武夷菌素作用机制的研究
摘要
武夷菌素是从不吸水链霉菌武夷变种(Streptomyces ahygroscopicus var.wuyiensis)的发酵液中分离出的活性成分，主要成分武夷菌素A是具有胞苷骨架的新型核苷类抗生素。本研究测定了武夷菌素的抗菌谱及其对番茄灰霉菌菌丝生长和分生孢子萌发的影响，探讨了武夷菌素抑制灰霉菌的作用机制，最后测定了武夷菌素对番茄幼苗体内四种抗病相关酶活性的影响。研究结果如下：
1．武夷菌素对供试的14种病原真菌和3种细菌具有不同程度的抑制作用。
2．武夷菌素纯品对番茄灰霉菌分生孢子萌发和菌丝生长的有效中浓度(EC_(50))分别为14.1μg/ml和18.0μg/ml。当武夷菌素的浓度达到100μg/ml时，完全抑制灰霉菌分生孢子的萌发。经武夷菌素(浓度≥10μg／ml)处理后的灰霉菌菌丝和刚萌发的分生孢子的致病性明显下降。光镜和电镜观察表明，武夷菌素(浓度≥25μg/ml)处理的灰霉菌菌丝和分生孢子的芽管出现形态结构的异常变化。灰霉菌菌丝生长的异常表现为分枝增多、膨大缢缩和液泡数目增多及合并等。武夷菌素(100μg/ml)处理灰霉菌后，菌丝顶端出现原生质渗漏的现象。灰霉菌分生孢子萌发的异常表现为芽管膨大及缢缩等。
3．灰霉菌菌丝被武夷菌素处理后，菌丝细胞膜的透性发生变化，具体表现为培养液的电导率随着武夷菌素浓度(浓度＞25μg/ml)的增加而增大。武夷菌素对灰霉菌菌丝麦角甾醇的合成影响不大。同位素标记掺入试验表明武夷菌素能抑制~(14)C-谷氨酸(蛋白质的前体物)的掺入，而不能阻止~3H-葡萄糖胺(几丁质的前体物)和~3H-腺嘌呤(核酸的前体物)的掺入，这表明武夷菌素可抑制菌丝蛋白质的合成，而对几丁质和核酸的合成没有影响。
4．武夷菌素能诱导番茄幼苗体内的抗病性相关酶如超氧化物岐化酶、过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶活性的增加。这表明在番茄上施用武夷菌素可能提高番茄幼苗的抗病性。
根据上述结果可以看出武夷菌素既能抑制番茄灰霉病菌，同时又能诱导番茄幼苗产生抗病反应，因而存在着较大的开发应用潜力。
Wuyiencin is a nucleotide antibiotic produced by Streptomyces ahygroscopicus var. wuyiensis. In this research, the inhibition and the mode of action of wuyiencin against Botrytis cinerea as well as other pathogens were characterized. The effect of this antibiotic on the activity of four resistance-related enzymes in tomato seedlings were also studied. The results include:
1. Wuyiencin exhibited an inhibitory ability to 14 species of phytopathogenic fungi and 3 species of bacteria (1 pathogen and 2 non-pathogens). Botrytis cinerea was more sensitive to the antibiotic than the other microorganisms.
2. Wuyiencin inhibited the growth of hyphae and the germination of conidia of B. cinerea and the value of 50% effective concentration (EC50) was 18.0 g/ml and 14.1 g/ml, respectively. It could completely inhibit the germination of conidia of B. cinerea when the concentration of wuyiencin reached 100 g/ml. The pathogenicity of hyphae and conidia of B. cinerea treated with wuyiencin ( 10 g/ml) was significantly attenuated. Microscopic observation showed that wuyiencin could interfer the normal development of hyphae of B. cinerea by the increased formation of branches and hyphal vacuoles. Leakage of cytoplasm from hyphal tips of B. cinerea treated with wuyiencin at 100 g/ml was also observed. Germ tubes of B. cinerea treated with wuyiencin at 75 g/ml were constricted in shape.
3. Wuyiencin was detected to influence on the permeability of the cell membrane of B. cinerea as shown by the increased electric conductivity in the cultural filtrates of the pathogen. However, it had no effect on sterol biosynthesis of this fungus. With 3H-glucosamine, 3H-adenine and 14C-glutamate as the precursors, incorporation experiments showed that wuyiencin significantly inhibited the protein synthesis, but had no significant effect on the synthesis of nucleic acid and chitin of B. cinerea.
4. The increased activities of superoxide dismutase, peroxidase, polyphenol oxidase and phenylalanine ammoniam-lyase in tomato seedlings upon the treatment by wuyiencin at 100 g/ml were detected. This suggests that wuyiencin could trigger the defense reactions of tomato plants.
A conclusion can be made that wuyiencin is a potential biofungicide for the control of B. cinerea as it not only can inhibit the pathogen directly, but also induce the resistance of tomato.
