| Bacterial wilt of tomato (BWT), a destructive soilborne disease caused by Ralstonia solanacearum, is a threat to tomato production and economical benefit. No effective measures have been developed yet. Environmentally friendly pesticide alternatives such as biolgical control have been suggested as integrated control strategies for BWT. Many beneficial microorganisms (BMs) have been screened for suppression of this disease; however, bio-products based on these BMs that could stably and efficiently control the disease are scare. In present study, BMs such as Bacillus amyloliquefaciens QL-18and Ralstonia pickettii QL-A6were isolated from the rhizosphere soil of healthy tomato plants. A bio-organic fertilizer (BOF) fortified with B. amyloliquefaciens QL-18and a liquid bio-product based on R. pickettii QL-A6were developed for suppression of BWT. A serial of experiments were conducted to test their control efficacies of BWT under greenhouse and field conditions. The possible control mechanisms were also discussed.The main results obtained were listed as follows:1. Field surveys on the epidemiolgy of BWT were undertaken in major tomato growing districts of the Qilin town, China. Disease incidences varied along with the crop seasons (spring and autumn) and previous crops (host and non-host plants). The pathogen was frequently detected from plant, soil and irrigation canals, not from irrigation water reservoir. Two phenotypically different colonies, fluidal (virulent R. solanacearum-Wke) and afluidal (avirulent R. solanacearum-liko) types were obtained from the rhizosphere soil using a modified semi-selective medium, South Africa (M-SMSA). Based on the results of the morpholgical characteristics, physiolgical and biochemical tests, amplication of R. solanacearum-specific genes,16S rRNA gene sequence and pathogenicity tests, the fluidal types were conformed as virulent R. solanacearum strains; and the afluidal types belong to R. pickettii and R. mannitolilytica.2. Thirty-seven out of503rhizobacteria isolated from the rhizosphere soil of healthy tomato plants showed inhibitory effects against R. solanacearum strain QL-Rs1115. B. amyloliquefaciens QL-18performed best in suppression of R. solanacearum in vitro and efficiently reduced the disease incidences and populations of R. solanacearum in the rhizosphere soil and crown of tomato plants growing in both of paddy soil and soil continuously cropped with tomato plants. B. amyloliquefaciens QL-18has the capacities of producing antibiotics, such as fengycin, subtilisin and iturin, and inhibiting the growth of the phytopathogens including R. solanacearum, R. solani, P. capsici, P. nicotianae, F. oxysporum f. sp. Niveum, F. oxysporum f. sp. Cucumuerimu, F. oxysporum f. sp. melonis and V. dahliae Kleb. B. amyloliquefaciens QL-18successfully colonized the surface of tomato roots; this was conformed by labeling B. amyloliquefaciens QL-18with gfp and detecting the fluorescent cells under a fluorescence microscope.3. Six composts, i.e., rapeseed cake compost (RCC), pig manure compost (PMC), mixture (1:1w/w) of RCC and PMC (R&P), chicken manure compost (CMC), cow dung compost (CDC), herb residue compost (HRC), and two peats, i.e., mosspeat (MP) and fenpeat (FP) were used as carriers of B. amyloliquefaciens QL-18. The results showed that RCC was the best carrier for B. amyloliquefaciens QL-18, because (1) B. amyloliquefaciens QL-18survived best in RCC, which could supply B. amyloliquefaciens QL-18with enough dissolvable organic carbon and nitrogen for multiplication;(2) RCC inhibited the growth of R. solanacearum. B. amyloliquefaciens QL-18could also survive well in the carrier FP, but the pathogen R. solanacearum survived in FP with large population (>105cfu/g dc) for more than60days, while R. solanacearum in RCC could not be detected15days after conservation. Thus, FP is unsuitable to be a carrier of a BM antagonistic against R. solanacearm.4. The effect of BOF, a bio-product of RCC fortified with B. amyloliquefaciens QL-18was tested on suppression of BWT under both of the greenhouse and field condition. The best biocontrol efficiency was obtained by application of BOF into substrate during the nursery phase of tomato seedling followed by a second application to pot soil when seedlings were transplanted in greenhouse. However, the suppressiveness in field depended on the crop seasons. Disease incidence in the spring crop season was significantly reduced, while a low suppressive effect was observed in the autumn crop season. Disease incidences were significantly and positively correlated with the initial (before transplanting) R. solanacearum population in the soil.5. Due to the good performances in colonization of the rhizosphere soil and stem of tomato, R. pickettii QL-A6was selected and developed as a liquid bio-product for suppression of R. solanacearum by use of the soil drench (SD) and stem injection (SI) methods in greenhouse. By the SI method, disease incidence was reduced by71.2%on the average with an inoculation dosage only about105cfu of R. pickettii QL-A6per plant. By the SD method, disease incidence was reduced by52.9%on the average but needed inoculation dosage was as high as about109cfu of R. pickettii QL-A6per plant. Thus, the SI method was chosen further test in field. The field disease incidence in R. pickettii QL-A6treated plots was8.8%at harvest time, while that in the sterilized water treated plots was as high as33.1%. It is concluded that direct injection of R. pickettii QL-A6in stem of tomato could be an alternative to chemical pesticides for biocontrol of R. solanacearum.6. The effects of application of BOF and injection of R. pickettii QL-A6alone or in combination on suppression of BWT were investigated under greenhouse and field conditions. The results showed that combined treatment achieved better control effects (70-86%). The control efficacies of application of BOF and R. pickettii QL-A6alone altered along with the crop seasons in field. The control efficacies of BOF treatment was high (54.7%) in spring crop season, while was low (30.5%) in fall crop season. The control efficacies of stem injection of R. pickettii QL-A6was high (80.0%) in fall crop season, while was low (31.6%) in spring crop season. As BOF was amended with the seedling substrate, B. amyloliquefaciens QL-18colonized in tomato rhizosphere with large population (>106cfu/g dw) and reduced the population of R. solanacearum in the rhizosphere soil. The delay of multiplication of R. solanacearum in the aboveground parts of plants may due to the systemically colonization of both shoots and roots after R. pickettii QL-A6cells were directly injected into the plant stem, in which R. pickettii QL-A6may form a series of biofilms or other barriers and compete against the pathogen for spaces and nutrients. |
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