Investigation into the emerging soil borne disease of peanut – Neocosmospora root rot Kylie M. Wenham Bachelor of Applied Science (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 School of Agriculture and Food Science i Abstract Neocosmospora root rot is an emerging soilborne disease of peanut (Arachis hypogaea L.) crops in Australia caused by Neocosmospora vasinfecta var. africana. The fungal pathogen was first identified in southern Queensland in 2005 following extensive damage to an irrigated peanut crop, and has since been found in all peanut growing areas in Queensland and New South Wales. Typical symptoms of the disease include extensive chlorosis and wilting of the plant starting from the main stem, and a blackened and decayed root system with reddish-orange perithecia of N. vasinfecta present on necrotic tissue of the main tap root and lateral root system as well as the pods. While the disease has been widely observed in agricultural production areas of Australia and is considered one of the most destructive pathogens of peanut, the biology, ecology and aetiology of N. vasinfecta is largely unknown therefore the biotic and abiotic factors that may influence the infectivity of the pathogen and severity of disease epidemics have not been identified. The purpose of this thesis is to investigate the biology, ecology and aetiology of N. vasinfecta var. africana and understand how these factors may contribute to the pathogenicity of the fungus. Due to limited knowledge of the biology of N. vasinfecta var. africana, a morphological and molecular approach was taken to determine the taxonomic identification and the morphological characteristics of the pathogen. The results of the morphological analysis of isolates collected from a range of peanut growing areas in Queensland identified the fungus to the species level as Neocosmospora vasinfecta. Further morphological analysis of the ascospores confirmed the characteristics of the ascospores to be consistent with the morphological description for the variety africana. Morphological analysis of isolates collected from peanut crops in Georgia were also consistent with N. vasinfecta however a MegaBlast search revealed the species variety to be vasinfecta which may account for the difference in pathogenicity seen between the two countries. Neocosmospora root rot has been identified in all peanut growing areas in Australia, however outbreaks of the disease have been sporadic and contained to isolated areas of peanut crops. An investigation into the behaviour of N. vasinfecta var. africana concluded that the fungus is likely to originate from foci of infected plants where soil inoculum is high and environmental conditions are conducive to infection, and is spread to neighbouring plants. N. vasinfecta var. africana has been identified as the most prevalent fungi isolated where plants have exhibited symptoms such as wilting, chlorosis, plant death and root decay, and therefore is considered to be a primary pathogen. There is the potential for N. vasinfecta var. africana to infect plants in conjunction with other pathogens such as peanut Tospovirus, which was evidenced by the presence of Tobacco Streak Virus (TSV) in the roots of peanut plants exhibiting symptoms of Neocosmospora root rot. Perithecia of N. vasinfecta ii var. africana were also present on the roots of peanut plants exhibiting symptoms of Capsicum Chlorosis Virus (CaCV) infection. Artificial infection of susceptible hosts with N. vasinfecta var. africana in glasshouse situations to reproduce the symptoms observed during outbreaks of the disease in the field is largely unsuccessful, and has cast doubt on the pathogenicity of the fungus. Based on the results of several experiments conducted, it is likely N. vasinfecta var. africana is an opportunistic pathogen that relies on a particular biotic or abiotic stress to trigger infection of plants by the fungus, however further work is required to identify this contributing factor. A seedling bioassay was developed for inoculation of peanut, chickpea (Cicer arietinum) and soybean (Glycine max) seedlings with N. vasinfecta under controlled conditions. This study found N. vasinfecta var. africana readily colonised germinating seed and 7-14 day old seedlings of all three species, leading to root decay and seedling death at 25-30°C and 85% relative humidity in a controlled environment chamber, therefore indicating the fungus may require specific conditions to initiate infection. The results of an investigation of the host range of N. vasinfecta var. africana found the fungus is able to colonise a variety of leguminous hosts including peanut, chickpea, soybean, mungbean (Vigna radiata), and non-leguminous cereal grain and fibre crop hosts including cotton (Gossypium hirsutum), corn (Zea mays), sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Colonisation of these plants by N. vasinfecta var. africana did not produce any typical symptoms of disease and did not have a detrimental effect on the growth and development of the host plant. Therefore it is possible N. vasinfecta var. africana has an endophytic capacity. Based on the results of survival studies of N. vasinfecta var. africana, it is apparent that it readily colonises the dried stems of peanut, chickpea, soybean, sorghum and corn particularly when buried under moist soil or sand, indicating saprophytic capability. This work has contributed to previously limited knowledge regarding the biology, ecology and aetiology of Neocosmospora vasinfecta var. africana as a soilborne pathogen of peanuts. This is the first major study to investigate Neocosmospora root rot as an emerging economically important disease of peanut and field observations indicate soil waterlogging may be a key trigger for the disease and the reason for sporadic epidemics of the disease to date. iii 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. iv Publications included in this thesis Wenham, K.M., Galea, V. J., Ryley, M.J., Wright, G.C., Bryden, W.L. (2014). Investigating the biology, epidemiology and management of Neocosmospora root rot of peanuts in Australia. In: Proceedings of the 8th Australasian Soilborne Disease Symposium. 8th Australasian Soilborne Disease Symposium 2014, Hobart, Australia, (47). 10-13 November 2014. Wenham, K.M., Galea, V. J., Ryley, M.J., Wright, G.C., Bryden, W.L. (2015). Investigating the biology, epidemiology and management of Neocosmospora root rot of peanut in Australia. In: Proceedings of the 47th American Peanut Research and Education Society Inc meeting. 47th American Peanut Research and Education Society Inc Meeting, Charleston SC, USA, (88). 14-16 July 2015. Wenham, K.M., Galea, V. J., Ryley, M.J., Wright, G.C., Bryden, W.L. (2015). Investigating the Pathogenicity of Neocosmospora species on germinating chickpea seedlings under controlled conditions. In: Proceedings of the Australasian Plant Pathology Society Conference. 2015 Australasian Plant Pathology Society Conference, Fremantle, Australia, (53). 14-16 September 2015. Wenham, K.M., Galea, V. J., Ryley, M.J., Wright, G.C., Bryden, W.L. (2015). Host range of Neocosmospora species causing root rot of legumes in Australia. In: Proceedings of the Australasian Plant Pathology Society Conference. 2015 Australasian Plant Pathology Society Conference, Fremantle, Australia, (173). 14-16 September 2015. Submitted manuscripts included in this thesis No manuscripts submitted for publication. Other publications during candidature No publications included. v Statement of parts of the thesis submitted to qualify for the award of another degree None. Contributions by others to the thesis Galea, V.J (Thesis Principal Advisor) for the experimental ideas and design, technical support, guidance, patience and revision of the whole thesis. Ryley, M.J (Thesis Advisor) for the experimental ideas and design, technical knowledge and support in my laboratory, glasshouse and fungal identification work and revision of the whole thesis.