1．武夷菌素对供试的14种病原真菌和3种细菌具有不同程度的抑制作用。
2．武夷菌素纯品对番茄灰霉菌分生孢子萌发和菌丝生长的有效中浓度(EC_(50))分别为14.1μg/ml和18.0μg/ml。当武夷菌素的浓度达到100μg/ml时，完全抑制灰霉菌分生孢子的萌发。经武夷菌素(浓度≥10μg／ml)处理后的灰霉菌菌丝和刚萌发的分生孢子的致病性明显下降。光镜和电镜观察表明，武夷菌素(浓度≥25μg/ml)处理的灰霉菌菌丝和分生孢子的芽管出现形态结构的异常变化。灰霉菌菌丝生长的异常表现为分枝增多、膨大缢缩和液泡数目增多及合并等。武夷菌素(100μg/ml)处理灰霉菌后，菌丝顶端出现原生质渗漏的现象。灰霉菌分生孢子萌发的异常表现为芽管膨大及缢缩等。
3．灰霉菌菌丝被武夷菌素处理后，菌丝细胞膜的透性发生变化，具体表现为培养液的电导率随着武夷菌素浓度(浓度＞25μg/ml)的增加而增大。武夷菌素对灰霉菌菌丝麦角甾醇的合成影响不大。同位素标记掺入试验表明武夷菌素能抑制~(14)C-谷氨酸(蛋白质的前体物)的掺入，而不能阻止~3H-葡萄糖胺(几丁质的前体物)和~3H-腺嘌呤(核酸的前体物)的掺入，这表明武夷菌素可抑制菌丝蛋白质的合成，而对几丁质和核酸的合成没有影响。
4．武夷菌素能诱导番茄幼苗体内的抗病性相关酶如超氧化物岐化酶、过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶活性的增加。这表明在番茄上施用武夷菌素可能提高番茄幼苗的抗病性。
根据上述结果可以看出武夷菌素既能抑制番茄灰霉病菌，同时又能诱导番茄幼苗产生抗病反应，因而存在着较大的开发应用潜力。
Wuyiencin is a nucleotide antibiotic produced by Streptomyces ahygroscopicus var. wuyiensis. In this research, the inhibition and the mode of action of wuyiencin against Botrytis cinerea as well as other pathogens were characterized. The effect of this antibiotic on the activity of four resistance-related enzymes in tomato seedlings were also studied. The results include:
1. Wuyiencin exhibited an inhibitory ability to 14 species of phytopathogenic fungi and 3 species of bacteria (1 pathogen and 2 non-pathogens). Botrytis cinerea was more sensitive to the antibiotic than the other microorganisms.
2. Wuyiencin inhibited the growth of hyphae and the germination of conidia of B. cinerea and the value of 50% effective concentration (EC50) was 18.0 g/ml and 14.1 g/ml, respectively. It could completely inhibit the germination of conidia of B. cinerea when the concentration of wuyiencin reached 100 g/ml. The pathogenicity of hyphae and conidia of B. cinerea treated with wuyiencin ( 10 g/ml) was significantly attenuated. Microscopic observation showed that wuyiencin could interfer the normal development of hyphae of B. cinerea by the increased formation of branches and hyphal vacuoles. Leakage of cytoplasm from hyphal tips of B. cinerea treated with wuyiencin at 100 g/ml was also observed. Germ tubes of B. cinerea treated with wuyiencin at 75 g/ml were constricted in shape.
3. Wuyiencin was detected to influence on the permeability of the cell membrane of B. cinerea as shown by the increased electric conductivity in the cultural filtrates of the pathogen. However, it had no effect on sterol biosynthesis of this fungus. With 3H-glucosamine, 3H-adenine and 14C-glutamate as the precursors, incorporation experiments showed that wuyiencin significantly inhibited the protein synthesis, but had no significant effect on the synthesis of nucleic acid and chitin of B. cinerea.
4. The increased activities of superoxide dismutase, peroxidase, polyphenol oxidase and phenylalanine ammoniam-lyase in tomato seedlings upon the treatment by wuyiencin at 100 g/ml were detected. This suggests that wuyiencin could trigger the defense reactions of tomato plants.
A conclusion can be made that wuyiencin is a potential biofungicide for the control of B. cinerea as it not only can inhibit the pathogen directly, but also induce the resistance of tomato.
