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We report here a rapid, efficient and simple method for the extraction of high molecular weight DNA from zoospores of Neocallimastix frontalis EB188. This anaerobic fungus, isolated from bovine digesta, effectively degrades plant fiber in vitro (Barichievich et al. 1990. Appl. Env. Micro. 56:43-48). Our interest in ruminal fungi stems from their ability to degrade wood materials (Joblin et al. 1989. FEMS Micro. Lett. 56:119-122) and their potential use in biomass saccharification. Zoospore DNA synthesis is of particular interest to our laboratory. It is these motile zoospores which colonize and degrade plant materials (Mountfort 1987. FEMS Micro. Rev. 46:501-508). To detail fully this metabolic event, it will be necessary to extract nucleic acids from zoospores. Such procedures have not been reported in the literature. Using acetone drying and enzymatic removal of cell walls, we have isolated high molecular weight DNA from very small amounts of culture. The procedure takes less than one hour and DNA yields are high. The DNA is readily cut with restriction endonucleases and religated efficiently but is otherwise stable. Electrophoretic analysis of the DNA confirmed the presence of repetitive sequences. This procedure will aid the study of DNA replication, DNA repair and DNA RFLP analysis of various strains using small (<1 >ml) cultures.

Experiments were performed to determine the effect of Aspergillus oryzae (AO) fermentation extract on zoospore development in the rumen fungus Neocallimastix frontalis EB 188. Powdered product, or liquid extract prepared from such powder, was added at the recommended value for supplementation in dairy cattle. Stationary and stirred cultures were periodically sampled and assayed for extracellular and intracellular protein and enzymes, gas production, zoospore production and maturation, and carbon source utilization. Soluble extract increased fungal physiology when grown in stirred vessels or stationary cultures. Treated cultures produced higher levels of enzymes (nearly double). Mobile zoospores matured into germination entities more rapidly in treated cultures, and when powdered product was used, nearly 3 times more motile zoospores were produced at 56 h of fungal growth. Levels of the intracellular enzyme malate dehydrogenase increased by 6-fold in the presence of powdered product. Product wheat bran carrier used as soluble extract or powder had very little effect on fungal cultures. Medium cellulose was completely hydrolyzed in all cultures but this occurred earlier in those containing AO treatment.

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