A study on the antimicrobal activity of 7 strains Sarcandra glabra endophytes including Lasiodiplodia pseudotheobromae.
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摘要:
目的 从7种草珊瑚内生真菌中筛选出具有广谱抑菌活性的菌株。 方法 采用菌丝生长速率法和最低抑制浓度法对7种草珊瑚内生真菌的发酵产物进行抑菌活性测定。 结果 7株草珊瑚内生真菌中，J-10较其余菌株不同溶剂萃取层对供试致病菌株表现出广谱的抑菌活性，其甲醇和乙酸乙酯萃取物在质量浓度为2 mg·mL−1时对罗汉果根腐病菌和甘蔗凤梨病菌抑菌率介于84.84%～88.51%。J-10为拟可可毛球二孢菌，其发酵产物的乙酸乙酯萃取物抑菌活性最为显著，对7种病原真菌的EC50值介于0.1995～1.1722 mg·mL−1，其中对玉米大斑病菌的毒力最强，对罗汉果根腐病菌的毒力最弱；J-10菌丝体甲醇提取物对7种病原真菌的EC50值介于 0.1255～9.4687 mg·mL−1，对茶轮斑病菌的毒力最强，对罗汉果根腐病菌的毒力最弱。同时，其乙酸乙酯萃取物对普通变形杆菌、伤寒沙门氏菌、罗汉果青枯菌、大肠杆菌、溶壁微球菌、蜡样芽孢杆菌、巨大芽孢杆菌、枯草芽孢杆菌、炭疽杆菌、藤黄微球菌、金黄色葡萄球菌11种致病细菌的抑菌效果优于甲醇和正丁醇萃取物，经24 h处理后的MIC介于0.125～2.5 mg·mL−1。 结论 拟可可毛球二孢菌具有抑菌广谱性，活性物质主要集中于乙酸乙酯萃取物中，属于中等极性。 Abstract:Object Endophytic Sarcandra glabra fungi exhibiting an antimicrobial activity were isolated and studied for application as potential resource of natural disease control agents. Method The in vitro methods based on pathogen mycelial growth rate and minimum inhibitory concentration of extracts of various solvents from the fermentation broths of 7 species of S. glabra known to have antimicrobial activities were applied to determine their potential for disease control on plants. Result Of the 7 strains of endophytic fungi, J-10, which was subsequently identified as Lasiodiplodia pseudotheobromae, showed a broad-spectrum antimicrobial activity against tested pathogens. For instance, at the concentration of 2 mg·mL−1, the methanol and ethyl acetate extracts from the J-10 fermentation broth displayed a bacteriostasis rate of 84.84-88.51% against Ceratocystis paradoxa and Fusarium solani. The ethyl acetate extract had the most significant antifungal activity with an EC50 against 8 pathogenic fungi ranging from 0.199 5 mg·mL−1 to 1.172 2 mg·mL−1, and the highest toxicity to Exserohilum turcicum but the lowest to Fusarium solani. The methanol extracts had EC50 against the same 8 pathogens ranging from 0.108 8 mg·mL−1 to 9.468 7 mg·mL−1 with the highest toxicity to Athelia rolfsii and the lowest to Fusarium solani. Furthermore, the ethyl acetate extract was more effective against Proteus vulgaris, Micrococcus lyssodeikticus, Staphyloccocusaureus, Escherichia coli, Salmonella typhi, Bacillus cereus, Bacillus megaterium, Bacillussubtilis, Bacillus anthraci, Micrococcus luteus (Schroeter) Cohn, Ralstonia solanacearum than either the methanol or n-butanol extract and rendered an MIC between 0.125 5 mg·mL−1 and 2.5 mg·mL−1 after 24 h of treatment on the pathogens. Conclusion L. pseudotheobromae showed a broad-spectrum antimicrobial activity against a variety of pathogens. The active components in the fermentation broth were of medium polarity and ethyl acetate soluble. -
图 1 7种草珊瑚内生真菌菌落形态
A~G依次为J-9、J-10、J-12、J-20、G-4、G-5、Y-8培养72 h菌落形态。
Figure 1. Colony morphologies of 7 endophytic fungi
A to G: morphology of J-9, J-10, J-12, J-20, G-4, G-5, and Y-8 colonies, respectively, cultured for 72 h.
