| The herbicide Atrazine is widely used in the control of weeds in corn farmland because of its low price and good herbicidal effect.Due to its biological toxicity and long residual time,atrazine has caused serious damage to soil quality.In addition,atrazine,which remains in the soil,can also affect the growth of aftercrop sensitive crops such as soybeans,thus threatening food security.In addition,the total phosphorus content in farmland soils in China is relatively high,but the available phosphorus content that can be us ed by plants is relatively low,which causes the unreasonable use of phosphate fertilizer and causes a series of pollution problems.In view of the practical problems in the above agricultural production process,this study selected Atrazine-degrading bacteria,Arthrobacter sp.DNS10 and phosphate-solubilizing bacteria Enterobacter sp.P1 as the strain resources.On the basis of clarifying the ability of strain P1 to dissolve phosphorus,the research focused on the growth and the atrazine degradation characteristics of compound strain under co-culture conditions,and the related synergistic degradation mechanism were discussed.At the same time,through pot experiment,the effect and biological mechanism of the above-mentioned strains on alleviating the stress of residual atrazine on soybean was further investigated.The above findings will provide the necessary theoretical basis and technical support for the in situ repair of complex microflora and the solution of rotation defects caused by atrazine.The main research results are as follows:?1?The phosphate-solubilizing bacteria P1 reached a stable phase after 18 h of culture,and the amount of bacteria reached?2.03±0.11?×108 cfu·m L-1.The p H of the culture solution decreased from 8.31±0.01 to 4.72±0.03 at 24 h.It was found that the strain P1 mainly dissolves inorganic phosphorus by secreting low molecular weight organic acids such as malic acid,succinic acid,?-ketoglutaric acid and citric acid.The effective phosphorus content in the 27 h culture medium reached the maximum value of 110.36±3.19 mg·L-1.?2?The interaction between the atrazine-degrading strain DNS10 and the phosphate-solubilizing bacteria P1 was evaluated.After co-culture of the above strains in the basic inorganic salt culture solution with 100 mg·L-1 atrazine as the sole nitrogen source for 48 hours,it was found that the degradation rate of the complex bacteria to atrazine reached 99.18±1.00%.The degradation rate of atrazine in strain DNS10 alone was only 38.57±7.39%.The concentration of cyanuric acid in the co-culture and single culture conditions was 63.91±3.34 mg·L-1 and 26.60±3.87 mg·L-1,respectively.In addition,the p H value of the co-culture treatment medium decreased from 6.78±0.02 to 5.10±0.29 at 36-48 h of culture,while the p H value of strain DNS1 and strain P1 alone did not change significantly in the above stage,and the co-culture conditions could be promote the growth and degradation of atrazine by two strains of microorganisms.In addition,the expression of the atrazine degradation-related genes trz N,atz B and atz C in co-culture treatments was 3.35-6.61,1.10-1.81 and 2.94-3.09 times that of the single strain DNS10 culture treatment at 36 h to 60 h.?3?Using the substrate utilization experiment,the potential characteristics of the substrate utilization of strain DNS10 and strain P1 under co-culture conditions were investigated.The results showed that strain P1 cannot degrade atrazine or utilize atrazine for growth,However,the OD600 of strain P1 increased from 0.024±0.001 and 0.025±0.001 to 0.043±0.002 and 0.043±0.000,respectively,under the condition that the atrazine metabolite ethylamine or isopropylamine was the sole nitrogen source.It was also found that the p H of the culture solution was decrease from 7.27±0.02 and 7.30±0.01 to 4.37±0.02 and 4.45±0.03,respectively.In addition,the study found that citric acid(0.3 mg·L-1)can increase the growth and degradation ability of strain DNS10,and the degradation rate is 6.43% higher than that without citrate.By separating and culturing the semi-permeable membrane,the two strains were able to utilize the substrate in the co-culture system while avoiding direct contact between the two strains to further verify that the strains DNS10 