The Study On Isolation And Identification Of The Effective Components Of Wuyiencin,the Grey Mould Prevention Mechanism And Impact On Soil Bacteria

Wuyiencin,a low toxicity,high efficiency,and broad-spectrum agricultural antibiotic,can prevent and control a variety of crops fungal diseases,and has been widely used in agricultural production.However,due to the unclear structure of the main active components,the research progress of disease prevention mechanism,chemical structure modification and transformation is slow,and the improvement of industrial production technology is restricted.In this study,the fermentation broth was adsorbed by macroporous adsorption resin HP-20 after filtration,oxalic acid precipitation,concentration and other pretreatment.And then,14 components were obtained,including 1 water-soluble component and 13 lipid-soluble components after Toyopearl HW-40C chromatography,Toyopearl HW-40F chromatography,silica gel column chromatography,Chromatorex ODS chromatography and preparation of liquid refining.The chemical structure of the water-soluble component was identified by ESI-MS?1H/13C-NMR?COSY?HMBC?HSQC and other techniques,and it was classified into glutamates.The antifungal activity test showed that it was effective against 13 plant pathogenic fungi,including Botrytis cinerea,Fulvia fulva,Fusarium oxysporum etc.Ten chemical structures of the 13 lipid-soluble components were identified:isophthalic acid,6"-O-methyl genistein 7-O-glucuronide,5-hydroxyiso-ph-thalic acid,3-methoxy-2-methyl-4H-pyran-4-one,uracil,3-thdroxy-5-methoxy-6-me-thyl-2,3-4H-pyran-4-one,4-methoxy-4-oxobutanioic acid,3-benzyl-6-isobulylidene,6-h-ydroxynicotinic acid.Among them,2-(3,7-dihydroxy-2-oxoindolin-3-yl)acetic acid is a new compound.This study further clarified the types of the active ingredients of wuyiencin,deepened the understanding of the active ingredients of wuyiencin,and laid a material foundation for the research on the mechanism of disease prevention,chemical structure modification and transformation,and the improvement of the effectiveness of disease prevention and resistance.Grey mould is an important plant disease,caused by Botrytis cinerea,resulting in serious economic loss.Wuyiencin has been demonstrated effectiveness against B.cinerea.However,the molecular mechanism of prevention and control of grey mould by wuyiencin is still unclear.In this study,it was found that the growth of B.cinerea treated by wuyiencin was slow,the aerial mycelium and pigment were reduced,the sporulation was significantly reduced,the mycelium was deformed,the thickness was different,the branch was reduced,the end of mycelium was expanded to form spherical bubbles,and the effect is more obvious with the increase of wuyiencin concentration.These changes were accompanied by differential expression(fold change>2.0)of 316 proteins identified by iTRAQ analysis(P<0.05).Among them,155 proteins were up-regulated and 161 proteins were down-regulated.The GO enrichment analysis showed that the three parts were mainly involved in molecular function,biological process and cellular component.27 key proteins were identified by PRM(parallel reaction monitoring),including 14 up-regulated(including carbohydrate metabolizing proteins and cell wall stabilizing proteins)and 13 down-regulated(including energy metabolism regulators,nucleotide/protein synthesis).A batch of candidate targets genes of B.cinerea were screened and identified,and the differentially expressed proteins and their related ways of inhibiting the growth and pathogenicity of B.cinerea were described in detail in this study.The molecular mechanism of prevention and control of grey mould by wuyiencin was preliminarily elucidated,which was helpful to develop a scientific and reasonable application method,so as to maintain the effectiveness of prevention and control of grey mould by wuyiencin.This study is great significant to enhance the innovation ability of biological pesticides with independent intellectual property rights in China,and also provides a new reference for the research on the pathogenic mechanism of grey mould.Antibiotic resistance genes(ARGs)have raised numerous concerns in recent years as novel environmental contaminants.At present,research on environmental contamination by antibiotics focuses on medical,animal husbandry,and aquaculture fields,with few studies on environmental contamination by agricultural antibiotics in the field of plant protection.The impact of wuyiencin on soil bacterial community structure and antibiotic resistance genes has not been reported.In the present study,pot experiments with four soil treatments(A,B,C and D)were set up in a greenhouse to plant tomatoes.Wuyiencin was sprayed in accordance with the recommended dose,and soil DNA was extracted at 7 time points.The bacterial communities were analyzed using 16s rRNA high-throughput sequencing and the absolute gene copy numbers and abundance of eight typical antibiotic resistance genes(tetG,tetM,tetX,ermB,ermF,mefA,sul?,sul?)and one integron I gene int? were detected by real-time PCR.The short term of wuyiencin did not significantly influence.The bacterial community structures were not significantly varied and the absolute gene copy numbers and ARG abundance were also not significantly influenced after the short-term use of wuyiencin.This study provided the theoretical basis for the evaluation of the environmental safety of wuyiencin and also provided the reference for other related researches on agricultural antibiotics.