant volatiles (Thomas and Waage 1996;

Dicke 1999; Groot and Dicke 2001).

Host plant resistance results from traits that reduce the attractiveness of plants to herbivores (antixenosis), reduce herbivore growth, fecundity and survival (antibiosis) or

136 increase the ability of plants to compensate for herbivory (tolerance) (Painter 1951, 1958).

Growing resistant varieties can indirectly affect natural enemies by modifying their behaviour or changing the nutritional quality of their host (van Emden 1995). In the present study, the evidence suggested that the potato cv. Anya showed both antixenosis and antiobisis resistance to M. persicae (Chapters, 4 - 7).

Studies on plant resistance to insect pests have shown that resistance varies with the age or growth stage of the plant (Slansky 1990). In the current experiments, the performance of M. persicae was consistently greater on lower mature leaves compared with upper younger leaves irrespective of cultivar and soil nutrition levels. Such observations are important considerations for the monitoring of M. persicae infestations and the efficacy of control methods.

No significant differences in percentage parasitism or the sex ratio of emerging parasitoids were found between cultivars but other parasitoid fitness parameters were negatively affected by the resistant cv. Anya; observations in agreement with Godfray

(1994) who suggested that host quality affects three components of parasitoid fitness: parasitoid survival, size and development time. It has been suggested that low host quality for herbivores (as a result of resistance) may have a greater impact on parasitoids than on predators since parasitoid larvae have to complete their development within or on a single host whereas predators can feed on multiple prey individuals (Vinson and Iwantsch 1980).

Many pest management practitioners have found that host plant resistance is fundamentally compatible with biological control (Verkerk et al. 1998a). Positive interactions result where natural enemies use herbivore-induced synomones emitted by plants as cues to find prey (Vet and Dicke 1992; Dicke et al. 2003; Arimura et al. 2005; D‟Alessandro et al.

2006). Partial plant resistance may also provide benefits for the third trophic level by reducing the growth rate of prey which in turn increases the duration of their availability to natural enemies (Feeny 1976; Price et al. 1980). In some cases, however negative interactions between plant resistance and biological control are caused by toxic secondary plant compounds which can be passed on through herbivores to their natural enemies (Hare

137

1992; Harvey et al. 2003). They may also be caused by plant physical factors which can impede natural enemy effectiveness (e.g. leaf toughness, cuticle thickness, trichomes (Price

1986)). Therefore it is important to integrate host plant resistance and biological control with precision.

10.2.3 Root herbivory

Attacks by herbivores can induce reflective changes in plant allelochemistry

(Bezemer et al. 2004, Kaplan et al. 2008a) and in the distribution of metabolites within plants (Kaplan et al. 2008b). These changes can also result in systemic resistance and affect performance and preference of secondary plant attackers (Stout et al. 2006).

Root-feeding nematodes can cause major damage on crop plants (Smiley et al.

2005; Fuller et al. 2008). The effects of root-feeding nematodes on above-ground herbivores can be negative due to the induction of systemic defences (Bezemer and van

Dam 2005) or by the lowering of amino-acid contents of leaves (Bezemer et al. 2005).

Positive effects may also occur, for instance due to the destruction by root-feeding nematodes of production sites of secondary metabolites in the roots (Kaplan et al. 2008a).

In the present work, below-ground herbivory by the root-knot nematode, M. javanica negatively affected plant quality by reducing plant biomass and M. persicae on nematode infested potatoes were observed to be less fecund, have a lower intrinsic rate of increase

(rm) and a prolonged developmental time on plants in both high and low nutritive composts.

Parasitoid (A. colemani) fitness related parameters (adult emergence, sex ratio and female hind tibia length) were also lower on aphids feeding on nematode infested plants but percentage parasitism was not affected by the presence of nematodes.

Root-knot nematodes establish and maintain permanent feeding cells (“giant cells”), which act as a nutrient sink, diverting nutrients for nematode development and reproducution (McClure 1977; Abad et al. 2009). Plant nutrient and water uptake are substantially reduced by the related damage to the root system, and infested plants are therefore weakened, are more sensitive to the level of soil nutrition and drought, and have

138 reduced yields (Abad et al. 2009). Changes in the host plant may therefore affect aphid performance and thus indirectly the performance of their parasitoids as discussed in

Sections 10.1 and 10.2.

Wurst and van der Putten (2007) reported that M. persicae produced fewer offspring on English plantain, P. lanceolata infected by root lesion nematodes (P. penetrans) compared with uninfected plants. Reduced performance of the aphid Rhopalosiphum padi on grasses has been related to reduce amino acid levels in leaves caused by root-feeding nematodes (Bezemer et al. 2005).

10.2.4 Conclusions

The present study has demonstrated the effects of plant quality and host plant resistance on herbivore-natural enemy interactions and the impact soil nutrition and below- ground herbivory can have on such above-ground multitrophic interactions. The effects of food plant quality on insect performance are usually less apparent on the parasitoid than on the herbivore (Gols and Harvey 2009). In the present study, while adverse effects of low soil nutrition, host plant resistance and root herbivory were seen on parasitoid fitness related parameters there was less effect on percent parasitism and wasps equally parasitised aphids on resistant and susceptible potato cultivars with and without root herbivory, although parasitism was greater on plants with high soil nutrition. Since vigorous, well nourished plants in combination with host plant resistance can better compensate for insect attack than plants under nutrient stress (Dosdall et al. 2004; Kalule and Wright 2005; Soglia et al. 2006; Sarfraz et al. 2009b), managing soil nutrition levels more precisely may be a useful enhancement for integrated cop and pest management systems.

10.3 Future work

Several areas of interest are identified here.

139

 To conduct multi-generation studies on aphid-parasitoid population dynamics to

determine whether an optimal level of soil nutrition can lead to an enhanced and

sustainable level of biological control.

 To investigate further the apparent antixenosis- and antibiosis-based resistance

mechanisms observed for cv. Anya. For example, entrainment experiments have

been conducted with Anya and Desiree to follow up the olfactometer studies with M.

persicae (Chapter 7) and identification of trapped volatile compounds will be

determined by GC-MS. Compounds of interest will be tested singly and in

combination using olfactometry and electro-antenogram methods.

Electrophysiological probing and feeding experiments with M. persicae are also

required to determine whether antixenosis in cv. Anya has a non-volatile component.

Chemical and molecular studies could also be used to identify differences between

cv. Anya and its related cvs Desiree and Pink Fir Apple that correlate with

differences in aphid preference and performance. The above studies would help to

identify traits for use in conventional plant breeding programmes or which may be

amenable to a genetic engineering approach.

 To investigate the impact of root-herbivory by plant parasitic nematodes on above-

ground multitrophic interactions under more natural, field related conditions,

including the effects of soil type and soil mositure levels.

140

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Appendices

Appendix 1. Foliage of potato, cv. Desiree

Appendix 2. Foliage of potato, cv. Anya

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Appendix 3. Foliage of potato, cv. Pink Fir Apple

Appendix 4. Foliage of potato, cv. Sante

From; http://www.europotato.org/full_image.php?image=./photos/sante_l.jpg&variety=Sant e).

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Appendix 5. Key growth stages of Ptato (From Johnson 2008)

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