Screening And Ability Of Saline-alkali Tolerant Rhizobia From Alfalfa

Alfalfa(Medicago sativa L.)is rich in nutrition and is known as the "king of forage grass".Saline alkali stress seriously affects the growth and development of alfalfa.The rhizobia-alfalfa symbiotic nitrogen fixation system is very sensitive to external stress,and salinity stress can inhibit nodule development and symbiotic nitrogen fixation.This study isolated rhizobia from alfalfa nodules,analyzed salinity tolerance and nitrogen fixation ability,evaluated the influence on the expression of nodules and nitrogen fixation,so as to provide high quality strains for the development of rhizobia microbial fertilizer and lay the foundation for alleviating the negative effects of salinity stress on alfalfa production.The main study results are as follows:1.Isolation and identification of rhizobiaTwenty-eight strains of rhizobia without color absorption on congo red YMA medium were preliminarily isolated and screened from alfalfa rhizobia,and 8 strains of rhizobia producing hydrolytic circle were screened on Ashby nitrogen-free culture with Na Cl 7% and p H 9.According to the bacterial morphology,16 S r DNA sequence analysis and physiological and biochemical detection,the eight strains were identified as Sinorhizobium meliloti.2.Analysis of strain characteristics of rhizobiumThe strain characteristics and salt tolerance of rhizobium were determined by kjeldahl method and molybdenum antimony anti colorimetry.The 8 strains of rhizobia isolated have the ability of nitrogen fixation,synthesis of indole acetic acid(IAA),production of ferritin and dissolution of inorganic phosphorus.The strains with strong nitrogen fixation ability are YL-25,YL-5,YL-39 and YL-38,and the strains with strong ACC deaminase activity are YL-25,YL-5 and YL-38.All 8 strains could synthesize IAA without L-Trp,and the amount of IAA synthesized by strain YL-38 was the highest.All 8 strains can synthesize ferritin and dissolve inorganic phosphorus in varying degrees.The isolated strains have strong salt and alkali tolerance.Except YL-28,other strains grew well at p H 7～9.When the salt concentration was 5%,the other strains grew well except YL-28 and YL-36.3.Effects of rhizobia on growth,nodulation and quality of alfalfaThe eight strains could promote the germination of alfalfa seeds under saline alkali stress,promote the growth of potted alfalfa and increase the number of root nodules.Among them,the growth promoting effect of strain YL-5 was the best.The plant height and aboveground dry and fresh weight increased by 40.05%,52.63% and40.72% respectively;The underground dry and fresh weight and root length increased by 72.41%,19.67% and 25.08% respectively.In terms of nodulation and nodule nitrogenase activity of alfalfa,YL-38 treatment had the best nodulation effect.The number of nodule per plant and fresh weight of nodule per plant increased by363.47% and 493.75% respectively,and the nitrogenase activity increased by295.74%.In terms of quality,compared with the control group,the crude protein and phosphorus contents of alfalfa treated with the strain increased,and the contents of neutral and acid detergent fiber decreased significantly.Among them,compared with the control,the neutral detergent fiber inoculated with YL-5,YL-25 and YL-38 strains decreased significantly,by 23.57%,21.55% and 22.60%.The acid detergent fiber of alfalfa treated with YL-25 and YL-38 decreased by 15.53% and 14.83%.4.Effects of rhizobium inoculation on nodulation and nitrogen fixation related gene expression of alfalfaThe expression of nodule signal pathway gene(NOD),nodule bean hemoglobin gene(LB)and glutamine synthase gene(GS)in alfalfa roots were detected by q RT-PCR.The results showed that compared with the control,the expression of NDD1,NOD2,LB1,LB2 and GS genes in alfalfa roots were up-regulated by inoculating strains YL-5,YL-25 and YL-38.The up-regulated expression of strain YL-38 was the most significant.Inoculation of rhizobium promoted the expression of nodulation and nitrogen fixation related genes of alfalfa,increased the accumulation of nitrogen,promoted the growth of alfalfa and improved the quality of alfalfa.