华东山茶花腐病病原菌分离鉴定

doi:10.3969/j.issn.1004-1524.2023.01.13

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (1): 121-127.DOI: 10.3969/j.issn.1004-1524.2023.01.13

华东山茶花腐病病原菌分离鉴定

普梅英(), 武自强, 张诗文, 李艳杰, 朱幼娇, 吴坤, 陈龙清, 王超()

西南林业大学国家林业和草原局西南风景园林工程技术研究中心,云南省功能性花卉资源及产业化技术工程研究中心,云南省森林灾害预警与控制实验室,云南昆明 650224

收稿日期:2022-04-07 出版日期:2023-01-25 发布日期:2023-02-21
通讯作者: *王超,E-mail: 47188127@qq.com
作者简介:普梅英(1997—),女,彝族,云南玉溪人,硕士研究生,研究方向为风景园林植物。E-mail: 670868861@qq.com
基金资助:
国家重点研发计划(2019YFD100100005);云南省教育厅科学研究基金(2021Y259);云南省教育厅科学研究基金(2021J0172);西南山地森林保育与利用教育部重点实验室开放基金(KLESWFU-202005);国家基础科学公共科学数据中心“世界山茶属植物品种数据库”(NBSDC-DB-03)

Isolation and identification of petal blight disease of Camellia japonica

PU Meiying(), WU Ziqiang, ZHANG Shiwen, LI Yanjie, ZHU Youjiao, WU Kun, CHEN Longqing, WANG Chao()

Southwest Research Center for Engineering Technology of Landscape Architecture (State Forestry and Grassland Administration), Yunnan Engineering Research Center for Functional Flower Resources and Industrialization, Key Laboratory of Forest Disaster Warning and Control in Yunnan Province, Southwest Forestry University, Kunming 650224, China

Received:2022-04-07 Online:2023-01-25 Published:2023-02-21

摘要/Abstract

摘要：

华东山茶(Camellia japonica L.)花腐病对华东山茶的观赏性影响极其严重,为明确华东山茶花腐病的致病菌及其生物学特性,提升华东山茶花期品质和观赏价值,采用组织分离法对云南省昆明市昆明植物园华东山茶的花腐病进行病原菌分离,并进行致病性测定、形态学和分子生物学鉴定。结果表明,从华东山茶品种‘五彩’(C. japonica ‘Wucai’)的病组织中分离得到1株菌株,其在PDA培养基上为白色菌丝,培养7 d长出黑色的分生孢子;将分离菌株回接于华东山茶品种‘五彩’健康花瓣上,接种后划破的花瓣病症较为明显,花朵失色变黄褐,严重时花瓣干腐或湿腐,并且产生分生孢子,与田间的症状一致;经过多引物(ITS、LSU、β-tubulin)合并系统发育分析,分离菌株与葡萄牙拟盘多毛孢(Pestalotiopsis portugallica)聚集在同一分支。综上可知,引起华东山茶花腐病的病原菌为葡萄牙拟盘多毛孢(P. portugallica),这是葡萄牙拟盘多毛孢(P. portugallica)侵染华东山茶花朵引起华东山茶花腐病的首次报道。

关键词: 华东山茶, 花腐病, 葡萄牙拟盘多毛孢, 致病性

Abstract:

The ornamental effect of Camellia japonica petal blight disease was very serious. In order to identify the pathogenic bacteria and biological characteristics of C. japonica petal blight disease and improve the quality and ornamental value of C. japonica, and provide the basis for prevention and control. The pathogen of petal blight disease of C. japonica from Kunming Botanical Garden in Kunming City was isolated by tissue separation method, and pathogenic bacteria were identified by pathogenicity assay, morphological and molecular biological identification. One strain was isolated from the C. japonica ‘Wucai’ diseased tissue, there was white hyphae on PDA medium and had black conidia in the later stage of culturing for 7 days. when it was connected back to the healthy petals, the disease of lacing petals after inoculation was more obvious, which could cause the flowers to become yellow and brown. When it was serious, it could make the petals dry rot or wet rot, and grew mycelium, which was consistent with the symptoms in the field and conformed to the pathogenicity determination of Koch’s rule. Based on the phylogenetic analysis of multiple primers (ITS, LSU, β-tubulin), Pestalotiopsis portugallica clustered in the same clade. It was confirmed that the pathogen of petal blight disease of C. japonica was P. portugallica. This was the first report that P. portugallica infected the flowers of C. japonica and caused petal blight disease.

