Unravelling the resistance mechanism of lettuce against Nasonovia ribisnigri Cindy J.M. ten Broeke Thesis committee Promotors Prof. dr. ir. J.J.A. van Loon Personal chair at the Laboratory of Entomology Wageningen University Prof. dr. M. Dicke Professor of Entomology Wageningen University Other members Prof. dr. ir. J. Bakker, Wageningen University Prof. dr. ir. B.P.H.J. Thomma, Wageningen University Dr. B.J. Vosman, Plant Research International, Wageningen University and Research Centre Dr. M. de Vos, Keygene B.V., Wageningen This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. Unravelling the resistance mechanism of lettuce against Nasonovia ribisnigri Cindy J.M. ten Broeke Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Thursday 5 September 2013 at 11 a.m. in the Aula. Cindy J.M. ten Broeke Unravelling the resistance mechanism of lettuce against Nasonovia ribisnigri. 244 pages. PhD thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in Dutch and English ISBN 978-94-6173-578-2 vi Abstract Abstract Aphids are serious pests of crop plant species, and host plant resistance is often the most effective and environmentally friendly control strategy to control these pests. One of these aphid pests is the black currant - lettuce aphid,Nasonovia ribisnigri (Mosely), an economically important pest of cultivated lettuce, Lactuca sativa L. Host plant resistance has been used since 1982 to control this aphid species and is mediated by the Nr-gene, originating from wild lettuceLactuca virosa L. However, this resistance is not effective anymore, sinceN. ribisnigri aphids virulent to the Nr-resistance have been reported since 2007. The aim of this thesis was to unravel the mechanism of resistance mediated by the Nr-gene against N. ribisnigri, by behavioural studies on the aphids on both resistant and susceptible lettuce, to allow lettuce breeders to accelerate their resistance breeding programmes. Although the exact mechanism of Nr-mediated resistance remains unknown, the data in this thesis provide insight into this mechanism. The active site of the Nr-mediated resistance is mainly located in the phloem and some resistance might be encountered by the aphids along the pathway to the phloem. The inability of the avirulent aphids to feed from the resistant plant could be caused by the failure of aphids to suppress the wound response of the sieve element. The resistance factor(s) are only produced in the shoot, because grafts with resistant shoots and susceptible roots were resistant, whereas grafts with susceptible shoots and resistant roots remained susceptible. An intact vascular system is needed for full resistance, because both detached leaves and leaf disks of resistant lettuce plants were less resistant. Variation in virulence was observed among populations of different geographical origin. Aphids from a highly virulent population performed equally well on both resistant and susceptible lettuce plants, whereas semi-virulent aphids performed better on susceptible lettuce plants. Both short-term and long-term virulence loss were observed for virulent aphid populations differing in virulence level, which indicates this virulence is associated with fitness costs. A possible mechanism underlying virulence in N. ribisnigri to the Nr- resistance is the presence of an effector protein in the salivary secretion of the aphids suppressing resistance. Virulent aphids seemed to actively suppress the resistance in lettuce against the avirulent aphids. The original donor or the Nr-resistance, L. virosa accession IVT 280, was tested as possible source of new resistance against the virulent biotypes of N. ribisnigri and was found fully resistant against virulent aphids, and can be exploited as a source of resistance in breeding for new resistance in cultivated lettuce. vii viii Table of contents Table of contents Abstract vii Chapter 1: General Introduction 1 Insect-plant interactions 2 Aphid-plant interactions 2 Aphid biology 2 Aphid feeding 4 Recording stylet penetration behaviour: the electrical penetration graph technique 7 Protection of plants against aphids 8 Chemical control 8 Biological control 9 Cultural practice 9 Host plant resistance 10 The Nasonovia – lettuce interaction 10 The Nasonovia – lettuce problem 10 Biological characteristics of Nasonovia ribisnigri 11 Lettuce 11 Nasonovia ribisnigri distribution 12 Protection of lettuce against Nasonovia ribisnigri 14 Chemical control 14 Behavioural disruption by semiochemicals 14 Biological control 15 Host plant resistance in lettuce 16 Appearance of a virulent biotype 17 Objectives of this thesis 17 Thesis Outline 18 Chapter 