Identification of the best method to isolate mycoparasitic Pythium species from vineyards soil

Abstract

Pythium species are recognised as important soilborne plant pathogens, however, some of these species (mycoparasitic Pythium species) are aggressive parasites of other fungi and oomycetes. These species have potential as biological control agents. However, there is limited information regarding the population diversity of mycoparasitic Pythium species present in New Zealand, especially associated with socio-economically important horticultural crops such as grapevines. The objective of this study was to determine the best method to isolate mycoparasitic Pythium species from soil collected from the Lincoln University vineyards. Three methods were tested: (1) soil dilution plating; (2) sclerotia baiting; and (3) pre-colonised fungal host baiting. (1) was done by plating 1:50 soil dilutions on Pythium selective media (CMA-PARP). (2) Sclerotinia sclerotiorum sclerotia were placed into soil water suspensions and the sclerotia transferred after 48 hr onto selective media. (3) 1.5 g of each soil sample was placed onto a pre-colonised host fungal culture plate of either Botrytis cinerea, Fusarium oxysporum, Ilyonectria liriodendra or Neofusicoccum parvum. The plates were observed microscopically after 7, 14 and 21 days for presence of characteristic oospores and isolation was carried out from any positive plates. Fusarium oxysporum pre-colonised plates and the sclerotia baiting method were the most effective at recovering mycoparasitic Pythium species from the soil samples. Isolates morphologically identified as Pythium oligandrum, as well as other unidentified mycoparasitic Pythium species were recovered. No mycoparasitic Pythium species were recovered on B. cinerea, I. liriodendra or N. parvum pre-colonised plates, with Trichoderma spp. and Clonostachys spp. recovered on B. cinerea and N. parvum plates. The species identity of the recovered isolates are currently being confirmed using Sanger DNA sequencing. These methods will be used to determine the diversity of mycoparasitic Pythium species in New Zealand vineyards.