Characterization and biocontrol potential of two chitinolytic bacteria, Pseudoduganella violaceinigra H-2 and Bacillus licheniformis CT-100 = 두 가지 키틴분해 균주 Pseudoduganella violacenigra H-2와 Bacillus licheniformis CT 100의 특성 및 생물학적 방제 잠재력

다국어 초록 (Multilingual Abstract)

Two chitinolytic bacterial strains were isolated from soybean culture soil and Cheonggukjang. 16S rRNA sequencing identified them as Pseudoduganella violaceinigra H-2 and Bacillus licheniformis CT-100. H-2 showed 2.5 U/mL of chitinase activity on day 3, whereas CT-100 had about 3.9 U/mL on day 6. H-2 chitinase was most at pH 4.0 and 50 °C, CT-100 chitinase was optimal at pH 4.0–6.0 and 60 °C, and both responded differently to metal ions. SDS-PAGE and native-PAGE analyses revealed strong chitinase activity bands at 39.2 kDa (H-2) and 62.7 kDa (CT-100). Analysis of sugar degradation patterns using (GlcNAc)1-6 as substrates indicated both strains-chitinases primarily acted endo-wise with exo-like activity appearing at later stages of the reaction. TLC/HPLC analysis using various colloidal chitin substrates revealed that GlcNAc was detected only when cicada-derived chitin was used. API test results showed that CT-100 could utilize a broader range of carbohydrates than H-2, which was unable to utilize N-acetyl glucosamine despite producing chitinase. Both strains produced extracellular enzymes, such as proteases. CT-100 also produced ammonia and siderophores, whereas H-2 showed strong PGP traits including IAA production. The culture solution and VOCs of CT-100 strongly suppressed Colletotrichum spp. conidial formation. In pepper seed germination experiments, the 100-fold diluted culture of CT-100 improved germination rate and early growth, and in in vivo experiments against C. acutatum, it also reduced disease incidence and lesion size. The se results suggest P. violaceinigra H-2 and B. licheniformis CT-100 are promising microbial resources for biological control and sustainable agriculture.

국문 초록 (Abstract)

본 연구에서는 콩 토양과 청국장으로부터 두 종의 키틴분해균을 분리하였으며 16s rRNA sequencing을 통해 Pseudoduganella violaceinigra H-2, Bacillus licheniformis CT-100으로 확인하였다. H-2는 3일차에 약 2.5 U/mL, CT-100은 6일차에 약 3.9 U/mL의 chitinase 활성을 나타냈다. pH, temperature, 금속 이온 조건 비교 결과 H-2는 pH 4.0과 50 °C, CT-100은 pH 4.0-6.0과 60 °C에서 활성이 가장 높았으며, 두 균주는 금속 이온에 대해 서로 다른 반응성을 보였다. SDS-PAGA 및 Native-PAGE 분석에서는 H-2와 CT-100에서 각각 39.2kDa와 62.7kDa의 강한 활성 밴드가 확인되었다. (GlcNAc)1-6을 기질로 한 당 분해 패턴을 분석 결과 두 균주의 chitinase는 주로 endo-acting 방식으로 작용하며, 반응 후반에는 exo-like 절단이 함께 관여하는 것으로 판단되었다. 다양한 기질의 Colloidal chitin을 이용한 TLC/HPLC 분석에서는 Cicada 기질에서만 GlcNAc가 검출되었다. API test 결과 CT-100은 H-2에 비해 다양한 탄수화물을 이용할 수 있었으며, H-2는 chitinase를 생산함에도 N-acetyl glucosamine을 이용하지 못하는 독특한 특성을 보였다. 두 균주는 protease 등 다양한 세포외 효소를 생산했으며, CT-100은 ammonia와 siderophore를, H-2는 IAA 생산 등 우수한 PGP 특성을 나타냈다. 또한 CT-100의 배양액과 VOCs는 Colletotrichum spp.의 분생포자 형성을 강하게 억제하였다. 고추 종자 발아 실험 결과 CT-10의 100배 희석 배양액이 발아율과 초기 생육을 향상시켰으며, C. acutatum에 대한 in vivo 실험에서도 병 발생률과 병반 크기를 감소시켰다. 따라서 P. violaceinigra H-2와 B. licheniformis CT-100이 생물 방제 및 지속가능 농업 실현을 위한 유망한 미생물 자원으로 사용될 수 있음을 확인할 수 있다.

목차 (Table of Contents)

• Content
• List of Figures ⅲ
• List of tables ⅳ
• Abstract ⅴ
• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 7
• 2.1. Isolation and identification of chitinase-producing bacteria 7
• 2.2. Analysis of chitinase production and enzymatic properties 7
• 2.2.1. Cell growth, protein content and chitinase activity assay 7
• 2.2.2. Effect of pH, temperature, and metal ions on chitinase activity 8
• 2.2.3. Gel electrophoresis, followed by CC and enzyme activity staining · 9
• 2.2.4. Analysis of chitin degradation products by TLC and HPLC 9
• 2.3. Evaluation of physiology and biochemical characteristics 11
• 2.3.1. Physiological characterization assays 11
• 2.3.2. Assessment of extracellular enzyme activities 11
• 2.4. Evaluation of plant growth promoting and biocontrol traits 12
• 2.4.1. In vitro assessment of plant growth promoting traits 12
• 2.4.2. Antagonistic activity against fungal pathogens 13
• 2.4.3. Seed germination assay 14
• 2.4.4. In vivo plant inoculation experience experiment 15
• 3. RESULTS AND DISCUSSION 17
• 3.1. Isolation and identification of chitinase-producing bacteria 17
• 3.2. Cell growth, protein content and chitinase activity 19
• 3.3. Effect of pH, temperature and metal ions chitinase activity 22
• 3.4. Gel electrophoresis followed by CBB and enzyme activity staining ·· 26
• 3.5. Degradation patterns of chitinase analyzed by TLC and HPLC 30
• 3.6. Physiological characteristic assay 37
• 3.7. Assessment of extracellular enzymes 40
• 3.8. In vitro assessment of plant growth promoting traits 42
• 3.9. Antagonistic activity of bacteria against fungal pathogens 44
• 3.10. Seed germination rate 47
• 3.11. Biocontrol of anthracnose 50
• 4. CONCLUSIONS 52
• References 54
• 국문초록 67
• RESEARCH PERFORMANCE 69