Study On Colonization Of High-Yield Nitrogen-Fixing Strains In Rhizosphere Or Phyllosphere Of Several Vegetables

Biological nitrogen fixation has the potential to replace nitrogen fertilizer,but nonsymbiotic nitrogen-fixing bacteria can only exert better nitrogen-fixing and promote growth in the rhizosphere or phyllosphere of the host plants.The colonization process of symbiotic nitrogen-fixing bacteria through the extracellular receptor recognition signal molecule and chemotaxis to the rhizosphere of leguminous plants has been reported,but studies on the colonization of non-symbiotic nitrogen-fixing bacteria in the rhizosphere or phyllosphere of crops,especially non-legume vegetables was limited.To reduce the amount of nitrogen fertilizer applied in agricultural production,we studied the colonization of high-efficiency non-symbiotic nitrogen-fixing bacteria in the rhizosphere and phyllosphere of vegetables.In this paper,three strains of high-efficiency nitrogen-fixing bacteria were screened out from 9 non-symbiotic nitrogen-fixing strains by the shake-flask experiment and nitrogenase activity evaluation experiment.The common vegetables in South China were selected as the tested materials and their leaf metabolites and root exudates were identified by GC-MS.The components of root exudates were selected to conduct chemotaxis,swarming,and in vitro experiments with two high-efficiency nitrogen-fixing strains.The allelopathic substances(signal molecules)of colonization for two strains were found.To study the effects of signal molecules on the colonization and growth promotion ability of high-fixing nitrogen-fixing bacteria,a potted pot experiment with comparative crops was carried out.The following conclusions were obtained:(1)The results of the shake-flask experiments indicated that Delftia tsuruhatensis 10492,Azotobacter chroococcum 22663,and Microbacterium sp 22660 had the highest ammonia nitrogen concentration in 9 non-symbiotic nitrogen-fixing bacteria after cultivating in 10% solution of nitrogen-poor soil for 7 days,reaching 2.25,2.09,1.61 mg / L.The acetylene reduction method was used to determine the nitrogenase activity of 22663 after cultivating in nitrogen-free medium for 4h for 2.94 nmol mg-1 h-1.Three highly efficient nitrogen-fixing bacteria were selected.(2)The signal molecule of strain 10492,mucic acid,was found in the common organic acids of root exudates.The number of colonies attracted by chemotaxis,swarming,and in vitro experiments reached 5.3 times,1.3 times,and 2.2 times of the control(water).P<0.05),the result of the pot experiment showed that the value of the growth-promoting index of the inoculated strain 10492 on the amaranth(its root exudates contains mucic acid)was more significant than that of the lettuce(its root exudates without mucic acid).The growth-promoting index was further increased after the addition of 30 ?M mucic acid in the root.On the total fresh weight index,inoculation 10492 can replace 7.85 mg/L of N in amaranth,inoculation 10492 with mucic acid can replace 11 mg/L of N;inoculation 10492 can be replaced 3.91mg/kg of N in lettuce,inoculating 10492 and adding mucic acidcan replace 3.92 mg/kg of N in lettuce.The abundance and expression of nif H in the rhizosphere and rhizosphere soil of amaranth are up-regulated,and amaranth is a suitable host plant for strain 10492.(3)The strain 22663 had about 3 times(p<0.05)chemotaxis response to the root exudates of the amaranth compared to the control(water),but had no positive chemotaxis response to the root exudates of the lettuce(water)(p>0.05).L-glutamic acid in the components of the root exudates of the amaranth has a strong recruitment and stimulation of biofilm formation of strain 22663.L-glutamate(30?M)in the chemotaxis,swarming,and the number of strains recruited in vitro 22663 higher than that of control(water)reached 2.9 times,0.2 times,and 7.4 times.L-glutamic acid(10?M)increased the biofilm formation of strain 22663 about 33 times compared with the control(water),while the root exudates of the lettuce no effect on strain 22663.In the potted soil experiment,the biomass,leaf chlorophyll of the vegetables,available nitrogen,and available nitrogen of rhizosphere soil increased after inoculated with strain 22663.The growth index of amaranth was higher than that of lettuce(p< 0.05),in terms of biomass,inoculum strain 22663 replaced N by about 10 mg/kg in the growth of amaranth and only replaced N 3.7 mg/kg in the growth of lettuce.The result of the q PCR showed that the abundance of nif H,nif D,and nif K genes of amaranth root strain 22663 reached 107,106,105 copies/g after inoculated strain 22663,while the abundance of those three genes in roots of lettuce was lower than the detection limit.After inoculated strain 22663,the abundance of nif H,nif D and nif K genes can be detected in the rhizosphere soil of both vegetables,the abundance of the rhizosphere soil of the amaranth is higher and the abundance of 16 s gene in the both vegetable roots and the rhizosphere soil can be increased.(4)It was found by the result of the pot experiment that the abundance of nif H,nif D,and nif K genes in amaranth leaves reached 106,105 and 105 copies/g after spraying inoculum strain 22663,while the abundance of nif H,nif D and nif K genes of Chinese cabbage leaves were lower than the detection limit.The growth promotion index of the strain 22663 on the amaranth was higher than that of the Chinese cabbage.It indicated that the strain could be colonized in the leaves of amaranth and could not be colonized in the leaves of Chinese cabbage.Amaranth was the appropriate host plant for the strain 22663 colonization in the leaves.