南太湖水体抑藻菌的筛选分离及其活性成分鉴定

doi:10.3969/j.issn.1004-1524.20230541

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (5): 1124-1133.DOI: 10.3969/j.issn.1004-1524.20230541

南太湖水体抑藻菌的筛选分离及其活性成分鉴定

陈瑶瑶(), 刘雪萍, 张绍勇, 王继栋, 王瑞俊, 张立钦^*()

湖州师范学院生命科学学院,浙江省媒介生物学与病原控制重点实验室,浙江湖州 313000

收稿日期:2023-04-26 出版日期:2024-05-25 发布日期:2024-05-29
作者简介:陈瑶瑶(1997—),女,山西太原人,硕士,研究方向为水产医学。E-mail:969816947@qq.com
通讯作者: * 张立钦,E-mail:zlqwz@zjhu.edu.cn
基金资助:
浙江省重点研发计划(2019C02030);湖州市自然科学基金(2020YZ06)

Isolation of strains with algicidal activity from South Taihu Lake in China and identification of active compounds

CHEN Yaoyao(), LIU Xueping, ZHANG Shaoyong, WANG Jidong, WANG Ruijun, ZHANG Liqin^*()

Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, College of Life Sciences, Huzhou University, Huzhou 313000, Zhejiang, China

Received:2023-04-26 Online:2024-05-25 Published:2024-05-29

摘要/Abstract

摘要：

采用稀释涂布法、改良分离培养基和16S rRNA基因序列对南太湖四季水体的可培养细菌进行分离、纯化与鉴定,筛选可抑制铜绿微囊藻(Microcystis aeruginosa)生长的活性细菌,并追踪其活性成分。结果表明:共分离得到420株可培养细菌,隶属于4门9纲20目35科66属192种,其中,黄杆菌属(Flavobacterium)、假单胞菌属(Pseudomonas)、短波单胞菌属(Brevundimonas)、芽孢杆菌属(Bacillus)的菌株数较多。16S rRNA基因序列结果显示,有20株细菌属于潜在新种。共分离到7株具有明显抑藻活性的细菌,其中,黄杆菌属菌株1-2-14的抑藻效果最佳,第7天时抑制率达71.52%。通过活性追踪,分离得到2个活性单体化合物cyclo(4-hydroxy-L-Pro-L-Leu)和pterodontriol B,其48 h抑藻活性分别为56.87%和67.81%。

关键词: 南太湖, 可培养细菌, 铜绿微囊藻(Microcystis aeruginosa), 抑藻活性

Abstract:

In the present study, culturable bacteria were isolated and purified from water samples of south Taihu Lake in four seasons by the dilution-plate method and modified isolation medium. These bacteria were identified using 16S rRNA gene sequence. Bacteria with algicidal activity against Microcystis aeruginosa were screened, and their active compounds were isolated and purified. It was shown that a total of 420 strains were isolated from water samples of south Taihu Lake in four seasons. They were classified into 192 species, 66 genera, 35 families, 20 orders, 9 classes and 4 phyla. Based on the isolated strain number, Flavobacterium, Pseudomonas, Brevundimonas and Bacillus were the majority at the genus level. There were 20 strains identified as potential new taxa by 16S rRNA gene sequence analysis. Seven strains showed obvious inhibitory activity against M. aeruginosa, of which Flavobacterium sp. 1-2-14 exhibited the strongest algicidal activity, as its inhibition rate reached 71.52% after co-cultured for 7 days. By adopting the tracing method of bioactivity, two compounds, cyclo (4-hydroxy-L-Pro-L-Leu) and pterodontriol B were isolated, and their inhibition rates against M. aeruginosa were 56.87%and 67.81% at 48 h, respectively.

Key words: south Taihu Lake, culturable bacteria, Microcystis aeruginosa, algicidal activity

中图分类号:

Q939.9
X524

陈瑶瑶, 刘雪萍, 张绍勇, 王继栋, 王瑞俊, 张立钦. 南太湖水体抑藻菌的筛选分离及其活性成分鉴定[J]. 浙江农业学报, 2024, 36(5): 1124-1133.

CHEN Yaoyao, LIU Xueping, ZHANG Shaoyong, WANG Jidong, WANG Ruijun, ZHANG Liqin. Isolation of strains with algicidal activity from South Taihu Lake in China and identification of active compounds[J]. Acta Agriculturae Zhejiangensis, 2024, 36(5): 1124-1133.

