高效苜蓿根瘤菌的筛选

Selection of Highly Effective Rhizobial Strains for Alfalfa

旨在利用celB基因标记技术从土著根瘤菌中筛选出与盛世苜蓿品种相匹配的有效性高、竞争性强的高效苜蓿根瘤菌株。从四川雅安、重庆北碚、万州采集野生苜蓿根瘤,采用平板划线法分离纯化获得93株纯菌株,通过与盛世品种在无氮水培液中进行结瘤试验和有效性分析并结合菌株的地理来源,初筛出了与对照相比有效性较高的4株苜蓿根瘤菌,分别为Y6-1-1、BB1-8-1-1、BB2-2-1-1和WZ-6-2-2。进一步利用celB基因标记技术对这4株菌进行竞争性研究,通过供体大肠杆菌HAMBI2356与根瘤菌接合的方法成功地将celB基因导入4个菌株中,对标记基因可能会对标记菌株产生的影响进行检测,结果表明标记基因在标记菌株内能稳定传代,而且对标记菌株的生长、竞争性及有效性都没有显著影响,从而证明celB基因标记技术可以作为一种简便、直观而又有效的检测手段用于追踪根瘤菌株在土壤中的竞争性。进一步通过田间试验比较分析标记菌与土著菌的竞争结瘤能力和固氮有效性,结果表明Y6-1-1、BB2-2-1-1、WZ-6-2-2与对照相比在占瘤率、瘤重、总瘤数上差异显著,占瘤率都在72%以上,显著提高了植株的干重、叶绿素含量、全氮量和产量,其中WZ-6-2-2的有效性最高、竞争性最强,可以用于苜蓿盛世品种的接种。

The aim of this experiment was to select highly competitive and effective rhizobial strains which were compatible with Shengshi alfalfa cultivar by using marker gene technique. The nodules from alfalfa were collected from Yaan in Sichuan,and Beibei,Wanzhou in Chongqing. 93 strains were isolated and purified. These strains were then inoculated to on the shengshi of alfalfa by pot experiment in Jensen＇s N-free solulion. By analysis of the result, 4 strains Y6-1-1, BB1-8-1-1, BB2-2-1-1 and WZ-6-2-2 were relatively more effective. These strains were selected for further sthdy of compedtiveness with soil indigenous rhizobia by using celB marker gene technique. By conjugation with Escherlchia coli HAMBI2356 which contains the celB gene, the celB gene were successfully introduced into the four strains respectively. The stability and impact of the insert celB gene on the rhizobial strains were evaluated by determination of generation time, pot inoculation test. The results showed that the marker gene was highly stable, and had no significant effect on the growth, competitiveness and symbiotic effectiveness of the rhizobial strains. So the celB gene marker technique can be used as a perfect means to track the competition of the rhizobial strains in soil. The selected four strains were further assessed for their competitiveness and effectiveness under soil conditions in field experiment. The resuits showed that three strains Y6- 1 -1, BB22-1-1, WZ6-2-2 among the four significantly increased the nodule weight, total nodule number, plant shoot dry weight, total nitrogen content, chlorophyll content and yield in comparison with the non-inoculation control. Their nodule occupancy was more than 72%. The best strain was WZ6-2-2, that could be used as inoculant for alfalfa shengshi cultivar.