图 2 7株内生真菌发酵产物对罗汉果根腐病菌和甘蔗凤梨病菌抑制活性菌斑图（抑菌率＞50 %）
A：凤（代表甘蔗凤梨病菌)；B：根（代表罗汉果根腐病菌）；C：J-9乙对凤（代表J-9乙酸乙酯萃取物对甘蔗凤梨病菌抑制效果，下同）；D：J-9甲对凤（甲代表甲醇）；E：J-10乙对根；F：J-10甲对根；G：J-10乙对凤；H：J-10甲对凤；I：J-12乙对凤；J：G-5乙对凤；K：G-5甲对凤；L：G-4正对根（正代表正丁醇）
Figure 2. Antimicrobial activity of secondary metabolites of 7 endophytic fungi against C. paradoxa and F. solani (bacteriostatic rate＞50%)
A: par, C. paradoxa; B: sol, F. solani; C: J-9e vs. par, inhibitory effect of J-9 ethyl acetate extract on C. paradoxa (same format for the following); D: J-9m vs. par, methanol extract; E: J-10e vs. sol; F: J-10m vs. sol; G: J-10e vs. par; H: J-10m vs. par; I: J-12e vs. par; J: G-5e vs. par; K: G-5m vs. par; L: G-4n vs. sol, n-butanol extract.
图 3 J-10发酵产物不同溶剂萃取层对7种植物病原真菌的抑制活性（菌斑图）
处理质量浓度均为2.5 mg·mL−1。A~G依次为玉米大斑病菌、辣椒炭疽病菌、茶轮斑病菌、贡柑链格孢菌、甘蓝黑斑病菌、甘蔗凤梨病菌、罗汉果根腐病菌；H~N依次为J-10甲醇萃取物对玉米大斑病菌、辣椒炭疽病菌、茶轮斑病菌、贡柑链格孢菌、甘蓝黑斑病菌、甘蔗凤梨病菌、罗汉果根腐病菌的抑制效果；O~U依次为J-10乙酸乙酯萃取物对玉米大斑病菌、辣椒炭疽病菌、茶轮斑病菌、贡柑链格孢菌、甘蓝黑斑病菌、甘蔗凤梨病菌、罗汉果根腐病菌的抑制效果；V为J-10正丁醇萃取物对罗汉果根腐病菌的抑制效果。
Figure 3. Antimicrobial activities of J-10 extracts against 7 plant pathogens (plaque diagram)
Treatment concentration at 2.5 mg·mL−1. A-G: E. turcicum, C. capsica, P. theae, A. citri, A. oleracea, C. paradoxa, and F. solani, respectively; H-N: inhibition effects of J-10 methanol extract on E. turcicum, C. capsica, P. theae, A. citri, A. oleracea, C. paradoxa, and F. solani, respectively; O-U: inhibitory effects of J-10 ethyl acetate extract on E. turcicum, C. capsica, P. theae, A. citri, A. oleracea, C. paradoxa, and F. solani, respectively; V: inhibitory effect of J-10 n-butanol extract on F. solani.
