[보고서]미생물 유래 천연물질을 이용한 수확 후 병해억제제 개발 및 산업화

이용훈

보고서 정보

주관연구기관

전북대학교
Chonbuk National University

연구책임자

이용훈

참여연구자

유상미 , 김동민 , 이하경 , 채종찬

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2012-02

과제시작연도

2011

주관부처

농촌진흥청

사업 관리 기관

농촌진흥청
Rural Development Administration

등록번호

TRKO201200009778

과제고유번호

1395021727

DB 구축일자

2013-05-20

표 Identification of bacterial strains by carbon source utilization, analysis of fatty acid composition and 16S rDNA sequence
표 Results of carbon source utilization pattern by API 50CHB kit
표 Incidence of green (■) and blue (□) mold on wounded mandarin fruits treated with different concentrations of B. amyloliquefaciens JBC36. Wounded mandarin fruits inoculated with 105 and 106 spores/ml of P. digitatum and P. italicum, respectively, followed by treatment with different concentrations of B. amyloliquefaciens JBC36. Disease incidence was recorded 5 DAI at 20°C and 90% RH. The same letters are not significantly different at P<0.05 according to DMRT. Vertical bars indicate standard deviation of the means.
표 Percent inhibition of mycelial growth of fungal pathogens by B. amyloliquefaciens JBC36.
표 Inhibition of fungal growth by B. amyloliquefaciens JBC36. (A) P. digitatum, (B) P. italicum, (C) P. expansum, (D) D. citri, (E) B. cinerea, (F) A. alternata, (G) P. citrophthora, (H) C. gloeosporioides, (I) G. candidum, (J) F. graminiarum.
표 Percent germination and germ tube length of P. digitatum and P. italicum spores in the presence of the B. amyloliquefaciens JBC36.
표 Inhibition of the mycelial growth of P. digitatum (a) and P. italicum (b) by CFS of JBC36. PDA mixed with each concentration of CFS was poured on the plates (5 cm diameter) and 10 ul of spore suspension was spotted in the center of the plates. Inhibition was expressed as the following equation: 100 ‐ [(radius of treatment)2 × 3.14/(radius of control)2 × 3.14] × 100.
표 Inhibition of the mycelial growth of P. digitatum by cell-free supernatants of JBC36.
표 Inhibition of the germination rate (a) and germ tube length (b) of P. digitatum by cell‐free supernatant of JBC36. CFS and spore suspension of P. digitatum (1 × 106 spores/ml) were added to 10% PDB. The percentage of spore germination was observed after 24 h incubation at 25℃. Germ tube length was measured with a light microscope at different concentration of the antagonist
표 Antifungal compound production according to growth stage
표 Yield and EC50 of SM from B. amyloliquefaciens JBC36 according to media
표 Stability of secondary metabolites according to the pH
표 Stability of secondary metabolites by treatment of various temperature. JBC316 was cultured in LB at 25℃ at 200rpm. After 48 hr incubation, bacterial cells were eliminated by centrifugation. The supernatants was incubated at 80℃, 100℃, and 121℃ for 1 hr, 1 hr, and 15 min. Fifty 50㎕ of treated supernatant was inoculated on the paper disks which were placed PDA media
표 Increase of control efficacy by combined treatment of JBC36 with secondary metabolites. Treatment: Secondary metabolite (5 mg/ml), bacteria (5 × 106 cfu/ml), pathogen (105 spore/ml)
$RP-HPLC chromatogram of antibiotic compounds produced by JBC36. The antifungal fraction obtained from gel filtration was eluted with Zorbax C18 column with linear gradient of 10-100% (V/V) acetonitrile/water with 0.04% trifluoroacetic acid. The antibiotic compounds were obtained at the retention time of 27-29, 36-44, and 73-77min.$ 표 RP-HPLC chromatogram of antibiotic compounds produced by JBC36. The antifungal fraction obtained from gel filtration was eluted with Zorbax C18 column with linear gradient of 10-100% (V/V) acetonitrile/water with 0.04% trifluoroacetic acid. The antibiotic compounds were obtained at the retention time of 27-29, 36-44, and 73-77min.
