番茄灰霉病菌拮抗稀有放线菌的分离及其抑菌物质分析
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摘要
为筛选新型有效的番茄灰霉病菌天然抑制产物,采用平板对峙法和菌丝生长速率法从44株采自辽宁各地的放线菌中筛选植物病原真菌拮抗放线菌,通过孢子萌发生长法和系统发育树分析番茄灰霉病菌拮抗放线菌的抑菌活性和分类地位,测试高抑菌活性菌株无菌发酵液活性物质的稳定性,并以高分辨率飞行质谱扫描鉴定稀有放线菌发酵液的抑菌产物。结果表明,筛选到13株具有抑菌活性的放线菌,其中SA32、SA33、SA37、SA39、SA45、SA51这6株菌对番茄灰霉病菌的生长及其孢子萌发有抑制效果,SA37和SA45对番茄灰霉病菌菌丝生长的抑制率分别高达75.13%和79.27%,对番茄灰霉病菌孢子萌发的抑制率分别高达86.25%和93.43%。系统发育树分析表明,SA45聚类于稀有放线菌北里孢菌属,其余5株菌均聚类于链霉菌属。对比高活性菌株SA37和SA45发酵液在-20～120℃、pH值1～14和3种酶(蛋白酶K、胰蛋白酶和胃蛋白酶)处理条件下的抑菌稳定性表明,SA45发酵液的抑菌活性和稳定性均高于SA37。高分辨率质谱分析不同溶剂提取的SA45活性物质表明,SA45发酵液中不存在已报道的活性物质。综上所述,北里孢菌SA45发酵液对番茄灰霉病菌具有高效和稳定的抑菌活性,可能存在新型抑菌物质,其菌株及发酵液具有较高的应用价值和研究价值。
In order to obtain new and effective natural antifungal products against Botrytis cinerea, 44 actinomycetes collected from Liaoning province were screened for antagonistic strains with antifungal activity.The antifungal activity and taxonomic status of antagonistic actinomycetes against Botrytis cinerea were analyzed by spore germination method and phylogenetic tree construction.Comparison tests of stability of active substances were performed between the aseptic fermentation broth of strains with significant antifungal activity.The antifungal products from fermentation broth of rare actinomycetes were identified by high resolution flight mass spectrometry.The results showed that 13 antifungal actinomycetes were isolated.SA32,SA33, SA37,SA39,SA45 and SA51 had antagonistic effects against the growth and spore germination of Botrytis cinerea.The inhibition rates of SA37 and SA45 against the mycelium growth of Botrytis cinerea were 75.13% and 79.27%, and their inhibition rates against the spore germination of Botrytis cinerea were 86.25% and 93.43%,respectively.Phylogenetic analysis showed that SA45 was clustered to rare actinomycetes Kitasatospora,and the other 5 strains were clustered to the genus Streptomyces.Under the conditions of-20—120 ℃,pH 1—14 and three enzymes(protease K, trypsin and pepsin),when high-activity strains SA37 and SA45 were compared,the antifungal activity and relative stability of SA45 fermentation broth were higher than those of SA37.The bioactive substances extracted from SA45 with different solvents were detected by high-resolution mass spectrometry,and the result showed that there was no reported active substance in fermentation broth of SA45.In summary,the fermentation broth of Kitasatospora sp.SA45 has efficient and stable antifungal activity against Botrytis cinerea,may contain new antifungal compounds,and its strain and fermentation broth have high application and research value.
In order to obtain new and effective natural antifungal products against Botrytis cinerea, 44 actinomycetes collected from Liaoning province were screened for antagonistic strains with antifungal activity.The antifungal activity and taxonomic status of antagonistic actinomycetes against Botrytis cinerea were analyzed by spore germination method and phylogenetic tree construction.Comparison tests of stability of active substances were performed between the aseptic fermentation broth of strains with significant antifungal activity.The antifungal products from fermentation broth of rare actinomycetes were identified by high resolution flight mass spectrometry.The results showed that 13 antifungal actinomycetes were isolated.SA32,SA33, SA37,SA39,SA45 and SA51 had antagonistic effects against the growth and spore germination of Botrytis cinerea.The inhibition rates of SA37 and SA45 against the mycelium growth of Botrytis cinerea were 75.13% and 79.27%, and their inhibition rates against the spore germination of Botrytis cinerea were 86.25% and 93.43%,respectively.Phylogenetic analysis showed that SA45 was clustered to rare actinomycetes Kitasatospora,and the other 5 strains were clustered to the genus Streptomyces.Under the conditions of-20—120 ℃,pH 1—14 and three enzymes(protease K, trypsin and pepsin),when high-activity strains SA37 and SA45 were compared,the antifungal activity and relative stability of SA45 fermentation broth were higher than those of SA37.The bioactive substances extracted from SA45 with different solvents were detected by high-resolution mass spectrometry,and the result showed that there was no reported active substance in fermentation broth of SA45.In summary,the fermentation broth of Kitasatospora sp.SA45 has efficient and stable antifungal activity against Botrytis cinerea,may contain new antifungal compounds,and its strain and fermentation broth have high application and research value.
