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목차

Abstract 6

I. 서언 20

II. 연구사 23

III. 재료 및 방법 31

1. 토양미생물의 항균활성 및 생육촉진 균주 선발 31

가. 식물병원성 곰팡이에 대한 향균활성 및 생육촉진 균주 선발 31

나. Bacillus amyloliquefaciens KB-MJK601 균주의 분류 및 동정 37

다. Bacillus amyloliquefaciens KB-MJK 601 균주의 2차 대사물질 유전자 확인 41

라. B. amyloliquefaciens KB-MJK601 균주의 배양 조건 및 균주 안정성 44

2. 식물병 방제 인산가용화 및 생육촉진 효과 47

가. 식물병 방제 효과 47

나. 인산가용화 효과 50

다. 생육촉진 효과 51

3. 제형화 및 실용화 52

가. 제형화에 따른 보조제 선발 52

나. B. amyloliquefaciens KB-MJK601 균주의 실용화 54

IV. 결과 56

1. 토양미생물의 항균활성 및 생육촉진 균주 선발 56

가. 식물병원성 곰팡이에 대한 항균활성 및 생육촉진 균주 선발 56

나. B. amyloliquefaciens KB-MJK601 균주의 분류 및 동정 67

다. B. amyloliquefaciens KB-MJK601 균주의 2차 대사물질 유전자 확인 80

라. B. amyloliquefaciens KB-MJK601 균주의 배양 조건 및 균주 안정성 85

2. 식물병 방제, 인산가용화 및 생육촉진 효과 101

가. 식물병 방제 효과 101

나. 인산가용화 효과 114

다. 생육촉진 효과 116

3. 제형화 및 실용화 120

가. 제형화에 따른 보조제의 선발 120

나. B. amyloliquefaciens KB-MJK601 균주의 실용화 132

V. 종합고찰 140

VI. 적요 146

VII. 참고문헌 149

Table 1 The selected organic cultivar regions for tomato, cucumber, chilli, apple, ginseng and peach to isolate beneficial rhizobial microorganisms 31

Table 2. Medium formulation contained tricalcium phosphate for the selection of stains harboring phosphate solubilization activity. 34

Table 3. Medium formulation contained carboxiymethyl cellulose for the selection of stain harboring cellulose decomposing activity. 35

Table 4. List of plant diseases for the selection of beneficial strains harboring antimicrobial activity against 14 kinds of plant pathogen. 36

Table 5. Gene specific primer sequences for the detection of the bacillomycin D, fengycin, iturin A, surfactin and zwittermicin A gene in B. amyloliquefaciens KB-MJK601. 42

Table 6. Gene Specific primer sequences for the detection of the 2,3 -butanediol and indole-3-acetic acid gene in B. amyloliquefaciens KB-MJK601. 43

Table 7. Composition of Komada's medium for the test of occurrence of bakanae disease of rice after treatment of B. amyloliquefaciens KB-MJK601. 48

Table 8. Yamazaki nutrient solution formula for the test of tomato leaf nutrient contents analysis after treatment of B. amyloliquefaciens KB-MJK601. 50

Table 9. Morphological characteristics of Bacillus strains from the selected areas. 57

Table 10. Relative growth rate of coco variety cherry tomato by isolated Bacillus strains. 58

Table 11. Selected bacteria showed different phosphate solubilization activity on potato dextrose agar-calcium phosphate medium and cellulose decomposing activity on Carboxymethyl cellulose... 60

Table 12. Growth inhibition effects against to 7 kinds of plant pathogenic fungi by selected microorganisms. 62

Table 13. Growth Inhibition effects of isolated B. amyloliquefaciens KB-MJK601 against to 14 kinds of plant pathogenic fungi in dual culture method. 65

Table 14. Different carbohydrate fermentation patterns of B. amyloliquefaciens KB-MJK601 and B. amyloliquefaciens MJ-1 tested by an API 50CHE kit. 70

Table 15. Comparison of cellular fatty acid compositions of strain B. amyloliquefaciens KB-MJK601 and B. amyloliquefaciens NBRC 15535T.(이미지참조) 79

Table 16. B. amyloliquefaciens and B. amyloliquefaciens subsp. plantarum biosynthestic genes corresponding to surfactin, fengycin, iturin A and bacillomycin D. 81

