| In this study, based on the analysis of the population dynamics of endophytic bacteria and antagonistic examination of its toward Fov and Vd in cotton within critical growth period, the endophytic baeterial strains with inhibition toward Verticillium wilt were screened step by step. Then colonization potential of a endophytic baeterial with better biocontrol efficacy toward Verticillium wilt was studied. The research might provide biocontrol agent and give the scientific principle for the control of cotton Verticillium wilt using endophytic bacteria. The main content of this researeh as follows:1.Population dynamics of endophytic bacteria and its antagonism toward Fov and Vd in cotton1.1 Population dynamics of endophytic bacteria in cottonTo explore population dynamics of endophytic bacteria in cotton, root; stem and leaf of cotton samples were surface-disinfested, and were subsequently used to isolate endophytic bacteria by diluting plate counting method. The population dynamics of endophytic bacteria in tissues showed:the population size of endophytic bacteria in root was significantly larger than that in leaf and stem; the populations at seedling stage were generally lower than those at the flowering/maturing stage in root, the populations in stem and leaf were fluctuant at different development stages, but variation law was not observed obviously. There were no significant differences of the population densities among 6 cotton cultivars in root; while the cultivar differences of it existed in stem and leaf. The results revealed that the population dynamics of endophytic bacteria were influenced by diffrent cotton cultivar, growth stage and tissue.1.2 Aantagonism of endophytic bacteria toward Vd and FovTo obtain endophytic bacteria strain antagonistic toward Verticillium dahliae Kleb (Vd), Fusarium oxysporium f.sp. Vasinfectum (Fov) from cotton.We assayed antagonism of the isolated endophytic bacteria (1306 isolates) toward three pathogens:Vd (V107, which is a highly virulent defoliating isolate and V396, which is a mildly virulent non-defoliating isolate), Fov (F108) by dual culture method. A total of 82,226,217 endophytic bacteria isolates were found significantly antagonistic toward V107, V396, F108, respectively.44 endophytic bacteria isolates doubly antagonized toward Both V107, V396 and F108 (DAEB). The proportion of endophytic bacteria antagonistic toward V107, V396, F108 was 12.66%,34.45%,31.66% in root; 1.20%,6.95%, 7.67% in stem; 2.56%,3.41%,4.6% in leaf. The examination of endophytic bacteria antagonistic toward Fov and Vd indicated that the proportion of endophytic bacteria antagonizing Vd and Fov in root was higher than that in stem/leaf, moreover, the amount of endophytic bacteria antagonizing toward V107 was less than that toward V396/F108.2 Molecular identification, antagonistic mchanism and security of DAEB2.1 Molecular identification of DAEB from cottonTo approximately character the categories of DAEB, we analyzed the 16 S rDNA sequence and BOX-PCR fingerprints of 39 DAEB isolates from cotton root. Based on 16S rDNA sequence analysis, All 39 DAEB isolates belong to two phyla: Bacteroidetes and Proteobacteria. Bacteria of the Proteobacteria dominated the collection of isolates, comprising 38 isolates, in which 17 were enterobacter spp. that existed in all root of cotton cultivars; 14 were Pantoea spp.; 4 were Erwinia sp.; 1 was Escherichia sp., Rhizobium sp., Klebsiella sp., Empedobacter sp. for each. Additionally,9 DBAEB isolates demonstrated≤97% sequence similarities with the most similar sequences of strain in the Ribosomal Database Project. These DAEB isolates might be potential novel species. Characterized by BOX-PCR fingerprints, these 39 DAEB isolates represented 35 different cluster types. So we could presume that in root of cotton, not only high diversity of DAEB endophytic bacteria exist, but also its rich types occur.2.2 Identification of HA02To clear category of HA02, we studyed on Its characteristics of morphology, cultivation, physiology, biochemistry, analysed 16S rDNA sequence, phylogenetic tree. Morphological, physiological and biochemical identification showed that HA02 belonged to Enterobacter sp.,16S rDNA sequences of HA02 matched with the most similar sequences of strain in database of EzTaxon server 2.1 were used for further identification, which showed the 16S rDNA sequences of HA02 shared 99.9% homologic with Enterobacter cancerogenus LMG 2693(z96078). and HA02, Enterobacter cancerogenus LMG 2693(z96078), Enterobacter asburiae JCM 6051 T(AB004744) sequencese constituted a branch in phylogenetic tree. Based on these results, it is considered that the HA02 belongs to Enterobacter sp.。3.Antagonistic mchanism and Security of DAEB.To explore the antagonistic mechanisms of DAEB, the agar diffusion method was used to detect extracellular chitinase, cellulase, xylanase, protease, pectinase, and siderophore secretion; colorimetric assay was used to detect for auxin determination. Eighteen out of 39 BAEB isolates showed protease activity; 15 isolates showed chitinases activity; however, cellulase, pectinase activity was detected each for only 1 isolate; xylanase activity was undetected; 10 isolates produced auxins; siderophores were produced for all isolates. The mechanism of DAEB antagonistic toward V107, V396 and F108 were complex, however, excreting siderophores may play a crucial role in itIn order to evaluate security of DAEB, germination experiment was carried out. The cotton seeds were inoculated by the 10 cfu ml-1 suspension of DAEB isolates. By improving seed germination or increasing the