西瓜新炭疽病病原菌分子鉴定及其生物学特性初探

doi:10.11924/j.issn.1000-6850.casb2021-0077

中国农学通报 ›› 2022, Vol. 38 ›› Issue (1): 131-136.doi: 10.11924/j.issn.1000-6850.casb2021-0077

所属专题：生物技术；植物保护；园艺

西瓜新炭疽病病原菌分子鉴定及其生物学特性初探

黄蔚¹(), 崔丽红¹(), 谢王超¹, 肖国强²

¹湘西民族职业技术学院,湖南湘西 416000
²永顺县高坪乡农技和农村合作经济服务站,湖南永顺 416000

收稿日期:2021-01-22 修回日期:2021-07-13 出版日期:2022-01-05 发布日期:2022-02-24
通讯作者: 崔丽红
作者简介:黄蔚,男,1973年出生,江西临川人,高级农艺师,本科,主要从事园艺作物栽培与病虫防治研究工作。通信地址：416000 湖南省湘西高新技术产业开发区武陵山大道3号湘西民族职业技术学院科技处,Tel：0743-8535328,E-mail： 127599136@qq.com。
基金资助:
2018年湖南省教育厅科学研究项目“湘西地区西瓜炭疽病生理小种鉴定及生物防治技术研究”(18C1706);2018年度州本级基础理论研究项目“F1代西瓜抗炭疽病新品种选育及示范推广”(2018SF2028)

Molecular Identification of a New Pathogen of Watermelon Anthracnose and Preliminary Study on Its Biological Characteristics

HUANG Wei¹(), CUI Lihong¹(), XIE Wangchao¹, XIAO Guoqiang²

¹Xiangxi Vocational and Technical College for Nationalities, Xiangxi, Hunan 416000
²Agricultural Technology and Rural Cooperative Economic Service Station, Gaoping Township, Yongshun County, Yongshun, Hunan 416000

Received:2021-01-22 Revised:2021-07-13 Online:2022-01-05 Published:2022-02-24
Contact: CUI Lihong

摘要/Abstract

摘要：

探究湘西地区西瓜炭疽病致病种类,为西瓜炭疽病防控提供理论依据。以湘西民族职业技术学院科技园种植的西瓜病叶为研究对象,对该病的病原菌进行分离提纯、形态学观察、柯赫氏法则验证、18S rDNA-ITS序列分析以及生物学特性研究。菌株C913菌落呈圆形,菌丝绒毛状,排列整齐,初为白色,后渐为灰白色。轮状排列分生孢子盘,并有橘红色分生孢子团。分生孢子圆柱形,两端钝圆,单胞,中间有隔,大小(9.8~17.5) μm× (4.5~5.9) μm。18S rDNA序列与GenBank中收录的4个胶孢炭疽菌相似性达到100%。其菌丝在温度为10~37℃时均能生长正常,但25℃时菌丝生长速度最快,菌落直径为78.28 mm,小于10℃或大于37℃时较慢。菌丝孢子在pH 5~11条件下均能生长,其中,pH 6时菌丝生长最快,菌落直径最大,为84.62 mm。以葡萄糖、蔗糖作为碳源培养基的菌落直径极显著高于其他处理,分别为63.4、62.2 mm;而乳糖明显不利于其菌丝生长(P<0.01),为33.1 mm,比对照少19.06%。(NH₄)₂SO₄则明显对C913菌丝生长有抑制作用。根据菌株的形态学特征、18S rDNA-ITS序列分析结果,鉴定该菌株C913为半知菌亚门腔孢纲黑盘孢目炭疽菌属的胶孢炭疽菌Colletotrichum gloeosporioides,这是国内外首次在西瓜病叶上发现该病原菌。且温度20~31℃、pH 6时最适菌丝生长,而在温度为25~31℃、pH 9时最适产孢。以葡萄糖、蔗糖为碳源的培养基菌落直径最大,其菌落直径依次为蛋白胨>NaNO₃>甘氨酸>谷氨酸>琼脂>(NH₄)₂SO₄。

关键词: 西瓜炭疽病, 18S rDNA-ITS, 生物学特性, 胶孢炭疽菌, 菌落直径

Abstract:

