The Influence And Mechanism Of Atrazine On Nitrogen Fixation Of Rhizobia-Soybean Symbionts

Corns and soybeans are the main grain crops grown in the black soil region of northeast China.As a result of production and economic factors,the northeast black soil area has seen a spike in maize cultivation and a sharp decline in soybean cultivation.Steadily improving soybean cultivation area and yield is a major strategic demand and historical mission in the field of agricultural production in northeast black earth.The symbiotic nitrogen fixation system formed by soybean and rhizobia in soil provides nitrogen for soybean growth,thus maintaining normal growth and physiological function of soybean.Therefore,attention to the formation of indigenous rhizobium-soybean symbiote and its nitrogen fixation is very important to guarantee the production of soybean in black soil.Atrazine is widely used as a herbicide in northeast black earth.Its residues in the soil can cause harmful effects on the growth of soybean grown after corn stubble.It is unclear whether atrazine will influence the formation and nitrogen fixation function of soybean-rhizobium symbionts.Therefore,the study intends to focus on the effects of atrazine stress on the formation of soybean-rhizobium symbionts and the nitrogen fixation function,the effects of atrazine stress on the rhizosphere and non-rhizosphere soil of soybean and the diversity characteristics of rhizobium in rhizobium-soybean symbionts,as well as the effects of atrazine on the composition of soybean root exudates and the regulation of interactions between soybean and indigenous rhizobium in black soil study the aspects.On this basis,the mechanism of regulating the resistance of soybean to atrazine was elucidated by multi-group technique.Relevant studies are helpful to further reveal the toxic effect and mechanism of atrazine on soybean from the perspective of the formation of rhizobium-soybean symbionts and nitrogen fixation which systematically improve the mechanism of alleviating toxic stress of atrazine on soybean.The main findings are summarized below:（1）The effect of different atrazine exposure conditions on the formation and growth of rhizobium-soybean symbionts in soybean and black soil was studied by potting experiment.The results showed that atrazine had obvious inhibitory effect on the number of nodules and dry weight after application.When the stress concentration increases to 7.5 mg·kg^-1,the dry weight of the nodules decreased by 10.10%.With the further increase of the atrazine exposure concentration(10,15,20 and 30 mg·kg^-1),the dry weight of the nodules decreased further.It is shown that there is a dose-effect relationship between atrazine and nodule growth.In addition,atrazine exposure alters the subcromatic structure of nodule cells and reduces the number of rhizobium cells in nodules.At the same time,atrazine(5,7.5,10,15,20 and 30 mg·kg^-1)also significantly inhibited leghemoglobin in soybean nodules,and its content decreased by 15.65%,27.44%,51.47%,72.24%,88.37%and92.09%,respectively.These results indicate that atrazine interferes with the growth of rhizobia-soybean symbionts and has a potential effect on the nitrogen fixation ability of nodules by reducing the area of infection of rhizobia in the nodules.（2）The changes of soybean growth and physiological index under different concentration of atrazine exposure were investigated by potting experiment.The results showed that atrazine significantly inhibited the growth of soybean seedlings(10,15,20 and 30 mg·kg^-1).When the exposure concentration increases to 7.5 mg·kg^-1,the soluble protein content in the leaves decreased by 20.39%.The soluble protein content in the leaves was further reduced with increasing atrazine exposure levels.The total nitrogen content in the leaves decreased by 18.73%,41.35%,56.52%and74.16%at atrazine exposure levels of 10,15,20 and 30 mg·kg^-1,respectively.The activity of glutamate synthase and glutamide synthase in plant leaves decreased with the increase of atrazine exposure concentration.When atrazine exposure is 7.5 mg·kg^-1,the activity of glutamate synthase（GOGAT）and glutamide synthase（GS）decreased by 10.39%and 10.74%respectively.With the further increase of atrazine stress concentration,nitrogen metabolic enzyme（GOGAT and GS）activity in plants decreased gradually.In conclusion,atrazine inhibits the nitrogen metabolism of soybean plants,interferes with the accumulation of soluble protein and total nitrogen content in plants and thus inhibits the growth of soybean.