An Endophytic Quinclorac-degrading Bacterium And Study On Its Application

Quinclorac is a new auxin herbicide developed by BASF,and mainly used for controlling barnyard grass and certain dicot grasses in paddy field. Due to the low dosage, long lasting period and special effective control of barnyard grass, quinclorac is widely used in rice field. However, due to high stability of quinclorac, degradation of quinclorac in nature is very slow and its residence time is long,which will bring potential hazard to the envioronment and cause residual phytotoxicity to sensitive crops.Especially in some areas of rice-tobacco rotation field, residues of quinclorac cause serious phytotoxicity to sensitive tobacco and have a serious effect on yield and quality of tobacco.In this paper,residue detection method of quinclrac in soil and its degradation dynamics were studied, and phytotoxicity caused by quinclorac to sensitive crops was determined;An endophytic quinclorac-degarading bacteria was isolated from root of tobacco grown in quinclorac contaminated soils and strain Q3 was identified based on morphological characteristics, bio log identification and 16S rDNA gene sequence analysis.Then the degradation characteristics of Q3 was studied. The microbial degrading products of quinclorac were analyzed and identified by liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） and possible degrading pathway of quinclorac was proposed.Bioremediation effect of Q3 on tobacco phytotoxicity was studied through pot experiment and field experiment,then effect of Q3 on activity of tobacco protective enzymes was discussed,which could provide theoretical basis for its field application.The whole genome DNA of the quinclorac degrading strain Q3 was sequenced and preliminary analysis was conducted, which would provide theoretical basis for study molecular mechanism of microbial degradation of quinclorac. The results were as follows:With simplified QuEChERS method for pretreatment of quinclorac in soil,HPLC method for detection of quinclorac in soil was established.When the addition concentration of quinclorac was 0.05～1.0mg/kg, recovery of quinclorac with QuEChERS method was between 90.17%～95.27%, and the relative standard deviation（RSD） was between 1.94%～3.22%,which could meet the requirement of regular residue analysis. Meanwhile, the operating procedure of QuEChERS method was simple and less organic solvent would be used,so the QuEChERS method is more suitable for pretreatment of quinclorac in soil.Quinclorac residue in soil was detected by HPLC-20AT equipped with a UV detector. The results showed that the degradation dynamics equation of low concentration（Recommended dose） of quinclorac was Cx=0.984.e-0.024t with a correlation index of 0.9719,and the half life was 28.88d; While the degradation dynamics equation of high concentration（Double recommended dose） of quinclorac was Cx=1.5518.e-0.026t with a correlation index of 0.9905,and corresponding half life of quinclorac was 26.65 d.The results of indoor bioassay indicated that quinclorac caused phytotoxicity symptom to tobacco is mainly leaf twist, and growth of tobacco would stop and die at last under quinclorac residue concentration of 1mg/kg. The results also showed that the critical concentration causing tomato and pepper phytotoxicity was 10-4mg/L, while it was 10-5mg/L for carrot and celery, and 10-3mg/L for cucumber. The sensibility level to quinclorac from high to low were carrot, celery, pepper, tomato and cucumber. The results indicated that quinclorac has a certain inhibiting effect on growth of these crops, so logical choice of rotation crops according to actual quinclorac residue concentration may help reduce phytotoxicity caused by herbicide residue.With mineral salt streak plate method, an endophytic quinclorac-degarading bacteria named Q3 was isolated from root of tobacco grown in quinclorac contaminated soils. Strain Q3 was a gram-positive bacterium. Based on morphologic properties, physiological-biochemical characteristics, biolog identification and 16S rDNA gene sequence analysis,strain Q3 was finally identified as Bacillus megaterium. Antibiotic sensibility test indicated that strain Q3 was resistant to penicillin and penbritin.Effect of initial quinclorac concentration, culture temperature, culture pH, inoculation size, rotate speed of the shaker and degradation time on Q3 growth and its degrading efficience were studied. The results showed that the optimum condition for Q3 growth and quinclorac degradation were as follows:initial quinclorac concentration of 20 mg/L, culture temperature of 30℃, culture pH of 8.0, inoculation size of 6%（Volume ratio）, shaker rotate speed of 180 rpm. At optimum conditions, more than 93% of quinclorac could be degraded by Q3 in 7 days.Microbial degrading products of quinclorac were analyzed and identified by HPLC-MS/MS. The results showed that three degradation products in all were detected in the microbial degradation process of quinclorac, which were respectively 3, 7-dichloro-8-methyl-quinoline,3-chlorin-8-quinoline-carboxylic and 8-quinoline-carboxylic. Based on the results of mass spectrum, we hypothesized that strain Q3 may employ multiple pathways to degrade quinclorac. One possible pathway is through the reduction of carboxyl and the other pathway is through dechlorination of quinclorac.Bioremediation effect of strain Q3 on tobacco phytotoxicity caused by quinclorac was studied through pot experiment and field plot experiment. Meanwhile, effect of Q3 on activity of tobacco protective enzyme（including SOD, CAT, POD and APX） and MDA content was analyzed. The results of pot experiment showed that leaf length, leaf width and plant height of tobacco with Q3 application were recovered to 89.49%,92.14% and 93.64% of those of control 30 days after transplanting; and 93.40%,91.70% and 92.29% 45 days after transplanting. The results of field plot experiment showed that leaf length, leaf width and plant height of tobacco with Q3 application were recovered to 90.49%,90.69% and 88.95% of those of control 11 days after Q3 application;and 96.92%、93.83% and 92.58% 22 days after Q3 application. Both pot experiment and field plot experiment showed that strain Q3 had perfect bioremediation effect against tobacco phytotoxicity caused by quinclorac. Meanwhile, strain Q3 could also improve the activity of tobacco protective enzymes such as SOD, CAT, POD and APX, and decrease the content of MDA in tobacco, thus alleviated the tobacco phytotoxicity caused by quinclorac.The whole genome of Bacillus megaterium Q3 was sequenced by PacBio RS system and whole genome sequence was obtained. After filtering, the original data of sequencing was jointed by HGAP software,then jointed sequence was adjusted repeatedly and the whole genome draft of Q3 was obtained. The Bacillus megaterium Q3 genome consists of a circular chromosome（5,153,539 bp） and a circular plasmid（76,260 bp）. GC content of the Q3 chromosome was 38%. After genome annotation by PROKKA software, the Bacillus megaterium Q3 genome encodes 5264 protein genes,172 RNA genes（including 130 tRNA genes and 42 rRNA genes）. Compared with genomes of other reported Bacillus megaterium strains, there are 4694 core genes,294 pan genes and 216 special genes in the genome of Bacillus megaterium Q3. There is a plasmid Q3_pl in the genome of Bacillus megaterium Q3,which is homologous with the WSH-002_p1（NC₀17139） plasmid in the genome of strain WSH-002. Compared with the WSH-002_p1 plasmid, there is only one gene BACMQ3₀5432 that is the special gene of strain Q3. Plasmid Q3_p1 has a complete rRNA gene set and 17 tRNA genes,and its structure is similar to the chromosome.