青钱柳产黄酮类物质真菌的分离与鉴定

【目的】对青钱柳产黄酮内生真菌进行鉴定,探究提取黄酮类物质的新方法。【方法】采用组织分离对青钱柳枝、叶、根、皮的内生真菌进行分离,用不同培养基进行培养,观察其形态特征进行初步分类;通过显色反应、薄层层析(TLC)、高效液相色谱(HPLC)对内生真菌的发酵产物进行分析,筛选产黄酮类物质的内生真菌,并测定总黄酮产量。根据菌株的形态学特征,结合真菌rDNA的居间序列(internal transcribed spacer, ITS)的系统进化分析鉴定。【结果】分离得到67株内生真菌,通过菌落形态和显微形态观察,隶属于3纲、4目、6科、10属;最终确定4株产黄酮物质的内生真菌:PZ06、CY12、CP06、PP03,其中黄酮产量最多的是菌株PZ06,产量为3.61 mg/L。分子鉴定结合4种菌株的形态学鉴定结果表明,PZ06、CP06、PP03均属于链格孢属(Alternaria),CY12属于正青霉属(Eupenicillium)。【结论】青钱柳产黄酮的优势菌群为链格孢属(Alternaria)。该研究对植物内生菌的开发利用有着重要参考意义,产黄酮类物质的内生真菌的发现为活性物质的提取提供了新方法。

Isolation and identification of endophytic fungi from Cyclocarya paliurus (Batal.) Iljinskaja

【Objective】This study aimed to identify the endophytic fungi produced flavonoids in Cyclocarya paliurus(Batal.) Iljinskaja and explore a new method to extract flavonoids. 【Method】The endophytic fungi in the branches, leaves, roots, and bark of C. paliurus(Batal.) Iljinskaja were isolated using the tissue separation method. By observing the morphological characteristics of the endophytes, the fermentation products of the endophytic fungi from C. paliurus(Batal.) Iljinskaja were identified using the chromogenic reaction method, thin-layer chromatography(TLC) and high performance liquid chromatography(HPLC). The endophytic fungi produced flavonoids were screened, and the total flavonoid production was determined. Moreover, the strains were identified based on the morphological characteristics and the phylogenetic analysis of the intervening sequences of the fungal rDNA. 【Result】In total, 67 strains of endophytic fungi were obtained;through the identification of morphological characteristics, they were identified to belong to three classes, four orders, six families and ten genera. Four endophytic fungi producing flavonoids were finally identified: PZ06, CY12, CP06 and PP03. The most abundant flavonoids, with a yield of 3.61 mg/L, were produced by strain PZ06. Molecular identification combined with morphological identification indicated that PZ06, CP06 and PP03 belong to the genus Alternaria, whereas CY12 belongs to the genus Eupenicillium. The dominant flavonoid-producing endophytic fungi of C. paliurus(Batal.) Iljinskaja belong to the genus Alternaria. 【Conclusion】 The study of endophytic fungi in C. paliurus(Batal.) Iljinskaja is a key significance for the development and utilization of endophytes. In addition to identifying the endophytic fungi, the present study also provides a novel method for the extraction of active substances from C. paliurus(Batal.) Iljinskaja plants.