大兴安岭凋落物中可培养真菌分离、鉴定及活性筛选

doi:10.11924/j.issn.1000-6850.casb2021-0849

摘要/Abstract

摘要：

本研究旨在森林凋落物中尝试分离与鉴定具有抗菌与抗氧化活性的真菌资源。以大兴安岭森林凋落物为材料,利用稀释涂布平板法分离凋落物真菌,通过内部转录间隔区序列(ITS)进行真菌鉴定,利用平板打孔药剂扩散法测试抗菌活性,通过TLC-DPPH法进行提取物的抗氧化活性测定。本研究共分离得到65株真菌,它们分别隶属于47个属,54个分类单元,其中有16株真菌的序列相似性≤98%。这16株真菌中有8株有抗细菌活性,有10株有抗真菌活性,有6株菌的粗提物有抗氧化活性。发现3株真菌同时具有抗菌、抗氧化活性,它们最相近菌属分别为Phaeosphaeria、Cephalosporium和Ophiobolus。大兴安岭真菌资源丰富,本研究为抗菌及抗氧化的真菌天然资源的开发,提供了基础数据与备选菌株。

关键词: 大兴安岭, 凋落物真菌, 抗菌活性, 抗氧化活性

Abstract:

The purpose of this study is to isolate fungi resources that have antibacterial and antioxidant activities from forest litter. The fungi were isolated from forest litter in the Greater Hinggan Mountains by a particle-spreading-plate method, identified by internal transcribed spacer sequence (ITS), tested by an agar-well-diffusion method for antibacterial activities, and determined by TLC-DPPH method for antioxidant activities. A total of 65 fungal strains are isolated, which belong to 47 genera and 54 taxa, and the sequence similarity of 16 strains is ≤ 98%. Among these 16 strains, the crude extracts of 8 strains have antibacterial activities, 10 strains have antifungal activities, and 6 strains have antioxidant activities. There are 3 strains that have antibacterial and antioxidant activities at the same time, and their closest genera are Phaeosphaeria, Cephalosporium and Ophiobolus, respectively. The Greater Hinggan Mountains are rich in fungi resources. This study can provide basic data and candidate strains for the development of antibacterial and antioxidant fungi natural resources.

Key words: the Greater Khingan Mountains, litter fungi, antibacterial activity, antioxidant activity

中图分类号:

S482.7

司璐, 吴彤, 甄锦程, 于洪佳, 刘瑶, 杨骁, 徐利剑. 大兴安岭凋落物中可培养真菌分离、鉴定及活性筛选[J]. 中国农学通报, 2022, 38(26): 118-123.

SI Lu, WU Tong, ZHEN Jincheng, YU Hongjia, LIU Yao, YANG Xiao, XU Lijian. Isolation, Identification and Activity Screening of Culturable Fungi from Litter in the Greater Hinggan Mountains[J]. Chinese Agricultural Science Bulletin, 2022, 38(26): 118-123.

