| Rape Sclerotinia stem rot is a fungal disease caused by sclerotinia sclerotiorum,which occurs widely in the rape seed planting area in our country,it has seriously affected the yield and safety of rapeseed crops.In recent years due to the large-scale use of chemicals,causing irreversible damage to the environment.Therefore it is necessary to find environmentally and friendly means of sustainable biological control.In recent years biological control has been considered as a promising approach to prevention and treatment due to its little pollution to the environment,and several studies have been devoted to commercialization.So far to December 2014,there are3335 biological pesticides have been registered.Among them,there are 376 kinds of biological pesticides,which are Paenibacillus and Bacillus.Since 2011,only Bacillussubtilis has 706 patents,82 patents of Paenibacillus.In this experiment,175 strains were randomLy screened from rape and wheat surface,tissues and rhizosphere soil.5 antagonistic strains were screened by plate confrontation method,Identify the 5strains by physiological and biochemical test.Plate confrontation with filtrate,protease production assay,and sequence analysis of 16 SrDNA and gyrB gene,BLAST homology comparison and cluster analysis,results as follows:1.Collect the rape and the wheat and the rhizosphere soil of them.Isolated 175 bacterial strains by using plate dilution method.5 antagonistic strains with strong antagonism against Sclerotinia sclerotiorum were obtained after screening by plate confrontation method.Named these 5 strains as Y10?T21?T22?T23?T24.Make a second screening by Subculture,the inhibition of Sclerotinia sclerotiorum rate:44.5%-66.7%.Among them,Y10 has the best antibacterial effects,its rates is 66.7%,and with the best stability of antagonism.2.Through the physiological and biochemical identification of antagonistic strains,the identification results showed that 5 strains were gram positive bacteria,facultative anaerobic or aerobic,catalase reaction was positive.By sequencing16 SrDNA and gyrB gene of these 5 strains of bacterial strains and analysis.Through16 SrDNA sequencing and analysis.The results showed that Y10 was Bacillus polymyxa,T21?T22?T23was B.amyloliquefac,T24 was B.mojavensis.By analysis the result of gyrB gene sequence forT21 ? T23 strains.BLAST and cluster analysis in NCBI,the results showed that T21?T23 was B.amyloliquefaciens.3.Study the mechanism of biocontrol for the 5 strains.Processing aseptic filtrate by high temperature(at 120?,40min),we found in high temperature the plate confrontation metabolites almost with no inhibitory effects on Sclerotinia sclerotiorum,the results show that antagonism may caused by living bacteria or enzymes,antibiotics and other materials which can't exsist in high temperature.Inoculation of antagonistic strains in defatted protein medium,all of them have transparent bands appeared.The results showed that 5 strains of bacteria could produce protease.4.Test of 5antagonistic strains of Sclerotinia sclerotiorum by separating leaves and indoor potted plant.First the 5 strains of antagonistic strains were made into the fermentation liquid and sterile filtrate and spraying on the leaf surface of rape 24 h before.Inoculated leaves block with the diameter of 6mm of Sclerotinia sclerotiorum.The results show that the rate of the antagonistic bacteria liquid to inhibit Sclerotinia sclerotiorum was 8.0%~65.0%.The rate of sterile filtrate of antagonistic strains was5% ~13.0%.The T23 strain has a very good effect on both antagonistic bacteria liquid and sterile filtrate of antagonistic strains.The pot experiment inoculat in the same method above.The leaves were acupunctured when inoculated with Sclerotinia sclerotiorum block.The results of pot experiment showed that the rate of antagonistic strains bacteria liquid to inhibit Sclerotinia sclerotiorumwas4.7% ~56.8%.The rate of sterile filtrate of antagonistic strainswas15.5% ~39.3%.The T22 strain has a very good effect on antagonistic bacteria liquid,the rate was 56.8%.The T24 strain has a very good effect on sterile filtrate of antagonistic strains,the rate was 39.3%.The experimental results showed that strain T23 has a very good inhibitory effect in most experiments... |
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