Antagonistic Effects of Bacillomycins, 32,33-didehydroroflamycoin and Lucensomycin against Anthracnose and Gray Mold as Cell Envelope Disruptors Identified from Microbial Secondary Metabolites = 미생물 이차대사산물에서 분리한 세포외피 파괴물질 bacillomycins, 32,33-didehydroroflamycoin과 lucensomycin의 탄저병 및 잿빛곰팡이병에 대한 길항작용

다국어 초록 (Multilingual Abstract)

A microbial metabolite library was screened for antifungals that disrupt cell envelope of plant pathogenic fungi by using adenylate kinase (AK)-based cell lysis assay. Bacillus sp. strain GYL4, Streptomyces sp. strain DY46 and Streptomyces sp. strain CA5 were selected for harboring potent AK-releasing activity in their culture extract. B. amyloliquefaciens strain GYL4 was isolated from leaf tissue of pepper plants (Capsicum annuum L.). The antifungal compounds disrupting fungal cell envelope produced by strain GYL4 were identified and also, endophytic feature of strain GYL4 was examined. An eGFP-expressing strain of GYL4 (GYL4-egfp) was constructed and reintroduced into pepper plants, which confirmed its ability to colonize the internal tissues of pepper plants. GYL4-egfp was observed in the root and stem tissues 4 days after treatment and abundantly found in the internal leaf tissue 9 days after treatment. Anthracnose symptoms were markedly reduced in the leaves of pepper plants colonized by GYL4. Bacillomycin derivatives as the active components of GYL4 displayed control efficacy on anthracnose development in cucumber (Cucumis sativus L. cv. Chunsim). S. rectiviolaceus strain DY46 was isolated from soil samples obtained from Danyang province. The cell extract of the strain DY46 markedly reduced disease incidence (by 20.0%) of gray mold on tomato fruits. 32,33-didehydroroflamycoin (DDHR) was identified from the culture extract as an antifungal component which showed inhibitory effects against the mycelial growth of various plant pathogenic fungi at concentrations of 8 to 64 µg mL-1. DDHR completely inhibited gray mold development on tomato fruits at a concentration of 1000 µg mL-1. DDHR (100 µg mL-1) reduced disease incidence (by 11.1%) of gray mold on tomato fruits inoculated with a concentration of 104 conidia mL-1. Also, DDHR showed short residence time (2 days) in tomato fruits treated with DDHR. S. plumbeus strain CA5 was isolated from soil samples obtained from Chuncheon province. The cell extract of the strain CA5 markedly reduced disease incidence (by 22.2%) of gray mold on grapes. Lucensomycin was identified as the active component which inhibited the mycelial growth of various plant pathogenic fungi at concentrations of 1 µg mL-1. Lucensomycin completely inhibited gray mold development on grapes at a concentration of 100 µg mL-1. These results shows that microbial metabolites with AK releasing activity would be a plenteous reservoir of fungicidal compounds for plant disease control, especially for post harvest disease control that require short-term residual activity along with potent efficacy.

국문 초록 (Abstract)

