| The sheath blight caused by Rhizoctonia cerealis infection is one of the most serious fungal diseases to wheat in the world.In recent years,chemical methods are often used to control wheat fungal soil borne diseases.Various chemical fungicides are used to kill or inhibit the pathogen of Wheat Sheath blight.However,chemical control is easy to cause drug resistance,which is not conducive to the control of Wheat Sheath Blight,but also aggravates environmental pollution Dyeing is a threat to human and animal health.It is an ideal choice to use biological pesticide to control wheat sheath blight.In this study,antagonistic bacteria were screened from soil,and the optimization of fermentation conditions,antibacterial mechanism and preparation of fungicides were carried out.(1)screening and identification of Antagonistic Bacteria of Rhizoctonia cerealis.In this study,119 strains with different morphologies were isolated from chernozem collected from the desert grassland of Pulan County in Ali Region of Tibet Autonomous Region by gradient dilution method.The results showed that there were 6 antagonistic bacteria to Rhizoctonia cerealis,among which xz18-3 had the most obvious bacteriostatic effect,with 36.57%bacteriostatic rate to Rhizoctonia cerealis,36.57% to Fusarium graminearum,36.57% to Fusarium graminearum,yellow Fusarium,false Fusarium graminearum and peristalsis Through morphological observation,physiological and biochemical characteristics analysis and molecular biological identification,strain XZ18-3 was identified as Bacillus subtilis and named as Bacillus subtilis XZ18-3.(2)Optimization of fermentation conditions of Bacillus subtilis XZ18-3.The optimum fermentation carbon source,nitrogen source,inorganic salt,inoculation quantity,pH value,fermentation time and rotational speed were obtained by single factor experiment: glucose2.0%,peptone 2.0%,MgSO4 0.5%,inoculation quantity 1.0%,pH7.0,fermentation time 60 h,rotational speed 100r/min.In order to determine its reliability,the fermentation conditions of Bacillus subtilis XZ18-3 were optimized on the basis of single factor.The main factors were inorganic salts,fermentation time and rotational speed by Plackett-Burman test design with the bacteriostasis rate(Y)as the response value.On the basis of Plackett-Burman experimentdesign,Box-Behnten experiment design was used to carry out three-factor and three-level response test.Regression equation analysis of the response value was carried out by using Design-Expert 8.0.6 software,and the optimal fermentation conditions of Bacillus subtilis were as follows: glucose 2.00%,peptone 2.00%,MgSO4 1.25%,inoculation quantity 1.00%,temperature 37℃,inoculation quantity 21.00%.pH6.97,rotational speed 180 r/min,fermentation time 48 h,under this condition,the bacteriostatic rate was 57.25%,which was56.55% higher than that before(36.57%).(3)study on the physical and chemical properties of fermentation broth of strain XZ18-3.The physical and chemical properties of the fermentation broth optimized by the culture conditions of XZ18-3 were tested.The results showed that the fermentation broth was relatively stable under ultraviolet and light conditions for a long time,and the protease treatment could also maintain a high stability.Its thermal stability and acid-base stability had little effect on the fermentation broth.The results showed that the fermentation broth of XZ18-3 strain had good physical and chemical properties,stable properties,bacteriostatic rate could be maintained at a stable level under relative extreme environmental conditions,and had a certain tolerance.(4)The antibacterial mechanism of antagonistic XZ18-3 on Rhizoctonia cerealis.Through the observation of light microscope and transmission electron microscope,it was found that the number of mycelium after the treatment of fermentation liquid was more,the color of mycelium was darker,and it was irregular and crossed,the cell membrane of mycelium appeared bulge,depression and rupture,some organelles in the cell disintegrated,forming a large number of blank areas.The metabolites produced by fermentation of XZ18-3 can inhibit the growth of mycelium,and the wet weight and dry weight of the control group were reduced by 53.08% and 30.00% respectively compared with the experimental group.The results showed that the XZ18-3 fermentation solution destroyed the cell membrane of Rhizoctonia cerealis,caused the leakage of soluble sugar,soluble protein,nucleic acid and other macromolecules in the cell,and led to the cell death.The enzyme in the fermentation metabolite of XZ18-3 strain was detected.The results showed that the strain produced protease,glucanase,amylase and cellulase,and the content of protease and glucanase was the highest.(5)preparation of water and wettable powder of Bacillus subtilis strain XZ18-3.The best formulation of XZ18-3 water agent of Bacillus subtilis was determined as follows: 0.10 mg /ml SDS,0.02 mg / ml sucrose,0.02 mg /ml sodium carboxymethylcellulose,0.14 mg/ml sodium citrate.The optimum formulation of Bacillus subtilis XZ18-3 wettable powder was determined as follows: Kaolin 30.0%,polyvinyl alcohol 4.0%,tween-80 8.0%,sorbitol 2.0%,polyethylene glycol 2.0%,Bacillus subtilis fermentation liquid 100%.All the indexes of the preparation were tested and reached the standard.The results of pot experiment showed that:The wettable powder has a good control effect on Wheat Sheath Blight,and the control effect can reach 60.29% after it is diluted 100 times.Moreover,it belongs to Bacillus subtilis,and It is a kind of safe microorganism without pathogenicity,which can be used as a green and environmentally friendly biocontrol agent to control wheat sheath blight. |
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