The Effects Of Atrazine On Soil Microbial Community Structure And Molecular Diversity

With distinct character of permanent organic pollutant, and also belonging toenvironmental hormone pollutants, atrazine has caused great concern for the soil pollution ithas brought. In addition to the European Union, atrazine is still widely used in the worldwhich will have a great influence to soil ecosystem. The present study systematicallyinvestigated the ecological effect of soil microorganism in many aspects from the microbialbiomass, soil microbial function and structure and their genetic diversity, which was lack ofreports so far.Attrazine was selected as objective atrazine for pollutants and the experiment wasperformed to confirm the variation of microbial biomass with the fumigation-extractionmethod, the soil microbes function, structure and the influence of genetic diversity by meansof the Biolog, T-RFLP and DGGE technology, through which to estimate the ecologytoxicological effects of atrazine to soil microorganism in simulating conditions. The presentstudy determined the ecotoxicity of atrazine and the response process and time of soilmicroorganism from biochemic and molecular levels with the combination of traditionalmethods and molecular biology technology and may provide a scientific basis and technicalsupport for ecological risk assessment of attrazine to soil microorganism. The main results areas follows:1. Through the microbial flat count experiment, we studied the quantitative variation ofsoil bacteria, fungi and actinomycetes after exposure to three tested concentrations of attrazine(0.1,1,10mg/kg). The results showed that these three soil microorganism were all inhibitedon days7,14,21,28, and42. And the inhibition exhibited a dose-dependant manner withattrazine enhancement. With the fumigating-extraction method, the effects of soil microbialbiomass carbon were also showed a similar situation after exposed to attrazine.2. The research to varies with functional diversity of soil microorganism under thecondition of attrazine pollution by the systemic BIOLOG ECO method, and the resultssuggested that the indoor conditions deal with low concentration of attrazine would not affectthe whole community of soil microbial activity. In the whole experiment cycle, the whole ofthe soil microbial activity (AWCD) was slightly inhibited with high concentrations of attrazine (10mg/kg), but exhibited no obvious difference for most of time. The similarsituation was also appeared in three diversity indexes including Shannon, Simpson andMcintosh at the same concentration of attrazine. As a result, the soil microbial communitiesrichness, the uniformity of advantage degree of and species were decreased. The main use ofcarbon source microbial species level for sugar were raised and its derivatives order wassubsequently metabolic and secondary metabolites, lipid and fatty acid kind, amino acid andits derivatives.3. The changes of genetic diversity to soil microorganism was investigated by theapplication of by PCR-DGGE technology. The variation of ill the soil bacteria, fungi andbacteria of the community structure are as follows:(1) In the investigation of soil microorganism by attrazine pollution, the lane/striprecognition analysis, lane map contrast analysis and cluster analysis was adopted and wefound that soil microorganism were hardly influenced, while their similarity and relationshipswith control groups were relatively low at high attrazine concentrations; with the calculationof diversity index by Shannon, Simpson Mcintosh, we found that in the whole experimentcycle, soil bacteria the species richness was little influenced, but high concentrations ofattrazine could improve the common advantage of bacterium group and the homogeneousdegree of the bacteria were better at low concentrations; Through the rubber cutting recyclestrip, links, transformation and the sequencing analysis, we found the predominant microbeswhich were represented by bacteria DGGE strip were mainly belong to five categories, theywere Proteobacteria, Firmicutes, Deinococcus-Thermus, Actinobacteria, Acidobacteria.Among Proteobacteria, α-Proteobacteria,β-Proteobacteria,γ-Proteobacteria were covered.In addition, in the attrazine pollution condition, the abundance of Firmicutes was increasedsignificantly.(2) The lane/strip recognition analysis, lane map contrast analysis and cluster analysiswere applied to investigate the Archaea bacteria. The results suggested that the similarity andrelationships were relatively lower compare with control groups with the attrazine increasing,which was in accordance with soil bacteria; with the calculation of diversity index byShannon, Simpson Mcintosh, we found that the bundance of Archaea bacteria was inhibitedwhile the dominance was improved at high attrazine concentrations. Through the rubber cutting recycle strip, links, transformation and the sequencing analysis, we found thepredominant microbes which were represented by bacteria DGGE strip were mainly belong tothree categories, they were Crenarchaeota, Euryarchaeota and Thaumarchaeota, among whichsix led showed a high similarity with ammonium oxidation ancient fungus (AOA) diagramand the nitrosation of warm bacteria (Nitrososphaera) similarity is extremely high. It wasobviously that the atrazin pollution participated in nitrogen cycle of bacterium group activity.(3)The lane/strip recognition analysis, lane map contrast analysis and cluster analysiswas used for the determination of attrazine to soil fungi. We found that relative highconcentrations would have slight effects to the class group. With the calculation of diversityindex by Shannon, Simpson Mcintosh, we found both the abundance of fungal and thedominance of the most common species were exhibited no marked effects. Through therubber cutting recycle strip, links, transformation and the sequencing analysis, we found thepredominant microbes which were represented by bacteria DGGE strip were mainly havethree categories, they were Ascomycota, Basidiomycota and Stramenopiles. In addition, theexistence of attrazine induced the activity or the relevant abundance of Myrothecium.4. The PCR-T-RFLP technology was used to analyse the effects of attrazine to thebacterium structure in soil. The results suggested that the predominant bacterial flora in soilmicroorganisms and their changes could be visualized achieved by means of T-RFLPexperiment. The decreased of the stability of microbial population may be ascribe to thedecrease of species diversity, there were no significant differences in other treatment groupsexcepting for Brillouin index (H) and Shannon index (H') on10mg/kg concentration attrazine(P<0.05). Contrast to other data in Phylogenetic Assignment Tool, T-RFs which play animportant role in map could be produced by the16S rDNA enzyme of strains such asProteobacteria, Firmicutes, Deinococcus-Thermus, Actinobacteria, and Acidobacteria. Theresults were in accordance with DGGE sequences. By using principal component analysis, nogreat effects on soil microorganisms were found after exposure to attrazine.