| Verticillium dahliae Kleb. can cause vascular wilt, a serious disease of many plant species, including eggplant(Solanum melongena L.) and cotton(Gossypium hirsutum L.)in many areas of the world. Verticillium wilt is one of the major constraints on cotton and eggplant production in China. Because of the special contributions of endophytic bacteria to biological control, it is attractive to use this type of bacteria as an alternative tool to control soilborne vascular wilt disease.An endophytic bacterial strain, Jaas edl,was isolated from the interior of eggplant (Solanum melongena L.)stems. The strain Jaas ed1and its cell-free filtrate had strong antifungal activity against Verticillium dahliae. According to the morphological and physiological characteristics and phylogenetic analysis of the16s rDNA sequence, it was identified as Bacillius subtilis. The sequence data for the16S rDNA gene of the strain Jaas edl has been deposited in GenBank with the accession number EF178293. The assay results using plates showed that endophytic B. subtilis Jaas edl produced some volatiles which strongly inhibited the mycelial growth of V. dahliae. In addition, the strain also had the capacity of producing siderophores and protease, but it didn’t produce chitinase. The characterization of crude extracellular antifungal metabolites indicated that the antifungal activity was thermostable, tolerant to alkalescence pH, untolerant to acid pH, unsensible to proteinase K and trypsin. The crude extracellular antifungal metabolites showed strong antifungal activity against10fungal pathogens and1bacterial pathogen. Observed through optics microscopy, the crude extracellular antifungal metabolites caused cell membrane of Verticillium dahlia swelled and couldn’t retain integrity, and then the intracellular substances leaked out. By approaches of ammonium sulphate precipitation, sephedex G-25molecular sieve chromatography, Cellulose DEAE-52ion-exchange chromatography, FPLC 300SB-C18reversion phase chromatography, antifungal substances were able to be isolated and purified from the culture of endophytic B. subtilis Jaas edl. The purified antifungal substances only showed one band with far lower than6.5KDa molecular weight compared with standard protein on the Tricine-SDS-PAGE. The Q-TOF2mass spectrum of purified antifungal substances revealed that two clusters each containing several molecules with the difference of14Da. According to the molecular weight, we speculated1cluster compounds with the doubly charged ion at [M+H] m/z=718.38,725.40,732.40were homologous compound of Fengycin family, and the other with the doubly charged ion at [M+H] m/z=722.37,729.37,736.39,743.39were homologous compound of surfactin-like compound family. After sequenced the amino acid by Q-TOF2, the compound with the doubly charged ion at [M+H] m/z=718.38was identified as C-14fengycinA.We used the Rif mutant strain of Jaas ed1R instead of strain Jaas edl to study endophytic colonization ability. We inoculated endophytic B. subtilis Jaas ed1R to the eggplant roots, the bacteria could enter the eggplants from the roots and traveled upward to the stems and leaves quickly. The results suggest that the strain prefers to colonize inside the vascular tissues, so it can move to the whole plant quickly. In greenhouse experiments, the strain cell suspension effectively controlled Verticillium wilt of eggplant, and its control efficiency was far more significant than that of the cell-free filtrate after inoculation of V. dahliae. Therefore, not only the antifungal substance produce by the endophytic B. subtilis Jaas edl had the biocontrol potential, but also that the colonization of the bacterial inside plants might be another major factor in their effectiveness in controlling Verticillium wilt. No disease appeared in any eggplant inoculated with endophytic B. subtilis Jaas edl cell suspension or its cell-free filtrate. Further studies are currently underway to test the practical of usage of strain Jaas edl working as a biocontrol agent against Verticillium wilt in a large scale field experiment.In addition, the endophytic bacterial strain Jaas cd (original name73a) against cotton verticillium wilt was identified and its application method for using in field was improved. According to the morphologlcal, physiological characteristics, Biolog identification and phylogenetic analysis, strain Jaas cd was identified as Paenibacillus polymyxa. The sequence data for the16S rDNA gene of the endophytic P. polymyxa Jaas cd has been deposited in GenBank with the accession number AY942618. The strain showed the strong antifungal activity against12fungal pathogens including Verticillium dahliae Kleb, Fusarium oxysporum f. sp. vasinfectum, Fusarium moniliforme and Rhizoctonia solani. Effects of endophytic bacterial strain Jaas cd, antagonistic bacterial strain JB52and fulivic acid on growth,yield and Verticillium wilt of cotton were investigated through single and combine applications, respectively. The results showed that the endophytic P. polymyxa Jaas cd was the best among all treatments. The efficiency of combine application with the strain Jaas cd and strain JB52and combine application with the strain Jaas cd and fulvic acid were weaker than that single application with the strain Jaas cd. Height and fresh weight of individual cotton seedlings was increased up to29.9%,45.2%respectively by the application with the endophytic P. polymyxa Jaas cd in pots. Yield of cotton was increased up to11.5%and the control efficiency against Verticillium wilt of cotton was up to51.17%in the end of July and49.90%at the beginning of September by the application with the endophytic P. polymyxa Jaas cd in field. The results in the field trial experiments showed that the disease indexes of cotton Verticillium wilt were decreased and the yields of cotton were increased by spraying the culture liquid of the endophytic P. polymyxa Jaas cd to cotton seeding beds before transplantation or watering seedling roots after transplantation. Spraying the culture liquid of the endophytic P. polymyxa Jaas cd to the cotton seeding bed before transplantation was more convenient to operate than watering the seedling roots after transplantation. |
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