Adenylate kinase (AK) 어세이를 사용하여 미생물 대사산물 라이브러리로부터 식물병원진균의 세포외피를 파괴하는 항균활성 물질을 탐색하였으며 배양 추출물에서 강한 AK 방출 활성을 보여주는 Bacillus sp. strain GYL4과 Streptomyces sp. strain DY46 그리고 Streptomyces sp. strain CA5 균주를 선발하였다. B. amyloliquefaciens strain GYL4 균주는 고추식물의 잎 조직으로부터 분리하였으며 GYL4 균주가 생산하는 진균의 세포외피를 파괴하는 항균활성 물질을 동정하고 또한 GYL4 균주의 내생적 특성을 조사하였다. eGFP를 발현하는 GYL4-egfp 균주를 구축하고 고추 식물 내부 조직에 정착하는 능력을 확인하기 위해서 고추 식물에 재도입하였다. GYL4-egfp 균주는 처리 후 4일 후에 뿌리 및 줄기 조직에서 관찰되었고 9일 후에 잎 조직에서 발견되었다. GYL4 균주가 정착한 고추 잎에서 탄저병 병징이 현저하게 감소하였다. GYL4 균주의 유효 성분인 bacillomycin 파생물질들은 오이 탄저병에 대한 방제 효과를 보여주었다. S. rectiviolaceus strain DY46 균주는 단양 지역에서 수집한 토양으로부터 분리하였다. DY46 균주의 세포 추출물은 토마토 잿빛 곰팡이병의 병 발생을 20.0%까지 현저하게 감소시켰다. DY46 균주의 배양 추출물로부터 8 에서 64 µg mL-1의 농도에서 다양한 식물병원진균의 균사생장 억제 효과를 보여주는 항균 물질인 32,33-didehydroroflamycoin (DDHR)을 동정하였다. DDHR은 1000 μg mL-1의 농도에서 토마토 잿빛곰팡이병을 완벽하게 억제하였다. 또한 1 mL 당 104 포자 농도로 접종한 토마토에서 DDHR (100 μg mL-1)은 토마토 잿빛 곰팡이병의 병 발생을 11.1%까지 감소시켰으며 DDHR을 처리한 토마토에서 DDHR은 짧은 잔류시간 (2일)을 보여주었다. S. plumbeus strain CA5 균주는 춘천 지역에서 수집한 토양으로부터 분리하였다. CA5 균주의 세포 추출물은 포도 잿빛 곰팡이병의 병 발생을 22.2%까지 현저하게 감소시켰다. Lucensomycin은 1 µg mL-1의 농도에서 다양한 식물병원진균의 균사생장을 강하게 억제하는 유효 성분으로 동정되었으며 lucensomycin은 100 μg mL-1의 농도에서 포도 잿빛곰팡이병을 완벽하게 억제하였다. 이러한 결과는 AK 방출 활성을 가지는 미생물 대사산물이 식물병 방제와 특히 강한 효과와 짧은 잔류 활성이 요구되는 저장병 방제를 위한 살균활성 물질의 풍부한 저장소가 될 수 있다는 것을 보여준다.

목차 (Table of Contents)