引文
[1] 高苇, 王勇, 张春祥. 番茄灰霉病拮抗细菌的筛选与鉴定[J]. 中国植保导刊, 2016, 36(8): 16-19,25.
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[3] 李红晓, 张殿朋, 赵洪新,等. 解淀粉芽孢杆菌MH71抗菌物质理化特性及对番茄灰霉病菌的抑菌活性[J]. 中国生物防治学报, 2016, 32(4): 485-492.
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[5] 赵新贝, 上官妮妮, 李月飞,等. 番茄灰霉病拮抗细菌18BS-12发酵条件的优化及防治效果[J]. 西北农林科技大学学报(自然科学版), 2016, 44(6): 116-124.
[6] 徐大勇, 李志栋, 李峰,等. 番茄灰霉病菌拮抗放线菌的筛选、鉴定及其活性评价[J]. 中国生物防治学报, 2012, 28(2): 298-302.
[7] 王美琴, 马林, 韩巨才,等. 番茄内生菌St24的鉴定及其对灰霉病的生防作用[J]. 应用生态学报, 2012,23(9): 2529-2535.
[8] 高菊芳. 生物农药的作用、应用与功效(一):微生物产物农药[M]. 世界农药, 2001,23(1): 1-6.
[9] 沈玲. 稀有放线菌的分离及其发酵产物的农用活性研究[D]. 杨凌:西北农林科技大学,2006.
[10] 宋洪允, 韩立荣, 冯俊涛,等. 94株土壤放线菌抑菌活性的初步筛选[J]. 西北农业学报, 2010, 19(12): 172-176.
[11] 朱正兵, 吕文静, 纪兆林,等. 土壤生防放线菌的分离及其对油菜菌核病菌的抑制作用[J]. 现代农业科技, 2008(12): 106,108.
[12] 张志斌, 敖武, 熊瑶瑶,等. 内生拮抗放线菌FRo2的鉴定及抑菌活性物质的分离[J]. 微生物学通报, 2014, 41(8): 1574-1581.
[13] BOUKAEW S, CHUENCHIT S, PETCHARAT V. Eva-luation of Streptomyces spp. for biological control of Sclerotium root and stem rot and Ralstonia wilt of chili pepper[J]. Biocontrol, 2011,56(3): 365-374.
[14] 崔志婧. 灰霉菌产孢类群的划分及其遗传多态性分析[D].上海:华东师范大学,2012.
[15] 毕朝位, 仇剑波, 周明国,等. 禾谷镰孢菌Fusarium graminearum Schwabe分生孢子萌发过程中的核相变化及有丝分裂过程观察[J]. 菌物学报, 2008, 27(6): 901-907.
[16] SHI D, AN R, ZHANG W, et al. Stilbene derivatives from Photorhabdus temperata SN259 and their antifungal activities against phytopathogenic fungi[J]. Journal of Agricultural & Food Chemistry, 2017,65(1): 60-65.
[17] WILLETTS H J. Morphology, development and evolution of stromata/sclerotia and macroconidia of the Sclerotiniaceae[J]. Mycological Research, 1997,101(8): 939-952.
[18] URAMOTO M, ITOH Y, SEKIGUCHI R, et al. A new antifungal antibiotic, cystargin: Fermentation, isolation, and characterization[J]. Journal of Antibiotics, 1988,41(12): 1763-1768.
[19] YEO, WOON-HYUNG, SI-KWAN K,et al. Isolation, physico-chemical properties, and biological activity of new thiopeptide antibiotics, kimorexins[J]. Journal of Microbiology and Biotechnology, 1994,4(4): 349-353.
[20] WERNER G, HAGENMAIER H, DRAUTZ H, et al. Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity[J]. Journal of Antibiotics, 1984,37(2): 110-117.
[21] 张孟, 王艳婷, 汪立平, 等. 海洋抑菌放线菌Y15的筛选、鉴定及其代谢产物理化性质[J]. 微生物学通报, 2017, 44(3): 513-524.
[22] OMURA S, TAKAHASHI Y, IWAI Y, et al. Kitasatosporia, a new genus of the order Actinomycetales[J]. Journal of Antibiotics, 1982,35(8): 1013-1019.
