The Effect Of Water-soluble Humic Material On Medicago Sativa Yield And Its Mechanism On Inducing Nodulation And Nitrogen Fixation Of Sinorhizobium Meliloti With M. Sativa

Alfalfa (Medicago sativa L.) is an excellent perennial leguminous forage of great economic and nutritive value. For now, the alfalfa supply falls short of demand seriously which restrains the development of animal husbandry in China. It could be benificial to increase alfalfa yield and reduce nitrogen fertilizer usage, improve soil fertility at the same time when inoculated alfalfa with well-matched highly effective rhizobia according to the alfalfa cultivars. Besides, previous studies in our lab found that water-soluble humic material (WSHM) produced by microbial degradation of lignite were effective in increasing yield of several graminaceous crops as well as soybean by inducing nodulation and nitrogen of soybean. In this work the symbiotic efficiency of two rhizobia Sinorhizibium meliloti CCBAU01199 and Sinorhizibium meliloti CCBAU01290 with M. sativa cv. Zhongmu No.l were tested; the effects and mechanism of WSHM in inducing alfalfa growth and nodulation with S. meliloti were studied; well-matched highly effective rhizobia specific to eight alfalfa cultivars from Inner Mongolia were isolated and screened. The results of this work provided theoretical basis for improving symbiotic efficiency and material basis for inoculating rhizobia with alfalfa widely.The results of field experiments showed that the alfalfa yield were significantly increased by inoculating alfalfa with the two rhizobia mentioned above. Furthermore, the yield of alfalfa which inoculated with S. meliloti CCBAU01199 were higher than that of S. meliloti CCBAU01290 indicating that S. meliloti CCBAU01199 matching better than S. meliloti CCBAU01290. Beides, the inoculation method were also affected the yield. The best inoculation method for 5. meliloti CCBAU01199 was "on-seed" inoculation and the yield were increased by 37.57% and 24.78% respectively in two consecutive years. The results of soil physical and chemical properties measurement showed that the total nitrogen, available nitrogen and organic matter were increased, but the available phosphorus and potassium were decreased by inoculating rhizobia with alfalfa. Thus, phosphatic and potassic fertilizer were needed during alfalfa cultivation. In addition, the alfalfa yield of 0.5 g/L WSHM spray treatment was similar with that of 5. meliloti CCBAU01199 inoculation and the yield were increased by 29.57% and 20.34% respectively in two consecutive years. The alfalfa yield of 5. meliloti CCBAU01290 inoculation and WSHM spray treatment was higher (9.16%) than that of S. meliloti CCBAU01290 inoculation only. The nodulation status of alfalfa were detected and showed that the nodule number, nodule fresh weight and nitrogenase activity of alfalfa with native rhizobia and inoculated rhizobia were both increased significantly by WSHM.The mechanism of WSHM in inducing symbiotic nitrogen fixation between rhizobia and alfalfa were studied using the model strain S. meliloti 1021. The results showed that the growth of S. meliloti 1021 were significantly enhanced by WSHM with cell density increased by 5.07 times. While, cell density of S. meliloti 8530 (1021, expR+) which contain an intact quorum sensing (QS) system were increased by 20-50 times by WSHM suggesting that the effects of WSHM were related with QS of rhizobia. The results of reverse transcription-qPCR (RT-qPCR) showed that the expression of QS related genes expR, sinI and sinR in S. meliloti 8530 were repressed significantly by WSHM. Furthermore, WSHM regulated the QS system of S. meliloti 8530 mainly by repressing expR gene expression. A hypothesis that protein QsrR mediated the effects of WSHM was proposed by analysing the working pattern of WSHM. The expR expression expression pattern in qsrR mutant strain confirmed the hypothesis mentioned above. The expR expression was upregulated and the repression effect of WSHM on expR expression was abolished in qsrR mutant strain. Electrophoretic mobility shift assay (EMSA) and bacterial-one-hybrid assay proved that QsrR could bind with expR promoter and repressed expR expression directly. WSHM strengthened the interaction between QsrR and expR promoter and downregulated expR expression further. A transcription regulator of expR—QsrR was identified for the first time in this study. WSHM repressed the QS system of S. meliloti 8530 and resulted in enhanced growth, repressed EPS II and AHLs production which were benificial to rhizobia colonization in the rhizosphere and weaken the host denfence reponses, and in favour of symbiotic nitrogen fixation ultimately. The results of this study laid important foundation in improving symbioc nitrogen fixation.74 strains (5. meliloti) were isolated from alfalfa nodules collected from Khorchin, Inner Mongolia. Well-matched highly effective rhizobia specific to eight alfalfa cultivars from Khorchin were screened and obtained 1～3 highly effective rhizobia for each alfalfa cultivars. The results of this work provided excellent strains for alfalfa inoculation in Khorchin.