Biological Control Of Cucumber Fusarium Wilt By Using REMI Mutant Strain H6 Of Trichoderma Aureoviride

Two hundred mutants which are stable and resist to 250μg/mL Hygromycin had been got and the mutant library for resistance had been screened based on constructing the T23 mutant library using REMI(Restriction Enzyme-mediated Integration)method and Trichoderma viride as the original strain.Finally,the H6 was selected as the engineering strain for the later researching.Moreover,we created the liquid and solid fermentation procedures through the orthogonal experimental design of nutritional factors in the medium and groping of the fomenter conditions.The results are as follows:1.The ingredients of solid fermentation seed medium is potato 200 g/L,glucose 20 g/L,MgSO₄·7H₂O 0.5 g/L,FeSO₄·7H₂O 0.007 5 g/L,MnSO₄ 0.002 5 g/L,ZnSO₄ 0.002 g/L,KH₂PO₄ 3.83 g/L,NaNO₃ 1.42 g/L,(NH₄)₂SO₄ 1.1 g/L,CaCl₂ 1g/L.The ingredients and radio of solid fermentation medium are as follows:wheat bran:humic acid sodium:corn residue:rice husk:straw powder=15:0.75:3:10:10,the radio between the solid materials and water is 1:0.5(w/v).The fermentation condition is as follows:thickness of the medium is 7cm,the inoculums of the seeds is 20%,the temperature is 28℃,the ventilation is 0.5-0.8wm,stir once every 12 h in the fomenter. The product of conidia can up to 1.64×10¹¹/g.The solid fermentation product can be used directly after drying using oven of 40℃.2.The ingredients of the liquid fermentation medium formula is potato 200 g/L, glucose 20 g/L,KH₂PO₄ 1g/L,MgSO₄·7H₂O 0.5 g/L,FeSO₄·7H₂O 0.01 g/L.The fermentation condition is pH4,the inoculums' of spore suspension is 0.2%(v/v),bottled ratio is 100mL/250mL conical bottle,the temperature is 28℃,the time is 3d.The product of chlamydospore can up to 1.1×10⁸/g which are higher than the current domestic and foreign reports of biomass.3.The method of making biological pesticides is as follows:The liquid fermentation product needed to be mixed with vector which contains diatomite and calcium carbonate, the radio is 5:1.Then put the mineral nutrients into it.Its weight is 10g which includes the following components:KH₂PO₄ 7 g,MgSO₄ 1 g,CaSO₄ 1g,(NH₄)₆MO₇O₂₄·4H₂O 0.5 g,H₃BO₃ 0.2g,MnSO₄ 0.1g,FeSO₄ 0.1g,ZnSO₄ 0.1g.Then we mixed and dried,at last we made it into granules(biological pesticides).4.The control effect to the Cucurbits Fusarium Wilt of the product of solid fermentation and the biological pesticides is 95%～100%and 95.8%respectively(the dosage is 8%).We can get the conclusion that the health-effects of biological pesticides are higher than the other.Moreover,the biological pesticides have a longer shelf life,so it can better meet the needs of production and promotion.5.Through the screening of application dose in and out of room,we got the best proportion is 8%(quality than to the soil).At the same time,after the measurement to the height and weight of cucumber and the acidity and salinity of rhizosphere soil,we fond the product alter solid fermentation can improve the nature of the soil and can promote the growth of cucumber.Furthermore,the trend to reduce soil acidity is clearer than to reduce soil salinity.So this reflects the multi-function of the Trichoderma mutants.In the future,they will have more potential for wider application and promotion.