[23] YOUNGRYUN C, KEECHEOL S, MO H K, et al. Kitasatospora cheerisanensis sp. nov., a new species of the genus Kitasatospora that produces an antifungal agent[J]. International Journal of Systematic Bacteriology, 1999,49(2): 753-758.
[24] 王云, 郑豪盈, 樊永欣,等. 一株北里孢菌株的分离鉴定及其对松材线虫的致病性[J]. 微生物学通报, 2014, 41(10): 2035-2042.
[25] MOMOSE I, WATANABE T. Tyropeptins, proteasome inhibitors produced by Kitasatospora sp. MK993-dF2[J]. Journal of Antibiotics, 2017,70(5): 542-550.
[26] PETTIT G R, TAN R, PETTIT R K, et al. Antineoplastic agents. 560. Isolation and structure of kitastatin 1 from an Alaskan Kitasatospora sp.[J]. Journal of Na-tural Products, 2007,70(7): 1069-1072.
[27] AIDA W, OHTSUKI T, LI X, et al. Isolation of new carbamate- or pyridine-containing natural products, fuzanins A, B, C, and D from Kitasatospora sp. IFM10917[J]. Tetrahedron,2009,65(1):369-373.
[28] TAKAHASHI Y, IWAI Y, OMURA S. Two new species of the genus Kitasatosporia, Kitasatosporia phosalacinea sp. nov. and Kitasatosporia griseola sp. nov[J]. Journal of General and Applied Microbiology, 1984,30(5): 377-387.
[29] MOON S S, HWANG W H, CHUNG Y R, et al. New cytotoxic bafilomycin C1-amide produced by Kitasatospora cheerisanensis[J]. Journal of Antibiotics, 2003,56(10): 856-861.
[30] 马超,朱洪磊,黄太伟,等.番茄灰霉病菌内生拮抗细菌的筛选及鉴定[J].山西农业科学,2018,46(3):437-440,452.
[31] 陈然, 李俊凯, 李黎,等. 小麦赤霉病生物防治研究进展[J]. 河南农业科学, 2014, 43(12): 1-5.
[32] 高先悦,高淑敏,刘少杰,等.山东省小麦赤霉病菌的种群组成及毒素化学型分析[J].华北农学报,2017,32(S1):37-42.
[33] 王慧慧, 张文忠, 芦明,等. 玉米大斑病的研究进展[J]. 天津农业科学, 2016, 22(12): 133-136.
[2] 杨利敏, 仝赞华, 郭立华,等. 番茄灰霉生防菌CQ的分子鉴定及其生防效果研究[J]. 中国生物防治学报, 2015, 31(6): 956-960.
[3] 李红晓, 张殿朋, 赵洪新,等. 解淀粉芽孢杆菌MH71抗菌物质理化特性及对番茄灰霉病菌的抑菌活性[J]. 中国生物防治学报, 2016, 32(4): 485-492.
[4] 牛贞福, 国淑梅, 张鹤,等. 防控番茄灰霉病的化学药剂和生防菌株筛选研究[J]. 东北农业科学, 2016, 41(3): 41-45.
[5] 赵新贝, 上官妮妮, 李月飞,等. 番茄灰霉病拮抗细菌18BS-12发酵条件的优化及防治效果[J]. 西北农林科技大学学报(自然科学版), 2016, 44(6): 116-124.
[6] 徐大勇, 李志栋, 李峰,等. 番茄灰霉病菌拮抗放线菌的筛选、鉴定及其活性评价[J]. 中国生物防治学报, 2012, 28(2): 298-302.
[7] 王美琴, 马林, 韩巨才,等. 番茄内生菌St24的鉴定及其对灰霉病的生防作用[J]. 应用生态学报, 2012,23(9): 2529-2535.
[8] 高菊芳. 生物农药的作用、应用与功效(一):微生物产物农药[M]. 世界农药, 2001,23(1): 1-6.
[9] 沈玲. 稀有放线菌的分离及其发酵产物的农用活性研究[D]. 杨凌:西北农林科技大学,2006.
[10] 宋洪允, 韩立荣, 冯俊涛,等. 94株土壤放线菌抑菌活性的初步筛选[J]. 西北农业学报, 2010, 19(12): 172-176.
[11] 朱正兵, 吕文静, 纪兆林,等. 土壤生防放线菌的分离及其对油菜菌核病菌的抑制作用[J]. 现代农业科技, 2008(12): 106,108.
[12] 张志斌, 敖武, 熊瑶瑶,等. 内生拮抗放线菌FRo2的鉴定及抑菌活性物质的分离[J]. 微生物学通报, 2014, 41(8): 1574-1581.
