3株分离自高寒草甸根际芽孢杆菌的分子鉴定及其生物活性

摘要

For the purpose of restoring degraded vegetation and improving the aboveground biomass of grass-land,the biological activity of three strains of Bacillus spp.bio-control agents were isolated and screened. Three strains isolated from an alpine meadow (Kobresia pygmaea)rhizosphere in Huangnan Autonomous Pre-fecture,Qinghai Province,were identified by 16S rDNA and gyrB partial sequence analyses.Antagonistic ac-tivity of strains to pathogenic fungi were determined by the flat confrontation test,and the growth-promoting activities of strains were checked by soaking plant seeds in strain suspension (106 cfu·mL-1 of cell concentra-tion).In addition,cellulose-degrading activities of strains were checked with carboxymethylcellulose (CMC) plate test,Gram staining,and 3,5-dinitrosalicylic acid (DNS)methods;moreover,cellulase coding genes of the strains were amplified and analyzed.The results showed that the isolate of HNC3 was identified as Bacillus pumilus,and isolates of HNC8 and HNC11 were identified as B.thuringiensis.The three strains presented distinct antagonistic activity to Fusarium graminearum S and Fusarium oxysporum (inhibition zone diameter ≥ 11 mm).Furthermore,the strains showed obvious germination and growth-promoting activity af-ter soaking the wheat seeds in Bacillus suspension.The effect of the isolate of HNC3 on the growth of wheat seedlings was significant;the rate of bud and root length growth increased by 55% and 80%,respectively.The cellulase activity of the isolate of HNC3 reached 367.51 U·mL-1 ,and its cellulase coding genes could be am-plified including lichenase (blg1S),mannosidase (ManSig),and xylanase (xynD,xynB).The three Bacillus strains used as bio-control agents all showed antagonistic,plant growth-promoting,and cellulose-degrading ac-tivities,which present future research opportunities into the potential of their application in the recovery of plateau grassland vegetation,as well as in the field of ecological animal husbandry.%为青海省退化草地植被恢复、提高地上生物量筛选优良的生防芽孢杆菌菌源,采用16 S rDNA及gyrB基因序列分析法,鉴定分离自青海省黄南州高寒草甸高山嵩草(Kobresia pygmaea)根围的3株芽孢杆菌菌株;平板对峙试验检测菌株拮抗病原真菌活性;菌悬液(细胞浓度为106 cfu·mL-1)浸种以检测菌株对植物催芽及促生效果;CMC、Gram染色法及DNS法检测菌株降解纤维素活性,并扩增菌株降解纤维素酶编码基因.结果表明,菌株HNC3被鉴定为短小芽孢杆菌(Bacillus pumilus),菌株HNC8、HNC11被鉴定为苏云金芽孢杆菌(Bacillus thuringiensis);3株菌株对小麦赤霉病菌(Fusarium graminearum)、瓜类枯萎病菌(Fusarium oxysporum)具有明显的拮抗活性(抑菌圈平均直径均≥11 mm);3株菌株对小麦(Triticum aestivum)种子具有明显的催芽及促生效果;小麦种子经菌株HNC3菌悬液处理后,芽长、根长增加率分别达55%、80%;菌株HNC3降解纤维素酶活高达367.51 U·mL-1,并在该菌株中扩增到地衣聚糖酶(Lichenase,blg1S)、甘露糖苷酶(mannosidase,ManSig)、木聚糖酶(xylanase,xynD、xynB)4个纤维素酶编码基因.分离自青藏高原高寒草甸根围的3株芽孢杆菌兼具拮抗、催芽、促生及降解纤维素活性,在高原草地植被恢复、生态农牧业中具有一定的研究及应用潜力.