披针叶黄华内生真菌的分离与鉴定

doi:10.11843/j.issn.0366-6964.2021.010.030

摘要/Abstract

摘要： 旨在探讨有毒植物披针叶黄华内生真菌种类与种群分布情况，本试验以采自青海的披针叶黄华为试验材料，用表面消毒法对样品消毒后进行内生真菌分离，结合形态学鉴定和5.8S rDNA-ITS序列分析对分离菌株进行种属鉴定，并应用MEGA7.0软件进行系统发育分析。结果显示，从披针叶黄华中共分离获得29种内生真菌，分属于7纲、9目、11科、12属。披针叶黄华内生真菌总相对分离频率为79.31%，其中，叶的内生真菌种类最多（41.38%），茎次之（37.93%），种子（13.79%）和根（6.90%）相对较少；根据系统发育分析，所有内生真菌分属于两大种群，种群间亲缘关系较近。综上表明，披针叶黄华内生真菌种类丰富，青霉属是优势菌属。

关键词: 披针叶黄华, 内生真菌, 分离鉴定, 系统发育分析, 青霉属

Abstract: To investigate the endophytic fungi species and population distribution of the poisonous plant, Thermopsis lanceolata, the experimental materials collected from Qinghai Province were disinfected with surface disinfection method and then endophytic fungi were separated. The morphological and 5.8S rDNA-ITS sequence analysis were used to identify these strains, and the MEGA7.0 software was used for phylogenetic analysis. The results showed that twenty-nine endophytic fungi were isolated from the Thermopsis lanceolata, which belong to 7 classes, 9 orders, 11 families and 12 genera. The total relative separation frequency was 85.42%. Endophytic fungi are most commonly isolated from leaves (41.38%), followed by stems (37.93%), seed (13.79%) and roots (6.90%). According to phylogenetic analysis, all endophytic fungi belong to two populations with close genetic relationship. The results indicated that Thermopsis lanceolata had abundant endophytic fungi species and Penicillium sp. was the dominant genus.

Key words: Thermopsis lanceolata, endophytic fungi, isolation and identification, phylogenetic analysis, Penicillium sp.

中图分类号:

S859.87

赵文星, 吴可欣, 郭梓豫, 张云昊, 唐丽辉, 刘奕伶, 莫重辉, 赵宝玉, 路浩. 披针叶黄华内生真菌的分离与鉴定[J]. 畜牧兽医学报, 2021, 52(10): 2986-2994.

ZHAO Wenxing, WU Kexin, GUO Ziyu, ZHANG Yunhao, TANG Lihui, LIU Yiling, MO Chonghui, ZHAO Baoyu, LU Hao. Isolation and Identification of Endophytic Fungi in Thermopsis lanceolata[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2986-2994.