• ABBREVIATIONS i
• ABSTRACT ii
• TABLE OF CONTENTS vii
• LIST OF TABLES xiii
• LIST OF FIGURES xv
• 1.Introduction 1
• 1.1.Need for development of novel fungicide in modern agriculture 2
• 1.2.Microbial secondary metabolite as source of novel fungicide 3
• 1.3.Fungicides of microbial origin used in agriculture 4
• 1.4.Target sites of fungi for development of novel fungicies 11
• 1.4.1.Fungal cell wall 12
• 1.4.2.Fungal cell membrane 17
• 1.5.Current strategies in screening antifungal compounds 23
• 1.6.Adenylate kinase assay 24
• 1.7.The objectives of the study 26
• 2.Evaluation of the Endophytic Nature of Bacillus amyloliquefaciens strain GYL4 and Its Efficacy in the Control of Anthracnose 28
• 2.1.Abstract 29
• 2.2.Introduction 30
• 2.3.Materials and Methods 33
• 2.3.1.Microbial culture extract library 33
• 2.3.2.Adenylate kinase assay 33
• 2.3.3.Isolation of endophytic Bacillus sp. strain GYL4 34
• 2.3.4.Identification of the strain GYL4 35
• 2.3.5.Evaluation of the disease control efficacy of GYL4 37
• 2.3.6.Colonization test using an eGFP-expressing GYL4 strain in pepper plants 38
• 2.3.7.Purification of antifungal compounds produced by strain GYL4 39
• 2.3.8.Structural elucidation of CL4-1, CL4-2, CL4-3, CL4-4 and CL4-5 41
• 2.3.9.Bioassays for antifungal activities of CL4-1, CL4-2, CL4-3, CL4-4 and CL4-5 42
• 2.3.10.Evaluation of disease control efficacy of CL4-1, CL4-2, CL4-3, CL4-4 and CL4-5 43
• 2.3.11.Statistical analysis 44
• 2.4.Results 46
• 2.4.1.AK activity of strain GYL4 46
• 2.4.2.Bacillus sp. strain GYL4 isolated from pepper plants (Capsicum annuum L.) 46
• 2.4.3.Disease control efficacy of the strain GYL4 on pepper anthracnose 48
• 2.4.4.Colonization of pepper plants by eGFP-expressing GYL4 48
• 2.4.5.Identification and structural elucidation of antifungal compounds CL4-1, CL4-2, CL4-3, CL4-4 and CL4-5 51
• 2.4.6.Antifungal activities of bacillomycin derivatives 56
• 2.4.7.Disease control efficacies of bacillomycin derivatives on cucumber anthracnose 58
• 2.5.Discussion 60
• 3.Identification and Disease Control Efficacy of Polyene Macrolide 32,33- didehydroroflamycoin Produced by Streptomyces rectiviolaceus strain DY46 against Gray Mold on Tomato Fruits 63
• 3.1.Abstract 64
• 3.2.Introduction 65
• 3.3.Materials and Methods 67
• 3.3.1.Microbial culture extract library 67
• 3.3.2.Adenylate kinase assay 67
• 3.3.3.Identification of strain DY46 68
• 3.3.4.Evaluation of disease control efficacy of the cell extract of strain DY46 70
• 3.3.5.Purification of antifungal compound produced by the strain DY46 71
• 3.3.6.Structural elucidation of DY46A 73
• 3.3.7.Bioassays for antifungal activity of DY46A 74
• 3.3.8.Evaluation of disease control efficacy of DY46A 75
• 3.3.9.Disease control efficacy of DDHR according to inoculum concentration 77
• 3.3.10.Residence time of DDHR in the tomato fruits 77
• 3.3.11.Statistical analysis 78
• 3.4.Results 79
• 3.4.1.AK activity of strain DY46 79
• 3.4.2.Isolation and identification of strain DY46 79
• 3.4.3.Evaluation of disease control efficacy of the cell extract of strain DY46 82
• 3.4.4.Antifungal compound produced by strain DY46 85
• 3.4.5.Structure of DY46A 85
• 3.4.6.Minimum inhibitory concentrations (MIC) of DDHR 87
• 3.4.7.Control efficacy on gray mold development 91
• 3.4.8.Disease control efficacy of DDHR according to inoculum concentration 91
• 3.4.9.Residence time of DDHR in the tomato fruits 94
• 3.5.Discussion 98
• 4.Identification and Disease Control Efficacy of Polyene Macrolide Lucensomycin Produced by Streptomyces plumbeus strain CA5 against Gray Mold on Grapes 102
• 4.1.Abstract 103
• 4.2.Introduction 104
• 4.3.Materials and Methods 108
• 4.3.1.Microbial culture extract library 108
• 4.3.2.Adenylate kinase assay 108
• 4.3.3.Identification of strain CA5 109
• 4.3.4.Evaluation of disease control efficacy of the cell extract of stain CA5 111
• 4.3.5.Purification of antifungal compound produced by the strain CA5 112
• 4.3.6.Structural elucidation of CA5A 114
• 4.3.7.Bioassays for antifungal activity of CA5A 115
• 4.3.8.Evaluation of disease control efficacy of CA5A 116
• 4.3.9.Statistical analysis 118
• 4.4.Results 119
• 4.4.1.AK activity of strain CA5 119
• 4.4.2.Isolation and identification of strain CA5 119
• 4.4.3.Evaluation of disease control efficacy of the cell extract of strain CA5 121
• 4.4.4.Antifungal compound produced by strain CA5 123
• 4.4.5.Structure of CA5A 123
• 4.4.6.In vitro and in vivo antifungal activity of lucensomycin 126
• 4.5.Discussion 134
• 5.Conclusion 138
• 6.Literature cited 142