作物杂志,2023, 第6期: 261–269 doi: 10.16035/j.issn.1001-7283.2023.06.036

• 植物保护 • 上一篇    

黄瓜枯萎病的拮抗放线菌菌株的分离与鉴定

熊玉婷1(), 郑璐瑶1(), 贾雯淇1, 李曼1, 陈佳宁1, 李葵花1(), 高玉亮2()   

  1. 1延边大学农学院,133002,吉林延吉
    2延边朝鲜族自治州农业科学研究院,133400,吉林龙井
  • 收稿日期:2023-05-08 修回日期:2023-06-05 出版日期:2023-12-15 发布日期:2023-12-15
  • 通讯作者: 高玉亮,研究方向为蔬菜病害防治,E-mail:xrk1211@163.com;李葵花为共同通信作者,研究方向为蔬菜栽培,E-mail:khli@ybul.edu.cn
  • 作者简介:熊玉婷,研究方向为蔬菜栽培,E-mail:2424696009@qq.com
  • 基金资助:
    吉林省科技厅项目(20210202114NC);国家自然地区科学基金(32160702)

Isolation and Identification of Antagonistic Actinomycetes Strains against Fusarium Wilt of Cucumber

Xiong Yuting1(), Zheng Luyao1(), Jia Wenqi1, Li Man1, Chen Jianing1, Li Kuihua1(), Gao Yuliang2()   

  1. 1College of Agricultural, Yanbian University, Yanji 133002, Jilin, China
    2Yanbian Academy of Agricultural Sciences, Longjing 133400, Jilin, China
  • Received:2023-05-08 Revised:2023-06-05 Online:2023-12-15 Published:2023-12-15