图 5 拟可可毛球二孢菌菌落生长形态
A、B、C、D分别为拟可可毛球二孢菌菌落生长24 h、38 h、5 d、20 d形态图。
Figure 5. Cultured L. pseudotheobromae colonies
A. B, C and D are the morphological diagrams of L. pseudotheobromae colony growth for 24 h, 38 h,5 d and 20 d.
表 1 7种供试植物病原真菌
Table 1. Seven species of plant pathogenic fungi for testing
植物病原真菌
Phytopathogenic fungi拉丁名
Latin name来源
Source辣椒炭疽病菌 Colletotrichum capsica 广西大学农学院植物病理室 茶轮斑病菌 Pesta-lotiopsis theae 广西大学农学院植物病理室 甘蓝黑斑病菌 Alternaria oleracea 广西大学农学院植物病理室 甘蔗凤梨病菌 Ceratocystis paradoxa 广西大学农学院植物病理室 玉米大斑病菌 Exserohilum turcicum 广西大学农学院植物病理室 贡柑链格孢菌 Alternaria citri 广西特色作物研究所 罗汉果根腐病菌 Fusarium solani 本实验室 
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表 2 11种供试病原细菌
Table 2. Eleven species of pathogenic bacteria for testing
致病细菌
Pathogenic bacteria拉丁名
Latin name来源
Source普通变形杆菌 Proteus vulgaris 桂林医学院微生物实验室 溶壁微球菌 Micrococcus lyssodeikticus 桂林医学院微生物实验室 金黄色葡萄球菌 Staphyloccocusaureus 桂林医学院微生物实验室 大肠杆菌 Escherichia coli 桂林医学院微生物实验室 伤寒沙门氏菌 Salmonella typhi 桂林医学院微生物实验室 蜡样芽孢杆菌 Bacillus cereus 桂林医学院微生物实验室 巨大芽孢杆菌 Bacillus megaterium 桂林医学院微生物实验室 枯草芽孢杆菌 Bacillussubtilis 桂林医学院微生物实验室 炭疽杆菌 Bacillus anthraci 桂林医学院微生物实验室 藤黄微球菌 Micrococcus luteus(Schroeter)Cohn 桂林医学院微生物实验室 罗汉果青枯菌 Ralstonia solanacearum 本实验室
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表 3 7种内生真菌发酵产物对罗汉果根腐病菌和甘蔗凤梨病菌的抑制效果
Table 3. Antimicrobial activity of fermentation products of 7 endophytic fungi against C. paradoxa and F. solani
内生真菌
Endophyte来源
Source指示菌株
Test strain抑菌率 Inhibition rate/% 甲醇提取物
methanol extract乙酸乙酯提取物
Ethyl acetate extract正丁醇提取物
n-Butanol ExtractJ-9 茎 罗汉果根腐病菌 32.17±1.07 26.44±1.06 13.22±0.69 甘蔗凤梨病菌 53.26±0.72 66.09±0.63 12.07±1.13 J-10 茎 罗汉果根腐病菌 85.17±0.47 88.51±1.63 11.49±1.63 甘蔗凤梨病菌 86.6±0.88 84.84±0.73 15.16±0.26 J-12 茎 罗汉果根腐病菌 1.59±0.00 1.59±0.00 1.59±0.00 甘蔗凤梨病菌 11.59±0.00 73.91±0.00 — J-20 茎 罗汉果根腐病菌 — 1.59±0.00 1.59±0.00 甘蔗凤梨病菌 48.91±1.19 — — G-4 根 罗汉果根腐病菌 — 27.98±0.21 61.76±1.74 甘蔗凤梨病菌 22.43±1.63 — — G-5 根 罗汉果根腐病菌 1.59±0.00 1.59±0.00 73.53±1.07 甘蔗凤梨病菌 86.23±0.81 92.75±0.90 — Y-8 叶 罗汉果根腐病菌 — 22.75±0.73 2.12±0.21 甘蔗凤梨病菌 — 42.89±0.23 — “—”表示发酵产物对指示菌株无活性。
“—” indicates inactivity of fermentation product against indicator strain.
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表 4 7种内生真菌发酵产物对3种致病细菌的抑制作用
Table 4. Antimicrobial activities of 7 endophytic fungi fermentation products against 3 pathogens
内生真菌
Endophyte指示菌株
Test strain革兰氏
GramMIC/（mg·mL−1） 甲醇提取物
Methanol extract乙酸乙酯提取物
Ethyl acetate extract正丁醇提取物
n-Butanol ExtractJ-9 溶壁微球菌 G+ 0.625 2.000 0.250 普通变形杆菌 G− 2.500 0.125 0.250 罗汉果青枯菌 G− 1.250 0.500 0.250 J-10 溶壁微球菌 G+ 0.500 0.125 0.625 普通变形杆菌 G− 0.125 0.125 2.500 罗汉果青枯菌 G− 0.250 0.125 1.250 J-12 溶壁微球菌 G+ 0.250 2.50 5.000 普通变形杆菌 G− 0.500 1.25 2.500 罗汉果青枯菌 G− 1.250 5.000 5.000 J-20 溶壁微球菌 G+ — 2.500 — 普通变形杆菌 G− 5.000 1.250 — 罗汉果青枯菌 G− — — — G-4 溶壁微球菌 G+ 4.000 2.500 — 普通变形杆菌 G− 2.500 5.000 — 罗汉果青枯菌 G− 1.000 1.000 4.000 G-5 溶壁微球菌 G+ 1.000 1.250 — 普通变形杆菌 G− 0.250 0.250 — 罗汉果青枯菌 G− 0.500 1.000 — Y-8 溶壁微球菌 G+ 0.250 4.000 2.000 普通变形杆菌 G− 2.000 0.250 0.500 罗汉果青枯菌 G− 1.250 0.500 0.500 “—”表示内生真菌发酵产物对指示菌株无活性。
“—” indicates inactivity of fermentation product against indicator strain.
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表 5 J-10发酵产物不同溶剂层对7种植物病原菌的抑制活性
Table 5. Antimicrobial activities of J-10 extracts against 7 plant pathogens
供试植物真菌
Tested plantpathogenic fungi萃取层
Extraction layer毒力回归方程
Toxicity regression equation相关系数
Correlation coefficient半效应浓度（EC50）
50% effective concent-ration95%置信限
95% confidence limit玉米大斑病菌
Exserohilum turcicum甲醇 y=5.197 1+0.661 7x 0.977 4 0.503 7 0.228 8−0.778 6 乙酸乙酯 y=5.911 7+1.302 3x 0.963 8 0.199 5 0.140 8−0.258 1 辣椒炭疽病菌
Colletotrichum capsica甲醇 y=4.802 5+0.932 3x 0.997 0 1.539 8 1.130 7−1.948 8 乙酸乙酯 y=5.356 8+0.552 4x 0.979 3 0.226 0 0.163 8−0.288 1 茶轮斑病菌
Pesta-lotiopsis theae甲醇 y=4.928 2+1.397 6x 0.990 3 0.125 5 0.820 2−1.430 8 乙酸乙酯 y=5.725 2+1.080 0x 0.968 5 0.213 1 0.147 4−0.278 8 贡柑链格孢菌
Alternaria citri甲醇 y=5.002 0+1.003 9x 0.996 2 0.995 9 0.642 7−1.349 0 乙酸乙酯 y=5.523 6+0.915 0x 0.986 3 0.267 8 0.197 4−0.338 1 甘蓝黑斑病菌
Alternaria oleracea甲醇 y=4.810 2+0.910 3x 0.994 4 1.616 2 1.184 7−2.047 7 乙酸乙酯 y=5.597 5+1.243 6x 0.982 3 0.330 8 0.242 9−0.418 7 甘蔗凤梨病菌
Ceratocystis paradoxa甲醇 y=4.930 7+1.369 3x 0.986 5 1.123 6 0.919 6−1.427 5 乙酸乙酯 y=5.607 6+1.174 5x 0.977 3 0.303 9 0.203 7−0.404 1 罗汉果根腐病菌
Fusarium solani甲醇 y=4.234 2+0.784 4x 0.998 3 9.468 7 5.576 9−13.360 4 乙酸乙酯 y=4.925 5+1.079 3x 0.983 8 1.172 2 0.863 5−1.480 9
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表 6 J-10发酵产物不同溶剂层对11种致病细菌的抑制活性
Table 6. Antimicrobial effects of J-10 extracts against 11 pathogens
供试细菌
Test strain革兰氏
GramMIC/(mg·mL−1） 甲醇提取物
methanol extract乙酸乙酯提取物
Ethyl acetate extract正丁醇提取物
n-Butanol Extract普通变形杆菌 G− 0.1250 0.1250 2.5000 伤寒沙门氏菌 G− 1.2500 1.2500 2.5000 罗汉果青枯菌 G− 0.2500 0.1250 1.2500 大肠杆菌 G− 0.1250 0.1250 1.0000 溶壁微球菌 G+ 0.5000 0.1250 0.6250 蜡样芽孢杆菌 G+ 1.0000 0.6250 0.3125 巨大芽孢杆菌 G+ 1.2500 0.6250 2.5000 枯草芽孢杆菌 G+ 5.0000 2.5000 5.0000 炭疽杆菌 G+ 1.0000 1.2500 2.5000 藤黄微球菌 G+ 0.6250 0.5000 2.5000 金黄色葡萄球菌 G+ 0.5000 0.2500 2.5000
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