표 LC/ESI-MS spectrum of antifungal compound 2. Three ion peaks at m/z 1448.88, 1462.84, and 1476.85 were identified as fengycin homologs with different side chain length.
표 LC/ESI-MS/MS spectra resulting from precursor ion of m/z 1462.84 [M+H]+ for compound2. The fragmentions at m/z 1080.53, 966.54, and 201.11 suggested the presence of β-hydroxy fattyacid-Glu, β-hydroxy FA-Glu-Orn, and Ala-Glu sequence. A series of b-type fragments at m/z 226.12, 389.18, and502.28 ascribable to the sequence of Pro-Gln-Tyr-Ile.
표 Effect of VOCs of B. amyloliquefaciens JBC36 on mycelial growth of P. digitatum and P. italicum
표 Inhibition of mycelial growth of P.digitatum and P.italicum by VOC from B. amyloliquefaciens JBC36. (A) P.digitatum with JBC36, (B) P. digitatum control, (C) P.italicum with JBC36, (D) P.italicum control
표 Percentage of disease incidence with JBC36.
표 Summary of biocontrol related-mechanisms
표 Natural resources for growing biological control agents and their antifungal compound production
표 Effect of culture media on antifungal activity of CFS of JBC36
표 Bacterial growth rate on culture resources. B. amyloliquefaciens strain JBC36 was cultured in LB (◆), NYDB (■), Cheonnyuncho stem powder (40 g/L, pH 4.5, ▲), Cheonnyuncho fruit power (40 g/L, pH 4.5, ×), Mandarin peel extract (100 g/L, pH 4.5, ●).
표 Difference of bacterial growth according to pH of media. LB (◇, ◆), NYDB (□, ■), Cheonnyuncho stem powder (40 g/L, △, ▲), Cheonnyuncho fruit power (40 g/L, ○, ●). Blank legend is pH 4.5 and black legend is pH 6.8.
표 Antifungal activity by CFS of B. amyloliquefaciens JBC36 against P. digitatum and P. italicum according to various culture resources and pH conditions
표 Antifungal activity of B. amyloliquefaciens JBC36 on P. digitatum and P. italicum according to culture resources and pH conditions. CFS was produced from (a) pH 4.5 and (b) pH 6.8 of (A) LB, (B) NYDB, (C) Cheonnyuncho stem powder, and (D) Cheonnyuncho fruit powder.
표 Antifungal activity by cell-free supernatants of B. amyloliquefaciens JBC36
표 Inhibition of the mycelial growth of P. digitatum (left 2 panel) and P. italicum (right 2 panel) by cell-free supernatants of B. amyloliquefaciens JBC36. CFS was produced from 20% (a) and 40% (b) of (A, C) Cheonnyuncho stem powder and (B, D) Cheonnyuncho fruit powder.
표 Inhibition of the mycelial growth of P. digitatum and P. italicum by cell-free supernatants of antagonists
표 Inhibition of the mycelial growth of P. digitatum (left) and P. italicum (right) by cell-free supernatants of JBC36. PDA mixed with conidial suspension was poured on the plates (9cm diameter), 50 ul of CFS was spotted in the paper disk.
표 Percent germination of P. digitatum and P. italicum spores by cell-free supernatants of B. amyloliquefaciens JBC36
표 Interaction between B. amyloliquefaciens JBC36 and P. digitatum (upper panel) or P. italicum (lower panel). (Left panel) Light microscope micrography of test with 25% of cell-free supernatant (Cheonnyuncho stem powder or Citrus peel extract) of B. amyloliquefaciens JBC36 in 20% PDB with conidial suspension (2 × 106 spores/ml) after 24 h incubation at 25℃. (Right panel) Light microscope micrography of conidial suspension (2 × 106 spores/ml) in 20% PDB without cell-free supernatant
표 Antifungal activity by cell-free supernatants of B. amyloliquefaciens JBC36 against P. digitatum
표 Inhibition of the mycelial growth of P. digitatum by cell-free supernatants of B. amyloliquefaciens JBC36 according to various culture resources
표 Variation of antifungal compound production in B. amyloliquefaciens JBC36 according to the culture material and organic solvent which used for extraction of the compound.
