黄瓜枯萎病的拮抗放线菌菌株的分离与鉴定

doi:10.16035/j.issn.1001-7283.2023.06.036

摘要/Abstract

摘要：

黄瓜枯萎病是严重影响黄瓜产量和品质的土传病害。从大棚土壤中分离出放线菌，对娄彻氏链霉菌（Streptomyces rochei）亲缘关系最近的G8菌株进行了黄瓜枯萎病菌的抑菌试验和大棚黄瓜枯萎病的防治试验。结果表明，从大棚黄瓜土壤中共分离出43个放线菌，其中31个属于链霉菌属；G8菌株可抑制黄瓜枯萎病菌丝生长，对大棚黄瓜枯萎病的防治效果达75.5%；基于16S rDNA和特异性引物PCR产物测序序列与NCBI-BLAST数据库比对，将G8菌株鉴定为娄彻氏链霉菌。

关键词: 黄瓜枯萎病, 放线菌, 拮抗菌, 娄彻氏链霉菌

Abstract:

Fusarium wilt of cucumber is a primarily soil-borne disease which lead to yield loss and quality decline in cucumber. Actinomycetes isolates were separated from cucumber-cultivated soil. G8 strain of which was closest relative to Streptomyces rochei. The cucumber Fusarium wilt of mycelia growth inhibition tests and biocontrol efficiency experiments in the greenhouse were employed using G8 strain of Streptomyces. The results showed that a total of 43 actinomycetes isolated from cucumber cultivated glasshouse soil, of which 31 isolates belonged to Streptomyces. The strain G8 inhibited Fusarium wilt of mycelial growth, and biocontrol effect on Fusarium wilt of cucumber was 75.5%. 16S rDNA primer and PCR amplification products using specific primer were aligned to NCBI-BLAST database clearly showed that G8 strain was a S.rochei.

Key words: Cucumber Fusarium wilt, Actinomycetes, Antagonistic strain, Streptomyces rochei

熊玉婷, 郑璐瑶, 贾雯淇, 李曼, 陈佳宁, 李葵花, 高玉亮. 黄瓜枯萎病的拮抗放线菌菌株的分离与鉴定[J]. 作物杂志, 2023 (6): 261–269

Xiong Yuting, Zheng Luyao, Jia Wenqi, Li Man, Chen Jianing, Li Kuihua, Gao Yuliang. Isolation and Identification of Antagonistic Actinomycetes Strains against Fusarium Wilt of Cucumber[J]. Crops, 2023(6): 261–269