参考文献 36

[1]	李勇, 赵晓瑞, 张学礼. 披针叶黄华的化学成分研究[J]. 农业科学研究, 2008, 29(4):68-69.LI Y, ZHAO X R, ZHANG X L. Study on chemical components of Thermopsis lanceolata[J]. Journal of Agricultural Sciences, 2008, 29(4):68-69. (in Chinese)
[2]	魏启华, 赵博光, 郭道森. 披针叶黄华中抑制植物病原真菌物质的分离鉴定[J]. 南京林业大学学报:自然科学版, 2001, 25(5):85-88.WEI Q H, ZHAO B G, GUO D S. Isolation and identification of a compound from Thermopsis lanceolata to inhibit plant pathogenic fungi[J]. Journal of Nanjing Forestry University:Natural Sciences Edition, 2001, 25(5):85-88. (in Chinese)
[3]	JIN D M, MA J J, MA W H, et al. Legumes in Chinese natural grasslands:species, biomass, and distribution[J]. Rangeland Ecol Manag, 2013, 66(6):648-656.
[4]	LI W J, LI J H, LU J F, et al. Legume-grass species influence plant productivity and soil nitrogen during grassland succession in the eastern Tibet Plateau[J]. Appl Soil Ecol, 2010, 44(2):164-169.
[5]	李勇, 王永梅, 魏彦明. 披针叶黄华碱成分分析研究[J]. 中兽医医药杂志, 2016, 35(5):42-44.LI Y, WANG Y M, WEI Y M. Component analysis of alkaloid in Thermopsis lanceolate[J]. Journal of Traditional Chinese Veterinary Medicine, 2016, 35(5):42-44. (in Chinese)
[6]	赵宝玉, 张靖飞, 哈斯巴图, 等. 披针叶黄华生物碱成分研究[J]. 西北农林科技大学学报:自然科学版, 2002, 30(3):9-12.ZHAO B Y, ZHANG J Y, HA S B T, et al. Studies on chemical constitution of alkaloid from Thermopsis lancenolate[J]. Journal of Northwest Sci-Tech University of Agriculture and Forestry:Natural Science Edition, 2002, 30(3):9-12. (in Chinese)
[7]	赵宝玉. 中国重要有毒有害植物名录[M]. 北京:中国农业科学技术出版社, 2015:137-138.ZHAO B Y. Directory of important poisonous and injurious plants in China[M]. Beijing:China Agriculture Science and Technology Press, 2015:137-138. (in Chinese)
[8]	赵夏纬, 王一峰, 马文梅. 高寒草地不同坡向披针叶黄华蒸腾速率与叶性状的关系[J]. 生态学报, 2019, 39(7):2494-2500.ZHAO X W, WANG Y F, MA W M. The relationship of transpiration rate with leaf characters of Thermopsis lanceolate in different slopes of an alpine meadow[J]. Acta Ecologica Sinica, 2019, 39(7):2494-2500. (in Chinese)
[9]	时永杰. 披针叶黄华[J]. 中兽医医药杂志, 2003(S1):130-131.SHI Y J. Thermopsis lanceolata[J]. Journal of Traditional Chinese Veterinary Medicine, 2003(S1):130-131. (in Chinese)
[10]	KARLOV D S, SAZANOVA A L, KUZNETSOVA I G, et al. The genetic diversity of microsymbionts from Thermopsis lanceolata growing in Mongolia[J]. Ecol Genet, 2019, 17(1):43-51.
[11]	林源, 王志昇, 李勇, 等. 披针叶黄华中2种生物碱单体的分离与鉴定[J]. 畜牧与饲料科学, 2014, 35(11):1-4, 7.LIN Y, WANG Z S, LI Y, et al. Isolation and identification of two kinds of alkaloids from Thermopsis lanceolate[J]. Animal Husbandry and Feed Science, 2014, 35(11):1-4, 7. (in Chinese)
[12]	孙大林, 赵清梅, 余永涛, 等. 披针叶黄华中2株蠕形分生孢子内生真菌的鉴定[J]. 湖北农业科学, 2015, 54(18):4473-4478.SUN D L, ZHAO Q M, YU Y T, et al. Identification of two helminthosporioid fungal endophytes from Thermopsis lanceolata[J]. Hubei Agricultural Sciences, 2015, 54(18):4473-4478. (in Chinese)
[13]	张起鹏, 王建, 赵成章, 等. 高寒干旱草原披针叶黄华植物群落土壤种子库与地上植被的关系[J]. 生态学报, 2017, 37(14):4619-4626.ZHANG Q P, WANG J, ZHAO C Z, et al. Relationships between the soil seed bank and above-ground vegetation of a Thermopsis lanceolata community in degraded alpine arid grasslands[J]. Acta Ecologica Sinica, 2017, 37(14):4619-4626. (in Chinese)
