Isolation Of Pgpr From Vinegar Residue Compost And Its Induction Of Host Defense Responses Against Fusarium Wilt Of Cucumber

Fusarium wilt of cucumber caused by Fusarium oxysporum f.sp.cucumerinum J.H.Owen(FOC)is one of the major destructive soilborne diseases,with the protection of soil,the pathogen can survive to a long term,repeately infects the plant and result in considerable yield losses.As there are no resisitant varieties and the chemical controls such as soil disinfestation with methyl bromide have been banned from agriculture because of environmental and food quality concerns.Using suppressive media as an approach of biological control to assist crop growth is becoming popular.Vinegar residue substrate is a new type of horticulture organic substrate made from waste residue produced by the vinegar industry.The current research showed that the vinegar residue substrate has suppressive effects on cucumber Fusarium wilt.However,the mechanism of its control of cucumber fusarium wilt is unclear.In this study,we evaluated its effects on plant growth and suppression phenomena in cucumber,isolated the potential biocontrol agents from vinegar residue substrate,studied the induction of host defense responses against fusarium wilt by the biocontrol agent NSY50,analyzed the positive roles of NSY50 in the response of cucumber roots to FOC inoculation with a proteomic approach.Main research results were as follows:1.Greenhouse experiments were carried out to evaluate the effect of vinegar residue substrate on the growth of cucumber seedlings and disease suppression.These results showed that:comparison with control(peat:vermiculite=2:1,v/v),the vinegar residue compost-amended media(vinegar residue compost mixed with peat and vermiculite in a 6:3:1 ratio,VRS)showed a significantly positive effect on the growth of cucumber seedlings and the suppression of the disease severity index.Total organic C,microbial biomass C,basal respiration,and enzyme activities increased significantly after vinegar waste compost amendment.The cucumber rhizosphere population of FOC was significantly lower in VRS than in the control.These results suggested vinegar residue compost application significantly affected the chemical,microbiological,and biochemical properties of traditional soilless culture with peat,indicating that the vinegar residue compost-amended media exhibited a high degree of suppression for cucumber wilt.2.Using the dilution plate method isolated 10 potential biocontrol agents from vinegar residue substrate;the 16S rDNA gene demonstrated that the biocontrol agents were Paenibacillus polymyxa,Bacillus amyloliquefaciens and Bacillus licheniformis.Based on the results of antagonistic activity experiments and pot experiment,an interesting strain of P.polymyxa(named NSY50)was selected for further research.Morphological,physiological,and biochemical characteristics indicated that this strain was positive for protease,cellulase and ?-glucosidase,and produced indole acetic acid and 1-aminocyclopropane-1-carboxylate deaminase(ACCD).3.Further study on the induction of host defense responses against FOC by P.polymyxa NSY50 with solution culture.NSY50 could apparently mitigate the injury caused by the FOC infection and maintain the stability of cell structures.Inoculation of cucumber seedling roots with FOC led to the formation of necrotic symptoms and extensive colonization within 9 days of infection.While pretreatment with NSY50(NSY50+FOC)resulted in relatively minor browning.NSY50 can significantly up-regulate the expression level of defense related genes PR1 and PR5 in cucumber roots at the early stages upon challenge with FOC.However,the gene expression levels of a set of defense-related genes,such as the plant nucleotide-binding site(NBS)-leucine-rich repeat(LRR)gene family(e.g.,Csa001236,Csa09775,Csa018159),26 kDa phloem protein(Csa001568,Csa003306),glutathione-S-transferase(Csa017734)and phenylalanine ammonia-lyase(Csa002864)were suppressed by pretreatment with NSY50 compared with the single challenge with FOC after 9 days of inoculation.4.The two-dimensional electrophoresis(2-DE)approach in conjunction with MALDI-TOF/TOF analysis revealed a total of 56 proteins that were differentially expressed in response to NSY50 and/or FOC.The application of NSY50 up-regulated most of the identified proteins that were involved in carbohydrate metabolism and amino acid metabolism under normal conditions,which implied that both energy generation and the production of amino acids were enhanced,thereby ensuring an adequate supply of amino acids for the synthesis of new proteins in cucumber seedlings to promote plant growth.Inoculation with FOC inhibited most of the proteins related to carbohydrate and energy metabolism and to protein metabolism.The combined inoculation treatment(NSY50+FOC)accumulated abundant proteins involved in defense mechanisms against oxidation and detoxification as well as carbohydrate metabolism,which might play important roles in preventing pathogens from attacking.Meanwhile,western blotting was used to analyze the accumulation of enolase(ENO)and S-adenosylmethionine synthase(SAMs).NSY50 further increased the expression of ENO and SAMs under FOC stress.In addition,NSY50 adjusted the transcription levels of genes related to those proteins.Taken together,these results suggest that NSY50 may promote plant growth and alleviate FOC-induced damage by improving the metabolism and activation of defense-related proteins in cucumber roots.