福建省铁皮石斛茎腐病病原菌鉴定及其生物学特性

姚锦爱; 黄鹏; 兰成忠; 侯翔宇; 余德亿

doi:10.19303/j.issn.1008-0384.2021.12.009

福建省铁皮石斛茎腐病病原菌鉴定及其生物学特性

doi: 10.19303/j.issn.1008-0384.2021.12.009

福建省作物有害生物监测与治理重点实验室/福建省农业科学院植物保护研究所/福建省作物有害生物绿色防控工程研究中心，福建　福州　350013

基金项目: 福建省农业科学院项目(DEC202002、CXTD2021016)；福建省农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021017、XTCXGC2021011)

详细信息

作者简介:
姚锦爱（1978−），女，硕士，研究员，主要从事植物病虫害生防方面的研究（E-mail：yaoja@163.com）

通讯作者:
余德亿（1972−），男，研究员，主要从事病虫害生物防治方面的研究（E-mail：yudy_2004@126.com）

中图分类号: S 436
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出版历程
- 收稿日期: 2021-10-20
- 录用日期: 2021-10-20
- 修回日期: 2021-11-19
- 网络出版日期: 2021-12-30
- 刊出日期: 2021-12-28

Identification and Biological Characteristics of Stem Rot Pathogen of Dendrobium officinale

Laboratory for Monitoring and Integrated Management of Crop Pests/Institute of Plant Protection, Fujian Academy of Agricultural Sciences/Fujian Engineering Research Center for Green Pest Management, Fuzhou, Fujian　350013, China

摘要

摘要: 目的明确福建省铁皮石斛茎腐病病原菌及其生物学特性。方法从福建省诏安县、松溪县等地的3个种植基地采集典型病株并分离病原菌，采用形态学结合分子生物学（ITS、EF1-α、ACT、RPB2和LSU基因序列分析）的方法鉴定其病原菌种类，并对该病原菌菌丝体在不同温度、pH值、光照条件、碳源、氮源等生物学特性进行研究。结果经鉴定该病原菌为尖孢镰刀菌（Fusarium oxysporum）。生物学特性研究表明其菌丝生长最适温度为30 ℃，pH为7，光照有利于菌丝生长，最适碳、氮源分别为淀粉和蛋白胨。结论分离鉴定了福建省铁皮石斛茎腐病病原菌为尖孢镰刀菌（Fusarium oxysporum），明确了病原菌生物学特性。
- 铁皮石斛 /
- 茎腐病 /
- 鉴定 /
- 尖孢镰刀菌 /
- 生物学特性
Abstract: Objective To isolate and identify and determine the biological characteristics of the pathogen that caused the stem rot disease on Dendrobium officinale. Method Typical diseased plants were collected from 3 farms in Zhaoan and Songxi for pathogen isolation. Morphological and molecular identification methods including gene sequencing of ITS, EF1-α, ACT, RPB2, and LSU were performed on the isolates. Biological characteristics of the pathogen in response to temperature, pH, light condition, carbon source, and nitrogen source were determined. Result The pathogen was identified as Fusarium oxysporum, which grew optimally at 30 ℃ and pH 7 under light on a medium using starch for carbon and peptone for nitrogen sources. Conclusion F. oxysporum was identified to be the pathogen that caused the stem rot disease on D. officinale in Fujian, and the optimal mycelial growth conditions determined.
- Dendrobium officinale /
- stem rot /
- identification /
- Fusarium oxysporum /
- biological characteristics

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图 1 铁皮石斛茎腐病的症状及病原菌的形态

注：A，自然发病症状叶片；B，菌落形态特征；C，分生孢子; D，致病性测定；E、F，接菌后茎秆症状。标尺=20 μm。

Figure 1. Symptom and morphology of stem rot on D. officinale

Note: A, typical symptoms on naturally infected leaf; B, colony of stem rot of Dendrobium officinale; C, conidia; D, pathogenicity identification; E-F, Symptom on inoculated leaves. Scales=20 μm.

下载: 全尺寸图片幻灯片

图 2 基于ITS/ACT/EF1-α/RPB2/LSU基因序列采用邻接法构建的系统发育树

Figure 2. Phylogenetic tree based on sequences of ITS/ACT/EF1-α/RPB2/LSU genes using neighbor-joining method

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图 3 不同温度对病原菌菌丝生长的影响

注：不同小写字母代表差异显著（P<0.05），下同。

Figure 3. Effect of temperatures on mycelial growth of F. oxysporum

Note: Different lowercase letters indicate significant difference (P<0.05), the same as below figures.

