Identification Of The Novel Strain Hansschlegelia Zhihuaiae S 113 And Molecular Ecology Of Bioremediation Of Sulfonylurea Herbicides-contaminated Soils By Strain S 113

Sulfonylurea herbicides are one of the most applied herbicides in the world and account for 4.9%of the global sale market of pesticides.Sulfonylurea herbicides degrade very slowly in natural environment,and residue of sulfonylurea herbicides in the environment is harmful to successive plants,causing great economical damage.Therefore,great attentions have been paid to the residue dynamics,ecological effects and degradation in environment.Since sulfonylurea herbicides have been invented and applied in the world for only 30 years,the studies on the residue dynamics,ecological effects and degradation of sulfonylurea herbicides in the environment have not been carried out extensively.Hence,study of the ecological effects of sulfonylurea herbicides on soil microorganisms and bioremediation of sulfonylurea herbicides-contaminated soils by bioaugmentation has significant meanings.In this study,strain S 113,a broad sulfonylurea herbicides degrading bacterium stored in our lab,was used as the material.A polyphasic taxonomic study including phylogenetic analysis based on the 16S rRNA gene sequence and determination of phenotypic characteristics was performed on strain S 113.The strain is gram-negative,aerobic,coccoid-shaped.The highest 16S rRNA gene sequence similarity was 96.8%,with Hansschlegelia plantiphila VKM B-2347T.The predominant respiratory quinine was ubiquinone 10(Q-10).The major fatty acids were C18:1?7c and C16;0.The dominant phospholipids were diphosphatidylglycerol,phosphatidylmonomethylethanolamine,phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine.The G+C content of the DNA was about 65.7 mol%.DNA-DNA hybridization experiments showed 44.9%relatedness for strain S 113-T with its closest relative,H.plantiphila VKM B-2347T.The results of our polyphasic taxonomic analysis indicate that strain S 113 represents a novel species within the genus Hansschlegelia,for which the name Hansschlegelia zhihuaiae sp.nov.is proposed.The type strain is S 113T(= DSM 18984T = CCTCC AB 206143T = KCTC 12880T).Study of the colonization of strain S 113 by culture method and molecular marker method were both unsuccessful.A set of specific real-time PCR primer was designed based on the nov.sulfonylurea herbicide esterase gene(sulE),which was cloned from strain S 113 and with very low similarity to other esterase genes.A piece of 309 bp unique DNA,named sulE5,was cloned and checked as the target DNA.Real-time PCR method was used to detect number dynamics of strain S 113 in sulfonylurea herbicide-contaminated soils and sulfonylurea herbicide-sensitive plants maize.It was found that strain S 113 could survive in the soils for about 2 weeks.When strain S 113 was inoculated into soil at theconcentration of 108 cfu·g-1 dry soil,the number of strain S 113 decreased gradually,and 1585 cfu·g-1 dry soil of strain S 113 was detected at day 7.Strain S 113 could also survive in the plant of maize for a short period of time.It was found that 501 cfu·g-1 of strain S 113 was detected in the root.I did not detected strain S 113 in stalk or leaf of maize,and did not detect any strain S 113 after 14 days neither in soil nor in the maize plants.Bioremediation of sulfonylurea herbicide-contaminated soils by strain S 113 showed that low concentration metsulfuron methyl-contaminated soil was easier to remediate;soil with lower pH and higher porosity was easier to remediate;sulfonylurea herbicide with higher pKa was easier to remediate.454 prysequencing method was also used to study the ecological effects of bioremediation of sulfonylurea herbicides-contaminated soils by bioaugmentation of strain S 113.A total of 305,365 valid reads and 136,809 OTUs(Operational Taxonomic Units)were obtained from the 71 soil DNA samples through 454 prysequencing analysis.All sequences were classified from phylum to genus according to the program Qiime.25 different phyla or groups were identified at the cutoff=0.25 level,and 604 different genera were identified at the cutoff=0.03 level.It was found that low concentration of metsulfuron methyl(0.1 mg·kg-1 dry soil)could stimulate the microbial diversity of soil.However,medium concentration(2 mg·kg-1 dry soil)decreased the microbial diversity,and high concentration(10 mg·kg-1 dry soil)dramatically decreased the microbial diversity.The microbial communities of soil applied with different concentrations of metsulfuron methyl were all significantly recovered at 28 d by the inoculation of strain S 113.The application of metsulfuron methyl in soil inhibited the gram-negative bacteria,photo synthetic bacteria,nitrifying bacteria and plant growth-promoting rhizobacteria;stimulated gram-positive bacteria.The percentage changes of common sulfonylurea herbicides-degrading genus such as Pseudomonas,Bacillus,and Streptomyces were also investigated.The percentage of Pseudomonas in soils applied with high concentrations of metsulfuron methyl increased by about 10 times,and increased by about 3-5 times when medium concentration of metsulfuron methyl was applied.The inoculation of strain S 113 reduced the increase of Pseudomonas,showing that the change of Pseudomonas could be used as the indicator for the contamination of soils by metsulfuron methyl,especially for alkaline soils.I used three different kinds of soil,sampled from orchard,vegetable garden and lawn,as matrix to study the ecological effects of the bioremediation of sulfonylurea herbicides-contaminated soils by strain S 113.It was found that the microbial communities of different soils applied with metsulfuron methyl were all significantly recovered at 28 d by the inoculation of strain S 113.The recovery percentage of microbial community in soils with neutral pH was higher than that in soils with high pH.The application of metsulfuron methyl changed the microbial community in the three kinds of soils towarding the same direction.Inoculation of strain S 113 can significantly recovered this tendency.Metsulfuron methyl(MSM),ethametsulfuron methyl(ESM)and bensulfuron methyl(BSM)were used to study the ecological effects of the bioremediation of sulfonylurea herbicides-contaminated soils by strain S 113.It was found MSM caused more changes of soil microbial communities than ESM and BSM.Strain S 113 could degrade BSM faster than ESM and MSM in soil,and could remediate BSM and ESM-contaminated soil microbial communities faster than MSM-contaminated soil.All the three kinds of sulfonylurea herbicides could inhibit the Photosynthesis and nitrification of soil bacteria.