표 Variation of antifungal compound production in B. amyloliquefaciens JBC36 according to the culture material and organic solvent which used for extraction of the compound.
표 Flow chart for the analysis of antifungal compound produced from MPE.
$RP-HPLC chromatogram of antifungal fractions produced by B. amyloliquefaciens JBC36. The antifungal fraction obtained from gel filtration was eluted with Zorbax C18 column with linear gradient of 10-100% (V/V) methanol with 0.04% trifluoroacetic acid.$ 표 RP-HPLC chromatogram of antifungal fractions produced by B. amyloliquefaciens JBC36. The antifungal fraction obtained from gel filtration was eluted with Zorbax C18 column with linear gradient of 10-100% (V/V) methanol with 0.04% trifluoroacetic acid.
$RP-HPLC chromatogram of antibiotic compounds produced by JBC36. The antifungal fraction obtained from gel filtration was eluted with Zorbax C18 column with linear gradient of 10-100% (V/V) acetonitrile/water with 0.04% trifluoroacetic acid.$ 표 RP-HPLC chromatogram of antibiotic compounds produced by JBC36. The antifungal fraction obtained from gel filtration was eluted with Zorbax C18 column with linear gradient of 10-100% (V/V) acetonitrile/water with 0.04% trifluoroacetic acid.
$LC/ESI-MS spectrum of each antifungal fraction. Ion peaks at m/z 1449.6, 1463.6, 1477.6 was identified as C15 fengycin. C16 fengycin, and C17 fengycin$ 표 LC/ESI-MS spectrum of each antifungal fraction. Ion peaks at m/z 1449.6, 1463.6, 1477.6 was identified as C15 fengycin. C16 fengycin, and C17 fengycin
표 Identification of bacterial strain JBC17.
표 Morphological and physiological characteristics of JBC17.
표 Suppression of green and blue molds of mandarins by treatment with P. putida JBC17 strains. P. digitatum (■) and P. italicum (□) at concentrations of 105 and 106 spores/ml, respectively, were inoculated on wounded mandarin fruits. P. putida JBC 17 was inoculated at concentration of 5 ⅹ107 cfu/ml and incubated 20℃ for 5 days. Vertical bars indicate standard deviation of the means
표 Incidence of green mold on wounded mandarin treated with different concentrations of P. putida JBC17. Wounded mandarin fruits inoculated with 105 spores/ml of P. digitatum, followed by treatment with different concentrations of P. putida JBC17. Disease incidence was recorded after 5 days incubation at 20℃ and 90% RH. Columns with the same letter are not significantly different (p<0.05) according to the LSD. Vertical bars indicate standard deviation.
표 Antifungal activity of JBC17 against fungal pathogens
표 Inhibition of mycelial growth of fungal pathogens by JBC17 in a dual culture test.
표 Percent germination and germ tube length of P. digitatum and P. italicum conidia in presence of JBC17.
표 Percent germination of P. digitatum spores on PTFE membranes in cylinders.
표 Summary for the mode of action of JBC36
표 Percentage of decay of mandarin fruits coated with different formulations.
표 Effect of different coating materials mixed with or without JBC17 on the disease severity caused by P. digitatum on mandarin fruits. The treatment was as follows
표 Phenotypic characteristics of the HM1
표 Suppression of apple grey mold rot by B. subtilis HM1. 105 spores/ml of C. acutatum (anthracnose disease) and B. cinerea (gray mold rot) were inoculated as a pathogenic fungi. 108 of bacterial cells and cell free culture broth containing 56 μg/ml of lipopeptides were treated. All treatment performed on wounded surfaces of apples.
표 Antagonistic activity of B. subtilis strain HM1 against various phytopathogenic fungi in vitro
표 Inhibition activity against various phytopathogenic fungi by cell free filtrates of B. subtilis HM1