引文
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67. Leroux P, Fritz R, Debieu D. Mechanisms of resistance to fungicides in field strains of Botrytis cinerea. Pest Manag Sci, 2002, 58(9): 876-888
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69. Mashi E I, Paul B. Secretion of beta-1, 3-glucanases by the yeast Pichia membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing grey mold disease of the grapevine. Curr Microbiol, 2002, 44(6): 391-395
70. Milling R J, Richardson C J. Mode of action the anilino-pyrimidine fungicide pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea. Pestic sci, 1995, 45:43-45
71. Nakashita H, Watanabe K, Hara O. Studies on the biosynthesis of bialaphos. Biochemical mechanism of C-P bond formation: discovery of phosphonopyruvate decarboxylase that catalyzes the formation of phosphonoacetaldehyde from phosphonopyruvate. J Antibiot, 1997, 50(3): 212-219
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73. Shirane N, Masuko M, Takeda R. Effects of SSf-126, a novel alkoxyiminoacetamide blasticide, on mycelial growth and oxygen consumption of Pyricularia oryzae. Plant Pathology, 1994, 44:636-640
74. Slawecki R A, Ryan E P, Young D H. Novel fungitoxicity assays for inhibition of germination-associated adhesion of Botrytis cinerea and Puccinia recondita spores. Applied and Environmental Microbiology, 2002, 68(2): 597-601
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76. Sugiyama M, Mochizuki H, Nimi O. Mechanism of protection of protein synthesis against streptomycin inhibition in a producing strain. J Antibiot, 1981, 34(9): 1183-1188
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80. Walker R, Emslie K, Allan E. Bioassay methods for antifungal activity by Pseudomnas antimicrobica against the grey mould pathogen Botrylis cinerea. Journal of Applied Bacteriology, 1996, 81: 531-537
81. Walker R, Innes C M J, Allan E J. The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea. Letters in Applied Microbiology, 2001, 32:346-348
82. Walker R, Emslie K A, Allan E J. The symbiosis of bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea. Letters in Applied Microbiology, 2002, 34(1): 42-47
83. Wilde L G. Effect of fluoride on superoxide dismutase (SOD) activity in germinating mung bean seedlings. Fluoride, 1998, 31(2): 81-88
84. Xu Z T, Li L, Li C S, Qi J S. Studies on chemical control of Botrytis cinerea in protected environment fields. The first Asian conference on plant pathology, Beijing, 2000. 334
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65. Huang L L, Kang Z S, Yan Y G, Zhang G Q. Effects of ergosterol biosynthesis-inhibiting fungicide triadimefon on the development of venturia inaequalis on apple leaves. Mycosystema, 2001, 20(2): 250-257
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67. Leroux P, Fritz R, Debieu D. Mechanisms of resistance to fungicides in field strains of Botrytis cinerea. Pest Manag Sci, 2002, 58(9): 876-888
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73. Shirane N, Masuko M, Takeda R. Effects of SSf-126, a novel alkoxyiminoacetamide blasticide, on mycelial growth and oxygen consumption of Pyricularia oryzae. Plant Pathology, 1994, 44:636-640
74. Slawecki R A, Ryan E P, Young D H. Novel fungitoxicity assays for inhibition of germination-associated adhesion of Botrytis cinerea and Puccinia recondita spores. Applied and Environmental Microbiology, 2002, 68(2): 597-601
75. Stehmann C, Kaptteyn J C, Ward M A D. Development of a cell-free assay from Botrytis cinerea an biochemical screen for sterol biosyntesis inhibitors. Pestic Sci, 1994, 40:1-8
76. Sugiyama M, Mochizuki H, Nimi O. Mechanism of protection of protein synthesis against streptomycin inhibition in a producing strain. J Antibiot, 1981, 34(9): 1183-1188
77. Sugiyama M, Paik S, Nomi R. Mechanism of self-protection in a puromycin-producing microorganism. J Gen Microbiol, 1985, 131(8): 1999-2005
78. Tanaka N. Studies on the mechanism of action of antibiotcs. Nippon Saikingaku Zasshi, 1987, 42(5): 707-716
79. Tomasz A. Penicillin-bingding Proteins in bacteria. Ann Intern Med, 1982, 96(4): 502-506
80. Walker R, Emslie K, Allan E. Bioassay methods for antifungal activity by Pseudomnas antimicrobica against the grey mould pathogen Botrylis cinerea. Journal of Applied Bacteriology, 1996, 81: 531-537
81. Walker R, Innes C M J, Allan E J. The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea. Letters in Applied Microbiology, 2001, 32:346-348
82. Walker R, Emslie K A, Allan E J. The symbiosis of bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea. Letters in Applied Microbiology, 2002, 34(1): 42-47
83. Wilde L G. Effect of fluoride on superoxide dismutase (SOD) activity in germinating mung bean seedlings. Fluoride, 1998, 31(2): 81-88
84. Xu Z T, Li L, Li C S, Qi J S. Studies on chemical control of Botrytis cinerea in protected environment fields. The first Asian conference on plant pathology, Beijing, 2000. 334
85．Yamaguchi H．微生物来源农药的最新进展．国外医药抗生素分册，1990，11(2)：137-141
86. Yee Y C, Kisslinger B, Yu V L, Jin D J. A mechanism of rifamycin inhibition and resistance in Pseudomonas aeruginosa. J Antimicrob Chemother, 1996, 38(1): 133-140
87. Yocum R, Rasmussen J, trominger J. The mechanism of action of penicillin: Penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase. J Biol Chem, 1980, 255(9): 3977-3986