2.10.17
PARASITISM OF SCLEROTINIA SCLEROTIORUM SCLEROTIA BY THE MYCOPARASITE PYTHIUM OLIGANDRUM

A METTE MADSEN and E DE NEERGAARD

Department of Plant Biology, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark

Background and objectives
Pythium oligandrum is a potential biological control agent. When looking at hyphal-hyphal interactions it has a wide host range [1], but whether it can parasitize the resting structures of the same fungi is not known. Sclerotia of the plant pathogen Sclerotinia sclerotiorum is reported to survive for about 5 ;years in soil, and seed lots harvested from infected crops are often contaminated with sclerotia of S. ;sclerotiorum. Hence, we have studied whether P. ;oligandrum can parasitize sclerotia of S. ;sclerotiorum. Zoospores of P. ;oligandrum are used as the inoculum source since they are able to orientate during encystment so that germ-tubes emerge towards hosts. As encysted zoospores, they are able to initialize mycoparasitism and to establish in natural soils [2].

Materials and methods
S. ;sclerotiorum sclerotia produced on PDA were classified according to their sizes (three groups), and 2X125 sclerotia of each size were inoculated in a natural soil with or without Pythium oligandrum zoospores (1.25X105/20 ;g soil). The germination ability of sclerotia was estimated at day 4 and day 12, as was the sclerotial colonization ability of P. ;oligandrum. The experiment was repeated three times and the data analysed by odds ratio analyses. Experiments similar to the soil-studies were performed in water. In water, where S. ;sclerotiorum sclerotia and/or P. ;oligandrum zoospores were present, the activities of extracellular N-acetyl-P-D-glucosaminidase (EC 3.2.1.30)(NAGase), P-glucosidase (EC 3.2.1.58) and cellobiohydrolase (EC 3.2.1.91), were estimated by the following substrates: p-nitrophenyl-N-acetyl-O-D-glucosaminidase, p-nitrophenyl-P-D-glucopyranoside and p-nitrophenyl-O-D-celloside. In addition, Loewe Blue Substrates were used to estimate the P-D-glucanase (EC 3.1.2.6), endo-chitinase (EC 3.2.1.14) and proteinase activities.

Results and conclusions
Treatment of a soil inoculated with S. ;sclerotiorum sclerotia using P. ;oligandrum zoospores had a significant effect on the germination of sclerotia. Between 76.6% and 79.4% were able to germinate when collected at day 4 from the untreated soil, while only 28.6-45.1% from the P. ;oligandrum treated soil were able to germinate. The germination ability of the large sclerotia was significantly higher than the germination of the small sclerotia. Microscopy and plating of sclerotia on agar showed that sclerotia in the P. ;oligandrum-treated soils were internally colonized with P. ;oligandrum. The germination ability of the sclerotia was further reduced when the incubation time was extended. When P. ;oligandrum zoospores were added to water with sclerotia, the germination ability of the sclerotia was significantly reduced, as in the soil, showing that P. ;oligandrum is able to parasitize the sclerotia both in presence and absence of other microorganisms. Studies of the activities of extracellular enzymes showed that the NAGase, endo-chitinase, 0-glucosidase, cellobiohydrolase and proteinase activities were higher when the two fungi were inoculated together than when they were inoculated separately. The inductions of these enzymes in co-inoculations of P. ;oligandrum and S. ;sclerotiorum indicate that they might be involved in the parasitism of the sclerotia, but also reflects that P. ;oligandrum was able to establish on the sclerotia in water. In addition, P. ;oligandrum was able to utilize five carbohydrates which are present in greatest amounts in sclerotia. Enzyme assays and microscopy showed that P. ;oligandrum was also able to establish and fulfil its life cycle in water with 'nutrients' released from sclerotia. In conclusion, P. ;oligandrum has potential to control diseases caused by S. ;sclerotiorum sclerotia. W intenfd to study mycoparasitism of S. ;sclerotiorum hyphae in planta to see whether certain mycoparasitism-related enzymes can be detected by isoenzyme gels when the two fungi are interacting.

References
1. Laing SAK , Deacon JW, 1991. Mycological Research 95, 469-479.
2. Madsen AM, Robinson HL, Deacon JW, 1995. Mycological Research 99, 1417-1424.