Investigating soilborne nectriaceous fungi impacting avocado tree establishment in Australia Louisamarie Elicano Parkinson Bachelor of Biotechnology (Honours Class 1) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Queensland Alliance for Agriculture and Food Innovation 1 Abstract Black root rot is a severe disease of nursery avocado trees and orchard transplants caused by soilborne fungal pathogens in the Nectriaceae family. The genera reported to be associated with black root rot are Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis and Ilyonectria. These genera have not been widely studied in avocado, although the disease causes significant commercial loss, with symptoms including black, rotten roots; tree stunting; leaf wilt; and rapid tree decline and death. This PhD research aims to i) identify the nectriaceous fungal species found in avocado roots in Australia, using morphological studies and molecular phylogenetic analyses of fungal gene sequences; ii) to perform pathogenicity tests on avocado seedlings and fruit to determine the pathogenic species; iii) to investigate whether the pathogens produce phytotoxic exudates which induce and facilitate disease symptom development; iv) and to use the generated gene sequence data to develop a molecular diagnostic for rapidly detecting the pathogens. Fungal isolates were obtained from symptomatic roots from sick and healthy avocado trees, nursery stock, young orchard transplants and mature established orchard trees from all growing regions in Australia, and from other host species. Bayesian inference and Maximum likelihood phylogenetic analyses of concatenated ITS, β-tubulin and histone H3 gene loci were used to identify and classify 153 Nectriaceae isolates in the genera Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis, Ilyonectria and Mariannaea. Three new species of Gliocladiopsis were identified and described as G. peggii, G. forsbergii and G. whileyi in a taxonomic study focusing on this genus. Comprehensive phylogenetic analyses also revealed additional new species and species complexes in each genus. Fungal species associated with black root rot of avocado in Australia was shown to include: species complexes of Calonectria ilicicola, Dactylonectria macrodidyma, D. anthuriicola, D. vitis, D. pauciseptata and Gliocladiopsis peggii, all containing putative novel species; D. novozelandica, putative novel and unresolved species of Ilyonectria, unresolved Gliocladiopsis sp., G. forsbergii, G. whileyi, Cylindrocladiella pseudoinfestans, Mariannaea humicola and a putative novel species of Mariannaea. Glasshouse pathogenicity tests confirmed fungal isolates of Calonectria ilicicola from avocado, papaya, peanut and custard apple caused significant root rot in avocado cv. Reed seedlings within 5 weeks of inoculation, inferring potential disease transmission between 2 different hosts. These isolates also caused significant tree stunting, wilted leaves and seedling death in this time period. A Calonectria sp. isolate from blueberry was also found to cause black root rot in avocado seedlings but not stunting. New disease records in Australia were established for Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata and D. anthuriicola as pathogens of avocado, causing black root rot in glasshouse experiments within 9 weeks of inoculation; however, stunting was not observed. Avocado isolates of Cylindrocladiella pseudoinfestans, Gliocladiopsis peggii and Ilyonectria sp., and a grapevine isolate of Ilyonectria sp. were confirmed as non-pathogenic to avocado seedlings. Pathogenicity experiments on avocado cv. Hass fruit showed necrotic lesions developed on fruit skin at mechanically wounded sites inoculated with Ca. ilicicola isolates from avocado and papaya, and D. macrodidyma and Ilyonectria sp. isolates from avocado. However only Ca. ilicicola isolates from avocado were able to infect and cause necrosis at non-wounded sites. Fungal culture filtrates (CF) of Calonectria ilicicola and Dactylonectria macrodidyma grown in potato dextrose broth (PDB) were tested for the ability to induce and facilitate phytotoxic symptom development in tomato seedling model plants, and in avocado cv. Reed leaves and fruit. Purified Brefeldin A (BFA), a known phytotoxin produced by Ilyonectria and Dactylonectria spp., was also tested for symptom development in tomato seedlings. Tomato seedlings treated with BFA or CF of Ca. ilicicola developed significant leaf wilt, chlorosis and necrosis by 12 days post treatment, compared to plants treated with water. However, seedlings treated with D. macrodidyma CF were not significantly different to plants treated with unamended PDB. Symptoms in plants treated with Ca. ilicicola CF were statistically similar to those treated with BFA, and plants of both treatments were developed symptoms more rapidly and severely than seedlings treated with D. macrodidyma CF or PDB. Glasshouse and in vitro trials on avocado leaves were inconclusive. Necrotic lesions developed on avocado fruit cv. Reed regardless of treatment and there was inconclusive evidence to suggest the facilitation of Ca. ilicicola or D. macrodidyma CF on disease symptom development by nectriaceous fungi. The necrotic lesions on fruit singularly treated with Ca. ilicicola CF were significantly larger than lesions formed on fruit treated with unamended media, suggesting that nectriaceous fungal exudates may potentially facilitate the exacerbation of post-harvest disease. The pathogens Calonectria iliciciola and Dactylonectria macrodidyma were selected for the development of a rapid, loop-mediated isothermal amplification (LAMP) diagnostic test. The collection of nectriaceous fungal DNA sequence data from three gene loci enabled the 3 identification of candidate genes containing unique fixed nucleotides for specific species detection. Histone H3 was selected for detecting D. macrodidyma and β-tubulin was selected for detecting Ca. ilicicola. Species-specific LAMP primers were designed from the unique fixed nucleotides of those genes. The LAMP diagnostic was sensitive and specific to 0.01ng/µl of fungal DNA and could detect fungal mycelia of Ca. ilicicola within 12–25 minutes and D. macrodidyma within 13–28 minutes. Detection was significantly faster when tested with pure DNA, with Ca. ilicicola detected within 11 minutes on average and D. macrodidyma detected within 12–20 minutes. Calonectria ilicicola was successfully detected in necrotic avocado roots of cv. Reed seedlings previously inoculated in the glasshouse. The PhD study has significantly expanded knowledge on the diversity of nectriaceous fungal species associated with avocado, identified pathogens which cause black root rot disease in Australian avocado trees, demonstrated a potential role of phytotoxic exudates in pathogenicity, and contributed towards improving disease management in the global avocado industry through the development of a rapid molecular diagnostic test. 4 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. 5 Publications during candidature Peer Reviewed Scientific Journal Articles Parkinson LE, Shivas RG, Dann EK (2017) Novel species of Gliocladiopsis (Nectriaceae, Hypocreales, Ascomycota) from avocado roots (Persea americana) in Australia. Mycoscience 58(2):95-102. http://dx.doi.org/10.1016/j.myc.2016.10.004 Conference Abstracts Oral Presentations L. E Parkinson, A. R. McTaggart, R. G. Shivas and E. K. Dann (2015) Investigating soilborne nectriaceous fungi associated with black root rot in avocado, 8th World Avocado Congress, 13–18 September, Lima, Peru, p41 L. E Parkinson, A. R. McTaggart, R. G. Shivas and E. K. Dann (2015) Investigating soilborne nectriaceous fungi associated with black root rot in avocado, Australasian Plant Pathology Conference 2015, 14–16 September, Fremantle, Australia L. E Parkinson, A. R. McTaggart, R. G. Shivas and E. K. Dann (2014) Investigating soilborne nectriaceous fungi associated with black root rot in avocado, 8th Australasian