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

图1

部分菌株的菌落形态 (a) G8菌株,(b) 无色杆菌属,(c) 贪噬菌属,(d) 慢生根瘤菌属,(e)~(i) 链霉菌属

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 110027T Streptomyces lydicamycinicus NBRC 110027T BBNO01000020 100.00
2 A2 草地链霉菌ch24T Streptomyces pratensis ch24T JQ806215 100.00
3 A3 西唐链霉菌NRRL ISP-5322T Streptomyces setonii NRRL ISP-5322T MUNB01000146 100.00
4 A4 德米托链霉菌MS 405T Streptomyces durmitorensis MS 405T DQ067287 99.78
5 A5 西唐链霉菌NRRL ISP-5322T Streptomyces setonii NRRL ISP-5322T MUNB01000146 100.00
6 A6 草地链霉菌ch24T Streptomyces pratensis ch24T JQ806215 100.00
7 A7 草地链霉菌ch24T Streptomyces pratensis ch24T JQ806215 100.00
8 A8 利迪链霉菌NBRC 110027T Streptomyces lydicamycinicus NBRC 110027T BBNO01000020 100.00
9 A9 利迪链霉菌NBRC 110027T Streptomyces lydicamycinicus NBRC 110027T BBNO01000020 100.00
10 A10 西唐链霉菌NRRL ISP-5322T Streptomyces setonii NRRL ISP-5322T MUNB01000146 100.00
11 A11 链霉菌NEAU-ML8T Streptomyces kronopolitis NEAU-ML8T KP050495 99.92
12 A12 西唐链霉菌NRRL ISP-5322T Streptomyces setonii NRRL ISP-5322T MUNB01000146 100.00
13 A13 珊瑚状放线菌IFO 14095T Actinocorallia libanotica IFO 14095T U49007 99.84
14 A14 西唐链霉菌NRRL ISP-5322T Streptomyces setonii NRRL ISP-5322T MUNB01000146 99.39
15 A15 草地链霉菌ch24T Streptomyces pratensis ch24T JQ806215 100.00
16 A16 慢生根瘤菌属PAC68T Bradyrhizobium jicamae PAC68T LLXZ01000092 99.76
17 A17 匈牙利链霉菌MBRL 251T Streptomyces hundungensis MBRL 251T JN560157 98.94
18 A18 盐屋链霉菌NRRL B-5408T Streptomyces sioyaensis NRRL B-5408T DQ026654 99.13
19 A19 盐屋链霉菌NRRL B-5408T Streptomyces sioyaensis NRRL B-5408T DQ026654 99.85
20 A20 德米托链霉菌MS 405T Streptomyces durmitorensis MS 405T DQ067287 99.76
21 A21 利迪链霉菌NBRC 110027T Streptomyces lydicamycinicus NBRC 110027T BBNO01000020 100.00
22 A22 贪噬菌属BAM-48T Variovorax boronicumulans BAM-48T AB300597 99.63
23 A23 利迪链霉菌NBRC 110027T Streptomyces lydicamycinicus NBRC 110027T BBNO01000020 100.00
24 A24 匈牙利链霉菌MBRL 251T Streptomyces hundungensis MBRL 251T JN560157 99.06
25 G1 假单胞菌属P50T Pseudomonas piscium P50T LR797558 99.92
26 G2 好望角链霉菌NRRL B-24240T Streptomyces speibonae NRRL B-24240T JNXM01000266 99.55
27 G3 亚洲假单胞菌RYU5T Pseudomonas asiatica RYU5T MH517510 98.13
28 G4 无色杆菌属LMG 1866T Achromobacter ruhlandii LMG 1866T CADIJL010000070 99.70
28 G5 剑菌属Casida AT Ensifer adhaerens Casida AT JNAE01000171 100.00
30 G6 好望角链霉菌NRRL B-24240T Streptomyces speibonaeNRRL B-24240T JNXM01000266 99.54
31 G7 米氏假单胞菌CIP 105470T Pseudomonas migulae CIP 105470T AF074383 99.50
32 G8 娄彻氏链霉菌NRRL B-2410T Streptomyces rochei NRRL B-2410T MUMD01000370 100.00
33 G9 白色链霉菌NRRL B-1305T Streptomyces albogriseolus NRRL B-1305T AJ494865 100.00
34 G10 无色杆菌属LMG 1866T Achromobacter ruhlandii LMG 1866T CADIJL010000070 99.78
35 G11 白色链霉菌NRRL B-1305T Streptomyces albogriseolus NRRL B-1305T AJ494865 100.00
36 G12 白色链霉菌NRRL B-1305T Streptomyces albogriseolus NRRL B-1305T AJ494865 100.00
37 G13 白色链霉菌NRRL B-1305T Streptomyces albogriseolus NRRL B-1305T AJ494865 100.00
38 G14 白色链霉菌NRRL B-1305T Streptomyces albogriseolus NRRL B-1305T AJ494865 100.00
39 G15 白色链霉菌NRRL B-1305T Streptomyces albogriseolus NRRL B-1305T AJ494865 100.00
40 G16 白色链霉菌NRRL B-1305T Streptomyces albogriseolus NRRL B-1305T AJ494865 100.00
41 G17 无色杆菌属LMG 1866T Achromobacter ruhlandii LMG 1866T CADIJL010000070 99.78
42 G18 珊瑚状放线菌JCM 8201T Actinocorallia aurantiaca JCM 8201T AF134066 99.22
43 G20 剑菌属Casida AT Ensifer adhaerens Casida AT JNAE01000171 100.00

图2

不同放线菌菌株16S rDNA系统发育树

图3

室内黄瓜枯萎病菌抑菌效果及大棚黄瓜枯萎病的防治效果 (a)和(b)为皿内抑菌试验,(a) PDA培养基上生长的黄瓜枯萎病菌(对照),(b) 含G8放线菌的PDA培养基上生长的黄瓜枯萎病原菌;(c)~(e)为大棚黄瓜药剂试验,(c) 无药剂处理(对照),(d) 多菌灵药剂处理,(e) G8放线菌处理

表2

黄瓜枯萎病发病率及防治效果

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

图4

G8放线菌在ISP2(左)及ISP3(右)培养基上的菌落形态

图5

G8放线菌DNA扩增 (a) 特异性引物扩增结果,(b) 16S通用引物扩增结果;1~4泳道为G8放线菌,5泳道为阴性对照,M: DL 2000标记

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 (%)
期望值
E-value
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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 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 0.0 99.65 8 184 045 CP039123.1
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