图/表 6

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门 Phylum	纲 Class	目 Order	科 Family	属 Genus	种数量 Species number	菌株数量 Strain number
变形菌门 Pseudomonadota	β-变形菌纲 Betaproteobacteria	伯克氏菌目 Burkholderiales	产碱菌科 Alcaligenaceae	产碱菌属Alcaligenes	1	10
			丛毛单胞菌科 Comamonadaceae	氢噬胞菌属Hydrogenophaga Simplicispira	3 1	5 2
				食酸菌属Acidovorax	3	4
				Curvibacter	1	2
				Aquincola	1	1
				Inhella	1	1
				大单胞菌属Macromonas	1	1
				Kinneretia	1	1
				Ramlibacter	1	1
			草酸杆菌科	紫色杆菌属Janthinobacterium	5	6
			Oxalobacteraceae	马赛菌属Massilia	1	2
				Undibacterium	1	2
			伯克氏菌科 Burkholderiaceae	Ralstonia	2	9
		奈瑟菌目	奈瑟氏菌科	Aquella	1	1
		Neisseriales	Neisseriaceae	福格斯氏菌属Vogesella	1	1
	α-变形菌纲	鞘脂单胞菌目	Sphingosinicellaceae	Sandarakinorhabdus	1	2
	Alphaproteobacteria	Sphingomonadales	赤杆菌科 Erythrobacteraceae	赤杆菌属Erythrobacter	4	16
			鞘脂单胞菌科	新鞘脂属Novosphingobium	7	10
			Sphingomonadaceae	鞘氨醇单胞菌属Sphingomonas	4	7
				鞘脂单胞菌属Sphingopyxis	4	6
				Sphingorhabdus	5	16
		柄杆菌目	柄杆菌科	短波单胞菌属Brevundimonas	10	21
		Caulobacterales	Caulobacteraceae	柄杆菌属Caulobacter	4	11
				PAC001798_g	1	1
				苯基杆菌属Phenylobacterium	1	4
		根瘤菌目	Boseaceae	包西氏菌属Bosea	2	3
		Rhizobiales	甲基杆菌科 Methylobacteriaceae	甲基杆菌属Methylobacterium	2	2
			硝化杆菌科 Nitrobacteraceae	阿菲波菌属Afipia	1	1
			叶杆菌科 Phyllobacteriaceae	中慢生根瘤菌属Mesorhizobium	1	1
			根瘤菌科 Rhizobiaceae	根瘤菌属Rhizobium	2	6
			黄色杆菌科 Xanthobacteraceae	黄色杆菌属Xanthobacter	1	1
		红杆菌目 Rhodobacterales	红杆菌科 Rhodobacteraceae	芽殖杆菌属 Gemmobacter	3	4
		红螺菌目	Azospirillaceae	Elstera	1	1
		Rhodospirillales		Niveispirillum	1	1
	γ-变形菌纲 Gammaproteobacteria	气单胞菌目 Aeromonadales	气单胞菌科 Aeromonadaceae	气单胞菌属Aeromonas	1	1
		着色菌目 Chromatiales	着色菌科 Chromatiaceae	Pararheinheimera	3	7
		溶杆菌目	Lysobacteraceae	溶杆菌属Lysobacter	2	5
		Lysobacterales		Pseudoxanthomonas	2	7
				Silanimonas	1	1
				寡养单胞菌属Stenotrophomonas	1	2
		假单胞菌目 Pseudomonadales	莫拉菌科 Moraxellaceae	不动杆菌属Acinetobacter	1	1
			假单胞菌科 Pseudomonadaceae	假单胞菌属Pseudomonas	24	56
拟杆菌门 Bacteroidota	噬几丁质菌纲 Chitinophagia	噬几丁质菌目 Chitinophagales	噬几丁质菌科 Chitinophagaceae	Lacibacter	1	1
	黄杆菌纲 Flavobacteriia	黄杆菌目 Flavobacteriales	黄杆菌科 Flavobacteriaceae	黄杆菌属Flavobacterium	21	77
			威克斯氏菌科	Cloacibacterium	1	2
			Weeksellaceae	伊丽莎白菌属Elizabethkingia	1	1
	鞘脂杆菌纲 Sphingobacteriia	鞘氨醇杆菌目 Sphingobacteriales	鞘脂杆菌科 Sphingobacteriaceae	土地杆菌属Pedobacter	1	2
芽孢杆菌门	芽孢杆菌纲	核衣细菌目	芽孢杆菌科	芽孢杆菌属Bacillus	12	29
Bacillota	Bacilli	Caryophanales	Bacillaceae	Falsibacillus	1	1
				Fictibacillus	3	9
				Niallia	2	2
				Priestia	2	5
				Rossellomorea	3	4
				微小杆菌属Exiguobacterium	1	2
			Caryophanaceae	Lysinibacillus	1	2
				Metasolibacillus	2	4
				Rummeliibacillus	1	1
			类芽孢杆菌科 Paenibacillaceae	短杆菌属Brevibacillus	1	1
	梭菌纲 Clostridia	Eubacteriales	毛螺菌科 Lachnospiraceae	毛螺菌属Lachnospira	1	1
放线菌门	放线菌纲	微球菌目	Dermabacteraceae	Brachybacterium	1	1
Actinomycetota	Actinomycetes	Micrococcales	微杆菌科	Agrococcus	1	1
			Microbacteriaceae	微杆菌属Microbacterium	8	9
		Micromonosporales	小单孢菌科 Micromonosporaceae	小单孢菌属Micromonospora	2	5
		分枝杆菌目 Mycobacteriales	Mycobacteriaceae 链霉菌科	分枝杆菌属Mycolicibacterium 链霉菌属Streptomyces	6 4	13 4
		Kitasatosporales	Streptomycetaceae

