| Quinclorac(3,7-dichloroquinoline-8-carboxylic acid)is a highly selective auxin herbicide.It is commonly used in rice production to control barnyard grass.Nevertheless,the widespread use of quinclorac(QNC)has led to large amount of residual in soil which do great harms to succeeding tobacco.The quality and yield of tobacco are greatly affected by the phytotoxicity of quinclorac.Microbial transformation and degradation are currently believed to be the most important strategy to remove the quinclorac in environment.This research aimed to screen bacteria capable of degradation quinclorac,study the degradation characteristics,identify the enzymes and metabolites involved in the biodegradation process,elucidate quinclorac biodegradation pathway and verify the bioremediation to damaged tobacco by quinclorac in soil.The main results were as follows:1.Effects of quinclorac to agronomic,physiological characteristics and ultrastructure of leaves of tobacco grown in pots were studied.leaf width,leaf length and plant height of were significantly inhibited by quinclorac applied in soil.The inhabitation rate of 3.343×10-2mg/kg quinclorac for leaf width in 40d was 57.10%,and 22.25%for leaf length.The results showed that threshold of quinclorac harm would be 1.33×10-2 mg/kg dried soil.The activity of SOD,POD and CAT of tobacco was decreased after quinclorac treatment.SOD and CAT were more sensitive to quinclorac than POD.Content of·OH-,O2·-and MDA in Leaves and roots increased with treatment time and increasing concentration of quinclorac.Compared with control,the level of endogenous hormones,IAA,GA3 and ZR decreased significantly,but the content of ABA was higher than the control group.Transmission electron microscope(TEM)observation showed that cell wall,lipid membrane,cytoplasm,vacuole and other organelles were abnormal in top stem cell of damaged tobacco.Chloroplasts in tobacco leaves were damaged seriously.Quinclorac does harm to protective enzyme,oxygen free radical,endogenous hormones and ultrastructure of tobacco.As a result,the growth of tobacco is inhibited.2.Strain HN36 and HN1 capable of removal quinclorac was obtained from a herbicide manufactory sewage sludge using gradient concentration of quinclorac enrichment strategy.Strain HN36 was considered to be Pseudomonas sp.,belonged to gram-negative bacteria,based on physiological and biochemical properties,morphology and a partial sequence of 16S rDNA.Strain HN1 was considered to be Agrobacterium sp.Strain HN36 could degrade 96.93%of quinclorac to undetectable level at the original concentration of 500 mg/L within 24 h.Strain HN36 was able to utilize a wide variety of aromatic compounds as carbon and energy source,including catechol,quinoline,benzoic acid,phenol and hydroquinone.Nevertheless naphthalene,phenanthrene and benzene could not be used to growth.The optimal temperature for quinclorac degradation was 35℃ and HN36 had the highest degradation rate at pH 7.3.The methods of SDS-PAGE and 2-DE were used to study the different proteins induced by quinclorac.Chlorocatechol 1,2-dioxygenas(1,2-CCD)and phthalate dioxygenase reductase(PDR)were identified and considered to be the key enzymes in the degradation of quinclorac.The activities change of 1,2-CCD and PDR in the culture process was accorded with the biodegradation of quinclorac and growth curve of HN36.It indicated that these enzymes were closely related to quinclorac degradation.1,2-CCD was verified by RT-PCR results.In addition,three metabolites,4,7-bichlorid quinolone,2-chlorine terephthalic acid and 2-amino-4 chlorobenzoic acid,were identified by GC-MS technique.The degradation pathway of quinclorac was proposed based on the obtained enzymes and degradation products.4.Strain HN36 was able to break down 0.085 mg/kg dried soil quinclorac in soil to undetectable level in 60d.To some extent,the degradation rate increased with increasing of inoculum size.The optimal inoculum amount was 5×109 CFU/kg dried soil.Quinclorac could inhibit the activity of catalase,peroxidase,urease,protease and dehydrogenase in soil.The activity of enzymes in soil increased with the application of strain HN36.The physiological characteristics of damaged tobacco by quinclorac were improved by HN36.Protective enzyme activities of tobacco increased and the content of,O2·-·OH and H2O2 decreased with HN36 added to soil.The level of MDA would be lower with a larger inoculum size.At the same time,Antioxidation activity of roots was stronger and the GSH content was higher.Thus growth of damaged tobacco was well improved.A similar results were obtained for bioremediation of different quinclorac concentration in soil.The inhibitation of leaf width,leaf length and plant height by quinclorac stress reduced when HN36 was applied to soil.Agronomic characteristics of repaired tobacco were similar with those of normal tobacco with a inoculum size of 5×1010CFU/kg dried soil.Cellar structure of damaged top leaves was repaired and ultrastructure of top stem and leaves was alleviated with application of HN36.Total sugar,reducing sugar,starch,potassium levels were as follows:phytotoxic tobacco>repaired tobacco>normal tobacco.The ratio of reducing sugar/total sugar,total sugar/nicotine,total nitrogen/nicotine of the normal tobacco and the repaired tobacco are more suitable than the phytotoxic one. |
