Construction Of Biodegradable Bactericides Chlorothalonil And Carbendazim Fusion Bacteria And Preliminary Study On Their Degradation Characteristics

With the increasing use of chemical pesticides such as chlorothalonil and carbendazim in China,more and more attention has been paid to their residues.Chlorothalonil is a broad-spectrum,protective fungicide widely used in agricultural production.There have been many reports on the detection of chlorothalonil in soil,water body,greenhouse and agricultural products.Carbendazim is a kind of broad spectrum,internal absorption fungicide,widely used in industrial and agricultural production,is one of the varieties with the largest production and dosage of fungicide.It has a long half-life in nature and can cause severe"triple"effects in mammals and other animals even at low doses.At present,photolysis and microbial degradation are the main ways to reduce the pollution of bactericides chlorothalonil and carbendazim in soil.Compared with photolysis,microbial degradation has the advantages of high treatment efficiency,low cost and no secondary pollution.Therefore,it is of great significance to screen out bacterial strains that can efficiently degrade the two fungicides for bioremediation of soil contaminated with fungicides.Protoplast fusion,one of the methods of microbial breeding,has the advantages of complete genetic material transfer,good orientation and high breeding efficiency,and can overcome the limitation of narrow degradation spectrum of single fungicide.The technology has been widely used in the study of pollutant treatment of engineered bacteria,and the results are remarkable.At present,the application of protoplast fusion technology in genetic breeding of bactericide Chlorothalonil and carbendazim degradation bacteria has not been reported.The research significance of this topic lies in the construction of multifunctional genetically engineered bacteria that can simultaneously degrade the fungicides chlorothalonil and carbendazim,which can be used for the bioremediation of mixed contaminated soil,and can be used for the bioremediation of chlorothalonil and carbendazim complex contaminated soil.Provide a new way to better solve the problem of environmental pollution caused by chlorothalonil and carbendazim.In this paper,bactericides chlorothalonil and carbendazim were used as research objects to isolate and identify the efficient degradation strains,and the growth and degradation characteristics of the strains were studied.In order to explore the growth conditions and degradation of the fusion strain,the protoplast fusion technology was used to construct the fusion strain which could degrade both chlorothalonil and carbendazim.The main results are as follows:1.In this paper,a chlorothalonil degrading strain named CTN-5 was isolated from chlorothalonil and carbendazim contaminated samples by enrichment culture method,and a chlorothalonil degrading strain CTN-16 was isolated from our laboratory.A carbendazim degrading strain was identified as MBC-3.The biodegradable strains were identified as Bordetella sp.CTN-16,Brucella sp.CTN-5and Microbacterium sp.MBC-3,respectively,by physiological and biochemical identification and phylogenetic analysis.2.Fusion bacteria of CTN-16 and MBC-3and CTN-5 and MBC-3 were constructed based on protoplast fusion technology.Antibiotic screening experiments showed that CTN-16 was sensitive to kanamycin,MBC-3 was resistant to ampicillin,CTN-5 was sensitive to geneticin,and MBC-3was resistant to ampicillin.Kanamycin and ampicillin were used as genetic markers to screen fusion bacteria,and the other group were used as genetic markers to screen fusion bacteria.Two strains of fusion bacteria,named BD2 and BD3,which can be stably subcultured for more than 8 generations in the medium containing two antibiotics,were obtained.3.Strain BD2 was identified as Enterobacter sp.BD2,a Gram-negative bacterium with a short rod shape.This species is different from its parental genus division,which may be due to the recombination of the 16S r RNA gene sequence or the recombination of the genome during the fusion process,and the specific mechanism needs to be further explored by genome sequencing and other means.The physiological and biochemical characteristics of the fusion strain BD2 were similar to those of strain CTN-16,but the physiological and biochemical characteristics of the fusion strain BD2 were similar to those of strain MBC-3,which suggested that the fusion strain BD2 might have inherited some genetic characteristics of the two parents.BD3 was identified as Brucella sp.BD3 and its physiological and biochemical characteristics were more similar to that of strain CTN-5.However,the same physiological and biochemical traits were also similar to those of strain MBC-3,which suggested that the fusion strain BD3 might have inherited some genetic characteristics of the two parents.4.According to the results of random amplified DNA polymorphism（RAPD）analysis,BD2 had both the same bands as CTN-16 and MBC-3 strains and specific bands different from their parents,and BD3 also had the same situation.It was proved that the genetic material of the two parental strains did exchange during protoplast fusion.The genetic similarity indexes of BD2 and BD3 were 0.571 and 0.428,0.778and 0.444,respectively.5.The optimum growth conditions of fusion bacteria BD2 in inorganic salt medium were as follows:inoculation amount 5%,culture temperature 30℃,p H 7.5;The optimum growth conditions of BD3 in inorganic salt medium were as follows:inoculation amount 5%,culture temperature 32℃,medium p H 7.5.Under these conditions,the degradation rates of BD2 cultured in 50 mg·L^-1for 5 days were 79.8%and 65.2%,respectively.Under the same culture conditions,the reduced solution rates of BD3 were 80.2%and 60.3%,respectively.