Effects Of Symbiotic Bacteria And Trace Elements On The Degradation Of Pesticide Residues By Paenibacillus Polymyxa

Ginseng（Panax ginseng C.A.Meyer）is a perennial herb of Araliaceae.It can be used for both medicine and food.increase yearly.However,because organochlorine pesticides are difficult to degrade,have a long half-life,are easy to accumulate in plants,and are used improperly,structurally stable organochlorines such as pentachloronitrobenzene（PCNB）can still be detected in ginseng from different production areas.Pesticides,even individual content exceeding the limit standard,have seriously affected the quality and safety of ginseng products.Therefore,it is imminent to find ways to quickly and efficiently degrade pesticide residues.The methods for degrading pesticide residues are mainly divided into physical,chemical and biological methods.However,the efficiency of physical methods is low,and chemical methods are prone to secondary pollution.Microbial degradation of pesticide residues has low cost,good effect,and is green,safe and pollution-free.It has become an important means of degrading pesticide residues.Among the ginseng endophytes isolated and identified earlier in our laboratory,Paenibacillus polymyxa has the best biological control effect,and has the effect of degrading pentachloronitrobenzene（PCNB）pesticide residues,but its degrading effect on PCNB remains to be seen.Increased,its degradation PCNB gene is not clear.Therefore,the purpose of this study is to clarify the effects of symbiotic bacteria and trace elements on the degradation of pesticide residues by Paenibacillus polymyxa,and to explore its candidate genes for degradation of PCNB and their pathways.The main research contents are as follows:1.Screening of symbiotic bacteria of Paenibacillus polymyxa Using 4 strains of ginseng endophytes including Paenibacillus polymyxa as experimental materials,the dilution coating plate method,inhibition zone test method,strain cross-streaking method,and plate streaking method were used as experimental materials.method and other methods to screen the ginseng endophytic strains symbiotic with Paenibacillus polymyxa and analyze their phylogenetic evolution by ITS method.The results showed that:Bacillus cereus can co-exist with Paenibacillus polymyxa,the total number of viable bacteria in the co-culture solution increased significantly,and the number of single colonies of Paenibacillus polymyxa also increased to 2.3times that of single culture（P<0.05）.However,Bacillus amyloliquefaciens and Bacillus subtilis cannot coexist with Paenibacillus polymyxa.Paenibacillus polymyxa and Bacillus cereus have the closest relationship and the most distant relationship with Bacillus subtilis,which is the reason for the symbiosis between Paenibacillus polymyxa and Bacillus cereus strains.It is clear that Bacillus cereus can co-exist with Paenibacillus polymyxa,laying the foundation for subsequent research.2.The effect of the combination of symbiotic bacteria on the efficiency of degrading pesticide residues by Paenibacillus polymyxa Using Bacillus amyloliquefaciens,Paenibacillus polymyxa,Bacillus subtilis,and Bacillus cereus as experimental materials,the degradation rate of PCNB,hexachlorobenzene,cis-chlordane,and trans-chlordane were used as evaluation indicators to compare the symbiotic bacteria effects of combined and single use with Paenibacillus polymyxa on the degradation rate of pesticides..The results showed that:the degradation rate of hexachlorobenzene,Bacillus subtilis single culture group was 45.98%,which was significantly higher than the single culture group of Bacillus amyloliquefaciens and Bacillus cereus（P<0.05）;After 4d reaction,the co-culture group of Paenibacillus polymyxa and Bacillus cereus was 62.84%,which was significantly higher than the single culture group of Paenibacillus（P<0.05）,the single culture group of Bacillus cereus（P<0.05）,and the single culture group of Bacillus amyloliquefaciens.Culture group（P<0.05）,Bacillus subtilis single culture group（P<0.05）.For the degradation rates of cis-chlordane and trans-chlordane,the Bacillus amyloliquefaciens group was 55.14%and 56.38%,respectively,which were significantly higher than the single culture group of Bacillus cereus and the co-culture of Paenibacillus polymyxa and Bacillus cereus group（P<0.05）,the Bacillus cereus single culture group and the Paenibacillus polymyxa and Bacillus cereus co-culture group were significantly higher than the Paenibacillus polymyxa single culture group and the Bacillus subtilis single culture group（P<0.05）.Different strains have different degradation rates for different pesticides.The combination of Paenibacillus polymyxa and Bacillus cereus is beneficial to the degradation of PCNB,and the degradation rate of PCNB can be increased by 31.93%（P<0.05）.3.Screening of active parts of Paenibacillus polymyxa degraded PCNB pesticide residue protein The degradation rate of PCNB pesticide was used as an evaluation index to evaluate the degradation rate of Paenibacillus polymyxa protein extracted by different saturated ammonium sulfate,and to screen the active protein sites of Paenibacillus polymyxa degraded PCNB,and evaluate the stability of the protein;compare the effect of the combination of symbiotic bacteria and Paenibacillus polymyxa on the degradation of PCNB;at the same time,through SDS-polyacrylamide gel electrophoresis technology,screen the co-culture of symbiotic bacteria and Paenibacillus polymyxa tpes of differential protein bands before and after.The results showed that the PCNB protein degraded by Paenibacillus polymyxa was 40%saturated ammonium sulfate extracted protein;the PCNB protein degraded by Paenibacillus polymyxa had good thermal stability,p H stability and UV stability.Co-cultivation of Paenibacillus polymyxa and Bacillus cereus significantly improved the degradation of PCNB protein by Paenibacillus polymyxa（P<0.05）;after co-cultivation of Bacillus cereus and Paenibacillus polymyxa,there were 6 differentially expressed protein strips Bands with molecular weights of approximately 25k Da,32k Da,40k Da,53k Da,60k Da and 110k Da.4.Effects of trace elements on the degradation of PCNB pesticide residues by Paenibacillus polymyxa Using the PCNB degradation rate as an index,evaluate the efficiency of four trace elements in degrading PCNB by Paenibacillus polymyxa;and then analyze the difference in the efficiency of trace elements to improve the efficiency of Paenibacillus polymyxa in reducing PCNB pesticide residues by high-throughput sequencing technology and q RT-PCR expressed genes and their pathways of action.The results showed that among the four trace elements Fe²⁺,Cu²⁺,Zn²⁺and Mn²⁺,Fe²⁺had the strongest effect on reducing pesticide residues of Paenibacillus polymyxa（P<0.05）.Fe²⁺elements can significantly regulate the expression level of 902 genes in Paenibacillus polymyxa,among which 311 genes were significantly up-regulated,mainly focusing on energy metabolism,transformation,cell community-prokaryotes,carbohydrate metabolism and xenobiotic metabolism 9 pathways with extremely significant enrichment（P<0.05）;combined with quantitative PCR analysis,10 genes were finally screened,which are:PPYC2＿RS14025、PPYC2＿RS07805、PPYC2＿RS00620、PPYC2＿RS04765、PPYC2＿RS22865、PPYC2＿RS22850、PPYC2＿RS04185、PPYC2＿RS26090、PPYC2＿RS19610 and PPYC2＿RS06530,the differential expression of these genes,is the main gene that Fe²⁺element improves the ability of Paenibacillus polymyxa to degrade PCNB pesticide residues.