Biological Control Potential of Bacillus amyloliquefaciens KB3 Isolated from the Feces of Allomyrina dichotoma Larvae

Nam, Hyo-Song;Yang, Hyun-Ju;Oh, Byung Jun;Anderson, Anne J.;Kim, Young Cheol

  • Received : 2015.12.24
  • Accepted : 2016.02.20
  • Published : 2016.06.01


Most biocontrol agents for plant diseases have been isolated from sources such as soils and plants. As an alternative source, we examined the feces of tertiary larvae of the herbivorous rhino beetle, Allomyrina dichotoma for presence of biocontrol-active microbes. The initial screen was performed to detect antifungal activity against two common fungal plant pathogens. The strain with strongest antifungal activity was identified as Bacillus amyloliquefaciens KB3. The inhibitory activity of this strain correlated with lipopeptide productions, including iturin A and surfactin. Production of these surfactants in the KB3 isolate varied with the culture phase and growth medium used. In planta biocontrol activities of cell-free culture filtrates of KB3 were similar to those of the commercial biocontrol agent, B. subtilis QST-713. These results support the presence of microbes with the potential to inhibit fungal growth, such as plant pathogens, in diverse ecological niches.


Allomyrina dichotoma;biosurfactants;cyclic lipopeptides;growth medium;insect feces


