| Bell pepper blight caused by Phytophthora capsici L. is one of the most important diseases of pepper growing in open and greenhouse in China. Application of biocontrol agent could reduce application quantity of chemicals for controlling plant disease and delay the development of fungicide resistance in pathogen population. However, development of biocontrol was limited because of the instability of biocontrol efficacy. A new control measure, which combined fungicide with biocontrol agent for disease, was attempted to develop. The approach could solve both problem of chemicals and shortage of biocontrol. This paper carried out a series of experiments on Phytophthora capsici as a target pathogen suppressed by antagonistic microbes, which screened, identificated. Additionally, it was involved in mechanism of biocontrol, combination of antagonistic agent and fungicide, and impact of heavy metal ions on antagonistic agent. The results showed as followed:Antagonistic microbes from soil were screened with dual incubation in vitro. One hundred five isolates were collected. Thirteen antagonistic agents from these were bioassayed in dual incubation suppressing seven pathogens, such as Phytophthora capsici, Collectotrichum capsici, Botrytis cinerea, Alternaria solani, Fusarium oxysporum f. sp. lycopersici, Fusarium graminearum and Mycosphaerella melonis. The results indicated that antagonistic activitiy of A001 was the strongest among 5 antinomycetes and B100 was a best bacterium isolate.In colonization experiment, antagonistic bacterium B100 could colonize along root surfacees of pepper successfully though its efficiency controlling pepper blight was lower than B148. Colonies of regained from root of pepper reached to 7.06×105 cfu/g (root weight) after 20 days of inoculation. The colonization of B148 was much less than that of B100 isolate.Thereby, isolate B100 was determined as a candidate for biocontrol P. capsici according to a screening method of suppression-colonization. The identification of B100 was based on the morphological characteristics, physiological and biochemical characteristics and analysis of 16S rDNA sequence comparison. The results indicated that B100 was a spore producing bacilliform bacterium, which owed to aerobic or facultative anaerobic, gram positive. It was the most similar with Paenibacillus polymyxa on physiological and biochemical characteristics. The 16S rDNA sequence of B100 possessed high identities compared with Paenibacillus polymyxa GB-465 that its accession number was AY359623 on GenBank, and identities reached to 99.9%. Similarly, B100 mostly closed to Paenibacillus polymyxa in Phylogenetic tree. Therefore, B100 was identified as a bacterium of Paenibacillus polymyxa.The growth condition of B100 and its antagonism to P. capsici were studied in vitro. The results showed that the PD medium, beef extract-yeast extract peptone sucrose broth or NA broth were optimum to growth of B100. The optimum original pH values of the medium broth were from 6.0 to 8.0 in 28℃. In antagonism test, the antifungal rank as followed:B100 suspension> sterilized suspension> filtrate. Light microscopic studies clearly showed the effect of B100 on hyphal morphology of P. capsici. Exposure to B100, phytopathogen produced swelled or increased divaricator hyphae, abnormal contraction of cytoplasm, lysis of hyphae and cell.The variations in three defense enzymes activities were determined in leaf and root of the 5-6 leaf-old seedlings of bell pepper, which had been treated with B100 at concentration of 108 cfu/ml. The results indicated that the activities of peroxidase and polyphenol oxidase were significantly induced by B100 in both leaf and roots of bell pepper. However, the activities of phenylalanine ammonia-lyase in leaf and root were no obviously induced by B100 compared with untreated plant.The joint action between B100 and Amistar (azoxystrobin), against P. capsici was determined in vitro. The synergistic effect each of Azoxystrobin and B100, which concentration was beyond 102 cfu/ml, suppress hyphae growth of P. capsici in that the inhibitions of observed was higher than those of expected. The same effect was observed based on isobole of 90% inhibition. The combined Azoxystrobin or metalaxyl with B100 against bell pepper Phytophthora blight would provide more effectiveness than fungicide alone respectively in field.The impacts of three fungicides and six heavy metal ions on both mycelium growth of Phytophthora capsici and reproduction of B100 were determined in vitro. The results showed that inhibition of flumorph or mefenoxam on mycelium growth of P. capsici was much more than that of azoxystrobin, and there was no inhibition on B100. Only the inhibition of Ag+ or Cu2+ at concentration of 10mg/L amending PDA media on growth of P. capsici was observed in six heavy metal ions. In experiment for B100, There were no obviously differences in the bacteria concentration of B100, which had been respectively treated by Cu2+, Bi3+ and Pb2+, compared with control. The concentrations of B100 in nutrient broth amended 0.1-1 mg/L of Cd2+ and 0.5mg/L Ag+ were obviously higher than that of control. However, the concentrations of B100 in broth amended Hg2+ at 0.5-1.0 mg/L was obviously lower than that of control. Then, heavy metal ions may decrease efficacy of biocintrol agent B100 in the field soil polluted by the ions. |
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