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Fungi and Actinomycetes Associated with Cysts of Heterodera Trifolii Goffart (Nematoda: Tylenchida) in Pasture Soils in New Zealand

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Fungi and Actinomycetes Associated with Cysts of Heterodera Trifolii Goffart (Nematoda: Tylenchida) in Pasture Soils in New Zealand FUNGI AND ACTINOMYCETES ASSOCIATED WITH CYSTS OF HETERODERA TRIFOLII GOFFART (NEMATODA: TYLENCHIDA) IN PASTURE SOILS IN NEW ZEALAND BY F. S. HAY and R. A. SKIPP New Zealand Pastoral Agriculture Research Institute Ltd., Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand A survey of ten pastures sites in the North Island of New Zealand was conducted to identify micro-organisms colonising cysts of clover cyst nematode (Heteroderatrifolii). A total of 707 isolates of fungi and actinomycetes was recovered from 488 surface-sterilised cysts plated onto water agar Ninety-three percent of cysts were colonised, with 39% containing more than one organism. The most commonly isolated organisms (and their frequency of occurrence) were species of acti- nomycetes (30% of cysts), Fusarium(26%), Verticillium(16%), Gliocladium(14%), Mortierella(9%), Paecilomyces(8%), Phialophora(7%), Penicillium(6%), Exophiala(5%), Acremonium(3%), Cylindrocarpon (2%) and Trichocladium(2%). Predominant species of these included Fusariumoxysporum, F. culmorum, F. solani, Verticilliumchlamydosporium, Gliocladium roseum, Mortierella alpina, Paecilomyceslilacinus, an unidentified Paecilomycessp., Exophialapisciphila and Trichocladiumopacum. Young white cysts were most commonly colonised by actinomycete species (38%), Fusariumspp. (12%) and Gliocladiumspp. (10%); older (brown) cysts by actinomycete spp. (24%), Verticilliumspp. (23%) and Fusariumspp. (20%), and very old (black) cysts by Fusariumspp. (41%), actinomycete spp. (31%) and Gliocladium spp. (19%). Many of the species isolated are known colonists of the roots of white clover (Trifolium repens)and some are known parasites of nematode eggs. Keywords:Heterodera trifolii, clover cyst nematode, biological control, nematophagous fungi, actinomycetes, Trifoliumrepens The clover cyst nematode (Heterodera trifolii Goffart, 1932) is widespread in pasture soils throughout New Zealand and many other countries (Skipp & Gaynor, 1987). It is an important pest of white clover (Trifolium repens L.), causing reduced plant establishment and persistence, yield and nitrogen fixa. tion. Control of nematodes in pastures by chemicals or by crop rotation and management practices is impracticable (Skipp & Gaynor, 1987). Currently, the breeding of white clover resistant to nematodes (Mercer et al., 1990) represent., the most promising means to achieve control in the future. However, the development of cultivars which combine resistance with suitable agronomic characteristics may take many years, and the resistance achieved may not be stable. Therefore, there is a need to develop alternative methods of control, ol methods which can be integrated with plant resistance. Surveys in other countries have found a range of fungi to be associated with cysts of several heteroderid nematode species (Rodriguez-Kabana & Morgan- Jones, 1988; Stirling, 1991). Some of these fungi parasitise nematode eggs or 377 sedentary females on the roots of host plants. There is some evidence that such fungi can influence nematode populations in the soil. Kerry (1982) reported that populations of the cereal cyst nematode (Heterodera avenae Wollenweber) were maintained below an economically damaging threshold by parasitic fungi in fields in which cereals had been continuously cropped for a number of years. Significant levels of fungal parasitism have also been observed in Heterodera glycines Ichinohe in fields continuously cropped with soybean (Glycine max (L.) Merr.) and in Heterodera schachtii Schmidt in beet monoculture (Morgan-Jones et al., 1981, Tribe, 1979). Many pastures in New Zealand have been established for a number of years, affording the opportunity for populations of fungal parasites to have reached an equilibrium with nematode populations at some sites. This paper reports a survey of pasture sites in the North Island of New Zealand to examine the types of fungi associated with cysts of H. trifolii as a preliminary to identifying those which influence nematode populations. z MATERIALS AND METHODS Soil samples were collected in August, 1990 from ten pasture sites in the North Island of New Zealand from Kaikohe, Dairy Flat (two sites), Ruakura, Gisborne, Whangaehu, Maraekaka, Palmerston North, Levin and Foxton. Samples consisting of vegetation and soil to a depth of 20 cm were taken with a corer (2.5 cm diam) or a spade and stored at 4°C until required. Approximately 500 ml of soil were extracted with a fluidising column similar to that described by Trudgill et al. (1973) and the overflow was passed through 600um and 180 pm sieves. Cysts retained on the 180 pm sieve were washed into Petri dishes and examined under a stereomicroscope at x 10 magnification. Cysts were sorted on the basis of colour into white, brown or black categories, correspond- ing to increasing cyst age. Some individuals in the 'white cyst' category may have been living females; however, most had attained a final shape and con- tained many eggs and so were considered to be cysts. They were then stored in sterile water at 4°C until they could be processed further (usually less than 2 days). Cysts in suspensions were poured into a Gelman Sciences filter flask apparatus fitted with a Whatman no. 1 filter which had been oven sterilised at 160°C for 4 h. Water was removed by vacuum aspiration and replaced with 15 ml of 0.3% NaOCI. After 2 minutes the NaOCI solution was drawn through the filter and cysts were rinsed with three changes of 10 ml of sterile water. When sufficient cysts were available, 21 cysts of each colour from each site were placed aseptically on tap water agar (1.5%) containing 10 pg/ml oxy- tetracycline in Petri plates. Isolations were made from a total of 105 white cysts, 210 brown cysts and 173 black cysts during the survey. Plates were incubated at 21 °C for up to 2 wk and examined under a stereomicroscope at x 60 magnifica- tion. Plates were examined every 2 to 3 days and fungal hyphae or spores .
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