图/表 9

图1

表1

43株菌株测序结果

序号 Number	菌株编号 Strain number	最相近种属 Most closely related species	登录号 Accession number	最大相似度 Maximum similarity (%)
1	A1	利迪链霉菌NBRC 110027^T Streptomyces lydicamycinicus NBRC 110027^T	BBNO01000020	100.00
2	A2	草地链霉菌ch24^T Streptomyces pratensis ch24^T	JQ806215	100.00
3	A3	西唐链霉菌NRRL ISP-5322^T Streptomyces setonii NRRL ISP-5322^T	MUNB01000146	100.00
4	A4	德米托链霉菌MS 405^T Streptomyces durmitorensis MS 405^T	DQ067287	99.78
5	A5	西唐链霉菌NRRL ISP-5322^T Streptomyces setonii NRRL ISP-5322^T	MUNB01000146	100.00
6	A6	草地链霉菌ch24^T Streptomyces pratensis ch24^T	JQ806215	100.00
7	A7	草地链霉菌ch24^T Streptomyces pratensis ch24^T	JQ806215	100.00
8	A8	利迪链霉菌NBRC 110027^T Streptomyces lydicamycinicus NBRC 110027^T	BBNO01000020	100.00
9	A9	利迪链霉菌NBRC 110027^T Streptomyces lydicamycinicus NBRC 110027^T	BBNO01000020	100.00
10	A10	西唐链霉菌NRRL ISP-5322^T Streptomyces setonii NRRL ISP-5322^T	MUNB01000146	100.00
11	A11	链霉菌NEAU-ML8^T Streptomyces kronopolitis NEAU-ML8^T	KP050495	99.92
12	A12	西唐链霉菌NRRL ISP-5322^T Streptomyces setonii NRRL ISP-5322^T	MUNB01000146	100.00
13	A13	珊瑚状放线菌IFO 14095^T Actinocorallia libanotica IFO 14095^T	U49007	99.84
14	A14	西唐链霉菌NRRL ISP-5322^T Streptomyces setonii NRRL ISP-5322^T	MUNB01000146	99.39
15	A15	草地链霉菌ch24^T Streptomyces pratensis ch24^T	JQ806215	100.00
16	A16	慢生根瘤菌属PAC68^T Bradyrhizobium jicamae PAC68^T	LLXZ01000092	99.76
17	A17	匈牙利链霉菌MBRL 251^T Streptomyces hundungensis MBRL 251^T	JN560157	98.94
18	A18	盐屋链霉菌NRRL B-5408^T Streptomyces sioyaensis NRRL B-5408^T	DQ026654	99.13
19	A19	盐屋链霉菌NRRL B-5408^T Streptomyces sioyaensis NRRL B-5408^T	DQ026654	99.85
20	A20	德米托链霉菌MS 405^T Streptomyces durmitorensis MS 405^T	DQ067287	99.76
21	A21	利迪链霉菌NBRC 110027^T Streptomyces lydicamycinicus NBRC 110027^T	BBNO01000020	100.00
22	A22	贪噬菌属BAM-48^T Variovorax boronicumulans BAM-48^T	AB300597	99.63
23	A23	利迪链霉菌NBRC 110027^T Streptomyces lydicamycinicus NBRC 110027^T	BBNO01000020	100.00
24	A24	匈牙利链霉菌MBRL 251^T Streptomyces hundungensis MBRL 251^T	JN560157	99.06
25	G1	假单胞菌属P50^T Pseudomonas piscium P50^T	LR797558	99.92
26	G2	好望角链霉菌NRRL B-24240^T Streptomyces speibonae NRRL B-24240^T	JNXM01000266	99.55
27	G3	亚洲假单胞菌RYU5^T Pseudomonas asiatica RYU5^T	MH517510	98.13
28	G4	无色杆菌属LMG 1866^T Achromobacter ruhlandii LMG 1866^T	CADIJL010000070	99.70
28	G5	剑菌属Casida A^T Ensifer adhaerens Casida A^T	JNAE01000171	100.00
30	G6	好望角链霉菌NRRL B-24240^T Streptomyces speibonaeNRRL B-24240^T	JNXM01000266	99.54
31	G7	米氏假单胞菌CIP 105470^T Pseudomonas migulae CIP 105470^T	AF074383	99.50
32	G8	娄彻氏链霉菌NRRL B-2410^T Streptomyces rochei NRRL B-2410^T	MUMD01000370	100.00
33	G9	白色链霉菌NRRL B-1305^T Streptomyces albogriseolus NRRL B-1305^T	AJ494865	100.00
34	G10	无色杆菌属LMG 1866^T Achromobacter ruhlandii LMG 1866^T	CADIJL010000070	99.78
35	G11	白色链霉菌NRRL B-1305^T Streptomyces albogriseolus NRRL B-1305^T	AJ494865	100.00
36	G12	白色链霉菌NRRL B-1305^T Streptomyces albogriseolus NRRL B-1305^T	AJ494865	100.00
37	G13	白色链霉菌NRRL B-1305^T Streptomyces albogriseolus NRRL B-1305^T	AJ494865	100.00
38	G14	白色链霉菌NRRL B-1305^T Streptomyces albogriseolus NRRL B-1305^T	AJ494865	100.00
39	G15	白色链霉菌NRRL B-1305^T Streptomyces albogriseolus NRRL B-1305^T	AJ494865	100.00
40	G16	白色链霉菌NRRL B-1305^T Streptomyces albogriseolus NRRL B-1305^T	AJ494865	100.00
41	G17	无色杆菌属LMG 1866^T Achromobacter ruhlandii LMG 1866^T	CADIJL010000070	99.78
42	G18	珊瑚状放线菌JCM 8201^T Actinocorallia aurantiaca JCM 8201^T	AF134066	99.22
43	G20	剑菌属Casida A^T Ensifer adhaerens Casida A^T	JNAE01000171	100.00

