Korean Journal of Organic Agriculture (한국유기농업학회지)

Korean Association of Organic Agriculture (한국유기농업학회)
권호 표지
DOI QR코드

DOI QR Code

Quantitative Analysis of Bacillus amyloliquefaciens GR4-5 in Soil

Bacillus amyloliquefaciens GR4-5 균주의 토양 내 정량 분석

  • 김다연 (농촌진흥청 국립농업과학원) ;
  • 김병용 (천랩) ;
  • 안재형 (농촌진흥청 국립농업과학원) ;
  • 원항연 (농촌진흥청 국립농업과학원) ;
  • 김성일 (강원도농업기술원 인삼약초연구소) ;
  • 김완규 (농촌진흥청 국립농업과학원) ;
  • 송재경 (농촌진흥청 국립농업과학원)
  • Received : 2015.11.11
  • Accepted : 2015.11.26
  • Published : 2015.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Bacillus amyloliquefaciens GR4-5 was isolated from the rhizosphere soil of Korean ginseng and displayed broad-spectrum suppression of ginseng root rot pathogens. The survivability of B. amyloliquefaciens GR4-5 in soil was investigated under three different conditions; indoor, outdoor - of which soil was put in 14 mL tube after treatment - and field environments. Soil samples were collected over a four-week period from three experimental designs, and assessed for 16S rRNA gene copy number by quantitative polymerase chain reaction (qPCR). In outdoor condition, the 16S rRNA gene copy number of Bacillus spp. was 8.35 log copies g $soil^{-1}$ immediately after the GR4-5 treatment. Two weeks later, the 16S rRNA gene copy number of Bacillus spp. (6.70 log copies g $soil^{-1}$) was similar to that of the control (6.38 log copies g $soil^{-1}$). In indoor condition, the 16S rRNA gene copy number of Bacillus spp. maintained in a certain level for a longer period than those in outdoor and field. The 16S rRNA gene copy number of Bacillus spp. in field experiment was reduced faster than that of outdoor condition. Our results show that B. amyloliquefaciens GR4-5 can survive in bulk soil for 1 week, indicating its potential use as a biocontrol agent following 7 day application intervals. This study presents that outdoor microcosm system design could be a useful method to assess easily the survivability of beneficial microorganisms.

인삼뿌리썩음병에 길항력이 있는 Bacillus amyloliquefaciens GR4-5 균주 처리 전 후의 토양 내 Bacillus spp. 밀도 변화를 qPCR을 이용하여 분석하였다. 실내배양시험에서는 GR4-5 균주 처리 직후부터 4주째까지 Bacillus sp. group의 유전자 수가 무처리구보다 약 100배 이상으로 유지되는 경향이었다. 실외매몰시험과 포장시험에서는 유의차는 없었지만 경향으로 보아 GR4-5 처리구와 무처리구의 B. subtilis group 유전자 수가 비슷한 수준이 되는데 걸리는 시간은 7일 내외로 나타났다. 토양에 접종된 미생물의 생존에는 환경요인이 큰 영향을 미치며 그 중에서도 온도와 미생물의 격리 정도가 가장 큰 인자로 추정된다. 또한 GR4-5 균주의 생존에는 토양의 수분 함량 변화보다는 균주 처리 방법에 의한 영향이 더 크게 작용하는 것으로 보인다. 본 연구결과를 고려하면, B. amyloliquefaciens GR4-5 균주를 생물적 방제제로 사용하고자 한다면 7일 간격으로 관주 처리하는 것이 식물병원균 억제 및 근권 정착에 유리한 환경을 조성할 수 있을 것으로 판단된다. 또한 본 연구에서 제작한 실외 마이크로코즘 시스템은 제어하기 어려운 외부 환경 요인을 최소로 하여 유용미생물의 토양 내 생존 패턴을 분석하기 위한 간편한 방법으로서 유용하게 사용될 수 있을 것으로 판단된다.

