海洋来源杂色曲霉次级代谢产物及其抗植物病原细菌活性

doi:10.3864/j.issn.0578-1752.2020.19.010

摘要/Abstract

摘要：

【目的】针对植物细菌病害日趋严重,并缺少有效防治药剂的问题,从前期筛选获得的具有抗菌活性且次级代谢产物丰富的海洋杂色曲霉（Aspergillus versicolor）D5发酵提取物中分离纯化单体化合物,分析鉴定其化学结构,并进行抗菌活性评价,阐明目标真菌的抗菌活性成分,为新颖结构抗细菌生物农药的研发提供先导化合物。【方法】综合运用正/反相硅胶柱层析、Sephadex LH-20凝胶柱层析和半制备高效液相色谱（HPLC）等方法分离纯化单体化合物,采用核磁、质谱等现代波谱分析方法对单体化合物进行结构鉴定;采用微量稀释法对化合物进行抗6种植物病原细菌燕麦食酸菌（Acidovorax avenae）、胡萝卜软腐欧文氏菌（Erwinia carotovora）、密执安棒形杆菌（Clavibater michiganensis）、丁香假单胞菌（Pseudomonas syringae）、青枯雷尔氏菌（Ralstonia solanacearum）和野油菜黄单胞菌（Xanthomonas campestris）活性评价,获得活性化合物的最小抑菌浓度（MIC）。【结果】从海洋杂色曲霉D5马铃薯葡萄糖液体发酵培养基乙酸乙酯提取物中分离鉴定了12个化合物,包括4个2-羰基-4-苯基喹啉生物碱,viridicatin（1）、3-O-methylviridicatin（2）、3,6-O-dimethylviridicatin（3）和3-O-methylviridicatol（4）;2个双氧代哌嗪生物碱,（+）-cyclopenol（5）和（-）-cyclopenol（6）;3个喹唑啉fumiquinazolines生物碱及其衍生物,versicoloid A（7）、chrysopiperazine C（8）和cottoquinazoline A（9）;以及3个蒽醌类化合物,versiconol（10）、averufin（11）和noraverufanin（12）。抗菌活性结果表明,2-羰基-4-苯基喹啉生物碱3,6-O-dimethylviridicatin（3）具有显著的抗植物病原细菌活性,对青枯雷尔氏菌和野油菜黄单胞菌的MIC分别为50和100 μg·mL^-1。初步的构效关系分析表明,C-6位置的甲氧基可能是该类化合物发挥抗植物病原细菌作用的关键基团。【结论】海洋杂色曲霉D5代谢产物丰富,能够产生结构多样的生物碱和蒽醌类化合物。从其发酵产物中分离鉴定了9个生物碱类化合物和3个蒽醌类化合物,其中3,6-O-dimethylviridicatin（3）对青枯雷尔氏菌和野油菜黄单胞菌具有显著的抗菌活性,有开发成为抗细菌生物农药的潜力。

关键词: 海洋真菌, 杂色曲霉, 生物碱, 蒽醌, 结构鉴定, 植物病原细菌

Abstract:

【Objective】 Plant bacterial diseases are becoming more and more serious, and lack of control pesticides. The objective of this study is to isolate bioactive compounds from the previously obtained marine-derived fungus Aspergillus versicolor D5 with antibacterial activity and abundant secondary metabolites, identify their chemical structures, evaluate their anti-phytopathogenic bacterial activity, so that to clarify the antibacterial components of the target fungus. This research will provide lead compounds for the discovery of new antibacterial pesticides.【Method】The compounds were isolated by silica gel column chromatography (CC), octadecylsilyl silica gel CC, Sephadex LH-20 CC, and semipreparative HPLC, and were identified by modern spectral analysis methods including NMR and MS spectra. In addition, the actibacterial activity of the isolated compounds towards six phytopathogenic bacteria, including Acidovorax avenae, Erwinia carotovora, Clavibater michiganensis, Pseudomonas syringae, Ralstonia solanacearum and Xanthomonas campestris, was evaluated by double dilution method to obtain the minimum inhibitory concentration (MIC).【Result】Twelve compounds were isolated and identified from the ethyl acetate extract of the marine-derived fungus A. versicolor D5 fermented by potato dextrose water media, including four 4-aryl-quinolin-2-one alkaloids, viridicatin (1), 3-O-methylviridicatin (2), 3,6-O-dimethylviridicatin (3), and 3-O-methylviridicatol (4), two dioxopiperazine alkaloids, (+)-cyclopenol (5) and (-)-cyclopenol (6), three fumiquinazolines alkaloids and their derivatives, versicoloid A (7), chrysopiperazine C (8), and cottoquinazoline A (9), and three anthraquinone derivatives, versiconol (10), averufin (11), and noraverufanin (12). Among them, a 4-aryl-quinolin-2-one alkaloid, 3,6-O-dimethylviridicatin (3) exhibited an obvious antibacterial activity against R. solanacearum and X. campestris with MIC values of 50 and 100 μg·mL^-1, respectively. The structure activity relationship analysis revealed that the methoxy group at C-6 might play an important role in anti-phytopathogenic bacterial activity.【Conclusion】The metabolites of A. versicolor D5 are abundant, which can produce alkaloids and anthraquinones with various structures. Among them, nine alkaloids and three anthraquinones were obtained from cultures of D5. 3,6-O-dimethylviridicatin (3) exhibited clear antibacterial activities towards R. solanacearum and X. campestris.

Key words: marine-derived fungus, Aspergillus versicolor, alkaloid, anthraquinone, structural identification, phytopathogenic bacteria

付兵,王美,刘建阳,林伟,张成省,赵栋霖. 海洋来源杂色曲霉次级代谢产物及其抗植物病原细菌活性[J]. 中国农业科学, 2020, 53(19): 3964-3974.

FU Bing,WANG Mei,LIU JianYang,LIN Wei,ZHANG ChengSheng,ZHAO DongLin. Secondary Metabolites from a Marine-Derived Fungus Aspergillus versicolor and Their Anti-Phytopathogenic Bacterial Activity[J]. Scientia Agricultura Sinica, 2020, 53(19): 3964-3974.

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参考文献 36

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化合物浓度 Concentration (μg·mL^-1)	加药Dose青枯雷尔氏菌 R. solanacearum OD₆₀₀值OD₆₀₀value	加药Dose野油菜黄单胞菌 X. campestris OD₆₀₀值OD₆₀₀value
200	0.0917	—
	0.0885	—
	0.0835	—
100	0.0902	0.0891
	0.0947	0.0790
	0.0958	0.0788
50	0.1364	1.0253
	0.1504	1.0450
	0.1448	1.0277
25	0.2496	1.0800
	0.2457	1.0953
	0.2458	1.0898
12.5	0.4333	1.0911
	0.4246	1.0891
	0.4340	1.1001
6.25	—	1.1058
	—	1.0913
	—	1.0943