Table 17. B. amyloliquefaciens KB-MJK601 and B. amyloliquefaciens sub sp. plantarum M27 biosynthestic genes corresponding to 2,3-butanediol and indole-3-acetic acid. 84

Table 18. Influence of different carbon sources on the formation of inhibition zone against C gloeosporioides and cell growth of B. amyloliquefaciens KB-MJK601. 86

Table 19. Influence of different nitrogen sources on the inhibition zone against C. gloeosporioides and cell growth of B. amyloliquefaciens KB-MJK601. 88

Table 20. Influence of different metal ion sources on the inhibition zone against C. gloeosporioides and cell growth of B. amyloliquefaciens KB-MJK601. 90

Table 21. Influence of different culture temperature on the inhibition zone against C. gloeosporioides and cell growth of B. amyloliquefaciens KB- MjK601. 92

Table 22. Influence of different pH on the inhibition zone against C. gloeosporioides and cell growth of B. amyloliquefaciens KB- MjK601. 94

Table 23. Biological control of Sclerotinia rot in lettuce by B. amyloliquefaciens KB-MJK601. 113

Table 24. Effect of B. amyloliqefaciens KB-MJK601 on the nutrient composition of leaves of cherry tomato plants grown with nutrient solutions. 115

Table 25. Evaluation of growth promotion of cherry tomato by inoculation of B.amyloliquefaciens KB-MJK601. 117

Table 26. Studies of rice seedling establishment and growth response after treatment of different concentration of B. amyloliquefaciens KB-MjK601. 119

Table 27. Mycelial growth inhibition rate by different composition of plant extract against Colletotrichum gloeosporioides in using dual culture. 121

Table 28. The test of dispersing ability of B. amyloliqefaciens KB-MJK601 spore by non-ionic surfactants concentration in soybean leaf. 123

Table 29. The test of adhesion ability of B. amyloliqefaciens KB-MJK601 spore on hot pepper leaf by several kinds of adhesion booster in concentration. 125

Table 30. The spore counting on hot pepper leaf after treatment of B. amyloliquefaciens KB-MJK601 with different adhesion booster concentration. 126

Table 31. Proportion of prototype for the field trial test against chestnut Colletotrichum gloeosporioides. 131

Fig. 1. The map of 10 selected areas (Hwaseong Anseong Asan Namwon Jinju-Si Geumsan Buan Hamyang Goesan Eumseong-Gun) in Korea to identify beneficial microorganisms in... 32

Fig. 2. Clear zone formation on phosphate solubilization activity on potato dextrose agar-calcium phosphate medium by isolated KB-M]K strains, Pseudomonas aeruginosa DSM50071T,...(이미지참조) 61

Fig. 3. Lear zone formation on cellulose agar plates isolated by KB-MJK strains and Pseudomonas aeruginosa DSM50071T after 5 days of incubation. 61

Fig. 4. Growth Inhibition zone of Rhizoctonia solani KACC40132, Sclerotinia minor KACC41068 and Colletotrichum gloeosporioides KACC43014 by B. amyloliquefaciens... 63

Fig. 5. The growth inhibition of Botritis cinerea(A) and Fusarium oxysporum(B) mycelium by B. amyloliquefaciens KB-MJK601 antifungal secondary metabolite and spore Scanning electron... 66

Fig. 6. Morphological phenotypes of strain B. amyloliquefaciens KB-MJK601 on TSA medium. 68

Fig. 7. Scanning electron micrographs (SEM) of the strain B. amyloliquefaciens KB-MJK601. 68

Fig. 8. 16S rDNA sequences (1,340 bp) of strain B. amyloliquefaciens KB-MJK601. 72

Fig. 9. Sequences alignments of the 16S rRNA sequences of B. amyloliquefaciens NBRC 15535T (A) and phylogenetic tree (B) with the selected B. amyloliquefaciens KB-MJK601....(이미지참조) 74

Fig. 10. Gyrase subunit B (gyrB) DNA sequence (1,023 bp) of strain B. amyloliquefaciens KB-MJK601. 75