Keywords

References

  1. Ahn, Y. J., H. J. Kim, S. H. Ohh, and S. Y. Choi. 1982. Effect of soil fumigation on growth, root rot and red discoloration of Panax ginseng in replanted soils. J. Ginseng Res. 6: 46-55.
  2. Bashan, Y., M. E. Puente, M. N. Rodriguez-Mendoza, G. Toledo, G. Holguin, R. Ferrera-Cerrato, and S. Pedrin. 1995. Survival of Azospirillum brasilense in the bulk soil and rhizosphere of 23 soil types. Appl. Environ. Microbiol. 61: 1938-1945.
  3. Bennett, A. J., C. Leifert, and J. M. Whipps. 2003. Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils. Soil Biol. Biochem. 35: 1565-1573. https://doi.org/10.1016/j.soilbio.2003.08.001
  4. Binnerup, S. J., D. F. Jensen, H. Thordal-Christensen, and J. Sorensen. 1993. Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique. FEMS Microbiol. Ecol. 12: 97-105. https://doi.org/10.1111/j.1574-6941.1993.tb00021.x
  5. Cao, P., S. S. Shen, C. Y. Wen, S. Song, and C. S. Park. 2009. The effect of the colonization of Serratia plymuthica A21-4 in rhizosphere soil and root of pepper in different soil environment. Res. Plant Dis. 15: 101-105. https://doi.org/10.5423/RPD.2009.15.2.101
  6. Chung, H. S. 1975. Studies on Cylindrocarpon destructans (Zins.) Scholten causing root rot of ginseng. Rep. Tottori. Mycol. Inst. 12: 127-138.
  7. Danhorn, T. and C. Fuqua. 2007. Biofilm formation by plant-associated bacteria. Annu. Rev. Microbiol. 61: 401-422. https://doi.org/10.1146/annurev.micro.61.080706.093316
  8. Doran, J. W. and M. R. Zeiss. 2000. Soil health and sustainability: managing the biotic component of soil quality. Appl. Soil Ecol. 15: 3-11. https://doi.org/10.1016/S0929-1393(00)00067-6
  9. Dutta, S. and A. R. Podile. 2010. Plant growth promoting rhizobacteria (PGPR): the bugs to debug the root zone. Crit. Rev. Microbiol. 36: 232-244. https://doi.org/10.3109/10408411003766806
  10. Geels, F. and B. Schippers. 1983. Selection of antagonistic fluorescent Pseudomonas spp. and their root colonization and persistence following treatment of seed potatoes. J. Phytopathol. 108: 193-206. https://doi.org/10.1111/j.1439-0434.1983.tb00579.x
  11. Haggag, W. and S. Timmusk. 2008. Colonization of peanut roots by biofilm-forming Paenibacillus polymyxa initiates biocontrol against crown rot disease. J. Appl. Microbiol. 104: 961-969. https://doi.org/10.1111/j.1365-2672.2007.03611.x
  12. Ho, W. and W. Ko. 1985. Soil microbiostasis: effects of environmental and edaphic factors. Soil Biol. Biochem. 17: 167-170. https://doi.org/10.1016/0038-0717(85)90110-5
  13. Hwang, J. M., K. C. Park, and S. J. Kim. 2010. Contents of soil microbial phospholipid fatty acids as affected by continuous cropping of pepper under upland. Korean J. Soil Sci. Fert. 43: 1012-1017.
  14. Jesser, K. J., H. Fullerton, K. W. Hager, and C. L. Moyer. 2015. Quantitative PCR analysis of functional genes in iron-rich microbial mats at an active hydrothermal vent system (Lo'ihi Seamount, Hawai'i). Appl. Environ. Microbiol. 81: 2976-2984. https://doi.org/10.1128/AEM.03608-14
  15. Jun, H. S., W. C. Park, and J. S. Jung. 2002. Effects of soil addition and subsoil plowing on the change of soil chemical properties and the reduction of root-knot nematode in continuous cropping field of oriental melon (Cucumis melo L.). Korean J. Environ. Agr. 21: 1-6. https://doi.org/10.5338/KJEA.2002.21.1.001
  16. Kim, B. Y., J. H. Ahn, H. Y. Weon, J. Song, S. I. Kim, and W. G. Kim. 2012. Isolation and characterization of Bacillus species possessing antifungal activity against ginseng root rot pathogens. Korean J. Pestic. Sci. 16: 357-363. https://doi.org/10.7585/kjps.2012.16.4.357
  17. Kim, J. S., S. W. Kwon, S. J. Lee, B. G. Jung, J. Song, S. J. Go, and J. C. Ryu. 1999. Analysis of microbial community structure in soil and crop root system: I. Analysis of bacterial community structure in the soil and root system of red pepper and tomato. Korean J. Soil Sci. Fert. 32: 319-325.
  18. Krzyzanowska, D., M. Obuchowski, M. Bikowski, M. Rychlowski, and S. Jafra. 2012. Colonization of potato rhizosphere by GFP-tagged Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44 shown on large sections of roots using enrichment sample preparation and confocal laser scanning microscopy. Sensors-Basel. 12: 17608-17619. https://doi.org/10.3390/s121217608
  19. Lee, Y. H. and S. T. Lee. 2011. Comparison of microbial community of orchard soils in Gyeongnam Province. Korean J. Soil Sci. Fert. 44: 492-497. https://doi.org/10.7745/KJSSF.2011.44.3.492
