| Cucumber downy mildew caused by Pseudoperonospora cubensis (Berk. and Curt.) Rostov) is one of the major global cucumber diseases which occurred in about 70 countries. At present the efficient measures to protect cucumber from Pseudoperonospora cubensis are planting resistant varieties and controlling the disease by chemical agents. The duration of chemical agents became more and more shorter even 3 to 4 days, and the pesticide residues is also seriously critical. Therefore, biocontrol as a alternative is a promising method to control Pseudoperonospora cubensis on cucumber.1. Isolation and screening of the antagonistic bacteria toward Pseudoperonospora cubensis on cucumberThe tested antagonistic bacteriacame from the cucumber-associated antagonist isolated in this experiment and the pool of Biological Control Agents (BCAs) in the lab.One hundred and seventy three bacteria strains were isolated from different cucumber microenvironment including rhizosphere soil, phyllosphere and endogenous leaves. The activity of each strain was measured as followed:activity of protease, chitinase and cellulose, and the production of addicted ferrite. Assignment value was given to each strain according to their enzyme activity. Based on assignment and cluster analysis,22 potential biocontrol agents were screened out for greenhouse and field experiments.72 bacterial strains in the BCA library were selected to develop the greenhouse experiment. The biocontrol efficiency of these bacteria ranged from 35.36 to 91.19% at 15 d post inoculation of pathogen. The 30 antagonistic bacteria initially spray on the cucumber leaves 8,15,22,27 d, the disease severity were significantly lower than the control group in the nature field of 2010. The control efficiency of 13 agents against cucumber downy mildew is 35.64～65.59% in 2011 when compared with the water control.lt shows that there are three single strains 1BS4, DYW14、3BY4 and a mixture CB28 have a high and stable biocontrol.The 11 antagonistic bacterial (108 CFU/mL) are sprayed on cucumber leaves 20 d later, the data shows that the plant height, leaf area, fresh weight were higher than control,and plant biomass growth increase 3.08-35.07%. The correlation analysis showed that there is a positive correlation coefficient of 0.51 between the total activity of flat and colonization ability and greenhouse control efficacy.2. The colonization of antagonists in cucumber and the phyllosphere bacteria diversity analysisUsing rifampicin as a selection marker, the colonization ability of antagonistic bacteria in cucumber plants were determined under greenhouse and field conditions. For all tested agents in different varieties, there is a similar colonization curve "low-high-low" until keeping stable no matter in the cucumber leaves or rhizosphere soil. For each bacteria in one variety, the cfu of bacteria in the cucumber leaves were always lower than that in the rhizosphere soil, especially in the 30 d.The colonization of the combination CB28 in the cucumber "Late Autumn" is similar with the single bacterial, there is no significantly completion between 3BS3 and 3BJN7.Under the field condition, the colonization of different antagonists were affected by chemicals. About 21 days post treatment, this effect by chemicals decreased even disappeared. The pathogen also has an impact on the colonization of antagonists, especially at the 7 days post treatment.At the same time,the antagonists can significantly improve the cucumber chlorophyll content of leaf. Thesoluble sugar, soluble protein and vitamin C content of fruits treated by antagonists significantly increased than that of the control.Based on the above results,1BS4 is a promising antagonist with the high and stable biocontrol efficacy and the improvement of fruit production and quality. After the strain was sprayed on cucumber leaves, it indicates that 1BS4 improves the cucumber phyllosphere priority of bacteria at 30 d post treatment by the PCR-DGGE analysis.3. Biocontrol of cucumber downy mildew and plant growth promotion by PopW in cucumber under greenhouse and field conditionsIn previous study, PopW, as a new harpin protein, isolated from the bacterium Ralstonia solanacearum ZJ3721, showed induced resistance to various plant diseased. In this study, we investigated and evaluated the biocontrol efficacy of Pop W against cucumber downy mildew caused by Pseudoperonospora cubensis. There are no inhibition between PopW and 12 bacterial by Oxford cup method.12 bacterial-PopW combination and 4 different concentrations of PopW were selected to developthe greenhouse experiments. The PopW at the concentration of 250 mg/L obtained the best biocontrol efficacy of 42.85% at the 15th day after pathogen inoculation under the greenhouse experiment. Meanwhile, PopW (250 mg/L) promoted the growth of cucumber and increased the content of chlorophyll in leaves of the plant. In the field experiments, the PopW was applied each 15 days with the same concentration in 2010, and used three times in total. The biocontrol efficacy of PopW (250 mg/L) reached 41.87%,48.36%,53.21% and 50.85% at the 8, 25, 35 and 48 d after first treatment of PopW, respectively. Based on no significant difference between PopW and its combination of 5BJ1, PopW was just used to do the growth promoting in the greenhouse and field biocontrol experiments in the 2011. The same concentration of PopW was applied every 10 days, and the biocontrol efficacy of it reached 47.07%,48.17%,50.59% and 54.48% at the day of 8,15,22 and 27 d after first application of PopW, respectively. The results indicated that the PopW has the potential to be an effective biocontrol agent against Pseudoperonospora cubensis and the resistance induced by PopW could keep 10-15 even more days. Furthermore, PopW both increased cucumber yield with 33.01% per individual plant and significantly improved quality of cucumber fruit with the increased content of soluble sugar, soluble protein, free amino acids and vitamin C.4. ConclusionIn this study, a few strains of the potential to control cucumber downy mildew are found through a series of indoor and outdoor screening strategies. They can keep the control efficacy stability in the field if it sprays on the cucumber leave intervals 7 to 10 d once time. The test of different strains colonization ability shows that they own a good adaptability on the plant.And they also can promote the cucumber growth and improve the quality of the fruit. The bacteria 1BS4 (Bacillus cereus) can imorove cucumber microbial community structure. In addition we also found that PopW has an excellent effect on the cucumber. |
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