Optimization Of Fermentation Conditions Of Bacillus Kochii DDWB And Preliminary Study On Its Nematicidal Components

Plant root-knot nematode disease is one of the most important soil-borne diseases.According to incomplete statistics,the damage caused by nematodes to major crops amounts to US$100 billion each year worldwide.Long-term extensive use of chemical agents increases the environmental pressure and causes prominent problems about fungicide resistance.Biological control,with the advantages of environmental friendliness and safe to people and animals,might be used as an alternative or substitute for chemical agents.In our previous study,a strain of Bacillus kochii DDWB was first found with strong nematocidal activity,through the screening of soil microorganisms.Thus far,Bacillus kochii has only been found in the gut and food of fruit flies,and no documents about its harmful to humans or animals has been reported.However,the optimization of the fermentation conditions and its active components were still unclear.Therefore,excellent fermentation conditions were screened by single factor method and orthogonal experiment design,and the stability of fermentation broth was determined,using the corrected mortality rate of Meloidogyne incognita and the value of OD_600nm of fermentation broth as indicators.The active nematocidal components were extracted by acid precipitation method and ammonium sulfate sedimentation protein,and the nematocidal mechanism was determined.The results were as follows:1.Single factor method was used to screen the best carbon source,nitrogen source and inorganic salt of DDWB fermentation medium.The best carbon source,nitrogen source and inorganic salt was sucrose,yeast extract and KCl,respectively.On this basis,the best additive amount of carbon source,nitrogen source and inorganic salt was 2%,1%and 4%,respectively.According to the test results of the two groups of orthogonal design,the best formula of the medium was sucrose 2%,yeast extract 1%,potassium chloride 2%.2.Single factor method was used to screen the fermentation conditions of DDWB.The results suggested that the optimal initial pH was 8,the optimal liquid volume of a 250 ml conical bottle was 150 ml medium,the optimal fermentation time was 48 h,the optimal rotation speed was 160 rpm,and the optimal fermentation temperature was 31℃.3.The stability of secondary metabolites in the fermentation broth of DDWB was determined.The results showed that acid or alkali conditions and UV irradiation could decrease the nematocidal activity of the fermentation broth.Once the time of UV irradiation exposure longer than 4h,the activity began to decrease.It was insensitive to temperature response and can be inherited stably.The results showed that the activity decreased significantly after 4℃could be stored for 60 days and 25℃for 9 days.4.After the fermentation broth was treated with concentrated hydrochloric acid,the precipitate was extracted with methanol to obtain the crude extract.When diluted 100 times,the corrected mortality rate of J2 of M.incognita was 94.73%.However,the activity of the crude extract was not stable.The corrected mortality rate of M.incognita decreased to 11.46%after 3 days storage at 4℃and 1.59%after 6 days storage.However,the pot experiment results showed that the control effect of the inactivated crude extract on cucumber root knot nematode reached 94.44%,and the control effect on chicory root knot nematode reached 82.24%,which was significantly better than that of the fermentation broth.It is speculated that methanol extract can induce cucumber resistance to M.incognita.The conjecture is verified by split root test.The results of nematode infection test showed that the infection activity of M.incognita to cucumber decreased after methanol extract treatment.5.The nematocidal activity of crude protein obtained by ammonium sulfate deposition was determined.The results showed that crude protein obtained by ammonium sulfate deposition with 0-30%saturation had a weak nematicidal activity against J2 of M.incognita,and the corrected mortality rate of J2 after 24 h treatment was only 15.38%.The crude protein obtained by ammonium sulfate deposition with 30-60%saturation had a strong activity against M.incognita,and the corrected mortality rate of M.incognita after 24 h treatment was 84.67%.