珍稀濒危植物华顶杜鹃叶斑病病原菌的分离与鉴定

doi:10.3969/j.issn.1004-1524.20230435

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (4): 837-845.DOI: 10.3969/j.issn.1004-1524.20230435

珍稀濒危植物华顶杜鹃叶斑病病原菌的分离与鉴定

何佳薇(), 黄乐琴, 卢振宇, 方瑾, 雷子阳, 张慧娟, 蒋明()

台州学院生命科学学院,浙江台州 318000

收稿日期:2023-04-06 出版日期:2024-04-25 发布日期:2024-04-29
作者简介:何佳薇(2002—),女,浙江绍兴人,本科生,研究方向为植物分子生物学。E-mail:2803865006@qq.com
通讯作者: *蒋明,E-mail:jiangming1973@139.com
基金资助:
国家级大学生创新创业训练计划(202210350029)

Isolation and identification of the pathogen causing leaf spot disease on a rare and endangered plant species Rhododendron huadingense

HE Jiawei(), Huang Leqin, LU Zhenyu, FANG Jin, LEI Ziyang, ZHANG Huijuan, JIANG Ming()

College of Life Sciences, Taizhou University, Taizhou 318000, Zhejiang, China

Received:2023-04-06 Online:2024-04-25 Published:2024-04-29
Contact: JIANG Ming

摘要/Abstract

摘要：

华顶杜鹃(Rhododendron huadingense)是浙江省特有的珍稀濒危植物,叶斑病是该植物的重要病害。本研究以华顶杜鹃叶斑病病叶为材料,在病原菌分离纯化、致病性鉴定的基础上,利用形态学和分子生物学手段确定病原菌的种类。结果表明,从病叶中分离到1株真菌,命名为HDDJ;柯赫氏法则验证结果表明,该菌能引起叶斑病,病斑特征与野外发病情况一致;HDDJ的菌落起初为白色,后变为灰白色,最后呈黑色,气生菌丝絮状,这些特征与葡萄座腔菌(Botryosphaeria dothidea)吻合;利用PCR方法克隆HDDJ肌动蛋白和微管蛋白基因片段,系统发育分析结果表明,它们均与葡萄座腔菌聚为一支。根据形态学与分子鉴定结果,确定引起华顶杜鹃叶斑病的病原菌为葡萄座腔菌。华顶杜鹃叶斑病病原菌的分离与鉴定为将来开展该病害的防治奠定了基础。

关键词: 华顶杜鹃, 叶斑病, 葡萄座腔菌, 分子鉴定

Abstract:

Rhododendron huadingense is a rare and endangered plant species endemic to Zhejiang Province, and leaf spot is an important disease of this plant. R. huadingense leaves infected with leaf spot disease were used as materials. Based on pathogen isolation, purification, and pathogenicity identification, both morphological and molecular biology methods were applied to determine the fungal pathogen. The results showed that one fungal strain designated HDDJ was isolated from the diseased leaves. Using Koch’s postulates, the fungus caused leaf spot disease, and the characteristics of the lesions were consistent with those observed in the field. The colonies of HDDJ were white, then gradually turned gray, and finally became black with fluffy aerial mycelium. The hyphal characteristics of HDDJ were identical to those of Botryosphaeria dothidea. Fragments of actin and tubulin genes were cloned by using PCR, and phylogenetic analysis results revealed that both were clustered with B. dothidea. According to the morphological and molecular identification results, it was confirmed that the fungal pathogen causing R. huadingense leaf spot disease was B. dothidea. Isolation and identification of R. huadingense leaf spot disease laid a foundation for this disease control in the future.

Key words: Rhododendron huadingense, leaf spot disease, Botryosphaeria dothidea, molecular identification

中图分类号:

何佳薇, 黄乐琴, 卢振宇, 方瑾, 雷子阳, 张慧娟, 蒋明. 珍稀濒危植物华顶杜鹃叶斑病病原菌的分离与鉴定[J]. 浙江农业学报, 2024, 36(4): 837-845.

HE Jiawei, Huang Leqin, LU Zhenyu, FANG Jin, LEI Ziyang, ZHANG Huijuan, JIANG Ming. Isolation and identification of the pathogen causing leaf spot disease on a rare and endangered plant species Rhododendron huadingense[J]. Acta Agriculturae Zhejiangensis, 2024, 36(4): 837-845.

