Allma Mater Studiiorum – Uniiversiità dii Bollogna DOTTORATO DI RICERCA IN Scienze e Tecnologie Agrarie, Ambientali e Alimentari Ciclo 27 Settore Concorsuale di afferenza: 07/D1 Settore Scientifico disciplinare: AGR/12 TITOLO TESI: DETECTION AND MOLECULAR CHARECTERIZATION OF VIRUSES INFECTING ACTINIDIA SPP. Presentata da: Roberta Biccheri Coordinatore Dottorato Relatore Giovanni Dinelli Carlo Poggi Pollini Correlatore Claudio Ratti Esame finale anno: 8 Maggio 2015 Acknowledgements Acknowledgements I would like to thank Claudio and Carlo, for providing me with the opportunity to learn so much. Thank you for all of your support and advice. I would like to thank, Associate Professor M. N. Pearson from University of Auckland and A. G Blouin from the New Zealand Institute for Plant & Food Research for their assistance and support during in my period in New Zealand where I learned so much. I really want to thank all my collegues of the lab in Bologna for all of their support and encouragement: Alice, Chiara, Roberta and Mattia. I really want to thank my family and my boyfriend for all of their love and for to have supported my choices, even if it has been really hard for them. i Table of Contents Table of Contents Acknowledgement i Chapter 1: General Introduction: 1 Kiwifruit origins 2 The genus Actinidia 4 Actinidia species in cultivation 7 Global kiwifruit industry 9 Production and marketing in Italy 13 Disease in Actinidia spp. 16 The aims of my study 19 References 21 Chapter 2: Virus infecting Actinidia spp.: 24 Viruses of kiwifruit 25 Non-Specialist viruses 29 Alfalfa mosaic virus and Cucumber mosaic virus 29 Ribgrass mosaic virus and Turnip vein clearing virus 31 Apple stem grooving virus 33 Cucumber necrosis virus 35 Actinidia virus X 36 Kiwifruit-Adapted viruses 37 Actinidia citrivirus 37 Actinidia virus A and Actinidia virus B 39 Disease-Inducing viruses 41 Cherry leaf roll virus 41 Pelargonium zonate spot virus 42 Mechanically transmitted viruses with no known natural vectors 43 Viruses with aphid vectors 44 Viruses with presumed mealy bug vectors 44 Viruses transmitted by seed and/or pollen 45 References 47 Chapter 3:Project paper: Characterization of Cucumber mosaic virus naturally infecting Actinida chinensis in Italy 53 Abstract 54 Introduction 54 Materials and methods 57 Source plants 57 Transmissions to herbaceous indicators 58 Table of Contents Purification of viral particles and determination of viral protein weight 58 Random RT-PCR and sequencing 59 Circular RT-PCR 60 Construction of full-length agroinfectius clones and leaf agroinfiltration 61 Sequence alignment and phylogenetic analysis 61 Results 62 Symptoms observed and transmissions to herbaceous indicators 62 Transmission electron microscopy (TEM) and determination of viral protein weight 62 Sequencing 65 Sequence alignment and phylogenetic analysis 66 Detection by RT-PCR 70 Agroinfectious clones 70 Discussion 71 References 74 Chapter 4: Project paper: First report of Pelargonium zonate spot virus infecting Actinidia 77 Abstract 78 Introduction 79 Materials and methods 82 Virus isolates and host plant observation 82 Transmissions to herbaceous indicators 82 Transmission electron microscopy (TEM) 83 Purification of the viral particles 83 Random-PCR amplification and sequencing 84 Circular RT-PCR and full-length amplifications 85 Serological and molecular detection 87 Identification of the PZSV encoded suppressor of RNA silencing 87 Results 88 Symptoms on host 88 Transmissions to herbaceous indicators 88 Purification of the Viruses 90 Random-PCR amplification and sequencing 90 Circular RT-PCR and full-length amplifications 91 Serological and molecular detection 93 Evaluation of RNA silencing suppressor activity of CP and MP proteins 94 Discussion 95 References 97 Table of Contents Chapter 5 Project paper: Identification and characterization of two new viral species from Actinidia chinensis 101 Abstract 102 Introduction 102 Materials and methods 104 Plants material 104 Double-stranded RNA extraction 104 Reverse transcriptase and amplification reactions 105 Sequencing data analysis 105 Terminal regions 106 Results 108 Complete genome sequence of Actinidia latent virus 108 Computational analysis of Actinidia latent virus genome 109 Phylogenetic analyses of Actinidia latent virus 115 Characterization of Kiwifruit associated totivirus 1 121 Discussion 125 References 130 Chapter 6: General discussion and conclusion 133 General Discussion 134 Do viruses pose a threat to kiwifruit? 