图/表 7

参考文献 31

[1]	HYDE K D, XU J, RAPIOr S, et al. The amazing potential of fungi: 50 ways we can exploit fungi industrially[J]. Fungal diversity, 2019, 97(1):1-136. doi: 10.1007/s13225-019-00430-9 URL
[2]	SCHMIT J P, MURPHY J F, Mueller G M. Macrofungal diversity of a temperate oak forest: a test of species richness estimators[J]. Canadian journal of botany, 1999, 77(7):1014-1027. doi: 10.1139/b99-055 URL
[3]	KHIRALLA A, MOHAMED I, THOMAS J, et al. A pilot study of antioxidant potential of endophytic fungi from some Sudanese medicinal plants[J]. Asian pacific journal of tropical medicine, 2015, 8(9):701-704. doi: 10.1016/j.apjtm.2015.07.032 URL
[4]	ALY A H, DE BB AB A, Proksch P. Fifty years of drug discovery from fungi[J]. Fungal diversity, 2011, 50(1):3-19. doi: 10.1007/s13225-011-0116-y URL
[5]	Skropeta, Danielle. Deep-sea natural products[J]. Natural product reports, 2008, 25(6):1131-1166 doi: 10.1039/b808743a pmid: 19030606
[6]	HONG J H, JANG S, HEO Y M, et al. Investigation of marine-derived fungal diversity and their exploitable biological activities[J]. Marine drugs, 2015, 13(7):4137-4155. doi: 10.3390/md13074137 URL
[7]	HUANG Z, NONG X, REN Z, et al. Anti-HSV-1, antioxidant and antifouling phenolic compounds from the deep-sea-derived fungus Aspergillus versicolor SCSIO 41502[J]. Bioorganic & Medicinal chemistry letters, 2017, 27(4):787-791. doi: 10.1016/j.bmcl.2017.01.032 URL
[8]	PANG X, ZHAO J Y, FANG X M, et al. Metabolites from the plant endophytic fungus Aspergillus sp. CPCC 400735 and their anti-HIV activities[J]. Journal of natural products, 2017, 80(10):2595-2601. doi: 10.1021/acs.jnatprod.6b00878 URL
[9]	ABDEL-WAHAB N M, SCHARF S, ÖZKAYA F C, et al. Induction of secondary metabolites from the marine-derived fungus Aspergillus versicolor through co-cultivation with Bacillus subtilis[J]. Planta medica, 2019, 85(6):503-512. doi: 10.1055/a-0835-2332 URL
[10]	TENNAKOON D, GENTEKAKI E, JEEWON R, et al. Life in leaf litter: Fungal community succession during decomposition[J]. Mycosphere, 2021, 12(1).406-429. doi: 10.5943/mycosphere/12/1/5 URL
[11]	姜雪薇, 马大龙, 臧淑英,等. 高通量测序分析大兴安岭典型森林土壤细菌和真菌群落特征[J]. 微生物学通报, 2021, 48(4):1093-1105.
[12]	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.
[13]	张哲栋, 梁晶, 李泽宇,等. 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.
[14]	邱天艺. 大兴安岭森林凋落物可培养真菌分离鉴定及物质分析:[D]. 哈尔滨: 黑龙江大学, 2021:28-31.
[15]	孟建宇, 杨帆, 冀锦华,等. 大兴安岭森林土壤中纤维素降解真菌的分离及产酶条件优化[J]. 黑龙江畜牧兽医, 2020(17):108-111,120,171.
[16]	YAN L, YE D, SHAO Z, et al. A Sterol and Spiroditerpenoids from a Penicillium sp. Isolated from a Deep Sea Sediment Sample[J]. Marine drugs, 2012, 10(2):497-508. doi: 10.3390/md10020497 URL
[17]	LIU D S, RONG X G, KANG H H, et al. Raistrickiones A-E from a Highly Productive Strain of Penicillium raistrickii Generated through Thermo Change[J]. Marine drugs, 2018, 16(6):1-11. doi: 10.3390/md16010001 URL
[18]	曾奇, 仲伟茂, 王发左. 深海来源真菌次级代谢产物研究进展[J]. 天然产物研究与开发, 2018, 30(3):501-514.
[19]	朱妍妍, 艾嫦, 张嘉,等. 具有生物活性的植物内生真菌次生代谢产物[J]. 化学进展, 2011, 23(4):704-730.
[20]	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(4):232-236. doi: 10.1016/j.myc.2019.03.002 URL
[21]	MÖLLER E M, 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
[22]	WHITE T J, BRUNS T, LEE S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J]. PCR protocols: A guide to methods and applications, 1990, 18(1):315-322.
[23]	周立刚. 植物抗菌化合物[M]. 北京: 中国农业科学技术出版社, 2005:74-82.
[24]	王梅霖, 张志国. 玫瑰黄酮的提取及抗氧化活性的研究[J]. 中国调味品, 2021, 46(7):157-160,170.
[25]	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]. 天然产物研究与开发, 2020, 32(3):453-463.
[26]	El-Demerdash A. Chemical diversity and biological activities of Phaeosphaeria fungi genus: a systematic review[J]. Journal of Fungi, 2018, 4(4):1-10. doi: 10.3390/jof4010001 URL
[27]	秦铭铭, 耿燕, 栾雪,等. 药食用真菌提取物抗氧化活性研究[J]. 食品与生物技术学报, 2017, 36(1):80-86.
[28]	Zhang H, Bai X, Wu B. Evaluation of antimicrobial activities of extracts of endophytic fungi from Artemisia annua[J]. Bangladesh journal of pharmacology, 2012, 7(2):120-123.
[29]	ZHANG H W, HUANG W Y, Chen J R, et al. Cephalosol: An antimicrobial metabolite with an unprecedented skeleton from endophytic Cephalosporium acremonium IFB‐E007[J]. Chemistry-A European Journal, 2008, 14(34):10670-10674. doi: 10.1002/chem.200801000 URL
[30]	CHIN N X, NEU H C. In vitro antimicrobial activity of the new antibiotic vermisporin[J]. European journal of clinical microbiology and infectious diseases, 1992, 11(8):755-757. doi: 10.1007/BF01989986 URL
[31]	TAYONE W C, TANAKA K, TAKADA N. Ophiobolides, Polyketides Isolated from Ophiobolus sp. KTC 2293[J]. Chiang Mai journal of science, 2016, 43(3):477-483.