（3）Combined with rpo B high-throughput sequencing results,the response regularity of rhizobium diversity in soybean nodules,rhizosphere soil and non-rhizosphere soil to atrazine stress was revealed.The results showed that Bradyrhizobium,Afipia,Rhizobium and Methylovirgula were the dominant species of rhizobium in soybean nodules exposed to atrazine.Further studies on the diversity of Bradyrhizobium,Afipia,Rhizobium and Methylovirgula at the species level showed that atrazine(7.5 mg·kg^-1 and 20 mg·kg^-1)could change the rhizobium community structure in rhizosphere soil of soybean.The abundance of Afipia geno sp.3 in nodules was inhibited by 80%.The abundance of Bradyrhizobium sp.AD1＿1＿nodule and Bradyrhizobium sp.Ec3.3 increased in nodules and rhizosphere soils,and they were the dominant species under atrazine exposure.The abundance of Rhizobium leguminosarum and Rhizobium sp.CF142 decreased in nodules and increased in rhizosphere soil,indicating that Rhizobium leguminosarum and Rhizobium sp.CF142were sensitive to atrazine stress.Correlation analysis between rhizobium community composition and physiological indexes of soybean showed that the abundance of Afipia sp.,Rhizobium sullae,Rhizobium sp.CF142 and Rhizobium leguminosarum was significantly positively correlated with the soluble protein content,total nitrogen content and the activities of nitrogen metabolization-related enzymes（glutamate synthetase and glutamine synthetase）in soybean leaves exposed to atrazine,suggesting that the decrease of soybean physiological indexes was related to the decrease of the abundance of these species in soybean nodules.In addition,atrazine-mediated changes in nitrate nitrogen content of rhizosphere soils had a greater effect on rhizobium diversity than ammonia nitrogen,soluble organic nitrogen and soluble organic carbon.The results showed that the interference of atrazine on physiological indexes of soybean seedlings was related to the changes of rhizobium community in the nodules and rhizosphere soil.（4）Untargeted metabolomics were used to study the composition of soybean root exudates and to regulate the interaction between soybean and rhizobium.The results showed that atrazine significantly changed the composition and content of soybean root exudates.The relative content of Daidzein and D-glucose decreased by 58.37%and 69.19%respectively after exposure to 20 mg·kg^-1 atrazine.The relative content of 4-hydroxysphinganine,Menthol,Adenosine,Methylprenalic acid,L-Lysine,Ectoine,Spermidine increased by 72.02%,56.28%,149.93%,129.11%,182.27%,69.74%and 141.16%respectively.Further study showed that root exudates could promote soybean seedling growth,root nodule development and nitrogen accumulation after receiving back into the planting system,and AC treatment（treatment of soybean root exudates without atrazine）had a more significant effect on soybean seedling growth than AT treatment(treatment of soybean root exudates exposed to 20 mg·kg^-1 atrazine).rpo B gene sequencing data showed that the root exudation-mediated community structure changed significantly.The relative abundances of Sinorhizobium sp.RAC02 and Rhizobium sp.ACO-34A increased in AC treatment group,but decreased in AT treatment group.In addition,correlation analysis shows that the abundance of Sinorhizobium sp.RAC02 was positively correlated with the number of nodules and the secretion level of Daidzein and D-glucose,indicating that the higher level of root exudates of Daidzein and D-glucose would help soybean seedlings recruit Sinorhizobium sp.RAC02 and promote the nodulation and growth of soybean.（5）The effects of Bradyrhizobium japonicum AC20 on the growth of soybean,rhizosphere soil microbial diversity and gene expression of soybean carbon and nitrogen metabolism were evaluated by inoculation of Bradyrhizobium Japonicum AC20.The results showed that strain AC20 could effectively alleviate the growth inhibition of atrazine on soybean.When inoculated with 1.2×10⁸CFU·m L^-1·plant^-1,the plant dry weight and root dry weight of soybean seedlings increased by 235%and 214%,respectively,compared with the treatment without adding bacterial solution.The expression level of psb A gene in chloroplast of soybean seedlings inoculated with AC20 was 1.4times that of those inoculated without rhizobia.In addition,strain AC20 significantly increased leghemoglobin in soybean nodules and total nitrogen content in leaves.These results indicated that strain AC20 alleviated the growth inhibition of atrazine on soybean by increasing nitrogen accumulation in seedlings and improving photosynthetic characteristics.Transcriptomic analysis showed that atrazine stress significantly down-regulated the expressions of genes related to the metabolic pathway of starch and sucrose in soybean seedlings（Gyma.06G011700 and Gyma.19G192800）,and the relative expressions of these key genes were up-regulated after the addition of strain AC20.The relative abundance of candidate＿division＿WPS-1 in the rhizosphere was positively correlated with genes related to the metabolic pathways of starch and sucrose.These results indicated that strain AC20 up-regulated the expression of genes related to starch and sucrose metabolic pathways by increasing the relative abundance of candidate＿division＿WPS-1 in the rhizosphere,and finally alleviated the inhibitory effect of atrazine on soybean growth.