门 Phylum	纲 Class	目 Order	科 Family	属 Genus	种数量 Species number	菌株数量 Strain number
变形菌门 Pseudomonadota	β-变形菌纲 Betaproteobacteria	伯克氏菌目 Burkholderiales	产碱菌科 Alcaligenaceae	产碱菌属Alcaligenes	1	10
			丛毛单胞菌科 Comamonadaceae	氢噬胞菌属Hydrogenophaga Simplicispira	3 1	5 2
				食酸菌属Acidovorax	3	4
				Curvibacter	1	2
				Aquincola	1	1
				Inhella	1	1
				大单胞菌属Macromonas	1	1
				Kinneretia	1	1
				Ramlibacter	1	1
			草酸杆菌科	紫色杆菌属Janthinobacterium	5	6
			Oxalobacteraceae	马赛菌属Massilia	1	2
				Undibacterium	1	2
			伯克氏菌科 Burkholderiaceae	Ralstonia	2	9
		奈瑟菌目	奈瑟氏菌科	Aquella	1	1
		Neisseriales	Neisseriaceae	福格斯氏菌属Vogesella	1	1
	α-变形菌纲	鞘脂单胞菌目	Sphingosinicellaceae	Sandarakinorhabdus	1	2
	Alphaproteobacteria	Sphingomonadales	赤杆菌科 Erythrobacteraceae	赤杆菌属Erythrobacter	4	16
			鞘脂单胞菌科	新鞘脂属Novosphingobium	7	10
			Sphingomonadaceae	鞘氨醇单胞菌属Sphingomonas	4	7
				鞘脂单胞菌属Sphingopyxis	4	6
				Sphingorhabdus	5	16
		柄杆菌目	柄杆菌科	短波单胞菌属Brevundimonas	10	21
		Caulobacterales	Caulobacteraceae	柄杆菌属Caulobacter	4	11
				PAC001798_g	1	1
				苯基杆菌属Phenylobacterium	1	4
		根瘤菌目	Boseaceae	包西氏菌属Bosea	2	3
		Rhizobiales	甲基杆菌科 Methylobacteriaceae	甲基杆菌属Methylobacterium	2	2
			硝化杆菌科 Nitrobacteraceae	阿菲波菌属Afipia	1	1
			叶杆菌科 Phyllobacteriaceae	中慢生根瘤菌属Mesorhizobium	1	1
			根瘤菌科 Rhizobiaceae	根瘤菌属Rhizobium	2	6
			黄色杆菌科 Xanthobacteraceae	黄色杆菌属Xanthobacter	1	1
		红杆菌目 Rhodobacterales	红杆菌科 Rhodobacteraceae	芽殖杆菌属 Gemmobacter	3	4
		红螺菌目	Azospirillaceae	Elstera	1	1
		Rhodospirillales		Niveispirillum	1	1
	γ-变形菌纲 Gammaproteobacteria	气单胞菌目 Aeromonadales	气单胞菌科 Aeromonadaceae	气单胞菌属Aeromonas	1	1
		着色菌目 Chromatiales	着色菌科 Chromatiaceae	Pararheinheimera	3	7
		溶杆菌目	Lysobacteraceae	溶杆菌属Lysobacter	2	5
		Lysobacterales		Pseudoxanthomonas	2	7
				Silanimonas	1	1
				寡养单胞菌属Stenotrophomonas	1	2
		假单胞菌目 Pseudomonadales	莫拉菌科 Moraxellaceae	不动杆菌属Acinetobacter	1	1
			假单胞菌科 Pseudomonadaceae	假单胞菌属Pseudomonas	24	56
拟杆菌门 Bacteroidota	噬几丁质菌纲 Chitinophagia	噬几丁质菌目 Chitinophagales	噬几丁质菌科 Chitinophagaceae	Lacibacter	1	1
	黄杆菌纲 Flavobacteriia	黄杆菌目 Flavobacteriales	黄杆菌科 Flavobacteriaceae	黄杆菌属Flavobacterium	21	77
			威克斯氏菌科	Cloacibacterium	1	2
			Weeksellaceae	伊丽莎白菌属Elizabethkingia	1	1
	鞘脂杆菌纲 Sphingobacteriia	鞘氨醇杆菌目 Sphingobacteriales	鞘脂杆菌科 Sphingobacteriaceae	土地杆菌属Pedobacter	1	2
芽孢杆菌门	芽孢杆菌纲	核衣细菌目	芽孢杆菌科	芽孢杆菌属Bacillus	12	29
Bacillota	Bacilli	Caryophanales	Bacillaceae	Falsibacillus	1	1
				Fictibacillus	3	9
				Niallia	2	2
				Priestia	2	5
				Rossellomorea	3	4
				微小杆菌属Exiguobacterium	1	2
			Caryophanaceae	Lysinibacillus	1	2
				Metasolibacillus	2	4
				Rummeliibacillus	1	1
			类芽孢杆菌科 Paenibacillaceae	短杆菌属Brevibacillus	1	1
	梭菌纲 Clostridia	Eubacteriales	毛螺菌科 Lachnospiraceae	毛螺菌属Lachnospira	1	1
放线菌门	放线菌纲	微球菌目	Dermabacteraceae	Brachybacterium	1	1
Actinomycetota	Actinomycetes	Micrococcales	微杆菌科	Agrococcus	1	1
			Microbacteriaceae	微杆菌属Microbacterium	8	9
		Micromonosporales	小单孢菌科 Micromonosporaceae	小单孢菌属Micromonospora	2	5
		分枝杆菌目 Mycobacteriales	Mycobacteriaceae 链霉菌科	分枝杆菌属Mycolicibacterium 链霉菌属Streptomyces	6 4	13 4
		Kitasatosporales	Streptomycetaceae

