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Botryosphaeria Infections in New Zealand Grapevine Nurseries: Sources of Inoculum and Infection Pathways

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Botryosphaeria Infections in New Zealand Grapevine Nurseries: Sources of Inoculum and Infection Pathways Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Botryosphaeria infections in New Zealand grapevine nurseries: Sources of inoculum and infection pathways A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Plant Pathology by Regina Billones-Baaijens Lincoln University 2011 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Plant Pathology Abstract Botryosphaeria infections in New Zealand grapevine nurseries: Inoculum sources and infection pathways by Regina Billones-Baaijens The botryosphaeriaceous fungi can cause decline, dieback and death of grapevines. Anecdotal evidence has indicated that these pathogens might be present in the young vines sold by propagation nurseries, so this study investigated their role in spread of this disease. Sampling of grapevine nurseries across New Zealand showed that botryosphaeriaceous infections were present in eight out of nine nurseries with infection incidence ranging from 5 to 63%. Of the 311 propagation materials and plants received, 23% were positive for botryosphaeriaceous infection, with a total of 120 isolates recovered. The highest incidence was in failed grafted plants (33%), followed by Grade 1 plants (28%), rootstock cuttings (19%), scion cuttings (17%) and Grade 2 plants (7%). For grafted plants, the majority of botryosphaeriaceous species (49%) were isolated near the graft unions while infections on rootstocks and scion cuttings were mostly from the middle and basal parts. Identification of isolates by morphological and molecular methods showed that the six species commonly found in vineyards also occurred in nurseries, being Neofusicoccum luteum (57%), N. parvum (18%), N. australe (8%), Diplodia mutila (8%), Botryosphaeria dothidea (5%) and D. seriata (3%), with one novel isolate of N. macroclavatum and two unidentified botryosphaeriaceous isolates. Pathogenicity tests using one-year-old Sauvignon blanc rooted canes and green shoots showed that the seven identified botryosphaeriaceous species from the nurseries were pathogenic but pathogenicity differed significantly between species and isolates within a species, with N. parvum being the most pathogenic in both assays (P≤0.001). Genetic variability analysis using UP-PCR showed that N. luteum isolates of different pathotypes were genetically diverse with intra- and inter-plant and nursery variability but no association between genotype and pathogenicity was observed. Susceptibility tests using three isolates each of N. luteum, N. parvum and N. australe against the most commonly used scion and rootstock varieties (six of each) found that all varieties were susceptible to the three species with 5C and SO4 being the most susceptible of the rootstock varieties, and Merlot and Pinot noir being the most susceptible of the scions. ii Investigations into the sources of inoculum conducted in three nurseries in 2009 using conventional and molecular methods showed that the mothervines used to provide cuttings for propagation were the most likely source of botryosphaeriaceous infections. Propagules were detected on the surfaces of cuttings and dead grapevine materials, as well as in rain- water run-off, but not in soil samples collected from the mothervine blocks. The different botryosphaeriaceous isolates recovered from canes were mostly sited within the bark suggesting presence of latent infections. Since the isolates from mothervine trunk and canes were distributed in isolated patches and the UP-PCR assessment showed that they belonged to multiple genotypes and species, this indicated that they were spread from external sources. Microscopy and plating assays were not able to detect botryosphaeriaceous contamination from any of the nursery propagation stages but molecular methods using multi-species primers detected botryosphaeriaceous DNA in samples from a wash pit, pre- cold storage hydration tanks, post-grafting hydration tanks, grafting tools and callusing medium from the different nurseries. An investigation into the probable survival of N. luteum conidia during the processing of cuttings showed that they adhered rapidly and could not be totally washed from the surfaces of cuttings after 0 h, with minimal recovery of conidia after 1, 2 and 4 h incubation at room temperature. During this time they germinated and colonised the periderm, phloem and xylem but not the pith of the cuttings. At storage temperatures of 2 and 8°C some conidia were able to germinate. At 2°C, conidial germination was 4% after 48 h, the number gradually increasing to 17% after 3 weeks. At 8°C, conidial germination was 23% after 24 h, the number gradually increasing to 34% after 72 h with no further increase observed up to 2 weeks. Cold storage did not kill all conidia, since conidia from 72 h and 3 weeks storage at 2°C had 84% and 33% viability, respectively, while those from 8°C storage had 72% and 39% viability after 72 h and 2 weeks storage, respectively. The N. luteum conidia stored at different temperatures were similarly pathogenic compared to freshly-harvested conidia but higher pathogenicity was observed on conidia stored at 2°C for 48 to 72 h. Overall results suggest that botryosphaeriaceous infection in new vineyards may originate from latent infection that began in the nurseries, thus management of this disease should start at the nursery level to prevent this pathogen from being carried over into new vineyards. Keywords: Botryosphaeriaceous species, Diplodia, Neofusicoccum, grapevine nurseries, inoculum sources, latent infections, pathogenicity, susceptibility, rootstocks, scions iii Acknowledgements Anyone who worked closely with me for the last three and half years when I was doing this research could tell that it required certain things to maintain any sort of relationship with me– including extraordinary understanding, incredible patience, and the ability to believe I‟m actually working when I‟m staring blankly at the wall. As this major milestone in my life comes to a close, I would like to thank each and everyone who contributed to its realisation. To my three supervisors, Dr. Marlene Jaspers, Dr. Hayley Ridgway and Dr. Eirian Jones for their steadfast guidance, encouragement and endless support to get me through this PhD research. Their patience and compassion have been a tremendous source of inspiration. To Winegrowers New Zealand and Lincoln University for the research funds and scholarship that supported my work from its inception to completion. To New Zealand Plant Protection Society for their generous scholarship grant; and ICPP for the travel funds that allowed me to attend and present my work at local and international plant pathology conferences. To the grapevine nursery owners and staff, whose names I cannot mention but whose invaluable contributions – plant materials, expertise and time – constituted this whole research. Without their cooperation and help, this research work could not have been completed. To the university academic, technical and administrative staff who were always there to lend a hand; Dr. Nick Dickinson, for his trust and support and for having me in his academic team; Candice Barclay, for her incredible patience in helping through the hundreds of favours that I asked so I can catch up with my work including proofreading this manuscript; Brent Richards, Leona Meachen, and Norma Merrick, for their helping hands in my greenhouse experiments; Chrissy Gibson, Gail Harkers, Emma Gambles, Marianne Duncan, and Linda Forbes for answering all my endless queries. To Dr. Chris Frampton, for providing me with countless answers on the statistical analyses and data interpretation involved in this research; Dr. Glen Creasy for helping me better understand the anatomy and physiology of grapevines. To Alix Allard, Jenny Brookes, Sarena Che Omar and Kate Henry, four incredible students I was very fortunate to work with. Their passion for learning had inspired to me to pursue a career in teaching; and to the very special people in our plant pathology-molecular group (they know who they are) who offered their friendship and made my postgrad life more interesting. To my sister Raquel, who‟s been reading through all my thesis manuscripts (MSc and PhD alike) and made sure everything make sense; my parents-in-law Cor and Christine for their unconditional support, particularly Cor‟s creative piece of gadget that helped me in my rainwater-trapping experiment; and for the rest of my family here in New Zealand and overseas who cheered for my triumphs and enriched my life with love, understanding and support. And to my husband Richard, for telling me to slow down when I‟m driving myself to the limit, but pushed me forward when I felt like giving up, for putting up with my tantrums when the work pressure built up, and for bringing new meaning to the words love and understanding. iv Table of Contents Abstract ...............................................................................................................................
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