| Bacillus is widely existed in natural environment,and produces abundant secondary metabolites.It has extremely strong resistance and environmental adaptability and is a class of microbial communities with practical and economic value.In this study,we screened and obtained biocontrol Bacillus strains F85 and T113 which have a good inhibitory effect on Plasmodiphora brassicae in laboratory.In addition,a strain BJ-1 with high antagonistic activity against Magnaporthe oryzae was found in a contaminated plate.On the basis,the biocontrol mechanisms of these Bacillus strains in suppression of canola clubroot and rice blast were studied,and the biocontrol effects of the Bacillus strains was comprehensively evaluated.The main results are shown as follows:1.Total 667 microbial strains were isolated from the rhizosphere soil of rapeseed in severely diseased fields in Dangyang City,Hubei Province.These included 323strains of bacteria,253 strains of fungi,and 91 strains of actinomycetes.Fusarium oxysporum and M.oryzae were used as indicators for P.brassicae.There were 54isolates with a inhibition zone width of more than 3 mm by dual culture test.Among these,20 strains were screened to inhibit the germination of rest spores of P.brassicae by 30-50%,and 16 strains caused root resting spores of P.brassicae inactivation rate of 40-55%.Two potential biocontrol strains F85 and T113,were found to have a control efficiency of more than 60%through the pot experiment.Strains F85 and T113 significantly inhibited the infection of root hair,and reduced the differentiation of primary plasminogen of P.brassicae and inhibited the formation of secondary zoosporangium.The bacterial cells of the strains F85 and T113 were rod-shaped,and Gram-staining reactions both showed positive.16S rDNA molecular identification and phylogenetic tree analysis both indicated that the strain F85 and T113 belong to Bacillus.Also these two strains were identified as Bacillus sp.based on Biolog Identification System.2.The strain BJ-1 with strong antagonism against M.oryzae was identified as Bacillus subtilis by traditional morphology,16S rDNA sequence analysis and the Biolog Identification System.The inhibitory effect on the mycelia of M.oryzae remained after culture filtrates of B.subtilis BJ-1 treated with different temperatures(4-100°C)and different pH(2-12).These indicated that the culture filtrates of BJ-1have strong heat stability and resistance to acidic and alkali conditions.The dual culture test showed that B.subtilis BJ-1 has a broad spectrum of inhibition,and also has inhibitory effect on mycelia growth of a variety of plant pathogenic fungus.The antibacterial zone of M.oryzae reached 22.7 mm.Moreover,hyphae of M.oryzae treated with 0.5%BJ-1 culture filtrates were degraded and cracked.culture filtrates of B.subtilis BJ-1 could cause significant malformation of germ tubes and inhibit appressorial formation of M.oryzae.We found that washed bacterial cells of BJ-11×10~8 CFU/ml and 5%culture broth and culture filtrates could completely suppress M.oryzae development on detached rice leaves.The results of pot experiments showed the disease index decreased to 30.7 with the control efficacy of 50.0%when sprayed with 10%culture broth of B.subtilis BJ-1.More importantly,While rice seeds were soaked with culture broth for 2 days and then planted,the disease index was only 15.8and the control efficacy reached to 74.3%and the seedling height increased significantly.Real-time PCR revealed that rice seed treated with BJ-1 broth could up-regulate significantly the expression of PAL and ICS1 in SA signal pathway,AOS2 gene in JA signaling pathway and two PR genes PR1a and PR10.These results showed that seed treatment with BJ-1 could induce expression of relative genes in SA and JA signaling pathway and host systemic resistance. |
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