大兴安岭北方森林凋落物真菌及其抗菌化合物

doi:10.11924/j.issn.1000-6850.casb2020-0135

中国农学通报 ›› 2021, Vol. 37 ›› Issue (6): 104-110.doi: 10.11924/j.issn.1000-6850.casb2020-0135

大兴安岭北方森林凋落物真菌及其抗菌化合物

张哲栋¹(), 梁晶², 李泽宇¹, 高思禹¹, 邱天艺¹, 单体江³, 徐利剑¹()

¹黑龙江大学现代农业与生态环境学院,哈尔滨 150080
²新疆农业科学院核技术生物技术研究所,乌鲁木齐 830091
³华南农业大学林学与风景园林学院/广东省微生物信号与作物病害防控重点实验室,广州 510642

收稿日期:2020-05-28 修回日期:2020-07-30 出版日期:2021-02-25 发布日期:2021-02-24
通讯作者: 徐利剑
作者简介:张哲栋,男,1995年出生,山西怀仁人,硕士研究生,研究方向：资源利用与植物保护。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学现代农业与生态环境学院,Tel：0451-86609487,E-mail：872810444@qq.com。
基金资助:
国家自然科学基金“大兴安岭林下凋落物慢速生长产孢真菌的隐秘代谢物及其抗菌活性”(31870528);黑龙江省博士后科研启动金“兰伯盘菌W17聚酮类代谢物抗水稻病原菌的研究”(LBH-Q15123);东北盐碱植被恢复与重建教育部重点实验室开放基金“水稻脂类代谢盐碱地胁迫响应关键基因及代谢途径网络构建”(20200522-4)

Litter Fungi in the Boreal Forests and Their Antibacterial Compounds in the Greater Khingan Mountains

Zhang Zhedong¹(), Liang Jing², Li Zeyu¹, Gao Siyu¹, Qiu Tianyi¹, Shan Tijiang³, Xu Lijian¹()

¹College of Advanced Agriculture and Ecological Environment, Heilongjiang University, Harbin 150080
²Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091
³College of Forestry and Landscape Architecture, South China Agricultural University/Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Guangzhou 510642

Received:2020-05-28 Revised:2020-07-30 Online:2021-02-25 Published:2021-02-24
Contact: Xu Lijian

摘要/Abstract

摘要：

为了研究大兴安岭森林凋落物真菌的抗菌活性,首先利用颗粒涂布平板法分离真菌,通过扩增与比对内部转录间隔区序列初步鉴定分离到的真菌。采用双平板打孔药剂扩散法检测抗菌活性,使用层析法分离目标菌株单体化合物。通过波谱学分析确定其化学结构,使用96孔板液体稀释法检测单体化合物的最低抑菌浓度。共分离得到88株真菌,其隶属于57个属、74个分类单元,其中18株真菌内部转录间隔区序列相似性≤97%。其中6株真菌的乙酸乙酯提取物具有抗菌活性,2株真菌KNFL008和KNFL040抗菌活性较强。经活性追踪分离,共得到4个单体化合物,分别为化合物A（1,3-2-羟基-5-十五烷基苯）、化合物B（麦角固醇）、化合物C（2-氨基-3H-苯氧嗪-3-酮）和化合物D（2-乙酰氨基-3H-苯氧嗪-3-酮）。对化合物A、B、C进行了最低抑菌浓度的测定,为3.13~200 μg/mL。化合物A和D为首次在真菌提取物中分离。本研究为进一步开发利用大兴安岭凋落物真菌资源打下了基础。

关键词: 大兴安岭, 凋落物, 真菌, 抗菌活性, 次级代谢产物, 化合物分离

Abstract:

