决明草内生真菌拮抗菌株筛选鉴定及桔青霉ZH-11抗菌物质分析

Screening and identification of the endophytic fungi antagonistic strain of Cassia tora Linn. and the analysis of antibacterial substances of Penicillium citrinum ZH-11

摘要:

[背景] 由于抗生素的滥用，导致“超级细菌”出现，寻找新的抗菌药物将有效地应对细菌耐药问题，因为大多数抗菌药物都是从微生物中发现的，所以药用植物内生真菌的研究拓宽了药用资源，并且具有巨大的应用价值。[目的] 对采自江西九江庐山植物园的决明草进行内生真菌分离，筛选出拮抗菌株，并对拮抗菌株的次级代谢产物进行分离，分析其抗菌物质理化性质，为新型抗菌物质的研究提供基础数据。[方法] 用管碟法筛选拮抗菌株，并根据形态学特征和分子生物学的方法鉴定菌株，采用硅胶柱层析、葡聚糖凝胶LH-20柱层析和RP-C18柱层析对其次级代谢产物进行分离，并用液质联用（高分辨飞行时间质谱）和能谱仪分析所得抗菌物质的分子量和分子式。[结果] 筛选到一株广谱拮抗活性菌株桔青霉ZH-11，通过管碟法显示其对大肠杆菌、金黄色葡萄球菌、苏云金芽孢杆菌、水稻黄单胞菌、枯草芽孢杆菌、地衣芽孢杆菌、白色念珠菌、蜡样芽孢杆菌这8株指示菌均有较好的抑菌效果。从桔青霉ZH-11次级代谢产物中分离得到纯化合物Y3，其分子量为410.169 1，分子式为C₂₄H₂₆O₆。当Y3浓度为10 μg/mL时，其对大肠杆菌和苏云金芽胞杆菌的抑菌圈直径分别为16.23 mm和17.27 mm。[结论] 菌株桔青霉ZH-11的活性物质与已知的来源于桔青霉类的抗菌活性物质不同，该研究结果为进一步挖掘桔青霉属的活性产物奠定基础，同时丰富了人们对决明草内生真菌的认识。

Abstract:

[Background] Considering the emergence of "super bacteria" due to the abuse of antibiotics, the search for new antibacterial drugs is necessary for effectively conquering the problem of bacterial resistance. Since most antibacterial drugs are found from microorganisms, the research on endophytic fungi in medicinal plants can expand the medicinal resources, so it has a promising future for application. [Objective] To isolate the endomycete from the Cassia tora Linn. collected from Lushan Botanical Garden of Jiujiang, Jiangxi Province; screen out the antagonistic strains; isolate the secondary metabolites of such strains and analyze the physical and chemical properties of their antibacterial substances to prepare for the research of new antibacterial substances. [Methods] The antagonistic strains were screened by the cup-plate method and then identified based on morphological characteristics and molecular biology. Their secondary metabolites were isolated by silica gel, Sephadex LH20 and RP-C18 column chromatography, and the molecular weight and molecular formula of antibacterial substances were analyzed by liquid chromatography-mass spectrometry (GCMS) and EDS spectrometer. [Results] Penicillium citrinum ZH-11, a broad-spectrum antagonistic strain was screened out. As shown by the cup-plate method, it had a good bacteriostatic effect on eight indicator bacteria such as Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, Xanthomonas oryzae, Bacillus subtilis, Bacillus licheniformis, Candida albicans and Bacillus cereus etc. Active pure compound Y3 was isolated from the secondary metabolite of endomycete Penicillium citrinum ZH-11 with a molecular weight of 410.169 1 and a formula of C₂₄H₂₆O₆. When the concentration of Y3 was 10 µg/mL, the diameter of the inhibition zone against Escherichia coli and Bacillus thuringiensis was 16.23 mm and 17.27 mm, respectively. [Conclusion] This active substance is different from the known antibacterial active substances derived from Penicillium citrinum. The results laid a foundation for further exploration of the active products of Penicillium citrinum, and expanded our recognition of the endophytic fungi in Cassia tora Linn.