2.10.6
VARIATION IN THE NEMATICIDAL ACTIVITIES OF AUSTRALIAN ISOLATES OF PAECILOMYCES LILACINUS

Ja-On Park1, GR Stirling2 and K Sivasithamparam1

1Soil Science and Plant Nutrition, Faculty of Agriculture, University of Western Australia, Nedlands, 6907, Western Australia; 2Biological Crop Protection, Moggill, 4070, QLD, Australia

Background and objectives
Paecilomyces lilacinus (Thom) Samson, known as an egg parasite, is a potential biological control agent of plant-parasitic nematodes, especially root-knot nematodes (Meloidogyne spp.). Isolates of this species can vary in their pathogenicity and cultural characteristics [1,2] causing inconsistent experimental results. Possible mechanisms involved in the biological control of nematodes include parasitism [3], antibiosis [4] and other factors associated with soil amendment [5]. The objective of this experiment was to compare variation among the isolates of P. ;lilacinus to understand their mode of action.

Materials and methods
A total of 21 isolates of P. ;lilacinus were obtained from Queensland soils [1] and two from high organic soils in Western Australia. Secondary metabolites were obtained from the fungal cultures in potato dextrose broth at 25C in a rotary shaker incubator. Metabolites were extracted from 7-day-old cultures and tested on axenically grown Caenorhabditis elegans. The mortality was determined every 12 ;h. To study inhibition of reproduction, eggs of C. ;elegans were exposed to the metabolites. Cultures which inhibited production of eggs after 7 ;days, were regarded as positive.

A clear zone around the colony of each isolate on agar with 0.5% gelatine [6] and 0.2% colloidal chitin [3] was considered to indicate protease and chitinase enzyme activity, respectively. An intracellular metabolite profile of each isolate was established with a thin-layer chromatography of culture filtrate after 7 ;days growth [7].

The isolates were screened for parasitic activity against eggs of M. ;javanica in three tests (water agar, filter paper and millet substrate)[1]. A comparison will be made of the parasitic potential of the fungal isolates before and after prolonged storage.

Results and conclusions
Isolates LS23, LS91, LS92 and LS103 immobilised C. ;elegans after 12 ;h exposure; isolates LS10, LS16, LS95, LS98 and LS99 immobilized C. ;elegans after 24 ;h exposure. In contrast, the remaining isolates showed no nematicidal activity after 48 ;h. Egg production of C. ;elegans was inhibited when exposed to isolates LS23, LS39, LS43, LS91, LS92 and LS103 for 7 days.

With the exception of one isolate (LS23), all isolates produced protease activity on the gelatine medium. Isolates LS8, LS24, LS94 and LS103 showed strong activity (clear zone: 5.63-5.75 ;cm/day), whereas isolates LS2, LS11, LS95 and LS96 showed very weak activity (clear zone: 2.38-2.88 ;cm/day) on the medium. The results of the chitinase activity on colloidal chitin medium showed that only isolates LS10, LS11, LS45, LS94 and LS 103 produced detectable activity around the colony after 3 ;weeks. There is some indication that isolate LS23 has a pronounced and different metabolite profile from the others. This is currently being investigated.

References
1. Stirling GR, West LM, 1991. Australasian Plant Pathology 20, 149-154.
2. Cabanillas E, Barker KR, Nelson LA, 1989. Journal of Nematology 21, 164-172.
3. Dackman C, Chet I, Nordbring-Hertz B, 1989. FEMS Microbiology and Ecology 62, 201-208.
4. Hallmann J, Sikora RA, 1996. European Journal of Plant Pathology 102, 155-162.
5. Miller PM, Sands DC, 1977. Journal of Nematology 9, 192-197.
6. Kunert J, Zemek J, Augustin J, Kuniak L, 1987. Biologia(Bratislava) 42, 695-705.
7. Filtenborg O, Frisvad JC, Svendsen JA, 1983. Applied and Environmental Microbiology 45, 581-585.