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图 4 不同pH对病原菌菌丝生长的影响

Figure 4. Effect of pHs on mycelial growth of F. oxysporum

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图 5 不同光照条件对病原菌菌丝生长的影响

Figure 5. Effect of light conditions on mycelial growth of F. oxysporum

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图 6 不同碳源对病原菌菌丝生长的影响

Figure 6. Effect of carbon sources on mycelial growth of F. oxysporum

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图 7 不同氮源对病原菌菌丝生长的影响

Figure 7. Effect of nitrogen sources on mycelial growth of F. oxysporum

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表 1 茎腐病菌鉴定所用 PCR引物

Table 1. PCR primers for pathogenic identification of stem rot disease

引物名称 Primer name	引物序列　　 Primer sequence	参考文献 References
ITS1	5′-TCCGTAGGTGAACCTGCGG-3′	White等^[11]
ITS4	5′-TCCTCCGCTTATTGATATGC-3′	White等^[11]
ACT-512F	5′-ATGTGCAAGGCCGGTTTCGC-3′	Carbone等^[12]
ACT-783R	5′-TACGAGTCCTTCTGGCCCAT-3′	Carbone等^[12]
EF1	5′-ATGGGTAAGGA(A/G)GACAAGAC-3′	O'Donnell 等^[13]
EF2	5′-GGA(G/A)GTACCAGT (G/C) ATCATGTT-3′	O'Donnell 等^[13]
frpb2-5f	5′-GAYGAYMGWGATCAYTTYGG-3′	Liu 等^[14]
frpb2-7R	5′-CCCATRGCTTGYTTRCCCAT-3′	Liu 等^[14]
NL1	5′-GCATATCAATAAGCGGAGGAAAAG-3′	Dagar等^[15]
NL4	5′-GGTCCGTGTTTCAAGACGG-3′	Dagar等^[15]

下载: 导出CSV

表 2 基于ITS、ACT、EF1-α、RPB2和LSU基因系统发育树登录序列

Table 2. Sequences of ITS, ACT, EF1-α, RPB2, and LSU genes for construction of phylogenetic tree

序列编号 No.	种名 Species	基因库登录号 Genbank accession number
序列编号 No.	种名 Species	真菌核糖体基因转录间隔区 Internal transcribed spacer ITS	真菌肌动蛋白基因序列 ActinACT	延伸因子基因 Elongation factor 1αEF1-α	RNA聚合酶Ⅱ大亚基基因 The second lar-gest RNA polymerase subunitRPB2	中科灵芝核糖体大亚基基因 Large subunitLSU
1	尖孢镰刀菌FJDO-1 Fusarium oxysporum FJDO-1	MK880499	MK895954	MW546934	MZ031970	MZ031390
2	尖孢镰刀菌 Fusarium oxysporum	MG198889	LT841228	MH828025	MT305136	JN938909
3	亚粘团镰刀菌 Fusarium subglutinans	KY318486	KU603821	KF467375	MT337642	KU604086
4	腐皮镰刀菌 Fusarium solani	KY318489	KM231194	KY123913	MT305182	JN939552
5	拟轮枝镰刀菌 Fusarium verticillioides	KX385055	KU603765	KF467376	KM396613	KU604047
6	藤仓镰孢菌 Fusarium fujikuroi	KX385058	KU603840	KY123914	MW086869	KU604102
7	接骨木镰刀菌 Fusarium sambucinum	DQ132833	KM231213	KM231941	JX171606	KM231682
8	镰刀菌属种 Fusarium verrucosum	KM231812	KM231212	KM231940	JX171613	KM231681
9	层出镰刀菌 Fusarium proliferatum	GU074010	KM231217	KF467371	MT305154	KU604106
10	松树脂溃疡病菌 Fusarium circinatum	MH862654	KM231215	KM231943	JX171623	KX306907
11	镰刀菌属种 Fusarium illudens	KM231806	KM231202	KM231934	JX171601	KM231673
12	赤壳菌 Neocosmospora rubicola	KU323637	KM231197	KM231928	KM232367	KM231667

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