| The genus Bacillus is comprised of Gram-positive,spore-forming,and rod-shaped organisms with an aerobic or facultatively anaerobic metabolism,were used in agriculture,husbandry,industry,medicine,environment because of their strong adapting ability with stress-resistance spore.In this research,1307 isolates were obtained from 28 soil samples of plant rhizospheres from different ecological environment including Haixi,Haidong,Haibei,Hainan,Huangnan,Yushu,Guoluo states of Qinghai Province,and 135 isolates were identified with biochemical method and molecular identification including BOX-PCR and ERIC-PCR fingerprint analysis,16S rDNA and gyrB gene sequence analysis.The results showed that 135 Bacillus strains were identified into 27 isolates of Bacillus amyloliquefaciens,12 isolates of L.sphaericus,11 isolates of B.simplex,8 isolates of B.pumilus,7 isolates of B.atrophaeu,7 isolates of B.lichenifor,mis,7 isolates of B.licheniformis,9 isolates of B.subtilis,6 isolates of B.thuringiensis,2 isolate of B.cereus,7 isolates of B.weihenstephanensis,7 isolates of B.axarquiensis,4 isolates of B.mojavensis,1 isolate of B.velezensis,1 isolate of B.malacitensis,and 22 isolates of Bacillus sp.could not be identified into specific species,which were concluded as new species isolated from Qinghai-Tibet Plateau.These identified strains will provide strains resource for further bio-control Bacillus screening and functional characteristic exploring.Act as important bio-control strain,Bacillus spp.could produce lipopeptide which were consisted of surfactin,Iturin and Fengycin,play important role during plant disease control,were considered as main bacteriostat ingredient.In this research,Xanthomonas oryzae pv.oryzae and Sclerotinia sclerotinorium were chosen as the indicator pathogenic bacteria and fungi,respectively,to check antagonistic activity of Bacillus spp.strain.Thereinto,348 isolates were found to be antagonistic to Sclerotinia sclerotiorum,311 isolates were found to be antagonisticto to Xanthomonas oryzae pv.oryzae,and 298 isolates were found to be antagonistic to both of these two kinds of pathogens(diameter of the inhibition zone ? 5mm).Moreover,19 bio-control strains were identified to check the lipopeptide component using MALDI-TOF-MS,the result showed that 11 strains including B.axarquiensis DGL6,B.amyloliquefaciens DCD2,B.amyloliquefaciens BS11,B.amyloliquefaciens GL18,B.amyloliquefaciens KKD2,B.amyloliquefaciens MD1,B.amyloliquefaciens DGL1,B.atrophaeus DCD1,B.malacitensis GH1,B.pumilus DR20 and B.amyloliquefaciens RYS41 could produced three kinds of lipopeptide including surfactin,fengycin,iturin A or bacillomycins D.Five strains including B.atrophaeus CKL1,B.atrophaeus TS1,B.amyloliquefaciens TS3,B.mojavensis KKD1 and B.amyloliquefaciens RYS12 could produce two kinds of lipopeptide.The results showed that antagonistic activity of bio-control strain have relativity with lipopeptide produced by strains themselves.There existed special Bacillus spp.resource adapting in ecological condition of high-altitude,strong UV,low temperature of Qinghai-Tibet Plateau.In this research,30 bio-control strains which were isolated from different ecological environments,were chosen to check their stress-resistance and growth-promoting characteristic.Firstly,seed germination and seedling growth characteristic promoted by bio-control strains were checked.The results showed that strains including B.axarquiensis DGL6,B.amyloliquefaciens BS11,B.mojavensis KKD1,B.amyloliquefaciens KKD2 and B.pumilus DR20 could improve seed germination and seedling growth of rice which present obvious growth-promoting effect.Low-temperature adapting characteristic of strains were checked,and the result showed that 13 strains could grow well in the condition of 4? and 10?were obtained,thereinto,5 strains including B.atrophaeus CKL1,B.atrophaeus CKL4,B.atrophaeus CKL5,B.atrophaeus,CKL6 and B.atrophaeus TS1 presented good aggrandizing characteristic and low-temperature adapting characteristic.The salt-resistance characteristic of bio-control Bacillus,were checked to screen stress-resistance strains,the result showed that 6 strains including B.atrophaeus CKL1,B.atrophaeus CKL4,B.atrophaeus CKL5,B.atrophaeus CKL6,B.atrophaeus TS1 and B.sonorensis GH2 could grow in the condition of salt medium contained 13%sodium chloride solution and presented obvious salt-resistance characteristic.31 bio-control strains were chosen to check cellulose-decomposition activity by means of plate transparency-circle method,the result showed that all of these bio-control strains own activity to decompose cellulose,thereinto,strains including B.amyloliquefaciens KKD2,B.sonorensis GH2,B.amyloliquefaciens BS11,B.amyloliquefacienDCD2,B.amyloliquefaciens DGL1,B.axarquiensis DGL6 and B.amyloliquefaciens RYS9 presented obvious cellulose-decomposition activity.According to growth-promoting,stress-resistance and cellulose-decomposition characteristic checking,excellent strains were obtained for developing bio-fertilizer adapting in Qinghai-Tibet Plateau,also high-activity cellulose-decomposition strains were obtained for feedstuff processing in animal husbandry industry.Almost all the transcripts sequence of special tissue or organ at the special condition could be obtained quickly using RNA-Seq,which could reflect functional status of special cell in the special condition dynamically.RNA-Seq is kind of high-technology method to explore gene difference expression at the transcription level.In this studying,difference expression genes after interaction between model bio-control B.amyloliquefaciens FZB42 and Oryza sativa subsp.indica 9311 were analyzed using RNA-Seq technology,and response reaction of plant root and leaf to bio-control Bacillus were explored according to GO function annotation and KEGG pathway analysis,the purpose is to reveal growth-promotion and disease-resistance mechanism of bio-control Bacillus.The analysis result showed that 379 genes of T-root(root sample)presented difference expression,thereinto,257 genes were up-regulate expressed and 122 genes were down-regulate expressed,while 719 genes of T-leaf(leaf sample)presented difference expression including 501 genes were up-regulate expressed and 218 genes were down-regulate expressed after 4 hours interaction between B.amyloliquefaciens FZB42 and Oryza sativa subsp.indica 9311.GO annotation and KEGG pathway analysis reveal that bio-control Bacillus could influence multi-metabolic pathway include plant-pathogen interaction,plant signal transduction,lipid metabolism,carbohydrate metabolism,nucleotide metabolism,amino acid metabolism,energy metabolism,biosynthesis of secondary metabolites,metabolism of terpenoids and polyketides,metabolism of cofactors and vitamins,genetic information processing.The analysis results showed that bio-control Bacillus could promote development and disease-resistance of plant according to influence relative genes regulation and expression in the signal transduction and metabolism pathway. |
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