[13] BOUKAEW S, CHUENCHIT S, PETCHARAT V. Eva-luation of Streptomyces spp. for biological control of Sclerotium root and stem rot and Ralstonia wilt of chili pepper[J]. Biocontrol, 2011,56(3): 365-374.
[14] 崔志婧. 灰霉菌产孢类群的划分及其遗传多态性分析[D].上海:华东师范大学,2012.
[15] 毕朝位, 仇剑波, 周明国,等. 禾谷镰孢菌Fusarium graminearum Schwabe分生孢子萌发过程中的核相变化及有丝分裂过程观察[J]. 菌物学报, 2008, 27(6): 901-907.
[16] SHI D, AN R, ZHANG W, et al. Stilbene derivatives from Photorhabdus temperata SN259 and their antifungal activities against phytopathogenic fungi[J]. Journal of Agricultural & Food Chemistry, 2017,65(1): 60-65.
[17] WILLETTS H J. Morphology, development and evolution of stromata/sclerotia and macroconidia of the Sclerotiniaceae[J]. Mycological Research, 1997,101(8): 939-952.
[18] URAMOTO M, ITOH Y, SEKIGUCHI R, et al. A new antifungal antibiotic, cystargin: Fermentation, isolation, and characterization[J]. Journal of Antibiotics, 1988,41(12): 1763-1768.
[19] YEO, WOON-HYUNG, SI-KWAN K,et al. Isolation, physico-chemical properties, and biological activity of new thiopeptide antibiotics, kimorexins[J]. Journal of Microbiology and Biotechnology, 1994,4(4): 349-353.
[20] WERNER G, HAGENMAIER H, DRAUTZ H, et al. Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity[J]. Journal of Antibiotics, 1984,37(2): 110-117.
[21] 张孟, 王艳婷, 汪立平, 等. 海洋抑菌放线菌Y15的筛选、鉴定及其代谢产物理化性质[J]. 微生物学通报, 2017, 44(3): 513-524.
[22] OMURA S, TAKAHASHI Y, IWAI Y, et al. Kitasatosporia, a new genus of the order Actinomycetales[J]. Journal of Antibiotics, 1982,35(8): 1013-1019.
[23] YOUNGRYUN C, KEECHEOL S, MO H K, et al. Kitasatospora cheerisanensis sp. nov., a new species of the genus Kitasatospora that produces an antifungal agent[J]. International Journal of Systematic Bacteriology, 1999,49(2): 753-758.
[24] 王云, 郑豪盈, 樊永欣,等. 一株北里孢菌株的分离鉴定及其对松材线虫的致病性[J]. 微生物学通报, 2014, 41(10): 2035-2042.
[25] MOMOSE I, WATANABE T. Tyropeptins, proteasome inhibitors produced by Kitasatospora sp. MK993-dF2[J]. Journal of Antibiotics, 2017,70(5): 542-550.
[26] PETTIT G R, TAN R, PETTIT R K, et al. Antineoplastic agents. 560. Isolation and structure of kitastatin 1 from an Alaskan Kitasatospora sp.[J]. Journal of Na-tural Products, 2007,70(7): 1069-1072.
[27] AIDA W, OHTSUKI T, LI X, et al. Isolation of new carbamate- or pyridine-containing natural products, fuzanins A, B, C, and D from Kitasatospora sp. IFM10917[J]. Tetrahedron,2009,65(1):369-373.
[28] TAKAHASHI Y, IWAI Y, OMURA S. Two new species of the genus Kitasatosporia, Kitasatosporia phosalacinea sp. nov. and Kitasatosporia griseola sp. nov[J]. Journal of General and Applied Microbiology, 1984,30(5): 377-387.
[29] MOON S S, HWANG W H, CHUNG Y R, et al. New cytotoxic bafilomycin C1-amide produced by Kitasatospora cheerisanensis[J]. Journal of Antibiotics, 2003,56(10): 856-861.
[30] 马超,朱洪磊,黄太伟,等.番茄灰霉病菌内生拮抗细菌的筛选及鉴定[J].山西农业科学,2018,46(3):437-440,452.
[31] 陈然, 李俊凯, 李黎,等. 小麦赤霉病生物防治研究进展[J]. 河南农业科学, 2014, 43(12): 1-5.
[32] 高先悦,高淑敏,刘少杰,等.山东省小麦赤霉病菌的种群组成及毒素化学型分析[J].华北农学报,2017,32(S1):37-42.
[33] 王慧慧, 张文忠, 芦明,等. 玉米大斑病的研究进展[J]. 天津农业科学, 2016, 22(12): 133-136.
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