[14]	林源, 王志昇, 李勇, 等. 披针叶黄华生物碱成分分析[J]. 安徽农业科学, 2014, 42(33):11660-11661, 11663.LIN Y, WANG Z S, LI Y, et al. Analysis on the contents of alkaloid in Thermopsis lanceolate[J]. Journal of Anhui Agricultural Sciences, 2014, 42(33):11660-11661, 11663. (in Chinese)
[15]	VINOGRADOVA V I, ISKANDAROV S, YUNUSOV S Y. An investigation of the alkaloids of Thermopsis lanceolata[J]. Chem Nat Compd, 1971, 7(4):440-442.
[16]	叶润, 陈颐辉, 蔡静, 等. 高纯度金雀花碱的提取工艺及含量测定[J]. 离子交换与吸附, 2020, 36(2):163-172.YE R, CHEN Y H, CAI J, et al. Extracing technology and content detemination of high purity cytisine[J]. Ion Exchange and Adsorption, 2020, 36(2):163-172. (in Chinese)
[17]	党苗苗, 丁海娥, 从丙超, 等. 药用植物内生真菌研究进展[J]. 农村经济与科技, 2020, 31(14):31-32.DANG M M, DING H E, CONG B C, et al. Advances in endophytic fungi of medicinal plants[J]. Rural Economy and Science-Technology, 2020, 31(14):31-32. (in Chinese)
[18]	夏飞, 上官晓雨, 冯思亮, 等. 乌头中内生真菌的分离鉴定及其抑菌活性和机制研究[J]. 陕西科技大学学报, 2019, 37(6):66-72.XIA F, SHANGGUAN X Y, FENG S L, et al. Study on isolation, identification and antibacterial activity and mechanism of endophytic fungi from Aconitum carmichaeli debx[J]. Journal of Shaanxi University of Science & Technology, 2019, 37(6):66-72. (in Chinese)
[19]	黄燕, 曾东强, 唐文伟, 等. 植物内生真菌生物活性成分研究进展[J]. 天然产物研究与开发, 2020, 32(8):1419-1437.HUANG Y, ZENG D Q, TANG W W, et al. Research advances of endophytic fungi bioactive components[J]. Natural Product Research and Development, 2020, 32(8):1419-1437. (in Chinese)
[20]	王景仪, 李梦秋, 李艳茹, 等. 药用植物内生真菌的多样性及生物功能研究进展[J]. 生物资源, 2020, 42(2):164-172.WANG J Y, LI M Q, LI Y R, et al. Research progress on diversity and biological function of endophytic fungi in medicinal plants[J]. Biotic Resources, 2020, 42(2):164-172. (in Chinese)
[21]	唐雪辉, 毛凯, 干友民, 等. 植物内生真菌的应用研究概况[J]. 草原与草坪, 2005(5):16-21.TANG X H, MAO K, GAN Y M, et al. Research and application of plant endophyte[J]. Grassland and Turf, 2005(5):16-21. (in Chinese)
[22]	谢海伟, 冯嘉琪, 付晓晴, 等. 药用植物内生真菌的研究进展[J]. 江苏农业科学, 2020, 48(14):1-6.XIE H W, FENG J Q, FU X Q, et al. Research progress of endophytic fungi in medicinal plants[J]. Jiangsu Agricultural Sciences, 2020, 48(14):1-6. (in Chinese)
[23]	刘建利, 林勤, 闫兴富. 宁夏地区苦豆子内生真菌的分离与初步鉴定[J]. 干旱地区农业研究, 2012, 35(5):193-199.LIU J L, LIN Q, YAN X F. Isolation and preliminary identification of endophytic fungi from Sophora alopecuroides in Ningxia[J]. Agricultural Research in the Arid Areas, 2012, 35(5):193-199. (in Chinese)
[24]	孙璐, 任祯慧, 宋润杰, 等. 毛序棘豆内生真菌分离鉴定及系统进化分析[J]. 畜牧兽医学报, 2018, 49(9):2025-2035.SUN L, REN Z H, SONG R J, et al. Isolation, identification and phylogenetic analysis of fungal endophyte in Oxytropis trichophora[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(9):2025-2035. (in Chinese)
[25]	周德庆. 微生物学实验教程[M]. 2版. 北京:高等教育出版社, 2006:26-372.ZHOU D Q. Experimental microbiology[M]. 2nd ed. Beijing:Higher Education Press, 2006:26-372. (in Chinese)