  1. Arguelles-Arias, A., Ongena, M., Halimi, B., Lara, Y., Brans, A., Joris, B. and Fickers, P. 2009. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb. Cell Fact. 8:63.
  2. Bajpai, V. K. and Kang, S. C. 2012. In vitro and in vivo inhibition of plant pathogenic fungi by essential oil and extracts of Magnolia liliflora Desr. J. Agr. Sci. Tech. 14:845-856.
  3. Ben Khedher, S., Boukedi, H., Kilani-Feki, O., Chaib, I., Laarif, A., Abdelkefi-Mesrati, L. and Tounsi, S. 2015. Bacillus amyloliquefaciens AG1 biosurfactant: putative receptor diversity and histopathological effects on Tuta absoluta midgut. J. Invertebr. Pathol. 132:42-47.
  4. Bodour, A. A. and Miller-Maier, R. M. 1998. Application for a modified drop-collapse technique for surfactant quantitation and screening of biosurfactant-producing microorganisms. J. Microbiol. Methods 32:273-280.
  5. Breznak, J. A. and Brune, A. 1994. Role of microorganisms in the digestion of lignocellulose by termites. Annu. Rev. Entomol. 36:453-487.
  6. Brune, A. 2003. Symbionts aiding digestion. In: Encyclopedia of insects, eds. by V. H. Resh and R. T. Carde, pp. 1102-1107. Academic Press, New York, NY, USA.
  7. Buensanteai, N., Yuen, G. Y. and Prathuangwong, S. 2008. The biocontrol bacterium Bacillus amlyliquefaciens KPS46 produces auxin, surfactin and extracellular proteins for enhanced growth of soybean plant. Thai J. Agri. Sci. 41:101-116.
  8. Cawoy, H., Bettiol, W., Fickers, P. and Ongena, M. 2011. Bacillus-based biological control of plant diseases. In: Pesticides in the modern world: pesticides use and management, ed. by M. Stoytcheva. InTech, Rijeka, Croatia.
  9. Chen, X. H., Koumoutsi, A., Scholz, R., Eisenreich, A., Schneider, K., Heinemeyer, I., Morgenstern, B., Voss, B., Hess, W. R., Reva, O., Junge, H., Voigt, B., Jungblut, P. R., Vater, J., Süssmuth, R., Liesegang, H., Strittmatter, A., Gottschalk, G. and Borriss, R. 2007. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat. Biotechnol. 25:1007-1014.
  10. Chen, Y., Yan, F., Chai, Y., Liu, H., Kolter, R., Losick, R. and Guo, J. H. 2013. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation. Environ. Microbiol. 15:848-864.
  11. Choudhary, D. K. and Johri, B. N. 2009. Interactions of Bacillus spp. and plants--with special reference to induced systemic resistance (ISR). Microbiol. Res. 164:493-513.
  12. Chowdhury, S. P., Hartmann, A., Gao, X. and Borriss, R. 2015. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42: a review. Front Microbiol. 6:780.
  13. Dietel, K., Beator, B., Budiharjo, A., Fan, B. and Borriss, R. 2013. Bacterial traits involved in colonization of Arabidopsis thaliana roots by Bacillus amyloliquefaciens FZB42. Plant Pathol. J. 29:59-66.
  14. Ernst, R., Arditti, J. and Healey, N. P. 1971. Biological effects of surfactants. I. Influence on the growth of orchid seedlings. New Phytol. 70:457-475.
  15. Gardener, B. B., Kim, I. S., Kim, K. Y. and Kim, Y. C. 2014. Draft genome sequence of a chitinase-producing biocontrol bacterium, Lysobacter antibioticus HS124. Res. Plant Dis. 20:216-218.
  16. Ghribi, D. and Ellouze-Chaabouni, S. 2011. Enhancement of Bacillus subtilis lipopeptide biosurfactants production through optimization of medium composition and adequate control of aeration. Biotechnol. Res. Int. 2011:653654.
  17. Gregersen, T. 1978. Rapid method for distinction of gram-negative from gram-positive bacteria. Eur. J. Appl. Microbiol. Biotechnol. 5:123-127.
  18. Ha, B. D. 2013. Isolation of antagonistic microorganism against plant pathogen from feces of Allomyrina dichotoma larva and large scale fermentation of Bacillus amyloliquefaciens KB3. M.S. thesis. Chonnam National University, Gwangju, Korea.
  19. Hornby, D. 1983. Suppressive soils. Ann. Rev. Phytopathol. 21:65-85.
  20. Kang, B. R. 2012. Biocontrol of tomato Fusarium wilt by a novel genotype of 2,4-diacetylphloroglucinol-producing Pseudomonas sp. NJ134. Plant Pathol. J. 28:93-100.
  21. Kim, J. C., Choi, G. J., Park, J. H., Kim, H. T. and Cho, K. Y. 2001. Activity against plant pathogenic fungi of phomalactone isolated from Nigrospora sphaerica. Pest. Manag. Sci. 57:554-559.
  22. Kim, P. I., Ryu, J., Kim, Y. H. and Chi, Y. T. 2010. Production of biosurfactant lipopeptides iturin A, fengycin and surfactin A from Bacillus subtilis CMB32 for control of Colletotrichum gloeosporioides. J. Microbiol. Biotechnol. 20:138-145.
  23. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.
  24. King, E. O., Ward, M. K. and Raney, D. E. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab Clin. Med. 44:301-307.
