| Wheat scab is one of the devastating diseases on wheat crops in recent years.Not only does it cause crop yield reduction,but it also produces toxins that are harmful to human and animal health.At present,chemical control is still a commonly control measure.Long-term use of chemical fungicides will not only cause environmental pollution,but also promote the synthesis of toxins.The use of endophytic bacteria to control plant diseases has attracted increasing attention.In this study,F.graminearum was used as the target to isolate,screen and identify antagonistic bacteria from different tissues of healthy Eucommia ulmoides.And the control efficacy of endophytic bacteria was examined against F.graminearum using pot culture and plot experiment.On this basis,the effect of antagonistic bacteria on the germination and deformity of F.graminearum conidia was studied,and the influence of antagonistic bacteria on DON production abilities of F.graminearum was analyzed.Then,we investigated the induced systematic resistance of the biocontrol strain DZSG23,as well as the ability of DZSG23 to colonize wheat.The results laid a theoretical foundation for the biological control of wheat scab and provide bio-control resources.The main findings are as follows:1.A total of 104 endophytic bacteria were isolated from the roots,stems and leaves of healthy Eucommia ulmoides by dilution coating method.104 strains of endophytic bacteria were belonged to 6 genera:Bacillus sp.,Paenibacillus sp.,Brevibacillus sp.,Burkholderia sp.,Cupriavidus sp,Lysinibacillus sp.,in which,39 strains of Bacillus endophytic bacteria,accounting for 37.50%of the total number of isolated endophytic bacteria,were widely distributed in the roots,stems and leaves of Eucommia ulmoides.2.The inhibitory effects of 104 endophytic bacteria on F.graminearum were determined by confront antibiotic culture experiment.The results showed that endophytic bacteria DZSG09,DZSG23 and DZSJ16 had better inhibitory effect on F.graminearum,and the inhibition rates were 66.11%,69.04%and 64.58%respectively.Moreover,pot culture and plot experiment showed that the DZSG23 had the best control efficacy.In plot experiment,strain DZSG23 had 58.80%and 47.26%control effects on wheat varieties of annong 1314 and yangmai 158 respectively.3.The antibacterial mechanism of strain DZSG23 was studied,and the results showed the strain DZSG23 could cause the abnormity and tortuosity of mycelial of phytopathogen and so on,and produce lipopetide metabolites.The secondary metabolites,lipopeptide and bacterial suspension of DZSG23 were found to have lower effects on the conidial germination of F.graminearum,but strong effects on the teratogenicity of pathogen.At 36 h,the deformity rates of conidia of F.graminearum treated with lipopeptide(11.306 mg/mL),suspension of DZSG23 strain(1×106 CFU/mL)and secondary metabolite(2.539 mg/mL)reached 97.63%,57.70%and 20.46%,respectively.The pathogenicity of malformed spores was decreased in different treatments.On the 20th day,the disease index of wheat inoculated with malfored spores,which was treated with the lipopeptide(11.306 mg/mL),suspension of DZSG23 strain 1×106CFU/mL)and secondary metabolites(2.539 mg/mL)were 27.00,30.36 and 25.52 respectively,and the disease indexes of them were significantly lower than that of the blank control(the disease index was 37.12).4.The effect of DZSG23 on DON production abilities of F.graminearum was detected by high performance liquid chromatography(HPLC).It was found that different treatments had effects on DON production abilities of F.graminearum.Compared with the DON content of the blank control(2250.30 μg/g),cyanolide(0.031 mg/mL)had greater influence on DON production abilities of F.graminearum,and the treated DON content of F.graminearum was only 1012.72 μg/g,which was 1237.58 μg/g lower than the blank control.Secondly,F.graminearum treated with secondary metabolites(4.767 mg/mL),the DON production was 1037.24 μg/g.The DON content of F.graminearum after the treatment of DZSG23 suspension(1×106 CFU/mL)was 1046.79 μg/g,which was significantly better than carbendazim treatment.While the lipopeptide has little effect on DON production abilities of F.graminearum,the DON production of F.graminearum was treated with lipopeptide 11.306 mg/mL and 20.876 mg/mL,were 2188.15 μg/g and 1602.52 μg/g respectively.In addition,it was found that the expression of Tri5 gene of F.graminearum in different treatments was positively correlated with its toxin-producing ability.Among them,the expression level of Tri5 gene of F.graminearum was significantly down-regulated after cyanolide(0.031 mg/mL),metabolites(4.767 mg/mL)and DZSG23 suspension treatment.It can be seen that the DZSG23 can effectively control the production of DON by F.graminearum.5.The colonizations of antagonistic bacteria DZSG23 in wheat seedling and spike were detected by antibiotic labeling.It was found that DZSG23 could stably colonize wheat in wheat seedling stage and surface of the spike,but could not enter the wheat spike.On the 34th day after inoculation with DZSG23,the colonization of DZSG23 in roots,stems and leaves of wheat plants at seedling stage reached 1.437×106 CFU/g,3.285×103 CFU/g and 2.377×103 CFU/g,respectively.After 15 days of inoculation with DZSG23 strain on the surface of spike,the surface microbial density per gram of spike tissue still reached 7.15×103 CFU/mL,which formed a harmonious symbiotic relationship with wheat.6.Induction systemic resistance(ISR)signaling pathway studies indicate that DZSG23 can up-regulate the key gene PR-1 of the SA signaling pathway and the key gene AOS of the JA signaling pathway,indicating that the antagonistic DZSG23 introduces wheat to cause salicylic acid(SA)and jasmonic acid(JA)signaling pathways,activates defensive genes in wheat to increase disease resistance.7.The transcriptome sequencing technology was used to analyze the differential expression of total RNA in the spike of different time after treatment with DZSG23.The differentially expressed genes related to disease resistance were screened according to the functional annotations.It was found that there were 38 overlapping genes in treatments at different time periods,mainly encoding Peroxidase,Glycosyltransferase,Pathogenesisrelated proteins,Serine/threonine-protein kinase and other proteins.The results showed that the introduction of antagonistic DZSG23 into wheat could induce the expression of disease resistance genes in wheat spike,which laid a foundation for further testing of disease resistance genes. |
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