Activity Study Of Pcr-dgge Of Fungal Diversity Of The Organic Litter Layer In Pinus Sylvestris And High-frequency Strains

Fungal diversity in Pinus sylvestris var. mongolica litter was investigated by PCR-DGGE coupled with traditional cultivation method and morphological examination. Meanwhile lignocellulosic enzyme activities of three high-frequently isolated mycelial fungi were examined by pure culture.21 fungi strains were isolated by traditional cultivation method including 5 unknown fungi. Most strains isolated are filamentous fungi. Besides, in PCR-DGGE, the total DNA was extracted directly from L layer, F1 layer, F2 layer and H layer litter respectively by Bead-Beating method. Then about 460bp rDNA fragments were obtained by Nested-PCR with two primer pairs nu-0817-5'+ nu-1536-3'and nu-0817-5'+ nu-1196-3'-GC. The amplification products were analyzed by denaturing gradient gel electrophoresis (DGGE). DGGE sequencing analysis shows that the dominant bands were Aureobasidium pullulans, Ascomycota sp., Sporobolomyces inositophilus, Desmazierella acicola, Pulchromyces fimicola, Uncultured Hypocreales and Geopyxis majalis. On the whole Sporobolomyces inositophilus is examined both in PCR-DGGE and traditional cultivation method. Other fungi species detected by two method are not overlapped and similar.Three high frequently isolated filamentous fungi from litter of Pinus were as the research object, the method of fermentation was used to assay the mass defect of zymolyte and the activies of enzymes including carboxymethyl cellulase (EC3.2.1.4), filter paper activity (EC3.2.1.91), laccase (EC 1.10.3.2) and manganese peroxidase (EC 1.11.1.13) in fermenting process, and try to comfirm the relationship between the activities of enzymes and the mass defect of zymolyte. The results showed that the mass defect of zymolyte caused by Alternaria sp. is the highest. Among the three fungi strains, Penicillium sp. can produce highest activity of MnP. The relation between the mass defect of zymolyte and the activities of enzymes is positive correlation. The three strains of mycelial fungi are all belong to lignocellulose-decomposing fungi of the group of functional fungi.