2:Performance and feeding behaviour of two biotypes of the black currant-lettuce aphid,Nasonovia ribisnigri, on resistant and susceptible Lactuca sativa near-isogenic lines 21 Abstract 22 Introduction 23 Materials and Methods 24 Plants and Aphids 24 Performance test 25 Population development 25 EPG recording 26 ix Statistics 27 Results 27 Performance data 27 Population development 28 EPG analysis of biotype Nr:0 on different NILs 31 EPG analysis of biotype Nr:1 on different NILs 31 EPG analysis for biotype Nr:0 on NIL S1 and NIL R2 31 EPG analysis for biotype Nr:1 on NIL S1 and NIL R2 32 EPG analysis for biotype Nr:0 and biotype Nr:1 32 Discussion 36 Differences among NILs 36 Differences between biotypes 36 Plant surface effects 36 Pathway phase 37 Phloem phase 37 Resistance mechanism 38 Virulent biotype Nr:1 39 Genetic basis of plant resistance and aphid virulence 40 Methodological limitations 41 Conclusion 42 Acknowledgements 42 Chapter 3: Feeding behaviour and performance of different populations of the black currant-lettuce aphid, Nasonovia ribisnigri, on resistant and susceptible lettuce 45 Abstract 46 Introduction 47 Materials and Methods 48 Plants 48 Aphids 49 EPG recording 49 Performance test 50 Reproduction test 50 Statistics 50 Results 51 Aphid populations on the cultivars 51 EPG parameters 51 Performance and reproduction 52 Aphid populations on the NILs 56 x Table of contents EPG parameters 56 Performance and reproduction 56 Discussion 60 Differences in virulence between populations on resistant lines 60 Differences between resistant lettuce cultivar and NIL 61 Development of virulence 61 Loss of virulence 62 Conclusion 64 Acknowledgements 64 Chapter 4: Resistance to a new biotype of the lettuce aphid Nasonovia ribisnigri in a Lactuca virosa accession 71 Abstract 72 Introduction 73 Material and Methods 74 Plants and Aphids 74 EPG recording 74 Performance 75 Reproduction test 75 Statistics 75 Results 76 Nr:0 76 Nr:1 Germany 83 Nr:1 Belgium 83 Nr:1 Perpignan 83 Nr:1 Paris 84 Discussion 84 Lettuce aphid behaviour and performance on susceptible L. virosa accessions 84 New resistance source against Nr:1 85 Utilising resistance against the Nr:1 biotype 86 Conclusion 87 Acknowledgements 87 Chapter 5: The effect of co-infestation by conspecific and heterospecific aphids on the feeding behaviour of Nasonovia ribisnigri on resistant and susceptible lettuce cultivars 93 Abstract 94 Introduction 95 xi Materials and Methods 96 Plants and Aphids 96 EPG recording 97 Performance test 98 Statistics 99 Results 99 Effects of feeding in an intraspecific group 99 Effects of Nr:1 on Nr:0 aphids 100 Effects of Nr:0 on Nr:1 aphids 100 Effects of Macrosiphum euphorbiae 106 Effects of Myzus persicae 106 Discussion 107 Living in a group 107 Defence suppression in lettuce 107 Induced defence in lettuce 109 The effect of heterospecific aphid species on N. ribisnigri 109 Conclusions 111 Acknowledgements 111 Chapter 6: The effect of rearing history on behaviour and performance of two virulent Nasonovia ribisnigri populations on two lettuce cultivars 119 Abstract 120 Introduction 121 Materials and Methods 122 Plants and Aphids 122 EPG recording 123 Performance test 123 Statistics 124 Results 124 Nr:1 Ge colonies on Corbana 124 Nr:1 Ge colonies on Terlana 124 Nr:1 Be colonies on Corbana 128 Nr:1 Be colonies on Terlana 128 Corbana versus Terlana 131 Nr:0 versus Nr:1 on Terlana 131 Discussion 131 Cost of virulence 133 Effect of different rearing plants 134 Conclusion 135 xii Table of contents Acknowledgements 135 Chapter 7: Feeding behaviour and performance of Nasonovia ribisnigri on grafts, detached leaves and leaf disks of resistant and susceptible lettuce 137 Abstract 138 Introduction 139 Materials and Methods 140 Plants and Aphids 140 Plant treatments 141 EPG recording 142 Performance test 143 Statistics 143 Results 144 Grafts 144 Detached leaves 148 Leaf disks 149 Discussion 149 Grafts 149 Excised leaves and leaf disks 150 Resistance factor 152 Conclusion 153 Acknowledgements 153 Chapter 8: General Discussion 159 Introduction: Host plant resistance against aphids 160 The Nr-gene 161 Signalling pathways 167 NBS-LRR genes 168 Factors influencing resistance 169 Virulence in aphids 169 Aphid effectors 170 Virulence in Nasonovia ribisnigri 172 Transcriptomics of gut and salivary gland tissue of Nasonovia ribisnigri 173 Symbionts 175 Resistance against pesticides 176 Alternative resistance 176 New host plant resistance in Lactuca virosa 176 Transgenic plants 177 xiii How to extend the durability of R-genes? 178 Conclusion 180 Future perspectives 182 References 184 Summary 209 Nederlandse Samenvatting