Combined Remediation Of Arbuscular Mycorrhizal Fungi And Hansschlegelia Zhihuaiae S113 On Bensulfuron-methyl Contaminated Soil

Bensulfuron methyl belongs to sulfonylurea herbicides with high efficiency,low toxicity,broad spectrum,and is widely used in China.Due to its slow digestion rate,the residue in the farmland soil is serious,which is easy to cause phytotoxicity to the susceptible crops.Therefore,the problem of remediating soil contaminated of bensulfuron-methyl is imminent,and the degradation of microorganisms is an important way to degrade bensulfuron-methyl.Hansschlegelia zhihuaiae S1 13 is a strain that selected in this laboratory,which can efficiently degrade bensulfuron-methyl.Arbuscular mycorrhizal fungi(AMF)Rhizophagus intraradices is a kind of beneficial microorganism commonly found in soil,which can promote host plants to absorb nutrients and promote plant growth well.In this paper,S1 13 and AMF were used as materials,and maize was selected as tested crops that were sensitive to bensulfuron-methyl and could be used as AMF-infected host.The strain S1 13,AMF and bensulfuron-methyl were added with different treatments to study the effects of different treatments on maize growth,the degradation rate of bensulfuron-methyl and the infection rate of AMF.The effect of AMF on the colonization of S113 in maize roots,and the dynamic changes of bacterial community in the rhizosphere soil of maize also have been studied.It provides a theoretical basis for the combination of pesticide-degrading bacteria and plantpromoting bacteria to remediate herbicide-contaminated soil.1.Remediation effect of AMF and strain S113 on bensulfuron-methyl contaminated soilWhen the concentration of bensulfuron-methyl was 3 mg·kg-1,the growth of maize could be restored to the CK level by adding 15 mL of S113 suspension(OD600=1.5).In the treatment of adding different combinations of S1 13,AMF and bensulfuron-methyl,only adding S113 has no significant effect on maize growth.Adding AMF has a significant growth promoting effect on the healthy maize.Bensulfuron-methyl could make a phytotoxic effect on maize,but adding S1 13 could eliminate this phytotoxicity.Adding AMF to the treatment that S1 13 eliminated the phytotoxicity could promote the growth of maize significantly.AMF could not eliminate the phytotoxicity of bensulfuron-methyl,but could make the maize grow slightly better.In the soil contaminated with bensulfuron-methyl,the residual concentration was 1.57 mg·kg-1 and the residual concentration of adding AMF treatment was 1.35 mg·kg-1 at 36 d.The addition of S1 13 resulted in faster degradation of bensulfuron-methyl.The residual concentration of bensulfuron-methyl was about 0.5 mg·kg-1 at 6 d,and no residue was detected at 12 d.The infection rate of AMF could reach 80-90%to healthy maize at 36 d,but it was only 20-30%under the condition of phytotoxicity.Bensulfuron-methyl phytotoxicity significantly reduced the AMF infection rate.2.Effects of AMF and cheA knockout on colonization of S113 in maize rhizosphereThe plasmid carrying the GFP gene was electroporated into S1 13 to emit green fluorescence.Under confocal scanning laser microscopy(CLSM),the effect of adding AMF on the colonization of S113 in maize root surface and rhizosphere was qualitatively observed.It was found that S1 13 could colonize on the root surface of maize whether it was inoculated with AMF or not.The fluorescence intensity decreased with time.Only weak fluorescence signals could be detected on the 15th day.The quantitative method of fluorescence quantitative PCR was used to investigate the effect of AMF on the colonization dynamics of S1 13 in maize root surface and rhizosphere soil quantitatively.The results showed that the density of S113 could reach 4.5×104 cells·g-1 on the root surface and 5.2×108 cells·g-1 soil in the rhizosphere soil at 5 d when inoculated 15 mL of S113 suspension(OD600=1.5).The strain density gradually decreased over time.The concentration of S113 in the rhizosphere soil inoculated with AMF was higher than that in the non-inoculated at 20 d.The number of S113 in the rhizosphere soil increased after the AMF infection rate increased,indicating that the infection of AMF could increase the number of strain S1 13 in the rhizosphere soil of maize.The histidine kinase CheA is an important component of bacterial motility and chemotaxis,and an important driving factor for bacterial colonization.After knocking out the gene cheA in S113,it could be found that strain S1 13 had chemotaxis ability to oxalic acid,while the chemotaxis ability of ?cheA-S113 was not obvious.By capillary experiment,it was found that the chemotaxis activity of ?cheA-S113 decreased.The amount of ?cheAS113 entering the capillary was only 5.7×103 CFU·mL-1,while the number of S113 was 20.8 ×103 CFU·mL-1.The number of colonizations of S113 and ?cheA-S113,which colonized on root surface and in rhizosphere soil,was quantitatively determined by qPCR.It was found that the initial density of S1 13 in the rhizosphere soil reached 3.8×108 cells·g-1 soil,and the strain ?cheA-S113 reached 3.6×108 cells·g-1 soil.The concentration of S113 was 1.6×104 cells·g-1 soil,while ?cheA-S113 was too low to be detected in the rhizosphere soil at 20 d,which indicated that S113 colonized in the rhizosphere was more than ?cheA-S113.The density of S1 13 on root surface was 4.9×104 cells·g-1,and ?cheA-S113 was 2.3×104 cells·g1 at 5 d.?cheA-S113 could not be detected at 15 d,and S1 13 could still be detected with 1.2×104 cells·g-1 at 20 d,which indicated that ?cheA-S113 had a lower density on the root surface than S113.3.Ecological effects of AMF and strain S113 on soil remediation of bensulfuronmethylThe high-throughput sequencing further explored the dynamic changes of bacterial community structure in rhizosphere soil samples taken at 12,24 and 36 d in the treatment of different combinations of strains S113,AMF and bensulfuron-methyl.Alpha diversity analysis showed that the exogenous addition of strains S113 and AMF could increase the diversity and abundance of bacterial communities in the rhizosphere soil of maize.The analysis of Beta diversity showed that,the four treatments had a big difference with the CK.The four treatments were S113,AMF,S113+BSM and S113+AMF.The reason maybe that the exogenous addition of strains S113 and AMF,result in changes in bacterial community structure probably.By making S113,AMF and bensulfuron-methyl as single-factor variable,the Beta diversity analysis showed that the difference was relatively little between the same treatments,and more between different treatments,after adding S1 13,AMF and bensulfuronmethyl.And the treatments of adding S1 13,AMF,bensulfuron-methyl,which had more difference with the 0 d sample,indicating that these three single-factor variables have an impact on the community structure.The relative abundance of species at the level of the phylum showed that S1 13 could promote the relative abundance of Actidobacteria.The relative abundance of the genus indicated that the relative abundance of Hansschlegelia was significantly increased at 12 and 24 d in all treatments with adding S1 13,indicating that S1 13 could survive at least 24 days in the rhizosphere soil.S1 13 could inhibit Nocardioides.The relative abundance of Pseudoduganella in the soil with bensulfuron-methyl was significantly increased,which might indicate that the strain in the genus could use bensulfuron-methyl as a carbon source.In the late stage of AMF infection,the relative abundance of Sphingomonas was inhibited.