[26]	魏景超. 真菌鉴定手册[M]. 上海:上海科学技术出版社, 1979.WEI J C. Manual of identification of fungi[M]. Shanghai:Shanghai Scientific and Technical Publishers, 1979. (in Chinese)
[27]	路浩, 卢围, 赵宝玉, 等. 我国主要疯草内生真菌分离鉴定及其进化分析[J]. 中国兽医学报, 2012, 32(12):1819-1823, 1827.LU H, LU W, ZHAO B Y, et al. Isolation and identification of endophytic fungus from the serious locoweed of China[J]. Chinese Journal of Veterinary Science, 2012, 32(12):1819-1823, 1827. (in Chinese)
[28]	陈宣, 王丹萍, 杨珺, 等. 海南安诺兰菌根表面消毒方法研究[J]. 现代农业科技, 2016, (3):179-180, 183.CHEN X, WANG D P, YANG J, et al. Study on surface disinfection of mycorrhiza from Anota hannanensis in Hainan province[J]. Modern Agricultural Science and Technology, 2016(3):179-180, 183. (in Chinese)
[29]	王进, 吕彪, 肖占文, 等. 苦豆子和披针叶黄华种子特性及硬实破除研究[J]. 农业工程学报, 2010, 26(S1):406-410.WANG J, LÜ B, XIAO Z W, et al. Seed characteristics and hard seed broken methods of Sophora alopecuroides and Thermopsis lanceolata[J]. Transactions of the Chinese Society of Agricultural Engineering, 2010, 26(S1):406-410. (in Chinese)
[30]	余永涛, 何生虎, 赵清梅. 宁夏苦豆子中产苦参碱内生真菌的分离与鉴定[J]. 中国农业科学, 2013, 46(13):2643-2654.YU Y T, HE S H, ZHAO Q M. Isolation and identification of matrine-producing fungal Endophytes from Sophora alopecuroides in Ningxia[J]. Scientia Agricultura Sinica, 2013, 46(13):2643-2654. (in Chinese)
[31]	黄恩霞, 张书航, 刘欣瑜, 等. 藏沙蒿内生真菌分离鉴定与系统进化分析[J]. 动物医学进展, 2020, 41(9):68-74.HUANG E X, ZHANG S H, LIU X Y, et al. Isolation, identification and phylogenetic analysis of endophyeic fungus in Artemisia wellbyi[J]. Progress in Veterinary Medicine, 2020, 41(9):68-74. (in Chinese)
[32]	陈青青, 聂倩文, 江健伟, 等. 白芨内生真菌的分离鉴定及其群落结构分析[J]. 长江大学学报:自然科学版, 2020, 17(6):75-81.CHEN Q Q, NIE Q W, JIANG J W, et al. Isolation and identification of Bletilla striata Endophytic fungi and the analysis of its community structure[J]. Journal of Yangtze University:Natural Science Edition, 2020, 17(6):75-81. (in Chinese)
[33]	张琪, 王嘉琦, 吕梦霞, 等. 青霉属真菌次级代谢产物的结构类型及其药用活性的研究进展[J]. 工业微生物, 2019, 49(2):56-65.ZHANG Q, WANG J Q, LÜ M, et al. Advances in structural types and medical activities of secondary metabolites of Penicillium fungi[J]. Industrial Microbiology, 2019, 49(2):56-65. (in Chinese)
[34]	张德武, 赵建元, 周佳琦, 等. 青霉属真菌Penicillium sp. CPCC 400786的抗病毒活性成分[J]. 菌物学报, 2020, 39(2):434-440.ZHANG D W, ZHAO J Y, ZHOU J Q, et al. Antiviral chemical constituents from the fungus Penicillium sp.CPCC 400786[J]. Mycosystema, 2020, 39(2):434-440. (in Chinese)
[35]	钱一鑫, 康冀川, 雷帮星, 等. 青蒿内生真菌Alternaria sp. (PQH12)提取物体外细胞毒活性及抗氧化活性的研究[J]. 天然产物研究与开发, 2014, 26(9):1458-1462, 1509.QIAN Y X, KANG J C, LIE B X, et al. Cytotoxic and antioxidant activities of extracts of endophytic fungus Alternaria sp. (PQH12) from Artemisia annua[J]. Natural Product Research and Development, 2014, 26(9):1458-1462, 1509. (in Chinese)
[36]	MEENA M, SAMAL S. Alternaria host-specific (HSTs) toxins:an overview of chemical characterization, target sites, regulation and their toxic effects[J]. Toxicol Rep, 2019, 6:745-758.