| Fusarium oxysporum Schlecht.is a ubiquitous soil-borne phytopathogen that causes vascular wilt on most widely cultivated crops.Controlling vascular wilt is difficult in agricultural practices and heavy application of chemical fungicides causes adverse effects on the environment and human health.Interest in fusarium wilt biological control based on application of antagonistic micro-organisms is renewed.However,limited survival time and inefficient colonization ability in soil restricted their use.New type of biocontrol agents is seriously needed to effectively control Fusarium wilt.Myxobacteria are gram-negative indigenious soil bacteria with complicated multicellular morphogenesis and behavior over their complex life cycle.Myxobacteria isolated from soils exhibited efficient preying on a broad range of soil bacteria and fungi.Based on their efficient survival and feeding characteristics,myxobacteria are considered to have great potential in the biocontrol.Corallococcus sp.EGB exhibits efficient predation on various plant pathogens,and a novel outer membrane protein GluM was identified to be involved in the predation of myxobacteria.In this research,we focused on the antifungal mechanisms of GluM towards F.oxysporum f.sp.Cucumerinum(FOC)and biological control of FOC by strain EGB in soil.1.GluM destroys the cell wall,actives the HOG pathway,causes the brust of ROS and induces apoptosis-like in FOC cellsResults showed that the minimum inhibitory concentration of GluM on spores'germination was 2.0 U/ml.2.0 U/ml of GluM treatments decrease the survial rate of spores and monohypha to 10.40%and 30.68%,respectively,after 12 h treatment,which were significantly lower that of zymolyase treatments(72.90%and 73.94%).Meantime,we also observed that the content of glycerol in FOC cells increased 2.6-fold and cell wall was obviously perforation after GluM treatment.After hog1 gene of HOG pathway knocked out,the intracellular glycerol content of ?hog1 mutants and FOC-WT strain exhibited no significant difference,while ?hog1 mutants exhibited decreased sensitivity toward GluM hydrolysis.However,we found that ?slt2 mutants were more sensitivity to GluM.The results showed that activation of the HOG pathway caused an increase in intracellular glycerol content,and the resulting increase in intracellular swell pressure of FOC exacerbated cell death due to cell wall destruction,and the addition of 1.0 M sorbitol reduced mortality by only about 13.2%.Further studies showed that the significant accumulation of intracellular ROS directly caused cell death,and the reducing substrances vitamin C and vitamin E could improve the tolerance of FOC to GluM treatment.Meanwhile,the mutant lacking the apl gene,which encodes a polypeptide resembling yeast YAP1-like transcriptional activators,which are involved in cellular responses to stress,displayed more sensitivity to GluM compared with wild-type strain.The apoptotic phenomenons including mitochondrial membrane potential dissipation,phosphatidyl serine externalization and DNA fragmentation were observed in GluM treated cells,and the apoptosis-like of FOC cells were induced finally.The results of protoplast processing indicated that the apoptotic phenomenon in FOC cells caused by GluM treatment was not induced by small molecules released by cell wall hydrolysis.So,above results indicated that the burst of ROS and the apoptosis-like caused by GluM treatment play an important role in killing FOC cells by GluM.2.Solid-state fermented Corallococcus sp.strain EGB exhibites efficient biocontrol of cucumber Fusarium wilt in pot-and field-scale experimentsStrain EGB liquid culture and SSFM(solid-state fermentation matrix)showed efficient biocontrol of cucumber fusarium wilt in two consecutive years' field experiments,and the biocontrol efficiencies were 65.96%(2015)and 53.94%(2016).The ecological mechanism of biocontrol of cucumber Fusarium wilt by strain EGB was studied by pot experiment.Results of pot experiment showed that 85.72%control efficiency was obtained from the treatment of SSFM.At the same time,the colonization of FOC and EGB in the rhizosphere of the cucumbers were dectected by qPCR.The results showed that Compared with the initial concentration of FOC in the soil(105 spores/g soil),the population of FOC after the application of SSFM was decreased by 70%,The results indicated that EGB could strongly inhibit the growth and reproduction of FOC in soil.SEM was used to observe the film forming ability of EGB in cucumber roots.Our results showed that the number of EGB that colonized the meristematic zone was much less than the number of EGB that colonized the elongation zone.In order to study the driving force of EGB colonization in cucumber roots,we analyzed the chemotactic ability of EGB to cucumber root exudates.The results showed that strain EGB exhibited chemotactic behavior to cucumber root exudates and it was more sensitive to 10-100 ?M maltose and 10-80 ?M maltitol than other compounds.3.Corallococcus sp.strain EGB can affect soil microbial community structureThe migration process of EGB in soil and the changes of microbial community in soil afte SSFM treatment were also studied.We used high-throughput technology to detect that abundance changes of microorganisms at different sampling points.The results showed that the number of Corallococcus in the natural soil was very low,however,after the application of the SSFM,the abundance of Corallococcus in the intermediate sampling point of the groups treated with the SSFM on both the 15th and 27th day significantly(p<0.05)increased.Notably,in contrast with natural soil,the abundances of Corallococcus in the rhizosphere soil of the samples on the 15th day were not significantly different;however,the abundances significantly(p<0.05)increased in the group treated with the SSFM after 27 days compared to 15 days.It suggested that the EGB had moved from the center of the pot toward the roots of the plants.The abundance of microorganisms between SSFM treated group and SSFM untreated group were compared.The results showed that the abundance of thirteen genius bacteria and five genius fungus were significantly increased in SSFM treated group,and many microorganisms among them have been reported to have the potential in biological control,such as,Bacillus,Pseudomonas and Thielavia.We also found the abundance of nineteen bacteria and twenty-seven fungus were significantly decreased,however,none of them have been reported to be related with plants' growth promoting or biological control.Therefore,we deduced that EGB,as a predatory myxobacteria,has the ablity to change the microbial community structure and make the soil develop in the healthy direction.4.GluM relieves the protection of ?-glucan from FGB1 and enhances the systemic resistance of plants to FOCPrevious report showed that FGB1 is a novel type of disulfide-bridge-containing lectin that specifically binds ?-glucan via ?-1,6-linked glucose moieties in a context of a ?-1,3 glucan backbone,FGB1 alters cell wall composition by interacting with ?-1,6-linked glucan to protect ?-glucan polymers from recognition and avoid ?-glucan triggered immunity in planta.From our study,we found that the degree of hydrolysis of ?-1,3-glucan by zymolyase was increased due to the hydrolysis of ?-1,6-glucan by GluM.Meantime,we found that the transcript levels of PR genes were significantly increased in cucumber roots treated with FOC and SSFM.Hence,we hypothesized that EGB could eliminate the protective effect of FGB1 on ?-glucan and promote the recognition of ?-glucan by plants for producing systemic rsistance to FOC.In conclusion,the mechanism of strain EGB against FOC was clarified in this research.?-1,6-glucan of FOC cell wall was hydrolyzed and the cell intergrity was damaged by the outer memberne ?-1,6-glucanase,GluM,screated from strain EGB.In this process,HOG pathway was activated and the accumulation of intracellular gelycerol exacerbated the damage of FOC cell wall.Meanwhile,the accumulation of ROS caused cell death,directly,and induced apoptosis-like of FOC cells.Strain EGB could colonize effectively in soil,control the number of pathogenic bacteria and regulate the structure of soil microbial community.The systematic resistance of plants to FOC was enhanced due to GluM relieved the protection of ?-glucan from FGB1.This study provides a theoretical and practical basis for the biocontrol of plants' fusarium wilt by Corallococcus sp.EGB. |
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