贵州岩溶山区野生天蓝苜蓿根瘤菌资源发掘、固氮特性及其多样性研究

天蓝苜蓿(Medicago lupulina)是一种广为分布的优良野生豆科饲草植物。为了发掘贵州岩溶山区天蓝苜蓿特定的根瘤菌资源,筛选获得固氮促生能力强、寄主识别范围广的高效抗逆天蓝苜蓿根瘤菌,从贵州省19个县(市)4个不同生态功能区采集天蓝苜蓿根瘤样品,经分离、纯化、菌体形态观察、16S rRNA基因序列比对分析,获得69个根瘤菌保存菌株。区系分析结果表明,分离菌株分属6属25种,其中中华根瘤菌属(Sinorhizobium)的分布频率高达55.71%,为优势属,草木樨中华根瘤菌(S.meliloti)分布频率达到38.51%,为优势种。选择来自不同地域、不同种类的天蓝苜蓿根瘤菌28株,采用百脉根盆栽回接方法,结合菌株固氮酶活性的测定,筛选出优良促生固氮根瘤菌9株,其中5个菌株的酶活力及植物促生效果与另外4株的差异均达显著(P<0.05)或极显著水平(P<0.01),为最优促生根瘤菌。本研究结果可为区域草业发展及生态环境建设提供共生微生物资源。

Research on Resource Exploration,Nitrogen Fixation Characteristics and Diversity of Rhizobia of Medicago lupulina in Karst Mountainous Area of Guizhou

Medicago lupulina is a kind of excellent wild leguminous forage plant widely distributed in the world. To explore the specific rhizobial resources of Medicago lupulina in karst mountainous area of Guizhou,and to screen out the rhizobial strains which possess a wide range of host recognition ability and strong nodule formation and nitrogen fixation capacity,the root nodule samples were collected from four different ecological functional areas in 19 counties (cities) of Guizhou Province. Through isolation,purification,observation of bacterial morphology,and 16S rRNA gene sequence analysis,69 rhizobium strains were obtained and preserved. The results of floristic analysis showed that all the preserved Medicago lupulina rhizobium strains belonged to 6 genera and 25 species,of which Sinorhizobium was the dominant genera and Sinorhizobium meliloti was the dominant species,with the distribution frequencies of 55.71% and 38.51%,respectively. 28 strains of different species of Medicago lupulina rhizobia were selected from different regions. Using Lotus corniculatus potted inoculation planting methods,combined with the determination of strain nitrogenase activity,9 efficient growth-promoting and nitrogen-fixing rhizobial strains were screened out,of which 5 strains' nitrogenase activity and growth-promoting effect were significantly different (P<0.05) or highly significantly different (P<0.01) with the other 4 strains,and they were identified as the best growth-promoting rhizobial strains. The results of this study could provide symbiotic microorganism resources for the development of regional prataculture and the construction of karst ecological environment.