Biocontrol Technology Of Soil-Borne Disease Of Potato Bacterial Wilt And Its Mechanism Of Action

Potato bacterial wilt(Ralstonia solanacearum) is a soil-borne disease that seriously suppressed the potato plant(Solanum tuberosum) growth worldwide and causes serious economic losses. A lot of methods have been used to prevent the disease, among which biocontrol has been proved to be the most promising one. Biological control is one of the important measures for overcoming soil-borne diseases, because of its effectiveness and environment-friendly use. Screening antagonists against pathogens is the vital work for the biocontrol of plant diseases.In this study, two bacterial strains LH23(Bacillus amyloliquefaciens) and LH36(Bacillus subtilis) with antagonistic activity against R. solanacearum, were selected from the rhizosphere soil of healthy potato plant grown in the field strongly diseased by bacterial wilt. Pot and field experiments were conducted to evaluate the LH23(Bacillus amyloliquefaciens) and LH36(Bacillus subtilis) strains and their derived bio-organic fertilizers (BIO23and BIO36) as potential biocontrol agents against potato bacterial wilt and the related mechanisms were also investigated. The main results obtained are listed as follows:(1) In this study, more than140bacterial strains (bacterial antagonists against R.solanacearum) were isolated from rhizosphere soil of potato, tomato, eggplant and banana. Cell-free filtrate of six antagonistic strains had much stronger antagonistic activity against four R. solanacearum strains with inhibitory zones over20mm. Based on antagonistic activity against four R. solanacearum strains and ten soil-borne fungal pathogens as well as biocontrol efficacy in the greenhouse, two bacterial strains LH23and LH36were selected out of many candidates as potential biocontrol agents.(2) Based on the results of morphologic characteristics, physiological and biochemical properties and phylogenetic analysis of16S rRNA, the strains LH23and LH36, were identified as Bacillus amyloliquefaciens and Bacillus subtilis, respectively. Measurement from laboratory tests showed that the LH23and LH36were able to produce growth-promoting substances indole acetic acid (IAA), siderophore, prolease, amylase, glucanase, antagonism of extracellular protein and dissolve P in medium. And the LH23and LH36could decrease the content of extracellular Polysaeeharides produced by R. solanacearum, suggesting that LH23and LH36were effective antagonists against the pathogen of potato bacterial wilt.(3) In order to find a suitable antagonist inoculation method, we compared the methods of root-dipping with soil-drenching in the aspects including rhizocompetence, and effect of promoting plant growth under greenhouse conditions. The drenching treatment resulted in a higher rhizocompetence and plant-yield increase, and this method was also easier to operate in the field on a large scale. Pot and field experiments were performed to investigate the promoting effects of LH23(Bacillus amyloliquefaciens) and LH36(Bacillus subtilis) strains and their derived bio-organic fertilizers (BIO23and BIO36) in potato growth. In greenhouse experiment, BIO23and BIO36significantly promoted potato plant growth, especially the root growth. The root fresh weight of BIO23and BIO36treatments were1.8and2.1times higher than those of control, respectively. The root lengths, root SurfArea, root AvgDiam and RootVolume of the plants treated with BIO23and BIO36were significantly higher than those of control. The potato yield increments on85days after the application of BIO23and BIO36were17.7%and10.8%, respectively as compared with OF (organic fertilizer) treatment in the field experiments.(4) Application of BIO23and BIO36decreased the incidence of bacterial wilt disease and increased potato yields. In one greenhouse experiment, the biocontrol efficiency of BIO23and BIO36applied were100%and100%, which were the mostly successful treatment while the application of the strains LH23and LH36alone resulted in a biocontrol efficiency of85.1%and81.1%, respectively. In another greenhouse experiment, the biocontrol efficiency of BIO23was84.6%, which was the most successful treatment and BIO36was the second in terms of the biocontrol efficiency (80.8%); the increased percentages of potato yields when compared with the control (chemical fertilizer) were63.5%(BIO23),64.7%(BIO36)34.8%(LH23),33.6%(LH36) and20.7%(OF). In field experiments, the biocontrol efficacies of BIO23and BIO36treatments were92.0%and84.0%, and the yield increases of BIO23and BIO36treatments were42.3%and28.8%, respectively, when compared with the OF treatment.(5) Bio-organic fertilizers (BIO23and BIO36) could alter the microbial community structure and improved plant health. The counts of bacteria and actinomyces in the rhizosphere soils were significantly increased in BIO23and BIO36treatments, whereas the fungi in the soil in the both treatments were decreased; the counts of antagonists, bacteria and actinobacteria in the rhizosphere soil were significantly increased in BIO23and BIO36treatments, whereas the counts of R. solanacearum and fungi in the soil in the both treatments were decreased in the greenhouse experiments. In addition, the changes in the microbial populations in field experiments were the same as those observed in the greenhouse experiment. In the pot experiment, Bio-organic fertilizers (BIO23and BIO36) significantly increased the activities of plant defence enzymes such as POD, CAT, SOD and PPO, in plant leaves, and decreased MDA content, and consequently relieved the symptoms of potato.In conclusion, our studies showed that the bio-organic fertilizer, an appropriate combination of organic fertilizer (OF) with the antagonists LH23or LH36used in the present study, could effectively suppress the potato wilt, promote the growth of potato plants and thus the quality of potato tubers, which mainly depends on producing many antibiotics and growth-promoting substances and the alternation of soil microbial community, after the successful colonization of the strains in soils. Results in this study would be useful in the developping new methods to better control potato bacterial wilt disease. Further researches should be done in the molecular microbilogical aspects of the functions performed by bio-organic fertilizers in remediation of continuously cropping obstacles and in the promotion of plant growth and health exerted by functional microbes added.