Study On Preservation And Molecular Identification Of Nematode-eating Fungi

Nematophagous fungi are important biocontrol strains of parasitic nematodes in animals and plants.The purpose of this topic is to study and discuss the preservation methods of nematode-eating fungi of different genera.In the short-term and long-term preservation of many strains,we can find one or more better preservation methods,which can maintain or improve the viability of the strains and maintain a good predation effect at the same time.After amplifying and sequencing the ITS sequences of 12 strains previously sampled and isolated from China,The sequences of these isolates were analyzed by phylogenetic analysis,the genetic relationship between these isolates and other similar strains was studied,and the isolates were identified.The research contents are as follows:The isolates were preserved in 6 ways: Agar slope preservation of 2% wheat bran extract,soil drying preservation,skimmed milk preservation of silica gel grains,adsorption preservation of modified silica gel particles,and freezing preservation(-18 ? and-80 ?).The survival and predation effect were examined after 6 months.The results show that the survival rate of isolates preserved in various ways for 6months is 100%,94.12%,96.43%,95.56,88.89,70.37%,respectively,and the predation rate is 87.31,77.08,81.48,97.67,75.00,63.16,respectively.Among them,the survival effect of test tube preservation was the best,followed by silica gel skimmed milk.The predation effect of modified silica gel adsorption preservation was the best,followed by test tube slope preservation.The survival and predation ability of the strain after 3-5 years preservation were studied by test tube slope method,freeze preservation method(-18 ? and-80 ?),vacuum freeze drying method,improved silica gel adsorption method,soil drying method and grain preservation in cone flask.The strains were preserved by freeze preservation(-18 ? and-80 ?),vacuum freeze drying and test tube slope preservation for 3 years.The predation effect on Haemonchus contortus L3 in sheep feces was determined by double plate culture in vitro.The results showed that the strain was best preserved by vacuum freeze drying.The reduction rates of Haemonchus contortus L3 during cryopreservation(-18 ? and-80 ?),vacuum freeze-drying preservation and test tube slope preservation were 88.81%,97.61%,75.20%,97.56%,93.99% 99.64%,87.36% 97.59%,respectively,and the reduction rates were 88.81%,97.61%,75.20%,93.99% 99.64%,87.36% 97.59%,respectively.Through DNA extraction,PCR amplification and sequencing of 12 isolates isolatedfrom feces and related media,the partial sequences of 18 s rDNA,28 s rDNA and all sequences of ITS1,5.8s rDNA and ITS2 were obtained.by GenBank multiple sequence alignment,phylogenetic analysis,and combined with some morphological results,the results showed that the 12 strains were all Arthrobothys.NPS027,PS015,NPS044,F046,BS087,BS012,NPH037,447,8 strains were Arthrospora oligospora,NPS014 were Arthrobothys thaumasia.The three strains of NPS033,NPS058 and NF015 were Arthrobothys musiformis.