136 Mitigating virus spread 137 Conclusions 138 References 139 Appendix A : Invited Review: Viruses of kiwifruit (Actinidia species) 140 Appendix B: Materials and method 141 Appendix C: Primers and Accession numbers 154 Appendix D: Sequence 158 - Chapter 1 - Chapter 1: General Introduction 1 - Chapter 1 - Kiwifruit origin The kiwifruit plant is native to eastern Asia and in 1900 it was just a plant growing in the hills and mountains of south-central China, between the Yangzi (Chang Jiang) and Pearl (Zhu Jiang) rivers (Datson & Ferguson, 2011) but palaeobiological studies estimate kiwifruit to be at least 20-26 million years old (Qian & Yu, 1991). One of the earliest descriptions of the plant and fruit (known then in China as mihoutao, monkey peach) was assigned to an author in the twelfth century Song Dynasty, who described kiwifruit as “ found in the valleys of the mountains; it is a vine with round, pubescent leaves, which grows by climbing over trees; in shape and size the fruit resembles an egg; its skin is brown; after the first frosts, it becomes sweet and edible,” as referenced by Ferguson (Ferguson, 1990b). Kiwifruit comprise more than 55 species and about 76 taxa belonging to the genus Actinidia, with a wide variability in fruit shape, size, colour and composition (Figure 1.1) (Ferguson, 1990a). Fig 1.1: Fruit of the Actinidia genus showing variation in flesh colour, size and shape. 2 - Chapter 1 - The original name of kiwifruit was ‘Chinese gooseberry’ and it was a name in common usage in New Zealand and elsewhere until it has been exported to the United States in 1959 . The idea to rename the fruit ‘kiwifruit’ is credited to Frieda Caplan, owner of Frieda’s Finest Produce Specialitiea, which was among the first company to import the fruit into the United States. With its brown furry skin, which resembled New Zealand’s iconic native bird the kiwi, Frieda suggested New Zealand growers to rename the fruit to get a better marketing response. Following this, the New Zealand fruit marketer Turners & Growers adopted this name and since then the name kiwifruit has achieved general acceptance across commercial, scientific and technical fields (Ferguson & Bollard, 1990). The name kiwifruit is now often used for all species within the genus Actinidia. Actinidia species were introduced to Europe, the U.S.A. and New Zealand in the late 19th and early 20th century (Ferguson & Bollard, 1990). New Zealand was largely responsible for the initial development and commercial growing of kiwifruit. In 1904, Isabel Fraser, returned from her travel in China, introduced the first kiwifruit seeds to New Zealand upon which the New Zealand kiwifruit industry was built. By 1910 the plants raised by a friend, Alexander Allison, produced the first fruit outside China. Actinidia deliciosa cv. Hayward was selected around 1925 and kiwifruit production started in New Zealand with the first commercial orchards established in 1930s and the first commercial exports of fruit of A. deliciosa started in 1953 (Ferguson & Bollard, 1990). Until 2000 A. deliciosa cv. Hayward was the backbone of the global kiwifruit industry and almost all the international trade in kiwifruit was of this sole cultivar. When facing overproduction in the early 1990s, the New Zealand industry innovated and assessed the commercial potential of another species, Actinidia chinensis (Ferguson & Huang, 2007). Domestication and breeding of firstly Actinidia deliciosa and more recently, A. chinensis, from wild germplasm resulted in a lot of commercially cultivated varieties worldwide distributed. The most important steps in the development of kiwifruit as a world commercial crop are summarized in Table 1.1. Until very recently, world trade in kiwifruit had developed from the stage with one predominant green-fleshed cultivar to one commercially important yellow-fleshed cultivar with other emerging yellow- 3 - Chapter 1 - fleshed cultivars and finally to the initial commercialization of red-centered yellow- fleshed cultivars. However, all this has changed with the arrival in most countries producing kiwifruit of the disease caused by Pseudomonas syringae pv actinidiae (Ferguson, 2013). Table 1.1: Important steps in the domestication and commercialization of kiwifruit. 1899 First plants of A. deliciosa grown outside China 1904 Plants of A. deliciosa first sold in England 1904 Seed of A. deliciosa arrive in New Zealand 1910 First fruit of A. deliciosa produced outside China ca. 1930 Establishment of the first commercial A. deliciosa orchard in New Zealand ca. 1930 Selection of the A. deliciosa cultivar ‘Hayward’ Late 1930 First commercial orchard of ‘Hayward’ 1952 First commercial coolstorage of A.deliciosa kiwifruit 1953 First commercial exports of A. deliciosa kiwifruit from New Zealand 1959 Invention of the name kiwifruit 1961 First cultivation of A. chinensis 1965 First commercial crop of A.deliciosa kiwifruit outside New Zealand 1975 Only ‘Hayward’ A. deliciosa fruit accepted for export from New Zealand 1982 First fruit of A. chinensis known