黄瓜根际芽孢杆菌亲缘辨识特性研究

Kin discrimination between Bacillus spp. strains from cucumber rhizosphere

[目的]依据群集运动表型和16S rRNA基因序列信息研究黄瓜根际芽孢杆菌亲缘辨识能力,进而形成直接合作的多细胞行为,找到解淀粉芽孢杆菌SQR9的近亲菌株。[方法]采用高温处理和稀释涂布的方法从黄瓜根际筛选芽孢杆菌,得到菌株后分析其16S rRNA基因测序结果,结合swarming平板对峙表型对芽孢杆菌类群进行亚家族分类。最后通过24孔细胞培养板静置培养、salkowski比色法分析吲哚乙酸(IAA)产量和植酸酶活性测定的方法评价SQR9近亲菌株的促生潜力。[结果]初筛共选出118株芽孢杆菌,复筛后确定为21株。菌株swarming对峙后表现融合、半融合和对峙3种表型。在融合的27组菌株里,25组有100%相同的16S rRNA基因,而其余2组的基因相似度较低。产生边界的组合中只有10%(142对中只有15对)的16S rRNA基因相似度大于99%;半融合的基因相似性为99.5%~100%。菌株SQR9的亲近菌株P2、W-4和N-2在生物膜形成、IAA分泌和植酸酶活性等方面都接近SQR9。[结论]初步证明swarming表型与16S rRNA有一定的相关性,菌株SQR9近亲菌株P2、W-4和N-2都是潜在的根际促生菌。

[Objectives] We investigated the ability of cucumber rhizosphere bacterium Bacillus spp. to discriminate kin from nonkin in the context of swarming and 16S rRNA gene squences,then formed a direct cooperative muticellular behavior, and screen the kin strains of Bacillus amyloliquefaciens SQR9. [ Methods ] The Bacillus spp. strains were isolated from plates spread with diluted solution of cucumber rhizosphere soil samples after heat shock. Then the sub family classification of those Bacillus spp. strains were identified based on 16S rRNA gene sequence analysis and swarming discrimination phenotype which were performed on swarming agar. We investigated the biocontrol potential of those kin strains of SQR9 through static culture biofilm assays in 24 well plate, IAA detection by salkowski reagent and the production of phytase. [ Results] 118 Bacillus spp. strains were isolated through primary screening from cucumber rhizosphere, and 21 Bacillus spp. strains were collected after secondary screening. Three different phenotypes （ boundary, intermediate and merging）were detected from Bacillus spp. strains swarms. Out of 27 merging swarm combinations,25 combinations had 100% identical 16S rRNA gene,while the other two showed lower nucleotide identity. In contrast, only 10% of strain pairs that formed clear boundary lines （15 combinations out of 142）had higher than 99% identical 16S rRNA gene. In addition, combinations showed an intermediate lines shared a genetic identity ranging from 99.5% to 100%. Three kin strains of SQR9（ P2,W-4 and N-2） showed similar abilities in biofilm formation, IAA production and phytase activities when compared with SQR9. [ Conclusions ] Present study preliminarily proved that swarm interaction phenotype correlates with 16S rRNA, and three kin strains of SQR9 （P2, W-4 and N-2）are potential plant growth-promoting rhizobacteria.