To explore the pathogenic species of watermelon anthracnose in western Hunan, and provide a theoretical basis for watermelon anthracnose prevention and control, we took the diseased leaves of watermelon grown in the Science and Technology Park of Xiangxi Vocational and Technical College for Nationalities as the research objects, isolated and purified the pathogen of the disease, and conducted morphological observation, Koch's rule verification, 18S rDNA-ITS sequence analysis and the biological characteristics analysis. The colony of strain C913 was round, hyphae of villiform and neatly arranged. It was white at first and then grayish white. The conidia discs were arranged in a ring shape, and there were orange-red conidia clusters. The conidia are cylindrical, blunt at both ends, single cell, with a septum in the middle, and the size was (9.8~17.5) µm× (4.5~5.9) µm. The 18S rDNA sequence was 100% similar to the four Colletotrichum gloeosporioides included in GenBank. The hyphae could grow normally when the temperature was 10-37℃, but the growth rate of the hyphae was the fastest at 25℃, and the colony diameter was 78.28 mm. The growth slowed down when the temperature was less than 10℃ or greater than 37℃. The hyphae spores could grow under the condition of pH 5-11. The hyphae grew fastest when the pH was 6 and the colony diameter was the largest, 84.62 mm. The colony diameter with glucose and sucrose as the carbon source medium was significantly higher than that of other treatments, 63.4 mm and 62.2 mm, respectively; while lactose was not conducive to the growth of mycelium (P<0.01), which was 33.1 mm, and 19.06% less than that of the control. (NH₄)₂SO₄ obviously inhibited the growth of C913 hyphae. According to the morphological characteristics of the strain and the results of 18S rDNA-ITS sequence analysis, the strain C913 was identified as Colletotrichum gloeosporioides of the genus Colletotrichum of the subphylum Coelospora. The pathogen is found on diseased leaves of watermelon for the first time. When the temperature is 20-31℃ and pH is 6, it is the most suitable condition for mycelial growth, and when the temperature is 25-31℃ and pH is 9, it is the most suitable condition for sporulation. The colony diameter is the largest in the medium with glucose and sucrose as carbon sources, and the colony diameter is in the order of peptone>NaNO₃>glycine>glutamic acid>agar>(NH₄)₂SO₄.

Key words: watermelon anthracnose, 18S rDNA-ITS, biological characteristics, Colletotrichum gloeosporioides, colony diameter

中图分类号:

S436

黄蔚, 崔丽红, 谢王超, 肖国强. 西瓜新炭疽病病原菌分子鉴定及其生物学特性初探[J]. 中国农学通报, 2022, 38(1): 131-136.

HUANG Wei, CUI Lihong, XIE Wangchao, XIAO Guoqiang. Molecular Identification of a New Pathogen of Watermelon Anthracnose and Preliminary Study on Its Biological Characteristics[J]. Chinese Agricultural Science Bulletin, 2022, 38(1): 131-136.

图/表 11

图1. 西瓜炭疽病病叶症状（田间采集）

图2. C913菌落（正面）

图3. C913分生孢子团

图4. C913病原菌分生孢子显微形态特征

图5. C913病原菌接种西瓜离体叶片7天后症状

图6. C913病原菌的18S rDNA序列PCR扩增产物电泳图

图7. C913菌株的18S rDNA序列的系统发育进化树

图8. 桃离体叶片接C913后4天（左）和梨离体叶片接种C913后4天（右）的症状

图9. 不同温度对C913菌株生长和产孢量的影响

图10. 不同pH培养基对C913菌株菌丝生长和产孢量的影响

碳源	平均菌落直径/mm	氮源	平均菌落直径/mm
乳糖	33.1±1.9fF	谷氨酸	40.3±1.0dD
甘露醇	57.5±2.0bB	硝酸钠	52.9±2.2bB
果糖	60.9±1.6abA	蛋白胨	65.5±1.2aA
葡萄糖	63.4±1.2aA	甘氨酸	48.5±1.3cC
蔗糖	62.2±1.7aA	硫酸铵	24.2±1.3fF
麦芽糖	52.7±2.5cC	CK	34.2±1.8eE
琼脂(CK)	40.9±1.3dD

表1. 不同碳源、氮源对C913胶孢炭疽菌菌丝生长的影响

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西瓜新炭疽病病原菌分子鉴定及其生物学特性初探

Molecular Identification of a New Pathogen of Watermelon Anthracnose and Preliminary Study on Its Biological Characteristics

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