length of (Shoot+Root), compared untreated control,9 isolates of the 39 DAEB had significantly high vigor index, conspicuously,7 isolates of the 39 DAEB inhibited the seedling growth significantly after 11 days late. These observations indicated that part of the DAEB isolates had growth promoting potential, and endophytic bacteria may produce plant growth phytohormones.4. Control of cotton Verticillium wilt V107 by DAEB4.1 Control of cotton Verticillium wilt by using DAEB in greenhouse trial.To primarily screen the DAEB that shared biocontrol potential toward V107, the trials of challenging inoculation and pot in greenhouse were carried out. Twelve out of 39 DAEB isolates inhibited Verticillium wilt over 50% in challenging inoculation. Ten DAEB isolates selected from challenging inoculation trial were used in pot trial, in which the isolates of HA02; CK06; and HA03 were effective in reducing Verticillium wilt over 50%. The greenhouse trial suggestted that partial DAEB isolates have biocontrol potential.4.2 Biocontrol efficacy of DAEB isolates HA02, CK06 in field trialTo find biocontrol efficacy of DAEB isolates, we designed drenching and drenching plus injecting treatment with inoculum HA02, CK06 in field trial. The leaf wilt and vascular discoloration indexes of HA02 were 33.3,66.7 with drenching treatment; 31.8,68.0 with drenching plus injecting treatment, the indexes of both the treatments were significantly less than that of control; percentage protection of two treatments were 45.8%,16.3%; 48.3%,14.8%, respectively. However, indexes of both the treatments with CK06 were equivalent to control, which were no any of biocontrol efficacy. Both the treatments of HA02 significantly increased plant height, branch, boll, and weight of boll than that of control; while both the treatments of CK06 had not such efficacy. We also found that there were no significant differences in biocontrol efficacy and characteristics related to yield between drenching and drenching plus injecting treatment. Field trial suggestted that only HA02 had better biocontrol efficacy, which could decrease disease index of Verticillium wilt; increase characteristics related to yield.5.CoIonization of HA025.1 Colonization of HA02-gfp in cottonIn order to explore the reason that HA02 had better biocontrol efficacy to V107, we detected quantitatively and qualitatively colonization of HA02 in cotton. Firstly, using CaCl2 method, we successfully acquired transformant HA02-gfp, that showed strong green fluorescence, stablely resistance to chloromycetin. HA02-gfp had similar growth curve in nutrient broth medium and antagonistic; biocontrol efficacy toward Vd as the parent HA02. Therefore, we researched colonization of HA02-gfp instead of the parent HA02. By seed soaked treatment, the population of HA02-gfp fluctuated between log10 2.74cfu/g- fw and log10 5.2cfu/g f-w in 1-40 day old seedling in root, which tended to increase from 1 to 10 day, then to decreace, but slightly to increase at 30-day-old seedling, and still had log10 2.74cfu/g- fw at 40-day-old seedling. The population of HA02-gfp in stem were significantly lower than that in root, which had also the same trend as that in root, however, could not be found at 40-day-old seedling. As HA02-gfp mainly colonized in cotton root, we only qualitatively studyed colonization aiming at cotton root. Stereoscopic fluorescence microscope showed that HA02-gfp were distributed in maturation zone of primary root, lateral root, juncture of primary and lateral root of 7-day-old treatment seedlings. The observation of HA02-gfp colonization under confocal laser scanning microscopic demonstrated that HA02-gfp mostly colonized in epidermis; big cystiform cavities that were surrounded by thickened-walls cell of cortex anear epidermis; also little colonized in intercellular spaces in maturation zone of cotton primary root of 7-day-old treatment seedlings. HA02-gfp still chiefly colonized in big cystiform cavities as the previous, hardly any of HA02-gfp were found in epidermis in maturation zone of cotton primary root of 12,17-day-old treatment seedlings. The colonization of HA02-gfp in root and stem could explain the reason that HA02-gfp possessed better biocontrol effcacy.5.2Quantitative colonization of HA02-gfp in other hostTo study colonization spectrum, population dynamics of HA02-gfp in root and stem of maize, weat, rice, rape, soybaen seedling were determined by diluting plate counting method. HA02-gfp could be detected in all the plant but rice in stem and root of 1-30 day-old seedling, after these host seeds were soaked. The populations in maize were more richer than others. The populations of these host tended to decreaced with time process. The population of HA02-gfp in root of weat, maize, rape were significantly higher than that in stem, while which in stem of soybaen were higher than in stem. The dynamic populaton of HA02-gfp in other host suggested that the hosts colonized by HA02-gfp were broad-spectrum, the population were influlenced by species, time, tissue.The relationship between endophytic bacteria and its host is a process of long-term evolution together. Endophytic bacteria have stable, protected niche, is saefty to human. As a biocontrol agent, its application will protect environment; reduce contamination caused by chemicals; improve biologic diversity of ecosystem in field;. keep ecological equilibrium. Study of the theory and application that endophytic bacteria control plant disease would not only plays an important role in biocontrol, but also provide a concept for sustainbally agricultural development. |
