| Nitrogen-fixing bacteria is an important part of different ecosystems.There are many kinds of nitrogen-fixing bacteria(NFB)with different trophic types.They can perform biological nitrogen fixation in three ways: symbiotic nitrogen fixation,associative nitrogen fixation,and free-living nitrogen fixation,which have the ability to coordinate nitrogen fixation and other multiple functions in different environments.However,it is a big challenge to regulate nitrogen-fixing ability together with other functions of NFB in root system of different crops at different times.Therefore,in order to construct a multifunctional community using different trophic NFB that can improves soil fertility,and promotes the growth of maize and soybean,free-living nitrogen-fixing bacteria(FNFB)and associative nitrogen-fixing bacteria(ANFB)were isolated from soil and corn roots.The symbiotic nitrogen-fixing rhizobia was isolated from root nodule of soybean before.The NFB were identified by 16 S r RNA and nif H gene,and then they were screened again with the abilities of nitrogenase activity,inorganic phosphorus-solubilizing,organic phosphorus-decomposing,cellulosedecomposing and IAA-producing.The combinations of " FNFB + ANFB + SNFB" were constructed and inoculated on plants.Then the effects of nitrogen-fixing bacteria combination on the growth,soil nutrients,soil enzyme activities and root bacterial community of the former crop-maize and the following crop-soybean were explored.The main results of this study are as follows:(1)In this study,162 bacterial strains of were isolated by using nitrogen-free medium,among which 28 strains were identified as the nitrogen-fixing bacteria,including six freeliving nitrogen-fixing bacteria and 22 associative nitrogen-fixing bacteria.The nitrogenase activity of Azotobacter spp.showed that of strain(3,7 and 9)could reach 273.72 nmol/mg·h,together with strain 9 and 12 also having the growth-promoting properties such as phosphatesolubilization,cellulose-solubilization and IAA production.Among the associative nitrogenfixing bacteria,the nitrogenase activity of strains M27(Klebsiella sp.)and M38(Herbaspirillum sp.)could reach 120.84 nmol/mg·h,and had higher IAA-producing potential.Strain 1 belongs to Variovorax sp.,and its nitrogenase activity was slightly higher than that of other associative nitrogen-fixing bacteria.The strains in the genus Variovorax are common growth-promoting and stress-resistant strains.The symbiotic nitrogen-fixing rhizobia(SNFB)2-f also has phosphate-solubilizing,phosphate-decomposing,cellulose-solubilizing and IAAproducing properties.(2)Rescreening according to the growth-promoting characteristics of nitrogen-fixing bacteria,two free-living nitrogen-fixing bacteria(9 and 12),three associative nitrogen-fixing bacteria(1,M27 and M38)and one symbiotic nitrogen-fixing bacteria(2-f)were selected to construct the synthetic communities based on "Free-living + Associative + Symbiosis" model.Each community with nitrogen-fixing ability contains three bacteria,and a total of six the synthetic communities were formed.The order of nitrogenase activities from lower to higher was AVE2 > AVE1 > AHE1 > AHE2 > AKE1 > AKE2;the IAA production capacity was AKE1 > AKE2 > AHE1 > AHE2 > AVE1 > AVE2;Potassium-dissolving capacity was AHE1 >AVE1 > AHE2 > AVE2 > AKE2 > AKE1.By the comparing of the potential plnat growthpromoting traits,three groups including AVE2,AKE1 and AHE1 were obtained and tested furtherly.(3)The experimental of plant inoculation with three recreened groups showed that all three groups could significantly increased biomass and total nitrogen of maize at initial stage of inoculation.However,at 45 days after inoculation,there was no significant difference between the inoculated group and the control.Soybeans were planted in the soil after maize removed.The groups of AVE2 and AHE1 significantly increased soybean biomass,and the groups of AVE2 and AKE1 significantly increased the total nitrogen content of soybean.It can be concluded that the nitrogen-fixing combinations can not only promote the nitrogen accumulation of maize but also increase the nitrogen content of soybean,and the introduction of legume into the planting system can change the trends of the weakened effect of growthpromoting function,and stimulate the plant growth-promoting potential of the nitrogen-fixing ability again.(4)The results of soil nutrient and enzyme activities showed that three groups continuly increased the soil organic matter and total nitrogen.At the initial stage of inoculation,the nitrogen-fixing bacterial communities significantly increased the contents of nitrate nitrogen,ammonium nitrogen and available phosphorus in corn soil,but the difference gradually disappeared at the final stage.When soybeans were planted in the soil after corn,the contents of ammonium nitrogen and available phosphorus were significantly increased.The nitrogenfixing combinations significantly increased the soil enzyme activity of maize at the initial stage of inoculation,and then the improvement effect gradually disappeared.However,after planting soybean,compared with the control,the soil enzyme activity increased to some extect.(5)By analyzing the bacterial community associated with plant roots,results showed that the inoculation nitrogen-fixing bacterial groups had a greater effect on the bacterial community structure of maize and soybean rhizosphere than non-rhizosphere,but had no effect on the endophytic bacterial community of soybean nodules.On the 30 th day after inoculation,the variation of bacterial community structure in the rhizosphere and nonrhizosphere of maize was great,and then the the dissimilarity of the bacterial community structure with the non-inoculated bacterial community decreased,and the bacterial community showed a recovery trend.However,the changes of bacterial community in soybean rhizosphere were different from those in maize rhizosphere.The dissimilarity between the bacterial community structure of soybean rhizosphere and the non-inoculated group was enlarged when nitrogen fixation combinations AVE2 and AKE1 were applied,and Pseudomonas significantly enriched in soybean rhizosphere.The combination AHE1 showed on differences with the control,which may be related to the unsuccessful colonization of the combination member Herbaspirillum sp.In this study,the nitrogen-fixing bacteria were selected and the synthetic communities containing nitrogen-fixing bacteria were contructed.Three communities were used to explore the plant-growth promoting and persistent effect on the nitrogen accumulation and growth of maize and soybean.The multi-dimensional growth-promoting ability of nitrogen-fixing bacteria combination was explored,which could provid theoretical support for planting diversification to improve the growth-promoting effect of beneficial bacteria. |
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