青钱柳产黄酮类物质真菌的分离与鉴定

doi:10.3969/j.issn.1000-2006.201812001

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (2): 26-34.doi: 10.3969/j.issn.1000-2006.201812001

所属专题：青钱柳叶有效成分专题

• 专题报道(执行主编李维林) • 上一篇下一篇

青钱柳产黄酮类物质真菌的分离与鉴定

寇晓琳¹(), 谢楠¹, 吴彩娥¹^,²^,^*(), 范龚健¹, 洑香香²

1.南京林业大学轻工与食品学院,江苏南京 210037
2.南京林业大学, 南方现代林业协同创新中心,江苏南京 210037

收稿日期:2018-12-03 修回日期:2019-11-19 出版日期:2020-03-30 发布日期:2020-04-01
通讯作者: 吴彩娥
基金资助:
国家林业局科学技术推广项目(2017(08));江苏高校优势学科建设工程资助项目(PAPD)

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

KOU Xiaolin¹(), XIE Nan¹, WU Caie¹^,²^,^*(), FAN Gongjian¹, FU Xiangxiang²

1. College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China
2. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China

Received:2018-12-03 Revised:2019-11-19 Online:2020-03-30 Published:2020-04-01
Contact: WU Caie

摘要/Abstract

摘要：

【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。

关键词: 青钱柳, 黄酮, 内生真菌, ITS序列分析, 扫描电镜, 真菌分离, 鉴定

Abstract:

【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 genusAlternaria, 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 fromC. paliurus (Batal.) Iljinskaja plants.

Key words: Cyclocarya paliurus(Batal.) Iljinskaja, flavonoids, endophytic fungi, ITS analysis, scanning electron microscope, isolation, identification

中图分类号:

S718.81

寇晓琳,谢楠,吴彩娥,等. 青钱柳产黄酮类物质真菌的分离与鉴定[J]. 南京林业大学学报（自然科学版）, 2020, 44(2): 26-34.

KOU Xiaolin, XIE Nan, WU Caie, FAN Gongjian, FU Xiangxiang. Isolation and identification of endophytic fungi from Cyclocarya paliurus (Batal.) Iljinskaja[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(2): 26-34.DOI: 10.3969/j.issn.1000-2006.201812001.

图/表 7

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纲classis	目order	科family	属genus	总量/株 total strain	分离频率/% separation frequency
			木霉属Trichoderma	13	19.4
			青霉属Penicillium	9	1.9
		丛梗孢科Moniliacea	拟青霉属Paecilomyces	4	6.0
			地霉属Geotrichum	2	4.5
半知菌纲Deuteromycetes	丛梗孢目Moniliales		链格孢属Alternaria	16	23.9
		束梗孢科Stilbellaceae	棒束梗霉属Stilbaceae	1	1.5
		瘤座孢科Tuberculariaceae	镰刀菌属Fusarium	10	14.9
	黑盘孢目Melanconiales	黑盘孢科Melanconiaceae	刺盘孢属Colletotrichum	6	8.9
丝孢菌纲Hyphomycetes	无孢菌目Agonomycetales	无孢菌科Agaricaceae	丝核菌属Rhizoctonia	1	1.5
结合菌纲Class Zygomycetes	毛霉目Mucorale	毛霉科Mucoraceae	根霉属Rhizoupus sp.	5	7.5

菌株 bacterial strain	异槲皮苷含量/ (μg·μL^-1) soquercitroside	槲皮素含量/ (μg·μL^-1) quercetin	山柰酚含量/ (μg·μL^-1) kaempferol	总黄酮含量/ (mg·L^-1) flavonoids of sample
CY12	0.016±0.003	0.021±0.006	0.076±0.054	1.42
PZ06	0.038±0.009	0.060±0.011	0.190±0.081	3.61
PP03	0.220±0.029	0	0	2.77
CP06	0.014±0.009	0.031±0.003	0	0.57

青钱柳产黄酮类物质真菌的分离与鉴定

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

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