菌株编码 Strain No.	最相似菌株的种属名(登录号) The closest typical strain (Accession No.)	序列相似度 Sequence similarity/%
1-2-14	Flavobacterium cupreum CCM 8825^T(MH100899)	98.20
1-1-6	Flavobacterium ureilyticum S-42^T(KY117464)	97.18
1-2-20	Simplicispira lacusdiani CPCC 100842^T(MG708102)	98.55
1-2-29	Rheinheimera chironomi K19414^T(DQ298025)	98.46
2-1-14	Novosphingobium taihuense T3-B9^T(AY500142)	97.93
2-1-18	Flavobacterium buctense T7^T(KP744923)	97.90
3-2-16	Flavobacterium phycosphaerae MK012^T(MN559947)	97.64
4-1-13	Flavobacterium dankookense DSM 25687^T(jgi.1108032)	97.63
4-2-2	Flavobacterium granuli DSM 17797^T(jgi.1107693)	98.54
4-2-7	Flavobacterium terrigena DSM 17934^T(FNYA01000017)	97.63
4-2-9	Flavobacterium saccharophilum DSM 1811^T(jgi.1107675)	97.63
5-2-2	Flavobacterium silvisoli RD-2-33^T(SJPE01000025)	98.15
D-12	Flavobacterium cheonhonense ARSA-15^T(GU295972)	97.32
E-18	Caulobacter soli Ji-3-8^T(MT026974)	94.19
T1-10	Flavobacterium cheniae NJ-26^T(EF407880)	97.65
T2-8	Rheinheimera aquatica GR5^T(GQ168584)	98.40
T2-10	Flavobacterium filum EMB34^T(DQ372981)	97.87
T5-7	Flavobacterium tiangeerense 563^T(EU036219)	96.55
H1-31	Ralstonia mannitolilytica LMG 6866^T(AJ270258)	98.38
H5-31	Aquincola tertiaricarbonis L10^T(DQ656489)	98.55

