凋落物真菌Berkleasmium sp.及其螺二萘类化合物抗菌活性的研究

doi:10.11924/j.issn.1000-6850.casb2021-0800

中国农学通报 ›› 2022, Vol. 38 ›› Issue (22): 115-120.doi: 10.11924/j.issn.1000-6850.casb2021-0800

所属专题：生物技术；植物保护

凋落物真菌Berkleasmium sp.及其螺二萘类化合物抗菌活性的研究

甄锦程¹(), 穆玉婷¹, 司璐¹, 于洪佳¹, 都婷婷¹, 单体江², 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:2021-08-19 修回日期:2022-05-23 出版日期:2022-08-05 发布日期:2022-08-22
通讯作者: 徐利剑
作者简介:甄锦程,男,1997年出生,黑龙江鸡西人,硕士研究生,研究方向：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： 2687334053@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);黑龙江大学研究生创新科研项目“特境真菌活性次级代谢产物研究”(YJSCX2021-217HLJU)

Studies on the Antimicrobial Activity of Litter Fungus Berkleasmium sp. and Its Spirobisnaphthalenes

ZHEN Jincheng¹(), MU Yuting¹, SI Lu¹, YU Hongjia¹, DU Tingting¹, SHAN Tijiang², XU Lijian¹()

¹College of Advanced Agriculture and Ecological Environment, Heilongjiang University, Harbin 150080
²College of Forestry and Landscape Architecture, South China Agricultural University/ Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Guangzhou 510642

Received:2021-08-19 Revised:2022-05-23 Online:2022-08-05 Published:2022-08-22
Contact: XU Lijian

摘要/Abstract

摘要：

为了分离鉴定森林凋落物中可培养的真菌,并发现具有抗菌活性的菌株,研究它们的主要抗菌代谢物,为进一步开发利用凋落物真菌资源提供备选菌株。本研究通过颗粒法分离森林凋落物中的可培养真菌,用内部转录间隔区(ITS)序列分析鉴定分离到的真菌,采用打孔药剂扩散法检测抗菌活性,利用柱层析法分离抗菌单体化合物,通过波谱学分析确定其化学结构。共分离得到63株真菌,它们隶属于36个属、48个分类单元,它们中有14株菌ITS相似性小于98.5%。通过抗菌活性筛选发现,11株真菌的乙酸乙酯提取物具有抗菌活性,其中Berkleasmium sp. SGSF622抗菌活性较强。通过PDA培养基对该株真菌进行发酵,获得乙酸乙酯提取物3 g。通过抗菌活性追踪分离,得到其含量最高的抗菌化合物622A。通过核磁共振及高分辨质谱分析,进一步确认该化合物为螺二萘类化合物Sch53825,它的分子式为C₂₀H₁₃ClO₅,其抗青枯劳尔氏菌的最低抑菌浓度为125~250 μg/mL。研究首次在Berkleasmium属真菌中分离到化合物Sch53825,并首次发现该化合物具有抗植物病原菌活性,这为进一步开发利用森林凋落物真菌资源提供了参考。

关键词: 凋落物真菌, Berkleasmium, 抗菌活性, 次生代谢, 化合物分离

Abstract:

The purpose of this study is to isolate and identify culturable fungi from forest litter, to find the strains with antimicrobial activity and to study their main antimicrobial metabolites, so as to provide alternative strains for further development and utilization of fungal resources in forest litter. This study used the particle separation method to isolate the culturable fungi in forest litter, internal transcribed spacer (ITS) sequence analyses to identify isolated fungi, well diffusion method to detect antimicrobial activity, column chromatography method to separate antimicrobial monomeric compounds, and spectrum analyses to determine their chemical structure of the monomeric compounds. A total of 63 fungal strains were isolated, belonging to 36 genera and 48 taxa, among which, 14 strains had less than 98.5% of ITS similarity. Ethyl acetate crude extracts of 11 strains had antimicrobial activity, and Berkleasmium sp. SGSF622 had strong antimicrobial activity. The fungus was fermented in PDA medium, and 3 g of ethyl acetate extract was obtained. By bio-guided separation of antimicrobial activity, antimicrobial compound 622A with the highest content was obtained. By NMR and HR-MS analyses, this compound was further identified as Sch53825 and the molecular formula was C₂₀H₁₃ClO₅. Its minimal inhibition concentration of antibacterial activity against Ralstonia solanacearum was 125-250 μg/mL. It is the first time to separate Sch53825 from fungi of genus Berkleasmium. The study can lay a foundation for further development and utilization of fungal resources from forest litter.

Key words: litter fungi, Berkleasmium, antimicrobial activity, secondary metabolism, compound separation

中图分类号:

S718.81

甄锦程, 穆玉婷, 司璐, 于洪佳, 都婷婷, 单体江, 徐利剑. 凋落物真菌Berkleasmium sp.及其螺二萘类化合物抗菌活性的研究[J]. 中国农学通报, 2022, 38(22): 115-120.

