Improvement Of Soybean Growth Under Atrazine Stress By Bio-organic Fertilizer DNBF10:Patterns And Mechanisms Of Action

The herbicide atrazine poses significant biological toxicity and persists for a long time,which severely hinders the growth of soybean,a crop that is highly sensitive in the rice-bean rotation system.Studies showed that the use of organic fertilizers can regulate the tolerance of crops to non-biological stressors;however,it is still unclear whether coupling atrazine-degrading microorganisms with organic fertilizers can effectively alleviate the inhibition of atrazine on soybean growth and related mechanisms.In this study,we conducted a pot experiment to compare the effects of applying organic fertilizer alone in soil versus applying biologically organic fertilizer（DNBF10）prepared by coupling organic fertilizer with Arthrobacter sp.DNS10 on the growth and physiology of soybean seedlings under atrazine stress.By using transcriptome technology,we investigated the response patterns of soybean leaf nitrogen metabolism pathways,atrazine detoxification,and transport pathways,and clarified the biological mechanisms of DNBF10 in regulating soybean tolerance to atrazine stress.This study provides novel insights and research approaches to address the issues of atrazine inhibition of soybean seedling growth and crop rotation disorders.This study’s main discoveries can be summarized as follows:（1）This study aimed to investigate the dose-response relationship of DNBF10 in alleviating the growth stress of soybean seedlings caused by atrazine.The results revealed that the application of DNBF10 and organic fertilizer led to significant improvements in the plant height and root length of soybean seedlings,when compared to the pollution treatment.The application of DNBF10 at a dose of 1483 mg·kg^-1（equivalent to 220.8 kilograms per acre of organic fertilizer field application rate）demonstrated the greatest alleviation effect,resulting in a 14.1±0.2%increase in plant height,a 4.7±0.1%increase in root length,a 36.1±0.3%increase in plant dry weight,and a 19.4±0.3%increase in root dry weight.Furthermore,the DNBF10 application treatment not only reduced the death and damage of soybean seedling root tip cells,but also significantly increased the total nitrogen,total phosphorus,and total potassium content in the leaves,when compared to the pollution treatment and organic fertilizer application treatment.Additionally,compared with the same dose of organic fertilizer application treatment,the DNBF10application treatment at a dose of 1483 mg·kg^-1increased the content of total nitrogen,total phosphorus,and total potassium by 22.0±1.0%,35.5±1.8%,and 44.8±2.8%,respectively.These findings indicate that DNBF10 is more effective than organic fertilizer in alleviating the inhibition of atrazine exposure on soybean seedling growth and leaf nutrient accumulation.（2）This study aimed to investigate the dose-response relationship of DNBF10 in alleviating the physiological stress of soybean seedlings caused by atrazine.The results revealed that the application of DNBF10 and organic fertilizer led to a gradual increase in the content of photosynthetic pigments in soybean leaves,compared to the pollution treatment.Specifically,the content of chlorophyll a and b in soybean seedlings treated with DNBF10 at a dose of 1483mg·kg^-1increased by 66.2±2.3%and 86.5±2.9%,respectively,which was significantly higher than that of organic fertilizer application treatment,indicating that DNBF10 can alleviate the inhibitory effect of atrazine on the synthesis of photosynthetic pigments in soybean seedling leaves.Furthermore,when the DNBF10 application dose was 1483 mg·kg^-1,the content of proline and soluble protein in soybean seedling leaves decreased by 32.7±0.9%and 23.2±0.5%,respectively,compared to the same dose of organic fertilizer treatment,indicating that DNBF10 has a better alleviating effect on the excessive production of proline and soluble protein induced by atrazine than organic fertilizer.Additionally,the MDA and ROS（O₂^-,H₂O₂）content in soybean seedlings decreased in the DNBF10 and organic fertilizer application treatments,compared to the pollution treatment.When the DNBF10 application dose was 1483 mg·kg^-1,the MDA,O₂^-,and H₂O₂content in soybean seedling leaves and roots decreased significantly by 25.6±0.5%,57.4±3.6%,45.3±1.8%and 31.5±1.3%,44.7±3.6%and 54.3±2.8%,respectively,compared to the same dose of organic fertilizer treatment.Moreover,the activities of SOD,POD,and CAT in soybean seedlings treated with DNBF10 application were similar to the control treatment and showed a better recovery effect compared to the same dose of organic fertilizer application treatment.These findings indicate that DNBF10 can better alleviate the oxidative stress caused by atrazine in soybean.（3）The study investigated the regulation of different concentrations of DNBF10 application on the residual characteristics and forms of atrazine in the soybean-soil system.The results showed that the concentrations of atrazine in both the soil and soybean seedlings decreased gradually with increasing applications of organic fertilizer and DNBF10,when compared to the WR.Notably,at DNBF10 application dose of 1483 mg·kg^-1,the content of atrazine in the soil and soybean significantly decreased by 67.0±5.9%,59.7±2.8%,and 81.0%±3.6%,75.2±4.2%,respectively,when compared to the pollution treatment and the same dose of organic fertilizer treatment.Compared with the WR,organic fertilizer can reduce the content of bound residual atrazine in the soil.When the DNBF10 application dose reached 1483 mg·kg^-1,the reduction of the available,organic extracted,and bound residual atrazine in the soil reached 74.9%±2.7%,70.3%±3.6%,and63.4%±1.7%,respectively.This indicates that DNBF10 can reduce the migration of atrazine from the soil to soybean by decreasing the atrazine content in the soil and changing the forms of atrazine in the soil,thereby decreasing the atrazine accumulation in soybean.（4）RNA-Seq sequencing technology was used to study and analyze the effect of DNBF10 on the transcriptome of soybean seedling leaves under atrazine stress.The results revealed that DNBF10 enhanced plant nitrogen metabolism by upregulating the expression of transporter enzyme-encoding genes in the soybean nitrogen metabolism pathway,which provided the necessary nutrients for soybean seedlings,compared to the pollution treatment and organic fertilizer application treatment.Additionally,the subfamilies of CYP82A1 and CYP82A3 in the cytochrome P450 protein family,and the subfamilies of ABCG and ABCC in the ABC transporter protein family were found to be related to atrazine detoxification and transport.DNBF10 restored the overexpression of these subfamily encoding genes induced by atrazine,resulting in the normalization of their expression levels.These findings indicate that DNBF10 reduced the accumulation of atrazine in soybean by restoring the overexpression of cytochrome P450 and ABC transporter encoding genes in soybean seedling leaves,ultimately reducing the stress caused by atrazine.Overall,DNBF10 can improve the tolerance and resistance of soybean seedlings under atrazine stress by regulating multiple genes in soybean seedling leaves,including transporter enzyme-encoding genes in the nitrogen metabolism pathway and cytochrome P450 and ABC transporter encoding genes related to atrazine detoxification.