Identification of mycoparasitic Pythium species associated with vineyard soils in Canterbury, New Zealand : A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Horticulture Science at Lincoln University

Abstract

Pythium species are recognised as important soilborne plant pathogens, however, some of these species are aggressive parasites of other fungi and oomycetes, and are referred to as mycoparasitic Pythium species. These have the potential to be developed as biological control agents against plant pathogens. However, there is limited information regarding the population diversity of mycoparasitic Pythium spp. present in New Zealand, especially associated with economically important horticultural crop such as grapevine. The objectives of this study was to determine the best methods to isolate mycoparasitic Pythium species from soil collected from the Lincoln University vineyards, and to use the best methods in a more extensive North Canterbury vineyard survey to identify mycoparasitic Pythium species diversity. Additionally, the in vitro host range and mode of parasitism of mycoparasitic Pythium spp. isolates recovered from North Canterbury vineyard soils were investigated. The first soil sampling was done in summer 2021 from the organically and conventionally managed vineyard sites at Lincoln university. Three isolation methods were tested (1) soil dilution plating, (2) sclerotia baiting, and (3) pre-colonised fungal host baiting. The soil dilution plating was done by plating 1:50 soil dilution on Pythium selective media (CMA-PARP). For the sclerotia baiting method Sclerotinia sclerotiorum sclerotia were placed into soil water suspensions and the sclerotia transferred after 48 h onto selective media. For the pre-colonised fungal host baiting method 1.5 g of each soil sample was placed onto a pre-colonised host fungal culture plate of Botrytis cinerea, Fusarium oxysporum, Ilyonectria liriodendri, Neofusicoccum luteum, and Neofusicoccum parvum. The plates were observed microscopically after 7, 14 and 21 days for the presence of characteristic oospores and isolation was carried out from any positive plates. The sclerotia baiting and F. oxysporum pre-colonised plate methods were the most effective at recovering mycoparasitic Pythium species from the soil samples. Dilution plating method was the least effective method for isolating mycoparasitic Pythium spp. More mycoparasitic Pythium spp. isolates were recovered from the organically managed vineyard. The species identity of the recovered isolates were confirmed as P. acanthicum and P. oligandrum using sequencies of the ITS and cox II gene regions. The six vineyards were surveyed during autumn for soil sampling which included the organic and conventional vineyard sites, Lincoln University, Pegasus Bay Wine (Waipara), Sherwood Estate Wines (Stirling Vineyard, Waipara), Cross Hares Vineyard (Tai Tapu) and Lone Goat Vineyard (Burnham). As for the summer sampling P. acanthicum and P. oligandrum were recovered from the autumn soil sampling. The host fungal range of the P. acanthicum and P. oligandrum isolates recovered was investigated using a pre-colonised host culture assay, with four fungal hosts used B. cinerea, F. oxysporum, I. Iiriodendri and N. parvum. Isolates of mycoparasitic Pythium spp. were inoculated on PDA plates pre-colonised with the fungal host and after 14 days incubation observed microscopically for the presence of characteristic oospores of the mycoparasitic Pythium spp. Presence of oospore on pre-colonised fungal host plate indicated growth of mycoparasite on host colony. Of the four hosts tested N. parvum and F. oxysporum were identified as being susceptible host, I. Iiriodendri was identified at being less susceptible, and B. cinerea was identified as a resistant host. Interactions on I. Iiriodendri pre-colonised host plates indicated that P. acanthicum isolates was less aggressive than P. oligandrum. Four types of parasitic reactions being hyphal lysis, cytoplasmic coagulation, hyphal thickening, and some coiling attempts were observed when interactions between hyphae of P. acanthicum and P. oligandrum and the different host pathogens on coverslip thinly coated with water agar. Pythium acanthicum and P. oligandrum had the same mode of parasitism. The findings of this study showed that both P. acanthicum and P. oligandrum were present in vineyard soils in the North Canterbury region of New Zealand and, with regards to grapevine trunk pathogens, have a similar host range and mode of parasitism. However further glasshouse and field experiments are required to determine their efficacy in reducing infection of grapevines by grapevine trunk pathogens.