表1

图2

图3

表2

图4

图5

表3

特异性引物PCR所得序列比对结果

类型 Description	学名 Scientific name	最高分 Max score	总分 Total score	匹配度 Query cover (%)	期望值 E-value	匹配占比 Percent identity (%)	序列长度 Accession length (bp)	编号 Accession
链霉菌E1N263 16S核糖体RNA基因，部分序列 Streptomyces sp. E1N263 16S ribosomal RNA gene, partial sequence	链霉菌E1N263 Streptomyces sp. E1N263	281	281	95	3e-71	97.59	1418	KX279607.1
娄彻氏链霉菌ADLK1 16S核糖体RNA基因，部分序列 Streptomyces rochei strain ADLK1 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	281	281	97	3e-71	97.04	1502	KT781124.1
链霉菌Zapt6 16S核糖体RNA基因，部分序列 Streptomyces sp. Zapt6 16S ribosomal RNA gene, partial sequence	链霉菌Zapt6 Streptomyces sp. Zapt6	281	281	95	3e-71	97.59	829	DQ164424.1
紫色链霉菌AC02 16S核糖体RNA基因，部分序列 Streptomyces violaceus strain AC02 16S ribosomal RNA gene, partial sequence	紫色链霉菌 Streptomyces violaceus	279	279	99	8e-71	96.00	799	OQ559569.1
安氏链霉菌PA06 16S核糖体RNA基因，部分序列 Streptomyces ambofaciens strain PA06 16S ribosomal RNA gene, partial sequence	安氏链霉菌 Streptomyces ambofaciens	279	279	99	8e-71	96.00	919	OQ559107.1
娄彻氏链霉菌AF03 16S核糖体RNA基因，部分序列 Streptomyces rochei strain AF03 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	279	279	99	8e-71	96.00	930	OQ558840.1
深海链霉菌CAP12 16S核糖体RNA基因，部分序列 Streptomyces koyangensis strain CAP12 16S ribosomal RNA gene, partial sequence	深海链霉菌 Streptomyces koyangensis	279	279	99	8e-71	96.00	1463	OQ557958.1
紫色链霉菌EC01 16S核糖体RNA基因，部分序列 Streptomyces violaceus strain EC01 16S ribosomal RNA gene, partial sequence	紫色链霉菌 Streptomyces violaceus	279	279	99	8e-71	96.00	950	OQ557514.1
海洋放线菌ODS10 16S核糖体RNA基因，部分序列 Streptomyces lusitanus strain ODS10 16S ribosomal RNA gene, partial sequence	海洋放线菌 Streptomyces lusitanus	279	279	99	8e-71	96.00	938	OQ557452.1
链霉菌A6Y6 16S核糖体RNA基因，部分序列 Streptomyces sp. strain A6Y6 16S ribosomal RNA gene, partial sequence	链霉菌 Streptomyces sp.	279	279	99	8e-71	96.00	1490	OQ547297.1
云南链霉菌P86染色体，全基因组 Streptomyces yunnanensis strain P86 chromosome, complete genome	云南链霉菌 Streptomyces yunnanensis	279	1959	94	8e-71	97.58	9 982 274	CP095749.1
娄彻氏链霉菌AC143 16S核糖体RNA基因，部分序列 Streptomyces rochei strain AC143 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	279	279	99	8e-71	96.00	1418	OQ519886.1
娄彻氏链霉菌Z331-A 16S核糖体RNA基因，部分序列 Streptomyces rochei strain Z331-A 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	279	279	99	8e-71	96.00	1169	OQ509989.1
放线菌X93-1 16S核糖体RNA基因，部分序列 Actinomycetia bacterium strain X93-1 16S ribosomal RNA gene, partial sequence	放线菌 Actinomycetia bacterium	279	279	99	8e-71	96.00	1399	OQ509821.1
放线菌378 16S核糖体RNA基因，部分序列 Actinomycetia bacterium strain 378 16S ribosomal RNA gene, partial sequence	放线菌 Actinomycetia bacterium	279	279	99	8e-71	96.00	1515	OQ509818.1
链霉菌AKT34 16S核糖体RNA基因，部分序列 Streptomyces sp. strain AKT34 16S ribosomal RNA gene, partial sequence	链霉菌 Streptomyces sp.	279	279	99	8e-71	96.00	1381	OQ506606.1