菌株编码 Strain No.	最相似菌株的种属名(登录号) The closest typical strain (Accession No.)	序列相似度 Sequence similarity/%
1-2-14	Flavobacterium cupreum CCM 8825^T(MH100899)	98.20
1-1-6	Flavobacterium ureilyticum S-42^T(KY117464)	97.18
1-2-20	Simplicispira lacusdiani CPCC 100842^T(MG708102)	98.55
1-2-29	Rheinheimera chironomi K19414^T(DQ298025)	98.46
2-1-14	Novosphingobium taihuense T3-B9^T(AY500142)	97.93
2-1-18	Flavobacterium buctense T7^T(KP744923)	97.90
3-2-16	Flavobacterium phycosphaerae MK012^T(MN559947)	97.64
4-1-13	Flavobacterium dankookense DSM 25687^T(jgi.1108032)	97.63
4-2-2	Flavobacterium granuli DSM 17797^T(jgi.1107693)	98.54
4-2-7	Flavobacterium terrigena DSM 17934^T(FNYA01000017)	97.63
4-2-9	Flavobacterium saccharophilum DSM 1811^T(jgi.1107675)	97.63
5-2-2	Flavobacterium silvisoli RD-2-33^T(SJPE01000025)	98.15
D-12	Flavobacterium cheonhonense ARSA-15^T(GU295972)	97.32
E-18	Caulobacter soli Ji-3-8^T(MT026974)	94.19
T1-10	Flavobacterium cheniae NJ-26^T(EF407880)	97.65
T2-8	Rheinheimera aquatica GR5^T(GQ168584)	98.40
T2-10	Flavobacterium filum EMB34^T(DQ372981)	97.87
T5-7	Flavobacterium tiangeerense 563^T(EU036219)	96.55
H1-31	Ralstonia mannitolilytica LMG 6866^T(AJ270258)	98.38
H5-31	Aquincola tertiaricarbonis L10^T(DQ656489)	98.55

菌株编码 Strain No.	相似菌株种属名(登录号) The closest typical strain (Accession No.)	序列相似度 Sequence similarity/%	抑制率 Inhibition rate/%
C-1	Priestia aryabhattai B8W22^T(EF114313)	100.00	42.75±1.74
2-1-3	Flavobacterium granuli DSM 17797^T(jgi.1107693)	99.40	45.01±2.70
H4-20	Sphingomonas leidyi ATCC 15260^T(AJ227812)	99.88	47.80±1.41
H1-13	Inhella inkyongensis IMCC1713^T(DQ664238)	99.43	58.75±2.55
T3-11	Brevundimonas terrae KSL-145^T(DQ335215)	99.33	64.37±6.43
A-1	Fictibacillus barbaricus V2-BIII-A2^T(AJ422145)	100.00	65.89±0.85
1-2-14	Flavobacterium cupreum CCM 8825^T(MH100899)	98.20	71.52±1.26

南太湖水体抑藻菌的筛选分离及其活性成分鉴定

Isolation of strains with algicidal activity from South Taihu Lake in China and identification of active compounds

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化合物 Compound	不同浓度(mg·L^-1)处理48 h的抑藻率The inhibition rates under different concentration(mg·L^-1) treatment for 48 h
化合物 Compound	0.5	1	2
1	20.34±2.13	40.27±2.99	56.87±1.67
2	24.11±2.07	47.65±3.89	67.81±3.35

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