图/表 8

图1 华顶杜鹃的正常叶片和感病叶片 A,正常叶片;B,病叶。

Fig.1 Normal and diseased leaves of Rhododendron huadingense A, Normal leaves; B, Diseased leaves.

图2 致病性鉴定 A,接种病原菌5 d时叶片的正面;B,接种病原菌5 d时叶片的背面。

Fig.2 Pathogenicity detection A, Front side of leaf at 5 d after inoculation with pathogen; B, Back side of leaf at 5 d after inoculation with pathogen.

图3 病原菌HDDJ的形态特征 A,培养2 d的菌落形态;B,培养5 d的菌落形态;C,光学显微镜下菌丝的形态。

Fig.3 Morphological characteristics of pathogenic fungus HDDJ A, Colony morphology of HDDJ after 2 days of cultivation; B, Colony morphology of HDDJ after 5 days of cultivation; C, Morphology of mycelium under a light microscope.

图4 基因片段的PCR扩增产物 M,Middle DNA Marker II;1,微管蛋白基因片段;2,肌动蛋白基因片段。

Fig.4 PCR products of different gene fragments M, Middle DNA Marker II; 1, Tubulin gene fragment; 2, Actin gene fragment.

图5 HDDJ肌动蛋白序列及其同源序列的多重比对 AY972118,葡萄座腔菌CMW8000菌株;HDDJ,葡萄座腔菌;AY972117,葡萄座腔菌CMW7779菌株;KF951843,菜豆壳球孢CPC21388菌株;MH712507,大戟壳球孢McBr39菌株;MK134684,榆球壳孢PB_11f菌株;AY624264,细凹色二孢CMW5870菌株;AY624265,细凹色二孢MW4900菌株;MK574917,可可毛色二孢HNWN02菌株;MZ723784,伊朗毛色二孢SZ-YAM2菌株。

Fig.5 Multiple alignment of HDDJ actin gene sequence and its homologous sequences AY972118, Botryosphaeria dothidea strain CMW8000; HDDJ, B. dothidea; AY972117, B. dothidea strain CMW7779; KF951843, Macrophomina phaseolina strain CPC21388; MH712507, M. euphorbiicola strain McBr39; MK134684, Sphaeropsis ulmicola strain PB_11f; AY624264, Diplodia scrobiculata strain CMW5870; AY624265, D. scrobiculata strain MW4900; MK574917, Lasiodiplodia theobromae strain HNWN02; MZ723784, L. iranensis strain SZ-YAM2.

图6 HDDJ微管蛋白序列及其同源序列的多重比对 HDDJ,葡萄座腔菌;MN370930,葡萄座腔菌SX03菌株;MG761773,葡萄座腔菌PPO-46523菌株;MK482382,葡萄座腔菌JZB310177菌株;KX505929,藤黄新壳梭孢CAA628菌株;ON773422,小新壳梭孢,LSCKS8-7菌株;KU976452,沙雷氏色二孢Mz-F44菌株;OL405580,Lasiodiplodia mahajangana 17b菌株;MW315842,大戟毛色二孢GB49菌株。

Fig.6 Multiple alignment of HDDJ tubulin gene sequence and its homologous sequences HDDJ, Botryosphaeria dothidea; MN370930, B. dothidea strain SX03; MG761773, B. dothidea strain PPO-46523; MK482382, B. dothidea strain JZB310177; KX505929, Neofusicoccum luteum strain CAA628; ON773422, N. parvum strain LSCKS8-7; KU976452, Diplodia seriata strain Mz-F44; OL405580, Lasiodiplodia mahajangana strain 17b; MW315842, L. euphorbiaceicola strain GB49.

图7 基于HDDJ肌动蛋白基因片段及其同源序列构建的系统发育树

Fig.7 The phylogenetic tree based on HDDJ actin gene fragment and it homologous sequences

图8 基于HDDJ微管蛋白基因片段及其同源序列构建的系统发育树

Fig.8 The phylogenetic tree based on HDDJ tubulin gene fragment and it homologous sequences

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珍稀濒危植物华顶杜鹃叶斑病病原菌的分离与鉴定

Isolation and identification of the pathogen causing leaf spot disease on a rare and endangered plant species Rhododendron huadingense

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