表3

表4

16S rDNA通用引物PCR所得序列比对结果

类型 Description	学名 Scientific name	最高分 Max score	总分 Total score	匹配度 Query cover (%)	匹配占比 Percent identity (%)	序列长度 Accession length (bp)	编号 Accession
娄彻氏链霉菌SCSIOZ-SH08 16S核糖体RNA基因，部分序列 Streptomyces rochei strain SCSIOZ-SH08 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2649	2649	99	99.86	1459	KC747476.1
娄彻氏链霉菌SCSIOZ-SH07 16S核糖体RNA基因，部分序列 Streptomyces rochei strain SCSIOZ-SH07 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2649	2649	99	99.86	1461	KC747475.1
娄彻氏链霉菌SCSIOZ-SH06 16S核糖体RNA基因，部分序列 Streptomyces rochei strain SCSIOZ-SH06 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2645	2645	99	99.72	1461	KC747474.1
娄彻氏链霉菌LN204 16S核糖体RNA基因，部分序列 Streptomyces rochei strain LN204 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2643	2643	99	99.72	1459	MH265958.1
娄彻氏链霉菌CB6J5 16S核糖体RNA基因，部分序列 Streptomyces rochei strain CB6J5 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2643	2643	99	99.72	1458	KJ531619.1
娄彻氏链霉菌SCSIOZ-SH12 16S核糖体RNA基因，部分序列 Streptomyces rochei strain SCSIOZ-SH12 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2643	2643	99	99.72	1457	KC747480.1
娄彻氏链霉菌SCSIOZ-SH09 16S核糖体RNA基因，部分序列 Streptomyces rochei strain SCSIOZ-SH09 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2643	2643	99	99.65	1448	KC747477.1
娄彻氏链霉菌SCSIOZ-SH05 16S核糖体RNA基因，部分序列 Streptomyces rochei strain SCSIOZ-SH05 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2643	2643	99	99.65	1455	KC747473.1
娄彻氏链霉菌xsd08098 16S核糖体RNA基因，部分序列 Streptomyces rochei strain xsd08098 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2643	2643	99	99.72	1461	FJ481055.1
娄彻氏链霉菌SCSIOZ-SH13 16S核糖体RNA基因，部分序列 Streptomyces rochei strain SCSIOZ-SH13 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2641	2641	99	99.65	1456	KC747481.1
链霉菌YDG17 16S核糖体RNA基因，部分序列 Streptomyces sp. YDG17 16S ribosomal RNA gene, partial sequence	链霉菌YDG17 Streptomyces sp. YDG17	2641	2641	99	99.79	1467	U621883.1
娄彻氏链霉菌UAE1-3 16S核糖体RNA基因，部分序列 Streptomyces rochei strain UAE1-3 16S ribosomal RNA gene, partial sequence	娄彻氏链霉菌 Streptomyces rochei	2639	2639	99	99.52	1519	MN795133.1
链霉菌B2KY 16S核糖体RNA基因，部分序列 Streptomyces sp. strain B2KY 16S ribosomal RNA gene, partial sequence	链霉菌 Streptomyces sp.	2639	2639	99	99.52	1484	MN625924.1
链霉菌RBST1-4 16S核糖体RNA基因，部分序列 Streptomyces sp. RBST1-4 gene for 16S ribosomal RNA, partial sequence	链霉菌 Streptomyces sp.	2639	2639	99	99.52	1519	LC489245.1
链霉菌SGAir0924染色体，全基因组 Streptomyces sp. SGAir0924 chromosome, complete genome	链霉菌SGAir0924 Streptomyces sp. SGAir0924	2639	15 834	99	99.65	7 653 753	CP027297.1
链霉菌SS52染色体，全基因组 Streptomyces sp. SS52 chromosome, complete genome	链霉菌SS52 Streptomyces sp. SS52	2639	15 834	99	99.65	8 184 045	CP039123.1

表4

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处理 Treatment	发病率 Incidentce rate (%)	防治效果 Control efficiency (%)
多菌灵药剂Carbendazim	76.5±2.9b	14.5±0.8
G8	21.9±2.7c	75.5±2.3^**
对照Control	89.4±2.9a