Fig. 11. Sequence alignments of gyrase subunit B gene of strain B. amyloliquefaciens KB-MjK601 with B. amyloliquejaciens strain NMSX4 (A) and phylogenetic tree (B) Numbers at branches... 77

Fig. 12. PCR detection of biosynthetic genes corresponding zwitterrnicin A (ZWIT, itrurin AUTUD, bacillomycin D(BACC, surfacin(SUR3, fengycin(FEND, fengycin(FENA1),... 82

Fig. 13. B. amyloliquefaciens KB-MJK601 of biosynthestic genes by PCR detection corresponding to the 2,3-butanediol and indole-3-acetic acid respectively.... 84

Fig. 14. Cell growth and antifungal activity profiles by B amyloliquefaciens KB-MJK601 in 5L jar fermentor culture B amyloliquefaciens KB-MJK601 was grown in medium containing 15% glucose, 06%... 96

Fig. 15. Changes of B. amyloliquefaciens KB-MJK601 sporation character and colony unit in cultivation hours. 98

Fig. 16. Stability of B. amyloliquefaciens KB-MJK601 in various range of pH. 99

Fig. 17. Stability of B. amyloliquefaciens KB-MjK601 in various range of NaC1 concentration. 100

Fig. 18. Occurrence of bakanae djsease of rice according to djfferent spore treatment on a Komada's meilium after five days seedlings. 102

Fig. 19. The effect of Bakanae diseased rice according to different spore concentration treatment on a Komada's medium after five days seedlings. 103

Fig. 20. The control effect of bakanae diseased rice by treatment of different concentration of B. amyloliquefaciens KB-MJK601 after 15 days seedlings. 105

Fig. 21. The control effect of bakanae diseased rice by treatment of different concentration of B. amyloliquefaciens KB-MJK601 after 15 days seedlings. 106

Fig. 22. Antifungal activity of B. amyloliquefaciens KB-MJK601 against rice sheath blight. 108

Fig. 23. Antifungal activity of B. amyloliquefaciens KB-MJK601 against rice sheath blight 109

Fig. 24. Biological control of cucumber Sclerotinia rot in cucumber by B. amyloliquefaciens KB-MJK601. 111

Fig. 25. Biological control of cucumber Sclerotinia rot in cucumber by B. amyloliquefaciens KB-MJK601. 111

Fig. 26. Biological control of Sclerotinia rot in lettuce by B. amyloliquefaciens KB-MJK601. 113

Fig. 27. Evaluation of root growth promotion of cherry tomato by inoculation of B. amyloliquefaciens KB-MJK601. 117

Fig. 28. The test of dispersing agent ability on the plant pathogenic fungi Rhizoctonia solani. 124

Fig. 29. The test of dispersing agent P.M.H.l.(polyalkyleneoxide modified heptamethy ltrisiloxane) ability on soybean leaf. 124

Fig. 30. Changes of B. amyloliquefaciens KB-MJK601 spore germination by several moisture agent concentration proportion. 127

Fig. 31. Selection of UV blocker agent to reduce damage of B. amyloliquefaciens KB-MJK601. 128

Fig. 32. The synergistic effect of plant extracts, Eucalyptus oil and microorganisms on mycelial growth inhibition of Colletotrichum gloeosporioides. 130

Fig. 33. Control value of spraying prototype different dilution times to chestnut tree jabong variety in 2012 and 2013. 133

Fig. 34. Control value of spraying prototype 500 times dilution to chestnut tree as early maturing variety, medium maturing variety and late flowering variety. 135

Fig. 35. Colletotrichum gloeosporioides diseased Jabong chestnut medium maturing after variety prototype 500 times dilution treatment and control. 135

Fig. 36. Control value of treatment of prototype 500 times dilution to chestnut tree(early maturing variety, Danteak) in 4℃ and 25℃ storage test. 137

Fig. 37. C. gloeosporioides diseased Danteak chestnut, an early maturing variety in 25℃ storage test during 14 days after prototype 500 times dilution treatment. 137

Fig. 38. Room temperature storage test of mediwn maturing variety Jabong chestnut after different dilution times treatment of prototype to chestnut tree. 139

Fig. 39. C. gloeosporioides diseased Jabong chestnut medium maturing variety in room temperature storage test after prototype 500 times dilution treatment. 139