To study the antimicrobial activity of forest litter fungi in the Greater Khingan Mountains, firstly, fungi were isolated by a method of spreading particles on plates, and the isolated fungi were preliminarily identified by the amplification and alignment of internal transcribed spacer sequences. The antibacterial activities were detected by a two-layer-plate diffusion method and monomeric compounds of targeted strains were separated by chromatography. Spectroscopic analyses were used to determine the chemical structures of the monomeric compounds. A 96-well plate liquid dilution method was used to detect their minimum inhibitory concentration. A total of 88 fungi were isolated, belonging to 57 genera and 74 taxa. Of them, the internal transcribed spacer sequence similarities of 18 fungi were not higher than 97%. Among them, the ethyl acetate extracts of 6 fungi had antibacterial activities, and the two fungal strains KNFL008 and KNFL040 had stronger antibacterial activities than the others. By a bioassay-guided separation, four monomeric compounds, A (1,3-2-hydroxy-5-pentaalkylbenzene), B (ergosterol), C (2-amino-3h-phenoxyzin-3-ketone) and D (2-acetylamino-3h-phenoxyzin-3-ketone), were separated. The minimum inhibitory concentration of compound A, B and C was tested as 3.13-200 μg/mL. Compound A and D were firstly isolated from fungal extracts. This study lays a foundation for the further resource development and utilization of litter fungi in the Greater Khingan Mountains.

Key words: the Greater Khingan Mountains, litter, fungus, antibacterial activity, secondary metabolites, compound separation

中图分类号:

S718.81

张哲栋, 梁晶, 李泽宇, 高思禹, 邱天艺, 单体江, 徐利剑. 大兴安岭北方森林凋落物真菌及其抗菌化合物[J]. 中国农学通报, 2021, 37(6): 104-110.

Zhang Zhedong, Liang Jing, Li Zeyu, Gao Siyu, Qiu Tianyi, Shan Tijiang, Xu Lijian. Litter Fungi in the Boreal Forests and Their Antibacterial Compounds in the Greater Khingan Mountains[J]. Chinese Agricultural Science Bulletin, 2021, 37(6): 104-110.

