| The sulfonylurea herbicide,bensulfuron methyl,has been widely used in agricultural production for this high activity and broad spectrum.With the extension of application,the residues of bensulfuron methyl with long half-life accumulated in the farmland soil,which caused a serious damage to the rotation sensitive crops and resulted in economic losses.Removal of bensulfuron methyl residues in agricultural soil has become an urgent problem to be solved.In this paper,Hansschlegelia zhihuaiae S113 was selected as the degrading strain,and maize which was susceptible to the bensulfuron methyl residues was selected as target crop.The colonization of S113 on maize roots surface and in rhizosphere,the biofilm formation in vitro,the chemotactic response of S113 towards maize root exudates and identification of organic acids in root exudates were studied.Comparison the contribution of degradation and biofim forming in eliminating the injury to maize was performed.The micro-ecological effects of the bioremediation in rhizosphere soil contaminated by bensulfuron methyl,the transcriptional analysis of S113 towards bensulfuron-methyl and maize root exudates were conducted.Two bacterial strains,named TULL-A and Q-4,which could promote the growth of maize cooperated with S113 were isolated from maize rhizosphere soil.These two strains were identified as novel species.1.The colonization of the strain S113 in maize rhizosphere,and the effects of rootexudates on S113The colonization of S113 on maize roots and in maize rhizosphere soil was determined qualitatively by fluorescence in situ hybridization(FISH)using the probe based on the sulfonylurea herbicide esterase gene suldE obtained from strain S113.The results obtained by confocal laser scanning microscope(CLSM)and scanning electron microscope(SEM)showed that the colonization mainly occurred on the meristem zone,elongation zone and mature zone of the main roots surface and the strain S113 could form the microcolonies on root surface.The quantitative analysis by fluorescence quantitative real time PCR(qPCR)revealed that S113 survived in rhizosphere soil for 15d,the number of S113 reached at 104-106 cells·g-1 maize root surface and 105-108 cells·g-1 rhizosphere soil.The number of S113 maintained higher in the early stage of inoculation,and decreased later.The maize root exudates were collected by water-soluble method,and the results showed that the maize root exudates could promote the growth of strain S113 and the degradation of bensulfuron methyl.The different kinds of organic acids in root exudates were identified and quantitated by high performance liquid chromatography(HPLC).L-malic acid,oxalic acid,tartaric acid,succinic acid and fumaric acid were detected in maize root exudates.The concentration of tartaric acid was the highest,which reached 409.1 μM·g-1 root.Under the condition contaminated by bensulfuron methyl,the secretion of organic acids in maize root exudates was seriously inhibited,and increased after the addition of S113.The strain S113 exhibited chemotactic response to the maize root exudates and the five detected organi acids,especially towards L-malic acid.The colonization assay showed that L-malic acid and fumaric acid could significantly promote the colonization of S113 on maize roots.2.The analysis of biofilm formed by S113 on injury elimination to maize growth andthe transcriptome of S113 response towards maize root exudatesThe medium for S113 forming biofilm was preliminarily confirmed by comparison of the biofilm formation in various media according to the mechanism of biofilm formation and the biofilm formed by S113 in 48-well culture plate.The structure of biofilm formed at the bottom of plates was observed by CLSM.The images presented that the biofilm formed by S113 possessed complex three-dimensional structure.The dynamic changes of the biofilm biomass and the opposite suspended cells were monitored quantitatively.The results showed that the biofilm formation began at 24 h and the stable biofilm was formed at 96 h,disassembled at 120 h.The biomass of suspended cells in the medium was also increased,maintained at 72 h,slightly decreased at 96 h due to the biofilm formation,reached the maximum at 120 h.The effects of biofilm on the removal of injury to the maize growth were studied using bacterial biofilm inhibitor triclosan to inhibite the biofilm formation by S113.The results indicated 0.2 mg·L-1 triclosan could effectively inhibit the biofilm formation by S113,while had no significant effects on the growth of maize and the degradation of bensulfuron methyl by S113.The growth of maize was seriously inhibited when treated with 3 mg·L-1 bensulfuron methyl,recovered obviously with the addition of S113 simultaneously.When the biofilm was inhibited by 0.2 mg·L-1 triclosan,the growth of maize was also recovered,but weaker than that without triclosan,the fresh weight of seedling and root showed significant differences compared with the treatment without triclosan.Triasulfuron,nicosulfuron and pyrazosulfuron could not be degraded by S113,however the colonization and biofilm formation on maize roots still maintained under the treatment