ZHEN Jincheng, MU Yuting, SI Lu, YU Hongjia, DU Tingting, SHAN Tijiang, XU Lijian. Studies on the Antimicrobial Activity of Litter Fungus Berkleasmium sp. and Its Spirobisnaphthalenes[J]. Chinese Agricultural Science Bulletin, 2022, 38(22): 115-120.

图/表 5

参考文献 27

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序号	菌株号	相似度/%	最相近菌株	最相近菌株登录号
1	SGSF602	96.75	Parapyrenochaeta acaciae	NR155674
2	SGSF607	93.39	Pleurophoma sp.	MN177616
3	SGSF608	93.09	Cephalosporium gramineum	MH858350
4	SGSF615	87.14	Pseudoarthrographis phlogis	MH327796
5	SGSF616	95.55	Cladophialophora sp.	MN328294
6	SGSF619	97.29	Leptosphaeria biglobosa	KM056324
7	SGSF620	96.60	Pyrenochaetopsis microspora	HM751085
8	SGSF622	90.67	Berkleasmium sp.	KF850381
9	SGSF624	93.54	Polyphilus sieberi	MG719688
10	SGSF638	97.14	Xylohypha curta	MH862169
11	SGSF639	92.97	Monocillium loricatum	NR156407
12	SGSF288	94.02	Pyrenochaetopsis microspora	HM751085
13	SGSF289	97.84	Oidiodendron eucalypti	MH327803
14	SGSF297	92.64	Verticillium nubilum	LT560383

序号	菌株号	相似度/%	最相近菌株	最相近菌株登录号
1	SGSF602	96.75	Parapyrenochaeta acaciae	NR155674
2	SGSF607	93.39	Pleurophoma sp.	MN177616
3	SGSF608	93.09	Cephalosporium gramineum	MH858350
4	SGSF615	87.14	Pseudoarthrographis phlogis	MH327796
5	SGSF616	95.55	Cladophialophora sp.	MN328294
6	SGSF619	97.29	Leptosphaeria biglobosa	KM056324
7	SGSF620	96.60	Pyrenochaetopsis microspora	HM751085
8	SGSF622	90.67	Berkleasmium sp.	KF850381
9	SGSF624	93.54	Polyphilus sieberi	MG719688
10	SGSF638	97.14	Xylohypha curta	MH862169
11	SGSF639	92.97	Monocillium loricatum	NR156407
12	SGSF288	94.02	Pyrenochaetopsis microspora	HM751085
13	SGSF289	97.84	Oidiodendron eucalypti	MH327803
14	SGSF297	92.64	Verticillium nubilum	LT560383

菌株号	串珠镰刀菌							立枯丝核菌						青枯劳尔氏菌
菌株号	PD发酵		Rice发酵		YES发酵		PDA发酵	PD发酵	Rice发酵	YES发酵		PDA发酵		PD发酵	Rice发酵		YES发酵		PDA发酵
SGSF602	-		-		-		-	-	-	-		-		-	++		++		+
SGSF607	-		-		-		-	-	-	-		-		+	-		+		++
SGSF608	-		-		-		-	-	-	-		-		-	-		+		++
SGSF616	-		-		-		-	-	-	-		-		-	-		+		+
SGSF619	-		-		-		-	-	-	-		+		-	-		++		-
SGSF620	-		++		-		-	-	++	-		-		-	++		-		-
SGSF622	-		-		-		+++	-	-	-		+++		+	+		+		+++
SGSF638		-		-		-	-	-	-		-	-	-			-		+		-
SGSF639		-		+		-	+	-	-		-	-	-			-		+		+
SGSF288		-		++		-	-	-	-		-	-	-			-		-		-
SGSF289		-		-		-	-	-	++		+	-	++			+		++		+

菌株号	串珠镰刀菌							立枯丝核菌						青枯劳尔氏菌
菌株号	PD发酵		Rice发酵		YES发酵		PDA发酵	PD发酵	Rice发酵	YES发酵		PDA发酵		PD发酵	Rice发酵		YES发酵		PDA发酵
SGSF602	-		-		-		-	-	-	-		-		-	++		++		+
SGSF607	-		-		-		-	-	-	-		-		+	-		+		++
SGSF608	-		-		-		-	-	-	-		-		-	-		+		++
SGSF616	-		-		-		-	-	-	-		-		-	-		+		+
SGSF619	-		-		-		-	-	-	-		+		-	-		++		-
SGSF620	-		++		-		-	-	++	-		-		-	++		-		-
SGSF622	-		-		-		+++	-	-	-		+++		+	+		+		+++
SGSF638		-		-		-	-	-	-		-	-	-			-		+		-
SGSF639		-		+		-	+	-	-		-	-	-			-		+		+
SGSF288		-		++		-	-	-	-		-	-	-			-		-		-
SGSF289		-		-		-	-	-	++		+	-	++			+		++		+

凋落物真菌Berkleasmium sp.及其螺二萘类化合物抗菌活性的研究

Studies on the Antimicrobial Activity of Litter Fungus Berkleasmium sp. and Its Spirobisnaphthalenes

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