Screening And Identification Of Ahls Degrading Bacteria And Their Biological Control Effects

Previous studies mainly focused on individual behaviors of microorganisms.However,the perception of microorganism behaviors has changed due to the discovery of quorum sensing(QS).QS means that microbial modulate the expression of specific genes which related to many biological functions through perceiving signal molecules.N-Acyl homoserine lactones(AHLs)is one kind of important signal molecules of Gram-negative bacteria.Quorum quenching(QQ)is a new therapeutic strategy against plant diseases,which is based on QS.QQ can be achieved by inhibiting the synthesis or detection of the signal molecules,or by enzymecatalyzed degradation or modification of the signal molecules.The interruption of QS through QQ can result in the decrease of pathogenicity and control of plant dieases because the pathogenic genes associated with pathogenic bacteria could not express.In the process of preventing and controlling of plant disease,the pathogen will not face the selective pressure,therefore it will not develop resistance.So,it is able to add new contents to environmentally friendly plant disease control method that seeking new strains with quenching activity and apply these strains to biocontrol of plant diseases.This study focuses on screening of AHLs-degrading bacteria from soil samples,and the main results include the following parts:(1)Soil samples were collected from Xinjiang province,Guangdong province,Hebei province,and so on,and the enrichment and isolation of AHLs-degrading bacterial isolates were conducted with AHLs as the sole source of carbon.367 strains which could grow in mineral salt medium(MSM)supplemented with AHLs were obtained after isolation and purification.18 strains which can degrade AHLs were verified by biosensor strain from 367 strains.These isolates belong to Pseudomonas,Acinetobacter,Enterobacter and Ochrobactrum,Enterobacter,Bacillus,Agrobacterium,Shewanella,Ochrobactrum,respectively.(2)Three QQ strains designated as D-2,W-7 and XJ-10 were selected for further study.Based on morphological identification,16 S rDNA analysis,physiological and biochemical characteristics,Biolog analysis,the three strains were identified as Ochrobactrum intermedium,Pseudomonas nitroreducens,Acinetobacter schindleri,respectively.(3)The substrate spectrum and degradation efficiency of strains D-2,W-7 and XJ-10 were performed.The results showed that strain D-2 had a broad substrate spectrum which could degrade 3OC6 HSL,3OC8HSL and 3OC12 HSL.In the experiment of degradation and growth,degradation of 3OC6 HSL by strain D-2 was positively correlated with the growth.In the MSM containing 3OC6 HSL as the sole source of carbon,strain D-2 rapidly entered the growth logarithmic stage without any lag phase.The most rapid degradation of 3OC6 HSL by strain D-2 occurred at 8~24 h,and the signal molecule were completely degraded within 32 h.Strain W-7 is capable of degrading 3OC12 HSL and diffusible signal factor(DSF).The degradation of 3OC12 HSL was positively correlated with the growth of strain W-7.In the MSM containing 3OC12 HSL as the sole source of carbon,strain W-7 rapidly entered the growth logarithmic stage without lag phase.The most rapid degradation of 3O126 HSL by strain W-7 occurred at 12~24 h,and 3OC12 HSL were completely degraded within 48 h.Strain XJ-10 was only able to degrade 3OC12 HSL.Degradation of 3OC12 HSL by strain XJ-10 at 12 h and 24 h were 85.71% and 98.8%,respectively.In summarize,degradation of AHLs was positively correlated with the growth of QQ strains.In the MSM containing AHLs as the sole source of carbon,QQ strains rapidly entered the growth logarithmic stage without lag phase.QQ strains have significant and rapid degradation effect on AHLs.(4)Inoculation experiments were carried out to evaluate the QQ ability and actual biological control effect of strains D-2,W-7 and XJ-10 on pathogenic bacteria whose pathogenicity modulated by AHLs.Strain D-2 has biological control effect on soft rot of potato and carrot caused by Pectobacterium carotovorum subsp.carotovora(Pcc).Compared with the treatment group inoculated with pathogenic bacterium and QQ strain D-2,the soft rot disease symptoms of control group inoculated with pathogenic bacterium only were more serious.The antagonistic test indicated that there was no antagonistic effect between D-2 and pathogenic bacteria,so the biological control effect of strain D-2 was not achieved through antagonism.Isolates W-7 and XJ-10 both have biological control effects on soft rot of potato caused by Dickeya zeae EC1.The plant disease symptoms were obviously reduced when co-inoculation with pathogenic bacteria and QQ strains,W-7 and XJ-10,than the control group inoculated with pathogenic bacteria only.The inoculation test also showed that QQ strains,D-2,W-7 and XJ-10 were safe to crops.(5)Investigation the degradation mechanism of AHLs by strain D-2.Based on the characteristic that the structural change of AHLs may occur in alkaline conditions,the degradation of AHLs by strain D-2 was attributed to lactonase.At the same time,the most QQ activity of strain D-2 was found in the cell crude extract.That is,QQ activity of strain D-2 against AHLs may be from the intracellular enzyme.In addition,degradation products of C6 HSL were verified with GC-MS,and the metabolic pathway was proposed.C6 HSL was first enzymatically degraded by lactonase to form acyl-homoserine,which was further degraded to produce N-cyclohexyl-propanamide and propionamide.These products were finally metabolized as carbon dioxide and water.