and strain P1 can be grown by using each other's metabolites under co-culture conditions.Results showed in co-culture treatment the OD600 of strain DNS10 was 0.428±0.036,the strain P1 was 0.093±0.006,but the OD600 of strain DNS10 in pure culture treatment only 0.320±0.025.In addition the strain P1 did not grow.Moreover,atrazine was completely degrade d in the co-culture treatment at 24 h,while the pure culture degradation rate of the strain DNS10 was only 88.10±1.36%.The p H of the culture solution inoculated with the strain P1 in the co-culture treatment decreased from 6.85±0.00 to 6.09±0.13.However,the culture solution of the inoculated strain P1 was not reduced by the pure culture treatment,further indicating that the two bacteria can utilize each other's metabolites.?4?The effects of combined repair of strain DNS10 and strain P1 on the growth and physiological status of soybean seedlings under the stress of atrazine were investigated.The results showed that the biomass of soybean seedlings increased by 7.26±0.51% compared with the repair of strain DNS10 alone.The accumulation of atrazine in t he leaves of soybean seedlings treated with combined repair was significantly lower than that of strain DNS10 alone.From the submicroscopic structure of soybean seedling roots and leaves,it can be seen that the structure and function of chloroplast and mitochondria in the combined repair treatment are more complete,and the contents of chlorophyll a and chlorophyll b in soybean leaves treated with combined repair are 0.36±0.02 mg·g-1 and 0.18±0.11 mg·g-1,significantly higher than the single repair treatment of strain DNS10.Atrazine stress causes significant membrane lipid peroxidation in soybean seedlings,which activates the antioxidant defense system in soybean seedling leaves.The contamination treatment of not inoculated with atrazine-degrading bacteria was significantly higher than that of untreated with atrazine,and the SOD,POD activities in soybean leaves were similar to uncontaminated treatment,indicating that combined repair can reduce the stress of atrazine on the leaves of soybean seedlings.Among them,the SOD activity in the combined repair treatment was 83.55±5.69 U·g-1FW,and the strain DNS10 alone was 10.95±8.85 U·g-1FW,indicating that the combined repair treatment was better than the strain DNS10 single repair treatment in relieving the atrazine stress.It has a better effect on the challenge response produced by soybean seedlings.In addition,by investigating the atrazine reduction law in each treated soil,it was found that the combination of strain DNS10 and strain P1 was better than the strain DNS10 single inoculation treatment,and the degradation rate of atrazine in the soil at the 18 th day of repair treatment can reach 92.09±1.74%.?5?RNA-Seq sequencing technology was used to study the response mechanism of soybean seedlings under the stress of atrazine.Meanwhile also research the transcriptional differences of key genes of soybean seedlings under the stress of atrazine by the combined repair of degradation bacteria DNS10 with phosphate-solubilizing bacteria and the single repair of strain DNS10.The results showed that the metabolic pathways related to soybean seedlings affected by atrazine stress mainly include: photosynthesis,photosynthesis antenna protein,biosynthesis of secondary metabolites,glyoxylic acid and dicarboxylic acid metabolism,porphyrin and chlorophyll metabolism,photosynthetic organisms,carbon sequestration and other pathways.Compound vaccination?DNS10+P1?can up-regulate phosphatidylinositol phospholipase C,?-1,inositol-1,3,4-triphosphate 5/6-kinase/inositol-tetraphosphate-1-kinase,calmodulin and other genes which is involved in the phosphatidylinositol signaling system pathway in soybean seedlings compared with single inoculation with DNS10.Combined repair of atrazine challenge soybean seedlings can down-regulate genes that involved in fatty acid degradation pathways to improve soybean quality,up-regulate the 2,2-?-hydroxylase gene that involved in the brassinosteroid biosynthesis pathway and the DNA-3-methyladenosylase I gene that participate in base excision repair pathways to increase the stress resistance of soybean seedlings. |
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