Fermentation Conditions Of Clonostachys Rosea 67-1, A Highly Efficient Biocontrol Agent Of Plant Diseases

As a kind of hyperparasites,Clonostachys spp.played an indispensible part in bio-controlling of plant diseases.Clonostachys rosea 67-1 as the experimental material was studied in this paper.The sporulation and resistance characters of spores in liquid medium were studied,the technological parameters of its submerged fermentation were adjusted,and the bio-control preparation we made was used in field trial.So it was sufficiently prepared to establish practical microbial pesticides product line.The results were as follows.1.The growth and sporulation of C.rosea 67-1 in PD liquid medium,which had 4 growing stages(spore germination,mycelial growth,conidia formation,clamydospores fonnation),were observed and analyzed on the basis of measuring of its physiological charactors.2.After analyzing and comparing the effects of various nutritious conditions on the growth of C. rosea 67-1,it was found that the optimum condition for the sporulation of chlamydospores had two key factors which was bean-cake flour and carbamide,whose optimum mixture ratio was 3:1.The optimum initial pH of sporogenous medium was 7,and the optimum inoculum concentration was 2% of 2×10~8/mL spore suspension.3.Comparing the spore suspension with chlamydospores in the optimized liquid medium with aerial spores eluted from the PDA plates after high temperature,drought and UV treatment,it was shown that:after treatment under 60℃for 30 min,the survival rate of the submerged spores descended to 76.7%,and the aerial spores could hardly survive;After two weeks' drying treatment, the survival rate of submerged spores and aerial spores were 89.2%and 29.3%respectively;similarly, the survival rate of submerged spores dropped to 72.6%,while aerial spores 19.7%,when exposed to UV for 1 min.Hence,it could be demonstrated that submerged spores had higher resistance to the stressing environments than those of aerial spores.4.After iterative adjustments in 16L in-situ fermentor,the spore concentration of C.rosea 67-1 got to 3×10~8/mL with 30%chlamydospores when it came to the the terminal of the submerged fermentation.The submerged fermentation technological parameters were amplified and applied in 500L heat steam sterilization fermentor,and the same results were attained.The middle stage tests were finished in the workshop,and it supplied the fermentation production for the preparation of living microbial pesticide.5.The biopesticide of C.rosea 67-1 spores with the concentration of 2×10~8cfu/g was gained after centrifugation and concentration from fermentation liquid and absorption of the carrier.After applying to the wheat seeds and seedlings,it has been demonstrated that the effect of the microbial pesticide was 13.7%and 5.9%higher than that of two chemical pesticides on biocontroling wheat sharp eyespot caused by Rhizoctonia cerealis at flowering stage.The results laid the foundation for the preparation of C.rosea 67-1 biocontrol agents,but there are still many issues to be resolved on broth post-processing.