표 Process of lipopeptides extraction.
표 MALDI-TOF MS analysis of lipopeptides from B. subtilis strain HM1. Fengycin, iturin and surfactin mass peaks were detected in crude extracts prepared from the culture filtrate of the strain HM1 grown in the LB medium
표 PCR amplification using gene specific primers for antibiotic biosynthesis genes from the strain HM1. Lane 1, fenA; lane 2, fenD; lane 3, fenE; lane 4, ituA1; lane 5, ituA4; lane 6, ituD; lane 7, srfAA, lane 8, srfAD gene.
$Purification of lipopeptides by gel filtration. Crude lipopeptides were separated into 120 fractions with equal volume. The antifungal activity was analyzed by in vitro assay on PDA.$ 표 Purification of lipopeptides by gel filtration. Crude lipopeptides were separated into 120 fractions with equal volume. The antifungal activity was analyzed by in vitro assay on PDA.
$HPLC chromatogram of lipopeptides extracted from culture supernatant of B. subtilis HM1. The eluted fractions were detected at absorbance of 210nm. Iturin was eluted at 40∼46min and fengycin was eluted at 54∼55min of retention time, while surfactin was detected at 73∼75min. 160 fractions were collected by every 30 sec.$ 표 HPLC chromatogram of lipopeptides extracted from culture supernatant of B. subtilis HM1. The eluted fractions were detected at absorbance of 210nm. Iturin was eluted at 40∼46min and fengycin was eluted at 54∼55min of retention time, while surfactin was detected at 73∼75min. 160 fractions were collected by every 30 sec.
표 Effect of lipopeptides on conidia germination of B. cinerea. The phyto pathogenic fungi were treated with the partially purified lipopeptide at 100μg/ml and the conidia germination was observed by optical microscopy after 48h of incubation. Representative pictures showing the effect of each lipopeptide treatment are given
표 Identification of fengycin A and B
표 Hydrolysis of lactone bond. (a) MALDI-TOF MS spectrum of before and (b) after hydrolysis.
표 MS/MS spectrum of purified fengycin before (A) and after (B) hydrolysis of the lactone bond by 1M KOH.
표 MALDI-TOF/TOF mass spectrum of protonated ions of m/ z 1464.8 (A) and m/ z 1492.8 (B) showing typical fragments of the fengycin A and B.
표 Lipopeptides concentration of B. subtilis HM1 in different synthetic media.
표 Lipopeptides production by B. subtilis HM1 grown in minimal media with different carbon and nitrogen sources
표 Growth curve of mixed culture strains
표 Inhibition of phytopathogenic fungi mycelial growth on potato dextrose agar by the bacterial isolates. (A); C .coccodes, (B); C. gloeosporioides, (C); C, dematium, B; B. subtilis HM1 , P; Pseudomonas sp., M: B. subtilis HM1 + Pseudomonas sp.
표 Fungicides and insecticides used in study
표 Inoculation position of dual culture assay.
표 Resistant patterns of selection strains for pesticides
표 Growth of useful bacteria in pesticides.
표 Antagonistic patterns of pesticides treatment strain of C. coccodes
표 Inhibition of C. coccodes mycelial growth on potato dextrose agar by the Bacterial + chemical mixture
표 Antagonistic patterns of pesticides treatment strain of C. gloeosporioides
표 Inhibition of C. gloeosporioides mycelial growth on potato dextrose agar by the Bacterial + chemical mixture
표 Antagonistic patterns of pesticides treatment strain of C. dematium
표 Inhibition of C. dematium mycelial growth on potato dextrose agar by the Bacterial + chemical mixture.
표 Coating formulations based on Canauba wax or parafilm oil. Paraffin oil based coating (POC) solution was prepared with 0.03% sorbitan monostearate in 0.1% paraffin oil. Carnauba wax based coating solution (CWC) was manufactured with 0.15% morpholine olate in 0.07% carnauba wax and 0.05% paraffin oil. Both solutions were prepared in 100 times concentration, and diluted right before use.