  25. Kupferschmied, P., Maurhofer, M. and Keel, C. 2013. Promise for plant pest control: root-associated pseudomonads with insecticidal activities. Front. Plant Sci. 4:287.
  26. Lee, B., Lee, S. and Ryu, C. M. 2012. Foliar aphid feeding recruits rhizosphere bacteria and primes plant immunity against pathogenic and non-pathogenic bacteria in pepper. Ann. Bot. 110:281-290.
  27. Li, H., Soares, M. A., Torres, M. S., Bergen, M. and White J. F. Jr. 2015. Endophytic bacterium, Bacillus amyloliquefaciens, enhances ornamental hosta resistance to diseases and insect pests. J. Plant Interact. 10:224-229.
  28. Martin, P. A. and Travers, R. S. 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl. Environ. Microbiol. 55:2437-2442.
  29. Mendes, R., Kruijt, M., de Bruijn, I., Dekkers, E., van der Voort, M., Schneider, J. H., Piceno, Y. M., DeSantis, T. Z., Andersen, G. L., Bakker, P. A. and Raaijmakers, J. M. 2011. Deciphering the rhizosphere microbiome for disease-suppressive bacteria. Science 332:1097-1100.
  30. Mezghanni, H., Khedher, S. B., Tounsi, S. and Zouari, N. 2012. Medium optimization of antifungal activity production by Bacillus amyloliquefaciens using statistical experimental design. Prep. Biochem. Biotechnol. 42:267-278.
  31. Moran, N. A., Russell, J. A., Koga, R. and Fukatsu, T. 2005. Evolutionary relationships of three new species of Enterobacteriaceae living as symbionts of aphids and other insects. Appl. Environ. Microbiol. 71:3302-3310.
  32. Nadarasah, G. and Stavrinides, J. 2011. Insects as alternative hosts for phytopathogenic bacteria. FEMS Microbiol. Rev. 35:555-575.
  33. Nihorimbere, V., Cawoy, H., Seyer, A., Brunelle, A., Thonart, P. and Ongena, M. 2012. Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499. FEMS Microbiol. Ecol. 79:176-191.
  34. Nitschke, M. and Pastore, G. M. 2006. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresour. Technol. 97:336-341.
  35. Olcott, M. H., Henkels, M. D., Rosen, K. L., Walker, F. L., Sneh, B., Loper, J. E. and Taylor, B. J. 2010. Lethality and developmental delay in Drosophila melanogaster larvae after ingestion of selected Pseudomonas fluorescens strains. PLoS One 5:e12504.
  36. Ongena, M. and Jacques, P. 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 16:115-125.
  37. Park, J. Y., Oh, S. A., Anderson, A. J., Neiswender, J., Kim, J. C. and Kim, Y. C. 2011. Production of the antifungal compounds phenazine and pyrrolnitrin from Pseudomonas chlororaphis O6 is differentially regulated by glucose. Lett. Appl. Microbiol. 52:532-537.
  38. Pérez-García, A., Romero, D. and de Vicente, A. 2011. Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture. Curr. Opin. Biotechnol. 22:187-193.
  39. Ruffner, B., Péchy-Tarr, M., Ryffel, F., Hoegger, P., Obrist, C., Rindlisbacher, A., Keel, C. and Maurhofer, M. 2013. Oral insecticidal activity of plant-associated pseudomonads. Environ. Microbiol. 15:751-763.
  40. Sambrook, J. and Russell. D. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
  41. Schroth, M. N. and Hancock, J. G. 1982. Disease-suppressive soil and root-colonizing bacteria. Science 216:1376-1381.
  42. Shanchez-Contreras, M. and Vlisidou, I. 2008. The diversity of insect-bacteria interactions and its applications for disease control. Biotechnol. Genet. Eng. Rev. 25:203-243.
  43. Siripornvisal, S. 2010. Biocontrol efficacy of Bacillus subtilis BCB3-19 against tomato gray mold. KMITL Sci. Tech. J. 10:37-44.
  44. Song, G. C., Lee, S., Hong, J., Choi, H. K., Hong, G. H., Bae, D. W., Mysore, K. S., Park, Y. S. and Ryu, C. M. 2015. Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation. New Phytol. 207:148-158.
  45. Sreerag, R. S., Jayaprakas, C. A., Ragesh, L. and Kumar, S. N. 2014. Endosymbiotic bacteria associated with the mealy bug, Rhizoecus amorphophallis (Hemiptera: Pseudococcidae). Int. Sch. Res. Not. 2014:268491.
  46. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30:2725-2729.
  47. Wang, Y., Lu, Z. X., Bie, X. M. and Fengxia, L. 2010. Separation and extraction of antimicrobial lipopeptides produced by Bacillus amyloliquefaciens ES-2 with macroporous resin. Eur. Food Res. Technol. 231:189-196.
  48. Weisburg, W. G., Barns, S. M., Pelletier, D. A. and Lane, D. J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703.
  49. Yun, D. C., Yang, S. Y., Kim, Y. C. and Kim, I. S. 2013. Identification of surfactins as aphicidal metabolite produced by Bacillus amyloliquefaciens G1. J. Korean Soc. Appl. Biol. Chem. 56:751-753.

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Supported by : Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET)