Isolation, Identification, and Antibacterial Activity of Endophytic Fungi from Paris qiliangiana
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摘要:
目的 从药用植物啟良重楼中分离鉴定内生真菌，研究其对常见病原细菌的抑菌活性，并分析其次级代谢产物，为利用传统药用植物内生菌开发新型抗生素药物提供新的思路。 方法 采用平板分离法分离纯化啟良重楼内生真菌，提取其基因组DNA，PCR扩增ITS序列并测序，鉴定内生真菌的种类；采用滤纸片法研究其次级代谢产物对12种供试菌的抑菌活性；采用GC-MS技术分析次级代谢产物的种类。 结果 从啟良重楼中分离了2株内生真菌，分别鉴定为Setophoma caverna CLZ1和Psilocybe sp.CLZ2；S. caverna CLZ1对肺炎克雷伯菌（Klebsiella pneumonia ATCC 13883）、藤黄微球菌（Micrococcus luteus ATCC 4698）、苏云金芽孢杆菌（Bacillus thuringiensis ATCC 10792）、多耐铜绿假单胞菌（Pseudomonas aeruginosa ATCC 9027）和耐甲氧西林金黄色葡萄球菌（Staphylococcus aureus ATCC 43300，MRSA）等5种供试菌有中度敏感抑制作用，其最小抑菌浓度（MIC）为0.78~3.13 mg·mL−1；Psilocybe sp.CLZ2对MRSA有中度敏感抑制作用，其MIC值为3.13 mg·mL−1。运用GC-MS从S. caverna CLZ1提取物中鉴定出36个化合物，包括吡啶（0.91%）、烟酰胺（0.61%）、邻苯二甲酸二丁酯（1.42%）、油酸甲酯（2.28%）和硬脂酸甲酯（0.83%）等5个抑菌活性化合物；从Psilocybe sp. CLZ2提取物中鉴定出44个化合物，包括吡啶（0.106%）和苯乙醇（0.073%）等2个抑菌活性化合物。 结论 从啟良重楼根茎中分离出内生真菌S. caverna CLZ1和Psilocybe sp.CLZ2，发现S. caverna CLZ1的次级代谢产物具有广抑菌谱，并从中鉴定出5个可能具有抑菌活性的次生代谢产物。 Abstract:Objective Endophytes from the medicinal Paris qiliangiana plants were isolated to identify effective antipathogenic metabolites secreted.The in vitro activity of their secondary metabolites might lead to the development of new antibiotics for disease control on plants. Method Endophytic fungi of P. qiliangiana plants were isolated in a disc diffusion method and identified by ITS gene sequencing. The secondary metabolites from each isolate were obtained by filter paper separation and tested for antibacterial activity against 12 pathogens. Active components were analyzed by GC-MS. Results Setophoma caverna CLZ1 and Psilocybe sp. CLZ2 were the two functional endophytic fungi identified. The former showed a broad spectrum against the tested gram-negative and gram-positive bacteria including Klebsiella pneumonia ATCC 13883, Micrococcus luteus ATCC 4698, Bacillus thuringiensis ATCC 10792, Pseudomonas aeruginosa ATCC 9027, and Staphylococcus aureus ATCC 43300. Their intermediate degree of bacteriostatic activities on the pathogens had the MICs ranging from 0.78 to 3.13 mg·mL−1. The latter exhibited a similar degree of inhibition against S. aureus at an MIC of 3.13 mg·mL−1. The GC–MS analysis identified 36 chemicals in the S. caverna CLZ1 culture filtrate that included pyridine, nicotinamide, dibutyl phthalate, 9-octadecenoic acid (Z)-, and methyl ester methyl stearate. In the Psilocybe sp. CLZ2 filtrate, 44 compounds, including antibiotic pyridine and phenylethanols, were found. Conclusion The endophytic fungi, S. caverna CLZ1 and Psilocybe sp. CLZ2, isolated from the roots of the new variety of P. qiliangiana displayed significant antibacterial property. -
Key words:
- Paris qiliangiana /
- endophytic fungi /
- bacteriostatic activity /
- GC-MS
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图 1 啟良重楼内生真菌菌落及菌丝形态
A、B: S. caverna CLZ1的菌落和菌丝体；C、D: Psilocybe sp.CLZ2的菌落和菌丝体。
Figure 1. Colony and mycelial morphology of endophytic fungi isolated from P. polyphylla
A and B: Colony and mycelial morphology of S. caverna; C and D: Colony and mycelial morphology of Psilocybe sp.
图 2 啟良重楼内生真菌系统发育树
Figure 2. Phylogenetic trees of endophytic fungi isolated from P. polyphylla
表 1 内生真菌的抑菌圈直径
Table 1. Diameter of inhibition zone of endophytic fungi ( n = 3)
供试菌
Strains抑菌圈直径 Diameter of inhibition zone/mm 卡那霉素 Kan 洞栖棘壳孢CLZ1
S.caverna.CLZ1裸盖菇菌属CLZ2
Psilocybe sp.CLZ2肺炎克雷伯菌 K.pneumonia 23.81±0.18 a 11.84±0.08 b 8.62±0.04 c 金黄色葡萄球菌 S.aureus 17.06±0.08 a — — 粪链球菌 E. faecalis 29.21±0.02 a — — 枯草芽孢杆菌 B. subtilis 25.74±0.70 a 8.54±0.08 b — 藤黄微球菌 M. luteus 26.49±0.15 a 11.79±0.30 b — 苏云金芽孢杆菌B. thuringiensis 27.14±0.01 a 11.56±0.29 b 9.26±0.13 c 耐甲氧西林金黄色葡萄球菌 MRSA — 12.15±0.30 a 11.82±0.42 a 多耐铜绿假单胞菌 P.aeruginosa 26.06±0.20 a 11.36±0.02 b 9.80±0.04 c 大肠杆菌 E. coli 15.95±0.30 a — — 耐甲氧西林表皮葡萄球菌 MRSE 29.06±0.19 a — 8.70±0.02 b 溶藻弧菌 I.alginolyticus 21.78±0.03 a 8.33±0.16 b 8.48±0.02 b 鲍曼不动杆菌 C.bammannii 24.30±0.15 a 8.27±0.19 b — 同行数据后不同小写字母表示差异显著（P＜0.05）。
Data with different lowercase letters in the same line mean significant difference at 0.05 level.
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表 2 内生真菌的MIC和MBC
Table 2. MICs and MBCs of endophytic fungi
供试菌
Strains最低抑菌质量浓度 MIC/(mg·mL−1) 最低杀菌质量浓度 MBC/(mg·mL−1) 洞栖棘壳孢
CLZ1 S. caverna.
CLZ1裸盖菇菌属
CLZ2 Psilocybe sp.
CLZ2卡那霉素
Kan洞栖棘壳孢
CLZ1 S. caverna.
CLZ1裸盖菇菌属
CLZ2 Psilocybe sp.
CLZ2卡那霉素
Kan肺炎克雷伯菌 Klebsiella pneumonia 0.78 3.13 0.031 1.56 6.25 0.063 金黄色葡萄球菌 Staphylococcus aureus — — — — — — 粪链球菌 Enterococcus faecalis — — — — — — 枯草芽孢杆菌 Bacillus subtilis 6.25 — 0.016 12.50 — 0.031 藤黄微球菌 Micrococcus luteus 1.56 — 0.002 3.13 — 0.002 苏云金芽孢杆菌 Bacillus thuringiensis 3.13 1.56 0.063 6.25 6.25 0.125 耐甲氧西林金黄色葡萄球菌 Staphylococcus aureus 1.56 3.13 — 3.13 3.13 — 多耐铜绿假单胞菌 Pseudomonas aeruginosa 1.56 6.25 0.063 6.25 6.25 0.063 大肠杆菌 Enterococcus coli — — 0.031 — — 0.031 耐甲氧西林表皮葡萄球菌 staphylococcus epidermidis — 12.50 0.008 — 12.50 0.008 溶藻弧菌 ibrio alginolyticus 6.25 12.50 0.016 12.50 12.50 0.016 鲍曼不动杆菌 cinetobacter bammannii 3.13 — 0.008 6.25 — 0.016
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表 3 啟良重楼内生真菌活性成分GC-MS鉴定
Table 3. GC-MS identification on active metabolites of endophytes isolated from P. polyphylla
类别
Category出峰时间
The peak
time/min名称
Name含量 Content/% 匹配度
Match/%药理活性
Pharma-
cological
activity参考文献
ReferenceCLZ1 CLZ2 酯类
Esters
(24)2.912 丙酸乙酯 Propanoic acid, ethyl ester 6.37 3.052 96.4 2.944 乙酸正丙酯 n-Propyl acetate — 2.618 95.2 3.079 甲酸丁酯 Formic acid, butyl ester — 0.088 90.7 3.089 丙酮酸甲酯 Propanoic acid, 2-oxo-, methyl ester 4.83 — 86.5 4.825 乙酸丁酯 Acetic acid,butyl ester — 0.091 90.5 5.803 1,2-乙二醇单乙酸酯 1,2-Ethanediol, monoacetate — 0.300 85.7 13.287 泛内酯 Pantolactone 0.42 0.173 90.9 16.151 甘油单乙酸酯 1,2,3-Propanetriol, 1-acetate 1.16 — 92.3 28.961 3-氯-丙酸,4-甲酰基苯酯
Propanoic acid, 3-chloro-, 4-formylphenyl ester— 0.076 91.8 30.671 癸酸乙酯 Decanoic acid, ethyl ester — 0.071 90.5 33.230 避蚊酯 Dimethyl phthalate 0.42 — 91.3 36.189 月桂酸甲酯 Dodecanoic acid, methyl ester — 0.163 86.8 39.063 月桂酸乙酯 Dodecanoic acid, ethyl ester — 0.179 88.2 43.407 2,4-二羟基-3,6-二甲基苯甲酸甲酯
Benzoic acid, 2,4-dihydroxy-3,6-dimethyl-, methyl ester6.01 — 96.7 49.326 邻苯二甲酸二异丁酯
1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester2.08 0.404 91.5 51.276 棕榈酸甲酯
Hexadecanoic acid, methyl ester— 0.881 92.2 51.286 14-甲基十五烷酸甲酯
Pentadecanoic acid, 14-methyl-, methyl ester4.71 — 97.8 52.526 邻苯二甲酸二丁酯 Dibutyl phthalate 1.42 — 95.3 抑菌 [30-31] 53.584 十六酸乙酯 Hexadecanoic acid, ethyl ester — 0.836 89.4 56.805 亚油酸甲酯
9,12-Octadecadienoic acid, methyl ester1.06 — 98.6 57.003 油酸甲酯
9-Octadecenoic acid (Z)-, methyl ester2.28 — 96.7 抑菌 [27] 57.831 硬脂酸甲酯 Methyl stearate 0.83 — 98.2 抑菌 [28] 66.629 4,5-二羧基-γ-十五内酯 Spiculesporic acid — 0.292 87.4 68.820 2-棕榈酰甘油
Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester2.31 — 89.7 醛类
Aldehyde
(13)4.451 正己醛 Hexanal — 0.385 87.5 7.640 2-甲基-2-己烯醛 2-Hexenal, 2-methyl- — 0.043 87.6 9.761 （Z）-2-庚烯醛 (Z)-2-Heptenal 3.41 0.405 96.4 13.757 苯乙醛 Benzeneacetaldehyde — 0.118 92 14.500 （E）-2-辛烯醛 (E)-2-Octenal 0.90 85.0 16.792 壬醛 Nonanal 0.38 0.075 86.9 18.875 2-壬烯醛,（E）- 2-Nonenal, (E)- 0.011 86.7 21.765 癸醛 Decanal 0.217 87.5 22.727 5-羟甲基糠醛
5-Hydroxymethylfurfural0.264 89.5 24.442 2-癸烯醛,(Z) 2-Decenal, (Z)- 5.77 95.0 26.600 十一醛 Undecanal 0.44 0.078 94.2 26.995 （E,E） -2,4-癸二烯醛 (E,E)- 2,4-Decadienal 1.85 0.307 91.1 31.216 十二醛 Dodecanal 0.54 92.8 烃类
Hydrocarbons
(17)3.18 1,1-二乙氧基乙烷 1,1-diethoxy-Ethane 0.09 0.098 90.2 3.789 甲苯 Toluene 0.073 89.6 4.376 正辛烷 Octane 3.84 — 94.9 6.166 乙苯 Ethylbenzene — 0.877 89.8 11.877 八甲基环四硅氧烷 Cyclotetrasiloxane, octamethyl- — 0.089 92.9 30.815 十四烷 Tetradecane 0.36 — 90.6 34.977 十二烷 Dodecane — 0.503 91.6 35.666 2,4-二（1,1-二甲基乙基）-苯酚
2,4-bis(1,1-dimethylethyl)-Phenol4.53 — 93.6 36.852 正十九烷 Nonadecane 0.32 — 85.6 38.946 十六烯 Cetene 1.57 — 91.9 39.234 1-十六烷 Hexadecane 0.68 — 91.2 43.535 正十七烷 Heptadecane 2.47 0.673 90.6 46.585 十八烯1-Octadecene 1.75 0.669 94.8 60.037 二十二烷 Docosane 1.53 3.399 94.3 63.045 正二十三烷 Tricosane — 6.288 96.7 65.846 二十四烷 Tetracosane 1.83 7.326 98.6 68.585 二十烷 Eicosane 1.67 — 91.3 醇类
Alcohols
(4个)3.981 2,3-丁二醇 2,3-Butanediol — 0.247 92.3 16.685 3-吡啶醇 3-Pyridinol 0.54 — 85.2 17.128 苯乙醇 Phenylethyl Alcohol — 0.073 85.5 抑菌 [29] 31.696 2,5-二甲基-1,4-苯二醇
2,5-dimethyl-1,4-Benzenediol9.38 — 92.8 酸类
Acids
(5)24.944 壬酸 Nonanoic acid — 0.082 85.5 29.704 正癸酸 n-Decanoic acid — 0.108 92.9 33.727 十一烷酸 Undecanoic acid — 0.113 87.9 37.872 十二烷酸 Dodecanoic acid — 0.226 89.1 45.987 十四烷酸 Tetradecanoic acid — 2.686 99.1 其他
Others
(4)3.393 吡啶 Pyridine 0.91 0.106 92.3 抑菌 [24] 11.359 2-戊基呋喃 Furan, 2-pentyl- — 0.061 90.9 14.831 苯乙酮 Acetophenone — 0.160 86.4 30.340 烟酰胺 Niacinamide 0.61 — 87.8 抑菌 [25]
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