  20. Liu, C., J. Sheng, L. Chen, Y. Zheng, D. Y. W. Lee, Y. Yang, M. Xu and L. Shen. 2015. Biocontrol activity of Bacillus subtilis isolated from Agaricus bisporus mushroom compost against pathogenic fungi. J. Agric. Food Chem. 63: 6009-6018. https://doi.org/10.1021/acs.jafc.5b02218
  21. Liu, Y. P., N. Zhang, M. H. Qiu, H. C. Feng, J. M. Vivanco, Q. R. Shen, and R. F. Zhang. 2014. Enhanced rhizosphere colonization of beneficial Bacillus amyloliquefaciens SQR9 by pathogen infection. FEMS Microbiol. Lett. 353: 49-56. https://doi.org/10.1111/1574-6968.12406
  22. Lopez-Gutierrez, J. C., S. Henry, S. Hallet, F. Martin-Laurent, G. Catroux, and L. Philippot. 2004. Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J. Microbiol. Methods. 57: 399-407. https://doi.org/10.1016/j.mimet.2004.02.009
  23. Lugtenberg, B. J. J., L. Dekkers, and G. V. Bloemberg. 2001. Molecular determinants of rhizosphere colonization by Pseudomonas. Annu. Rev. Phytopathol. 39: 461-490. https://doi.org/10.1146/annurev.phyto.39.1.461
  24. Mahaffee, W. F. and P. A. Backman. 1993. Effects of seed factors on spermosphere and rhizosphere colonization of cotton by Bacillus subtilis Gb03. Phytopathology. 83: 1120-1125. https://doi.org/10.1094/Phyto-83-1120
  25. Ohh, S. H. and C. S. Park. 1980. Studies on Phytophthora disease of Panax ginseng CA Meyer; its casual agent and possible control measures. J. Ginseng Res. 4: 186-193.
  26. Ongena, M. and P. Jacques. 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 16: 115-125. https://doi.org/10.1016/j.tim.2007.12.009
  27. Park, J. W., S. Jahaggirdar, Y. E. Cho, K. S. Park, S. H. Lee, and K. S. Park. 2010. Evaluation of Bacillus subtilis native strains for plant growth promotion and induced systemic resistance in tomato and red-pepper. Korean J. Pestic. Sci. 14: 407-414
  28. Park, J. W. and D. E. Crowley. 2005. Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE. Biotechniques. 38: 579-586. https://doi.org/10.2144/05384ST04
  29. Raupach, G. S. and J. W. Kloepper. 1998. Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology. 88: 1158-1164. https://doi.org/10.1094/PHYTO.1998.88.11.1158
  30. Seong, K. Y., M. Hofte, J. Boelens, and W. Verstraete. 1991. Growth, survival, and root colonization of plant growth beneficial Pseudomonas fluorescens ANP15 and Pseudomonas aeruginosa 7NSK2 at different temperatures. Soil Biol. Biochem. 23: 423-428. https://doi.org/10.1016/0038-0717(91)90004-4
  31. Shin, J. H., B. D. Yun, H. J. Kim, S. J. Kim, and D. Y. Chung. 2012. Soil environment and soil-borne plant pathogen causing root rot disease of ginseng. Korean J. Soil Sci. Fert. 45: 370-376. https://doi.org/10.7745/KJSSF.2012.45.3.370
  32. Simons, M., H. P. Permentier, L. A. de Weger, C. A. Wijffelman and B. J. Lugtenberg. 1997. Amino acid synthesis is necessary for tomato root colonization by Pseudomonas fluorescens strain WCS365. Mol. Plant Microbe Interact. 10: 102-106. https://doi.org/10.1094/MPMI.1997.10.1.102
  33. Szczech, M. and M. Shoda. 2006. The effect of mode of application of Bacillus subtilis RB14-C on its efficacy as a biocontrol agent against Rhizoctonia solani. J. Phytopathol. 154: 370-377. https://doi.org/10.1111/j.1439-0434.2006.01107.x
  34. Van Veen, J. A., L. S. Van Overbeek and J. D. Van Elsas. 1997. Fate and activity of microorganisms introduced into soil. Microbiol. Mol. Biol. Rev. 61: 121-135.
  35. Vandenhove, H., R. Merckx, H. Wilmots and K. Vlassak. 1991. Survival of Pseudomonas fluorescens inocula of different physiological stages in soil. Soil Biol. Biochem. 23: 1133-1142. https://doi.org/10.1016/0038-0717(91)90025-F
  36. Weller, D. M. 1988. Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu. Rev. Phytopathol. 26: 379-407. https://doi.org/10.1146/annurev.py.26.090188.002115
  37. Xu, Z. H., J. H. Shao, B. Li, X. Yan, Q. R. Shen and R. F. Zhang. 2013. Contribution of bacillomycin D in Bacillus amyloliquefaciens SQR9 to antifungal activity and biofilm formation. Appl. Environ. Microbiol. 79: 808-815. https://doi.org/10.1128/AEM.02645-12
  38. Yamada, M. 2001. Methods of control of injury associated with continuous vegetable cropping in Japan: crop rotation and several cultural practices. Jpn. Agric. Res. Q. 35: 39-45. https://doi.org/10.6090/jarq.35.39
  39. Yu, Z., Y. Zhang, W. Luo and Y. Wang. 2014. Root colonization and effect of biocontrol fungus Paecilomyces lilacinus on composition of ammonia-oxidizing bacteria, ammoniaoxidizing archaea and fungal populations of tomato rhizosphere. Biol. Fertil. Soils. 51: 343-351.
  40. Zhu, Y. Z., D. S. Park, M. R. Cho, J. H. Hur and C. K. Lim. 2005. Suppression of Meloidogyne arenaria by different treatments of Pasteuria penetrans. Korean J. Pestic. Sci. 9: 437-441.