兰州地区白三叶根瘤菌分离鉴定与筛选

Isolation,identification and screening of rhizobium strains from white Clover in Lanzhou

为了筛选出兰州地区白三叶(Trifolium repens)高效共生固氮的根瘤菌,从兰州市安宁区绿化带白三叶根瘤内分离出6株疑似根瘤菌。通过形态学、生理生化和16S rRNA序列的系统进化分析来确定这6株菌的分类地位,并对鉴定为根瘤菌的菌株通过回接试验筛选高效根瘤菌。结果表明,所分离的6株菌16S rRNA序列与Rhizobium anhuiense、R.laguerreae、R.leguminosarum和R.sophorae的相似性最高(99.77%~99.92%),且聚集在一个分支,是根瘤菌属的成员;回接试验显示,接种根瘤菌GAU-00001、GAU‐00002、GAU‐00005和GAU‐00006的白三叶结瘤数分别是18.2、16.7、17.7和19.2;株高分别是(15.2±0.3)、(14.3±0.3)、(14.6±0.6)和(15.1±0.2)cm;鲜重分别是(0.324±0.002)、(0.318±0.005)、(0.315±0.005)和(0.321±0.027)g;干重分别是(0.06±0.007)、(0.062±0.027)、(0.06±0.01)和(0.062±0.01)g;粗蛋白含量分别是(21.27±0.49)%、(20.2±0.26)%、(20.7±0.31)%和(21.37±0.37)%;粗脂肪含量分别是(8.24±0.01)%、(8.11±0.16)%、(8.23±1.0)%和(8.25±0.23)%,各项指标均显著高于CK、GAU‐00003和GAU‐00004的处理。由此,菌株GAU‐00001和GAU‐00006是最适宜接种兰州白三叶的根瘤菌菌株。

In order to screen out high‐efficiency of nitrogen‐fixation rhizobia with Lanzhou white clover,six strains of candidate rhizobia were isolated from white clover nodules in the green belt of Anning District,Lanzhou.The taxonomic status of these six strains was determined by morphological,physiological,biochemical and systematic evolution analysis of 16S rRNA sequences;the strains identified as Rhizobia were screened for their efficiency by inoculation experiment.The results showed that the 16S rRNA sequences of the six strains had the highest similarity(99.77%~99.92%)with Rhizobium anhuiense,R.laguerreae,R.leguminosarum,and R.sophorae,and clustered together.All six strains were members of the genus Rhizobium.The inoculation experiment showed that the number of nodules inoculated with GAU‐00001,GAU‐00002,GAU‐00005,and GAU‐00006 were 18.2,16.7,17.7,and 19.2,respectively;the plant heights were(15.2±0.3),(14.3±0.3),(14.6±0.6),and(15.1±0.2)cm,respec‐tively;fresh weight were(0.324±0.002),(0.318±0.005),(0.315±0.005),and(0.321±0.027)g,respectively;dry weight were(0.06±0.007),(0.062±0.027),(0.06±0.01),and(0.062±0.01)g,respectively;crude protein were(21.27±0.49)%,(20.2±0.26)%,(20.7±0.31)%,and(21.37±0.37)%,respectively;crude fat were(8.24±0.01)%,(8.11±16)%,(8.23±1.0)%,and(8.25±0.23)%,respectively.All indicators were significantly higher than CK,inoculated with rhizobia GAU‐00003 and GAU‐00004.Therefore,the strains GAU‐00001 and GAU‐00006 were the most efficient rhizobium strains for Lanzhou white clover.