| Rhizoctonia solani is studied in order to screen biocontrol strain with an inhibitory effect from soil.Isolation and screening of biocontrol strain is the beginning of microbial pesticide research,for more,through morphology,the physiological and biochemical characteristics as well as molecular biology,classification status can be confirmed and it provide basics for development and utilization.Through study its fermentation conditions,the production of biocontrol strain of antibacterial material increased.To master the physical and chemical properties of antibacterial substances,providing the basis for further purification.Understanding about the antibacterial mechanism of bio-control strains may provide theoretical support for the future of the development of biological pesticide,eventually to achieve efficient and safe rice sheath blight.1.Screening and identification of biocontrol strain to Rhizoctonia solaniThe soil sample from several region of paddy field were collected from Liaoning,Jilin and Heilongjiang province.Three methods flat growth restraint,mycelial growth and spore germination were used to screen biological control agents to Rhizoctonia solani.Three effective biological control potential isolates including one high antibacterial activity fungus JLF6-2 and three bacteria JLN3-2,SYN6-3 and JLN3-8.They were screened from 211isolates which were separated and purified from the soil sample.Separately,the inhibitory action of bacteriostatic ring were 38.30 mm,18.30 mm,33.30 mm and 22.50 mm.The growth inhibition rate of mycelial were respectively 77.30%,63.29%,80.35%and 100%.The germination inhibition rate of sclerotium were 86.67%,83.33%,80.00%and 61.35%dividually.The vitro experiment results showed that in protective treatment the JLN3-2 could even reach 94.92%with treatment for 24h,In therapeutic treatment the JLN3-2 could reach74.58%with treatment for 8h.The test of indoor pot plantingresults showed that the highest control effect of JLN3-8 was 86.42%.All these four isolates was based on develop character and morphological characteristics,physiological and biochemical test as well as molecular biology assay.The isolate JLF6-2 was identified as Penicillium bilaiae.Also,JLN3-2 was identified as Lysinibacillus fusiformis,SYN6-3 was identified as Bacillus amyloliquefaciens and JLN3-8 was identified as Brevibacillus laterosporus.2.Study of biocontrol bacteria fermentation conditionsSingle factor test and orthogonal test were used to study the fermentation conditions.The results showed the optimum fermentation culture medium formula to 3 biocontrol bacteria strains were yeast extract 5g,sucrose 20g,peptone 10g,sodium chloride 5g,distilled water1000m L.The best fermentation conditions to JLN3-2 were fermentation time 48h,temperature 28℃,pH7,inoculation amount 5%,inoculating age 20h,culture volume80m L/250mL,table speed 150r.min-1.The best fermentation conditions to SYN6-3 were fermentation time 48h,temperature 28℃,pH 7,inoculation amount 5%,inoculating age 20h,culture volume 120mL/250mL,table speed 150r.min-1.The best fermentation conditions to JLN3-8 were fermentation time 48h,temperature 28℃,pH 8,inoculation amount 5%,inoculating age 20h,culture volume 120mL/250mL,table speed 150r.min-1.3.The physical and chemical properties of biocontrol bacteria fermented product and evaluation on the safety of cropsThe physical and chemical properties determination results show that the aseptic filtrate of 3 bacteria had good thermal stability,under 80℃with 10-120 min after all had good antibacterial activity,but on the 120℃high temperature caused inactivation during processing.The aseptic filtrate of 3 bacteria strains in neutral and weak alkali environment high antibacterial activity,had good stability.The aseptic filtrate of 3 bacteria strains had light stability and storage stability under the condition of low temperature preservation,could be long-term preservation.Safety determination results indicated the aseptic filtrate of 3 bacteria strains had a certain impact on germination rate and had a certain role in promoting on the radicle and bud growth.The results of the fermentation filtrate on plant safety showed that 3 strains of bacterial fermentation broth did not produce scab on the leaves of 8 test crops,and the color and lustre of the tested crops had no significant difference compared with the control group,indicating that the 3 strains of bacteria resistant fermentation broth were relatively safe to the tested crops.3 bacteria strains had wide antimicrobial spectrum.4.The preliminary research on the mechanisms of biocontrol bacteriaBiocontrol bacteria antibacterial material had strong inhibitory effecton rice sheath blight mycelium and sclerotium growth,making the mycelium deformity,protoplast condensation or dissolved.And antimicrobial substances could inhibit the germination of rice sheath blight sclerotia.The aseptic filtrate has obvious improvement to rice defense enzyme activity and active oxygen content.The aseptic filtrate of JLN3-2,SYN6-3 and JLN3-8 could effectively induce PAL and POD and PPO enzyme activity in rice leaves with a longer effect.T-SOD and CAT enzyme activity had also improved,but the effect period is shorter.Biocontrol bacteria also induced O2-and H2O2 content in rice leaves increased,so as to improve its resistance to disease. |