图/表 9

参考文献 27

[1]	Kotra L P, Mobashery S. β-Lactam antibiotics, β-lactamases and bacterial esistance[J]. Elsevier, 1998,96(3):139-150.
[2]	Radu S, Kqueen C Y. Preliminary screening of endophytic fungi from medicinal plants in malaysia for antimicrobial and antitumor activity[J]. The Malaysian journal of medical sciences: MJMS, 2002,9(2):23-33. URL pmid: 22844221
[3]	Ebel R. Terpenes from Marine-Derived Fungi[J]. Marine Drugs, 2010,8(8):2340-2368. doi: 10.3390/md8082340 URL pmid: 20948911
[4]	Huber F M, Gottlieb D. The mechanism of action of griseofulvin[J]. Canadian Journal of Microbiology, 1968,14(2):111-118. doi: 10.1139/m68-019 URL pmid: 5689440
[5]	Denning D W. Echinocandins and pneumocandins-a new antifungal class with anovel mode of action[J]. Journal of Antimicrobial Chemotherapy, 1997,40(5):611-614. doi: 10.1093/jac/40.5.611 URL
[6]	Waxman D J, Strominger J L. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics[J]. Annu Rev Biochem, 1983,52:825-869. doi: 10.1146/annurev.bi.52.070183.004141 URL pmid: 6351730
[7]	Godtfredsen W, Jahnsen S, Lorck H, et al. Fusidic acid: a new antibiotic[J]. Nature, 1962,193:987. doi: 10.1038/193987a0 URL pmid: 13899435
[8]	Negishi S, Cai-Huang Z, Hasumi K, et al. Productivity of monacolin K (mevinolin) in the genus Monascus[J]. Hakko Kogaku Kaishi, 1986,64:509-512.
[9]	Ypema H L, Gold R E. Kresoxim-methyl: Modification of a Naturally Occurring Compound to Produce a New Fungicide[J]. Plant Disease, 1999,83(1):4-19. doi: 10.1094/PDIS.1999.83.1.4 URL pmid: 30845437
[10]	Amal H A, Abdessamad Debbab, Peter Proksch. Fifty years of drug discovery from fungi[J]. Fungal Diversity, 2011(50):3-19. doi: 10.1007/s13225-011-0116-y URL
[11]	刘庆, 吴彦, 何海. 森林凋落物研究进展[J]. 生态学杂志, 2004(1):60-64.
[12]	凌华, 陈光水, 陈志勤. 中国森林凋落量的影响因素[J]. 亚热带资源与环境学报, 2009,4(04):66-71.
[13]	田兴军, 立石贵浩. 亚高山针叶林土壤动物和土壤微生物对针叶林分解的作用[J]. 植物生态学报, 2002,26(3):257-263.
[14]	张晶, 张惠文, 李新宇, 等. 土壤真菌多样性及分子生态学研究进展[J]. 应用生态学报, 2004,15(10):1958-1962.
[15]	刘博洋, 朱美齐, 李泽宇, 等. 大兴安岭森林凋落物真菌及其次生代谢物活性研究[J]. 中国农学通报, 2019,35(29):103-108.
[16]	杨立宾, 隋心, 朱道光, 等. 大兴安岭兴安落叶松林土壤真菌群落特征研究[J]. 中南林业科技大学学报, 2017,37(12):76-84.
[17]	潘学仁, 李思, 姚乃荣, 等. 大兴安岭阿里河林区食药用菌资源[J]. 中国食用菌, 1998(05):16-18.
[18]	杨秀丽, 闫伟. 兴安杜鹃菌根真菌分离与鉴定[J]. 北方农业学报, 2016,44(05):54-58.
[19]	李泽宇, 张志, 邱天艺, 等. 来源于大兴安岭多年冻土可培养真菌及其发酵物的生物活性[J/OL]. 天然产物研究与开发:1-15. http://kns.cnki.net/kcms/detail/51.1335.Q.20200326.1633.026.html, 2020-05-06.
[20]	SaghaiMaroof M A, Soliman K M, Jorgensen R A, et al. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics[J]. PANS, 1984,81(24):8014-8018.
[21]	Liang J, Liu B Y, Li Z Y, et al. Myxotrichum albicans, a new slowly-growing species isolated from forest litters in China[J]. Mycoscience, 2019,60(2019):232-236.
[22]	Möller E, Bahnweg G, Sandermann H, et al. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues[J]. Nucleic acids research, 1992,20(22):6115-6116. doi: 10.1093/nar/20.22.6115 URL pmid: 1461751
[23]	赵江林, 徐利剑, 黄永富, 等. TLC-生物自显影-MTT法检测滇重楼内生真菌中抗菌活性成分[J]. 天然产物研究与开发, 2008(01):28-32,51.
[24]	Tsuge N, Mizokami M, Imai S, et al. Adipostatins A and B, new inhibitors of glycerol-3-phosphate dehydrogenase[J]. The Journal of antibiotics, 1992,45(6):886-891. doi: 10.7164/antibiotics.45.886 URL pmid: 1500355
[25]	Kwon H C, Zee S D, Cho S Y, et al. C Cytotoxic ergosterols from Paecilomyces sp. J300[J]. Archives of Pharmacal Research, 2002,25(6):851-855. doi: 10.1007/BF02977003 URL pmid: 12510837
[26]	Kinjo J E, Yokomizo K, Awata Y, et al. Structures of phytotoxins, AV-toxins C, D and E, produced by zonate leaf spot fungus of mulberry[J]. Tetrahedron Letters, 1987,28(32):3697-3698.
[27]	Maskey R P, Li F C, Qin S, et al. Chandrananimycins A ~ C: Production of Novel Anticancer Antibiotics from a Marine Actinomadura sp. Isolate M048 by Variation of Medium Composition and Growth conditions[J]. The Journal of Antibiotics, 2003,56(7):622-629. doi: 10.7164/antibiotics.56.622 URL pmid: 14513905

