Bacillus amyloliquefaciens KB-MJK601 균주의 항균활성 및 식물 생육 촉진 효과

다국어 초록 (Multilingual Abstract)

The eco-friendly biological control of crop plants using benign soil microorganisms is one of the promising approaches to reduce the abuse of chemical pesticides and fertilizers in agricultural fields, which are often expensive and adverse effects on humans. With these advantages, the beneficial effects on plants of plant growth promoting rhizobacteria have been exclusively explored in the world. Moreover, their prominent antimicrobial activity has been proposed as a promising eco-friendly biological controller in agricultural fields to effectively reduce severe crop diseases. In this thesis, I successfully identified a Bacillus amyloliquefaciens KB-MJK601 as an excellent antimicrobial activity and plant growth promotion, isolated from cultivated soil in Korea. I tested the possibilities in the valuable utilities of the identified Bacillus strain in crop plants for growth promotion and biological control of their severe phyto-diseases. I also investigated their potential for antimicrobial activities against four kinds of plant pathogens.

1. To screen potential plant growth promoting rhizobacteria, antimicrobial activities, phosphate solubilization activity and cellulose decomposing activity, sixty bacterial candidate strains were successfully isolated from agriculturally cultivated soil in different area of South Korea.
2. Among the 60 candidates, I selected an excellent strain, KB-MJK601, as a plant growth promoting rhizobacterium (PGPR) having abilities for plant growth promotion and higher antifungal activity. The DNA sequence analysis of 16S rDNA and gyrB gene indicates that the KB-MJK601 shares similar genome sequences with B. amyloliquefaciens. I also tried to identify of this strain with API 50CHE system, showing positive signals for acid production from L-arabinose, Cellobiose, Inulin, D-turanose. A profiling of fatty-acid composition of the KB-MJK601 displays high 15 : 0 anteiso (39.65%) and 15 : 0 iso fatty-acid content (23.31%). Based on these results, KB-MJK601 is identified as a species of B. amyloliquefaciens.
3. The morphology of B. amyloliquefaciens strain KB-MJK601 shows gram-positive rods with 0.5∼0.7×1.5∼2.6 μm diameter. The optimum growth conditions of B. amyloliquefaciens KB-MJK601 is in medium containing 1.5% glucose, 0.6% Soybean, 1% Yeast extract, 0.1% CaCl2, 0.1% KH2PO4, pH 7.0 at 30℃ for 120 hours under air flow (2 vvm) with 200 rpm agitation in 5L jar fermenter. I found that the maximum growth of the Bacillus strain was up to 7.6 x 1010 cfu/ml and 100% sporation for 120 hours in a 5000 L fermenter. The growth condition of B. amyloliquefaciens KB-MJK601 is determined in the range of 2∼8 % NaCl , pH 6∼8 and 30∼40 ℃ of temperature.
4. To confirm if the selected B. amyloliquefaciens strain KB-MJK601 could produce general antibiotic effecters of Bacillus species including iturine, surfactin and fengycin, their bio-synthetic genes were analyzed in their genome by Polymerase Chain Reaction(PCR). I found that the genome contains the bio-synthetic genes for iturine A and C, surfactin and fengycin. Moreover, B. amyloliquefaciens KB-MJK601 strain has an ability to produce plant hormone 2,3-butanediol, a potential volatile compound involved in plant growth promotion and induced systemic resistance (ISR).
5. The abilities of B. amyloliquefaciens KB-MJK601 as a biological control agent for several representative plant pathogens were also investigated. In case of rice bakanae disease, the 35.6% reduction compared to control treatment was observed in the rice samples which were treated with 1×109 cfu/ml. With treatment of 1/500 dilution of 1×1010 cfu/ml of the B. amyloliquefaciens KB-MJK601, 57.6, 80.5 and 80.9% reduction rates were detected in rice sheath blight, cucumber sclerotinia rot and lettuce sclerotinia rot diseases, respectively.
6. The capacity of plant growth promotion by enhancing phosphate solubilization of B. amyloliquefaciens KB-MJK601 was evaluated. Inoculation of B. amyloliquefaciens KB-MJK601 significantly enhanced root development and biomass of tomato seedling. Seedling establishment and growth rates were also effectively increased by B. amyloliquefaciens KB-MJK601 inoculation. Finally, I confirmed the phosphate solubilization activity of the Bacillus strain by using potato dextrose agar-calcium phosphate medium.
7. For field application, the B. amyloliquefaciens KB-MJK601 strains were mixed with botanical active ingredient (Eucalyptus oil). I also tried to increase the bio-agent efficacy by adding adjuvant containing a polyalkyleneoxide modified heptamethy itrisiloxane 5% for a dispersing agent, 0.5 % sodium PCA for a moisturizer and Lowilite 62 for a UV-Blocker. The mixed bio-control agent containing B. amyloliquefaciens KB-MJK601 strains was directly applied to chestnut Colletotrichum gloeosporioides with 1/500 dilution. The disease severity was greatly suppressed by the bio-agent application as 58.6% to 67% compared to control value in both early and medium maturing variety and late flowering variety.
8. With these prominent effects on plant growth promotion, improvement of soil properties and disease controls, the B. amyloliquefaciens KB-MJK601 strain and its related natural products have been successfully applied in agricultural fields in Korea and other countries. Taken all together, I concluded that my work provides highly advanced biological approaches to eco-friendly controls of crop productivity and agricultural applications.

목차 (Table of Contents)

• I. 서언 1
• II. 연구사 4
• Ⅲ. 재료 및 방법 12
• 1. 토양미생물의 항균활성 및 생육촉진 균주 선발 12
• 가. 식물병원성 곰팡이에 대한 항균활성 및 생육촉진 균주 선발 12
• 나. Bacillus amyloliquefaciens KB-MJK601 균주의 분류 및 동정 18
• 다. Bacillus amyloliquefaciens KB-MJK601 균주의 2차 대사물질 유전자 확인 22
• 라. Bacillus amyloliquefaciens KB-MJK601 균주의 배양 조건 및 균주 안정성 25
• 2. 식물병 방제, 인산가용화 및 생육촉진 효과 28
• 가. 식물병 방제 효과 28
• 나. 인산가용화 효과 31
• 다. 생육촉진 효과 32
• 3. 제형화 및 실용화 33
• 가. 제형화에 따른 보조제 선발
• 나. Bacillus amyloliquefaciens KB-MJK601 균주의 실용화 35
• Ⅳ. 결과 37
• 1. 토양미생물의 항균활성 및 생육촉진 균주 선발 37
• 가. 식물병원성 곰팡이에 대한 항균활성 및 생육촉진 균주 선발 37
• 나. Bacillus amyloliquefaciens KB-MJK601 균주의 분류 및 동정 48
• 다. Bacillus amyloliquefaciens KB-MJK601 균주의 2차 대사물질유전자 61
• 라. Bacillus amyloliquefaciens KB-MJK601 균주의 배양 조건 및 균주 안정성 66
• 2. 식물병 방제, 인산가용화 및 생육촉진 효과 82
• 가. 식물병 방제 효과 82
• 나. 인산가용화 효과 95
• 다. 생육촉진 효과 97
• 3. 제형화 및 실용화 101
• 가. 제형화에 따른 보조제 선발 101
• 나. Bacillus amyloliquefaciens KB-MJK601 균주의 실용화 113
• Ⅴ. 종합고찰 121
• Ⅵ. 적 요 127
• Ⅶ. 참고문헌 130