Abstract
Cucumber is a common vegetable all over the world; however, its production is seriously affected by cucumber downy mildew caused by Pseudoperonospora cubensis. Endophytes have great potential to control such fungal diseases. The aim of the study was to isolate, screen and identify endophytic bacteria, which show antagonistic activity to downy mildew, in cucumbers. Here, surface sterilized leaves were used for endophytic bacteria isolation. All the isolates were further screened for the antagonistic activities against P. cubensis by leaf discs assay, separate leaves assay and sporangia releasing inhibition assay. The control efficacies of the endophytic bacteria (screened above) were finally evaluated through field tests. In this study, a total of 81 endophytic bacteria were isolated from cucumber leaves sampled at blossoming and fruiting stages, respectively, among which, six (strains CE1, CE8, CE9, Y1, Y2 and Y22) were tested for their strong antagonistic activity in vitro. In the field test, strain CE8, identified as belonging to Bacillus sp. based on 16S rDNA phylogenetic analysis, showed a strong control efficacy of 42.1 %, a value almost two times as high as that using 69 % metalaxyl-mancozeb WG, a widely used antifungal chemical in China. Based on the results above, CE8 was finally screened out for its great potential in cucumber downy mildew control, implicating a powerful value of the strain in the production practice.
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Al-Bader, D., Eliyas, M., Rayan, R., & Radwan, S. (2012). Air-dust-borne associations of phototrophic and hydrocarbon-utilizing microorganisms: promising consortia in volatile hydrocarbon bioremediation. Environmental Science and Pollution Research, 19, 3997–4005.
Anith, K. N., Radhakrishnan, N. V., & Manomohandas, T. P. (2003). Screening of antagonistic bacteria for biological control of nursery wilt of black pepper (Piper nigrum). Microbiological Research, 158, 91–97.
Chun, J., & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. International Journal of Systematic Bacteriology, 45, 240–245.
Cohen, Y. (1981). Downy mildew of cucurbits. In D. M. Spencer (Ed.), The downy mildews (pp. 341–354). New York: Academic.
Cohen, Y., & Rotem, J. (1971). Field and growth chamber approach to epidemiology of Pseudoperonospora cubensis on cucumbers. Phytopathology, 61, 736–737.
Fan, Y. G., Zhu, L. X., & Gong, M. F. (2012). The biological control efficacy of the strains H1 and H2 against cucumber downy mildew. Northern Horticulture, 7, 149–150.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791.
Heery, D. M., Gannon, F., & Powell, R. (1990). A simple method for subcloning DNA fragments from gel slices. Trends in Genetics, 6, 173.
Iwata, Y. (1949). Studies on the invasion of cucumber plants by downy mildew. Annals of Phytopathological Society of Japan, 13, 60–61.
Kumar, N., & Singh, K. P. (2011). Use of Dactylaria brochopaga, a predacious fungus, for managing root-knot disease of wheat (Triticum aestivum) caused by Meloidogyne graminicola. Mycobiology, 39, 113–117.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In E. Stackebrandt & M. Goodfellow (Eds.), Nucleic acid techniques in bacterial systematics (pp. 115–175). Chichester: Wiley.
Li, Q. Q., Lu, J. Y., Lin, W., Chen, Y. N., Lu, T. J., Xie, Y. L., et al. (2004). Isolation of pepper endophyte antagonists against Ralstonia solanacearum. Journal of Guangxi Agricultural and Biological Science, 23, 304–306.
Malfanova, N., Kamilova, F., Validov, S., Chebotar, V., & Lugtenberg, B. (2013). Is L-arabinose important for the endophytic lifestyle of Pseudomonas spp.? Archives of Microbiology, 195, 9–17.
Miao, Z. Y., Zhao, K. H., Liu, C. Y., Liang, C. H., Wang, H., & Lv, G. Z. (2009). Identification of the cucumber endobacterial strain B504 and its biocontrol effects against the cucumber wilt. Plant Protection, 35, 73–77.
Palti, J., & Cohen, Y. (1980). Downy mildew of cucurbits (Pseudoperonospora cubensis): the fungus and its hosts, distribution, epidemiology, and control. Phytoparasitica, 8, 109–147.
Pastor, N., Carlier, E., Andrés, J., Rosas, S. B., & Rovera, M. (2012). Characterization of rhizosphere bacteria for control of phytopathogenic fungi of tomato. Journal of Environmental Management, 95, S332–S337.
Pimenta, R. S., Silva, J. F., Buyer, J. S., & Janisiewicz, W. J. (2012). Entophytic fungi from plums (Prunus domestica) and their antifungal activity against Monilinia fructicola. Journal of Food Protection, 75, 1883–1889.
Rabeeth, M., Anitha, A., & Srikanth, G. (2011). Purification of an antifungal endochitinase from a potential biocontrol agent Streptomyces griseus. Pakistan Journal of Biological Science, 14, 788–797.
Rainey, F. A., Ward, R. N., Kroppenstedt, R. M., & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. International Journal of Systematic Bacteriology, 46, 1088–1092.
Shimizu, M., Yazawa, S., & Ushijima, Y. (2009). A promising strain of endophytic Streptomyces sp. for biological control of cucumber anthracnose. Journal of General Plant Pathology, 75, 27–36.
Sorkhoh, N. A., Ali, N., Dashti, N., Mailem, D. M. A., Awadhi, H. A., Eliyas, M., et al. (2010). Soil bacteria with the combined potential for oil utilization, nitrogen fixation, and mercury resistance. International Biodeterioration and Biodegradation, 64, 226–231.
Sun, Z. B., Zhang, H., Yuan, X. F., Wang, Y. X., Feng, D. M., Wang, Y. H., et al. (2012). Luteimonas cucumeris sp. nov., isolated a from cucumber leaf. International Journal of Systematic and Evolutionary Microbiology, 62, 2916–2920.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.
Thomas, C. E. (1996). Downy mildew. In T. A. Zitter, D. L. Hopkins, & C. E. Thomas (Eds.), Compendium of cucurbit diseases (pp. 25–27). Paul: APS Press.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882.
Urban, J., & Lebeda, A. (2006). Fungicide resistance in cucurbit downy mildew-methodological, biological and population aspects. Annals of Applied Biology, 149, 63–75.
Yumoto, I., Hirota, K., Yamaga, S., Nodasaka, Y., Kawasaki, T., Matsuyama, H., et al. (2004). Bacillus asahii sp. nov., a novel bacterium isolated from soil with the ability to deodorize the bad smell generated from short-chain fatty acids. International Journal of Systematic and Evolutionary Microbiology, 54, 1997–2001.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 31170035) and the Natural Science Foundation of Shandong (No. ZR2010CQ042). We thank Mr. Yunfu Wang for providing cucumber plants and Dr. Wenqiao Wang and Runjie Meng (Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences, Baoding, China) for their technical assistance. We also thank Ms Lauren Stirling (Chinese Academy of Agricultural Sciences) and Dr. Kai Ding (Department of Civil and Environmental Engineering, Villanova University, USA) for their assistance in the correction of the manuscript.
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Sun, ZB., Yuan, XF., Zhang, H. et al. Isolation, screening and identification of antagonistic downy mildew endophytic bacteria from cucumber. Eur J Plant Pathol 137, 847–857 (2013). https://doi.org/10.1007/s10658-013-0293-5
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DOI: https://doi.org/10.1007/s10658-013-0293-5