高效液相类型	分析型高效液相色谱	半制备型高效液相色谱
色谱柱	0.5 cm直径的ODS C-18反相硅胶柱	1 cm直径的ODS C-18反相硅胶柱
流动相	甲醇、水	甲醇、水
流速	1 mL/min	2.5 mL/min
柱温	25℃	25℃
固定检测波长	230 nm、280 nm	210 nm、254 nm
洗脱阶段1	0~4 min,10%甲醇等度	0~4 min,10%甲醇等度
洗脱阶段2	4~28 min,10%~90%甲醇梯度	4~35 min,10%~90%甲醇梯度
洗脱阶段3	28~35 min,90%~100%甲醇梯度	35~50 min,90%~100%甲醇梯度
洗脱阶段4	35~60 min,100%甲醇等度	50~70 min,100%甲醇等度

高效液相类型	分析型高效液相色谱	半制备型高效液相色谱
色谱柱	0.5 cm直径的ODS C-18反相硅胶柱	1 cm直径的ODS C-18反相硅胶柱
流动相	甲醇、水	甲醇、水
流速	1 mL/min	2.5 mL/min
柱温	25℃	25℃
固定检测波长	230 nm、280 nm	210 nm、254 nm
洗脱阶段1	0~4 min,10%甲醇等度	0~4 min,10%甲醇等度
洗脱阶段2	4~28 min,10%~90%甲醇梯度	4~35 min,10%~90%甲醇梯度
洗脱阶段3	28~35 min,90%~100%甲醇梯度	35~50 min,90%~100%甲醇梯度
洗脱阶段4	35~60 min,100%甲醇等度	50~70 min,100%甲醇等度

菌株号	相似度^*/%	最相近菌株^*	登录号^*
KNFL008	96	Myxotrichum stipitatum	MH378419
KNFL015	97	Myxozyma sp.	MN719070
KNFL018	95	Atrocalyx bambusae	MT146478
KNFL024	96	Chloridium virescens	MT146479
KNFL028	92	Hydropisphaera erubescens	MK182930
KNFL033	84	Scolecobasidium humicola	MK205294
KNFL038	95	Infundichalara microchona	MK205295
KNFL040	96	Acrospermum sp.	MT146474
KNFL047	94	Cladophialophora minutissima	MK205296
KNFL049	94	Polyphilus sieberi	MK205298
KNFL051	94	Infundichalara microchona	MT146472
KNFL060	95	Chalara hyalocuspica	MK205300
KNFL063	83	Radulidium epichloes	MK205301
KNFL066	94	Helotiales sp.	MK205302
KNFL075	92	Leohumicola sp.	MK205303
KNFL078	86	Chaetothyriales sp.	MK182929
KNFL084	96	Xylaria frustulosa	MT146476
KNFL087	84	Degelia gayana	MT146477

菌株号	相似度^*/%	最相近菌株^*	登录号^*
KNFL008	96	Myxotrichum stipitatum	MH378419
KNFL015	97	Myxozyma sp.	MN719070
KNFL018	95	Atrocalyx bambusae	MT146478
KNFL024	96	Chloridium virescens	MT146479
KNFL028	92	Hydropisphaera erubescens	MK182930
KNFL033	84	Scolecobasidium humicola	MK205294
KNFL038	95	Infundichalara microchona	MK205295
KNFL040	96	Acrospermum sp.	MT146474
KNFL047	94	Cladophialophora minutissima	MK205296
KNFL049	94	Polyphilus sieberi	MK205298
KNFL051	94	Infundichalara microchona	MT146472
KNFL060	95	Chalara hyalocuspica	MK205300
KNFL063	83	Radulidium epichloes	MK205301
KNFL066	94	Helotiales sp.	MK205302
KNFL075	92	Leohumicola sp.	MK205303
KNFL078	86	Chaetothyriales sp.	MK182929
KNFL084	96	Xylaria frustulosa	MT146476
KNFL087	84	Degelia gayana	MT146477

菌株	大肠埃希氏菌	青枯劳尔氏菌
阳性对照	+++	+++
KNFL008	-	++
KNFL018	+	-
KNFL024	-	+
KNFL040	++	+++
KNFL075	-	+
KNLF078	-	+

大兴安岭北方森林凋落物真菌及其抗菌化合物

Litter Fungi in the Boreal Forests and Their Antibacterial Compounds in the Greater Khingan Mountains

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