with these three sulfonylurea herbicides.The addition of S113 possessed little effect on the growth of maize under the condition contaminated with 3 mg·L-1 triasulfuron,nicosulfuron and pyrazosulfuron.The results showed that the elimination of injury to the growth of maize mainly attributed to the degradation process in rhizosphere,and the biofilm formation on roots also promoted the elimination of injury.The maize root exudates could significantly promote the biofilm formation by S113,and the transcriptome analysis of the genes regulated by maize root exudates under static biofilm formation condition was also performed through the high-throughput transcriptional sequencing.The results showed that the function of regulated genes mainly belonged to the metabolism of nutrients,including carbohydrate metabolism(alcohol dehydrogenase,adh2,citrate synthase,gltA),amino acid metabolism(acetyl-CoA C-acetyltransferase,atoB,ectoine hydroxylase,ectD),lipids metabolism(acyl-CoA thioesterase,tesA),cofactors and vitamins metabolism(dihydroneopterin aldolase,folB),genetic information processing(poly A polymerase,pcnB)signal transduction(two-component system),membrane transport(ABC transporters),cell motility and chemotaxis(chemotaxis protein,motA,flagellar assembly,flgE,fliF,flgH,flgB),Activation of genes involved in metabolism(leading to a growth promotion)and cell motility at 48 h,expression of genes responsible for exopolysaccharide production at 96 h could enhance S113 biofilm formation.The results could further explain the chemotactic response of S113 towards maize root exudates and the mechanism of the promotion of S113 growth and biofilm formation by root exudates.3.Researches on degradation of bensulfuron methyl in maize rhizosphere soil and itsecological effectsWith root irrigation of S113 into the maize rhizosphere soil contaminated with bensulfuron methyl(0.067,0.5 and 3 mg·kg-1-s·dry weight),the degradation efficiency of bensulfuron methyl and the maize growth indexes were measured.The results indicated that the natural degradation rate in maize rhizosphere soil treated wtih 3 mg·kg-1 bensulfuron methyl was 31.5%.Bensulfuron methyl could not be detected in rhizosphere soil after inoculation of S113,indicating that the addition of S113 could significantly promote the removal of bensulfuron methyl in maize rhizosphere soil,and eliminate the injury to maize growth.There were no significant differences in maize growth indexes between the treatment with S113 and control.At the same time,the dynamics of bacterial community in rhizosphere soil collected at 5,10,15,20 d were investigated.The analysis results of Alpha and Beta diversity of bacterial in maize rhizosphere soil showed that the diversity and abundance of the rhizosphere bacterial community were reduced by bensulfuron methyl with different concentrations.When the strain S113 was added to the rhizosphere,it had restorative effect.The results of relative abundance of bacteria in the genus in which the bensulfuron methyl degrading bacteria existed showed relative abundance of the genus Bacillus,Rhodococcus and Brevibacillus increased in the samples treated with bensulfuron methyl as the time went by,probable due to bensulfuron methyl served as growth material for the bacteria in these three genus.The relative abundance of Hansschlegelia was not significantly changed.However,in the samples treated with bensulfuron methyl and S113,the relative abundance of the genus Bacillus,Rhodococcus and Brevibacillus increased first and then slightly decreased as the time went by.The relative abundance of Hansschlegelia gradually decreased.4.Isolation and identification of two new bacterial species in maize rhizosphere soilIn the process of colonization investigation,four bacterial strains which could cooperate with S113 to promote the growth of maize were isolated,and two of them were named as TULL-A and Q-4.Sequences comparison based on 16S rRNA gene showed that the strain TULL-AT had the highest similarity to Mangrovibacter plantisponsor MSSRF40T(99.56%).Based on the multiple genotypic and phenotypic data,strain TULL-AT is identified as a novel species of the genus Alangrovibacte,for which the name Mangrovibacter yixingensis sp.nov.is proposed.The type strain is TULL-AT(=ACCC 19709T =KCTC 42181T).Sequences comparison based on 16S rRNA gene showed that the sequence of strain Q-4T had the highest similarity to Pedobacter zeaxanthinifaciens TDMA-5T(97.40%),followed by Pedobacter xixiisoli S27T(95.8%).On the basis of genotypic and phenotypic data,strain Q-4T is identified as a novel species of the genus Pedobacter,for which the name Pedobacter nanyangensis sp.nov.is proposed.The type strain is Q-4T(=KCTC 42442T=ACCC 19798T).This paper revealed the bensulfuron methyl degradation mechanism by S113 in maize rhizosphere soil,based on the colonization and biofilm formation on maize roots,the interrelation between S113 and root exudates,combined with transcriptome and high throughput sequencing analysis,provided theoretical foundation for the bioremediation of farmland soil contaminated with bensulfuron methyl. |
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