序号	菌株号	最相近菌种*	相似性*
1	SL098	Phaeosphaeria sp.	93.89%
2	SL503	Cephalosporium gramineum	92.03%
3	SL506	Knufia tsunedae	97.98%
4	SL514	Peniophora sp.	95.16%
5	SL518	Pseudeurotium sp.	96.99%
6	SL521	Acremonium psammosporum	94.32%
7	SL523	Fusicladium eucalyptigenum	89.23%
8	SL542	Capronia acutiseta	92.64%
9	SL544	Pseudoarthrographis phlogis	87.00%
10	SL708	Polyphilus sieberi	94.21%
11	SL710	Pezicula sp.	93.63%
12	SL721	Berkleasmium sp.	91.79%
13	SL723	Ophiobolus cirsii	95.01%
14	SL724	Exophiala moniliae	96.81%
15	SL726	Piniphoma wesendahlina	93.43%
16	SL728	Herpotrichia parasitica	95.54%

序号	菌株号	最相近菌种*	相似性*
1	SL098	Phaeosphaeria sp.	93.89%
2	SL503	Cephalosporium gramineum	92.03%
3	SL506	Knufia tsunedae	97.98%
4	SL514	Peniophora sp.	95.16%
5	SL518	Pseudeurotium sp.	96.99%
6	SL521	Acremonium psammosporum	94.32%
7	SL523	Fusicladium eucalyptigenum	89.23%
8	SL542	Capronia acutiseta	92.64%
9	SL544	Pseudoarthrographis phlogis	87.00%
10	SL708	Polyphilus sieberi	94.21%
11	SL710	Pezicula sp.	93.63%
12	SL721	Berkleasmium sp.	91.79%
13	SL723	Ophiobolus cirsii	95.01%
14	SL724	Exophiala moniliae	96.81%
15	SL726	Piniphoma wesendahlina	93.43%
16	SL728	Herpotrichia parasitica	95.54%

菌株号	丁香假单胞菌						青枯劳尔氏菌
菌株号	P	N	S	Y	R	A	P	N	S	Y	R	A
SL098	—	—	—	++	—	—	+++	—	—	++	—	—
SL503	—	—	—	—	—	—	+	++	—	++	++	++
SL521	—	—	—	—	—	—	—	—	—	++	—	—
SL523	—	—	—	—	—	—	—	—	—	+	—	—
SL721	—	—	—	—	—	—	++	++	—	—	—	—
SL723	—	—	—	—	—	—	+++	—	—	—	+++	++
SL726	—	—	—	—	—	—	—	—	—	+	—	—
SL728	—	—	—	—	—	—	—	—	—	—	—	++

菌株号	丁香假单胞菌						青枯劳尔氏菌
菌株号	P	N	S	Y	R	A	P	N	S	Y	R	A
SL098	—	—	—	++	—	—	+++	—	—	++	—	—
SL503	—	—	—	—	—	—	+	++	—	++	++	++
SL521	—	—	—	—	—	—	—	—	—	++	—	—
SL523	—	—	—	—	—	—	—	—	—	+	—	—
SL721	—	—	—	—	—	—	++	++	—	—	—	—
SL723	—	—	—	—	—	—	+++	—	—	—	+++	++
SL726	—	—	—	—	—	—	—	—	—	+	—	—
SL728	—	—	—	—	—	—	—	—	—	—	—	++

菌株号	立枯丝核菌						大斑病凸脐蠕孢菌
菌株号	P	N	S	Y	R	A	P	N	S	Y	R	A
SL098	—	—	—	++	+	—	+	+	—	+++	—	—
SL503	—	+	+	—	—	++	—	+	+	+	+	+
SL521	—	—	—	—	—	—	—	—	—	+++	—	—
SL533	—	—	—	—	—	—	—	—	—	++	—	—
SL544	—	—	—	—	—	—	—	++	—	—	—	—
SL708	+	+	+	—	+	—	—	—	—	—	—	—
SL710	—	—	—	++	+	++	—	—	—	++	—	—
SL723	++	—	—	—	+	+	++	—	—	—	++	—
SL726	—	—	—	+	—	—	—	—	—	—	—	—
SL728	—	—	—	—	—	+	—	—	—	—	—	—