표 Percentage of decay of mandarin fruits coated with different formulations
표 Effect of different coating materials mixed with or without JBC36 on the severity of green mold on mandarin fruits. The treatment was as follows
표 Effect of coating materials on population dynamics of B. amyloliquefaciens JBC36. The antagonistic bacteria (5×107 cfu/ml) were mixed with carnauba based coating solution (■), paraffin oil based coating solution (▲), and distilled water (◆), and viable bacterial cells were assayed at 1, 7 and 14 days after incubation at 4℃. Points represent the means of three replications and vertical bars indicate standard deviation of the means.
표 Biofilm formation by B. amyloliquefaciens JBC36 with different formulations. Formation in polystyrene microtiter plate was observed at 590 nm. Results represent the mean and standard deviation of a representative experiment
표 Percent germination and germ tube length of P. digitatum spore in the presence of different concentrations of carnauba wax. The percentage of spore germination and germ tube length was recorded with a light microscope after 24 h incubation in PDB (10%) at 25℃. Control means 1 × 106 spores/ml of P. digitatum in 10% PDB. Vertical bars indicate standard deviation of means.
표 Dynamics of the B. amyloliquefaciens JBC36 population on mandarine fruits with different formulations. The mandarin fruits were immersed in carnauba based coating solution (■), paraffin oil based coating solution (▲), and distilled water (◆) with JBC36 (5×107 cfu/ml). The bacterial population was assayed at 1, 7 and 14 days after incubation at 20℃, 90% RH. Points represent the means of three replications and vertical bars indicate standard errors of the means
표 Inhibition of mycelial growth according to molecular weight and concentration of chitosan
표 Effects of COS molecular weight and concentration on mycelial growth of P. digitatum, P. italicum, and P. expansum. Mycelial growth of (A) P. digitatum and (B) P. italicum on PDA at concentration of 0, 0.05, 0.1, 0.5, 1% (left to right) of MCOS (M.W. 3,000-5,000) and (C) P. expansum on PDA added with HCOS (M.W. 5,000-10,000) of COS. The photos were taken 7 DAI.
표 Percent germination and germ tube length of P. italicum spores at various concentrations of COS
표 Effect of MCOS solution on survival of B. amyloliquefaciens JBC36. Survival of antagonistic bacteria in (■) 0.5% and (▲) 1% of COS. Control in D.W. (◆).
표 Percentage of decay of mandarin fruits coated with chitosan with B. amyloiquefaciens JBC36 at 1×107 cells/ml. Wounded mandarin fruits inoculated with 105 spores/ml of P. digitatum followed by treatment with different concentrations of B. amyloliquefaciens JBC36 after 21 days incubation at 20℃ and 90% RH. Control means green mold only. Columns with the same letter are not significantly different (p < 0.05) according to the LSD. Vertical bars indicate standard deviation.
표 Process for microcapsulation of bacterial strains
표 Processes for amelioration of microcapsulation of bacterial strains
표 Process for wettable powder type-biological control formulation
표 Composition of experimental groups.
표 Formulations produced by microcapsulation of biological control agents.
표 Electron micrograph of capsulations observed by transmission electron microscope
표 Change of microcapsule size according to the concentration of emulsifer.
표 Change of microcapsule size according to the speed of stirring.
표 Final products of wettable type formulation of biocontrol agents.
표 Effects of JBC36 on mice

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