Key words: Camellia japonica, petal blight disease, Pestalotiopsis portugallica, pathogenicity

中图分类号:

普梅英, 武自强, 张诗文, 李艳杰, 朱幼娇, 吴坤, 陈龙清, 王超. 华东山茶花腐病病原菌分离鉴定[J]. 浙江农业学报, 2023, 35(1): 121-127.

PU Meiying, WU Ziqiang, ZHANG Shiwen, LI Yanjie, ZHU Youjiao, WU Kun, CHEN Longqing, WANG Chao. Isolation and identification of petal blight disease of Camellia japonica[J]. Acta Agriculturae Zhejiangensis, 2023, 35(1): 121-127.

图/表 4

图1 菌株23h形态特征观察 a,田间健康花症状;b,田间感病花症状;c,菌落;d,分生孢子;e、f,室内离体接种后花腐症状。

Fig.1 Morphological characteristics of strain 23h a, Healthy flowers in field; b, Symptom on flowers in field; c, Colony morphology; d, Conidium; e and f, Symptom on Camellia japonica flowers after inoculation.

表1 引物序列

Table 1 Primer sequences

基因名称 Gene name	引物 Primer	序列 Sequence(5'→3')
ITS^[17]	ITS1	TCCGTAGGTGAACCTGCGG
	ITS4	TCCTCCGCTTATTGATATG
LSU^[18]	NL1	GCATATCAATAAGCGGAGGAAAAG
	NL4	GGTCCGTGTTTCAAGACGG
β-tubulin^[19-20]	BT-2A	GGTAACCAAATCGGTGCTGCTTTC
	BT-2B	ACCCTCAGTGTAGTGACCCTTGGC

图2 菌株23h的ITS、LSU和β-tubulin序列电泳图 a、b、c为引物ITS、LSU、β-tubulin序列的电泳图;M为DL2000 marker.

Fig.2 Electrophoresis map of ITS, LSU and β-tubulin gene sequences in strain 23 h a, b, c were electrophoretic map of primer ITS, LSU, β-tubulin; M was the DNA marker of DL2000.

图3 基于ITS、LSU和β-tubulin序列的菌株23h多基因系统发育进化树

Fig.3 The phylogenetic tree of strain 23h based on ITS,LSU and β-tubulin gene sequence

参考文献 32

[1]	李丕睿, 刘永芝, 翟敏, 等. 山茶属华东山茶(Camellia japonica L.)研究进展[J]. 植物遗传资源学报, 2016, 17(6): 1022-1030.
	LI P R, LIU Y Z, ZHAI M, et al. Recent advances in Camellia japonica L[J]. Journal of Plant Genetic Resources, 2016, 17(6): 1022-1030. (in Chinese with English abstract)
[2]	吴雅文, 张宁, 白天, 等. 中日两国山茶花的渊源及异同[J]. 世界林业研究, 2015, 28(4): 81-84.
	WU Y W, ZHANG N, BAI T, et al. Origin and differences of Camellia between China and Japan[J]. World Forestry Research, 2015, 28(4): 81-84. (in Chinese with English abstract)
[3]	WANG Y, ZHUANG H, SHEN Y, et al. The dataset of Camellia cultivars names in the world[J]. Biodivers Data J, 2021, 9: e61646. DOI URL
[4]	王建伟, 钱显明, 张炳欣. 浙江省山茶花病害[J]. 植物病理学报, 1990, 20(2): 89-92.
	WANG J W, QIAN X M, ZHANG B X. The diseases of Camellia in Zhejiang Province[J]. Acta Phytopathologica Sinica, 1990, 20(2): 89-92. (in Chinese with English abstract)
[5]	何美仙. 山茶花主要病害的发生及防治[J]. 内蒙古林业科技, 2005, 31(1): 51-52.
	HE M X. Occurrence and control of main diseases of Camellia[J]. Inner Mongolia Forestry Science & Technology, 2005, 31(1): 51-52. (in Chinese)
[6]	杨英. 山茶花常见病虫害及其防治对策[J]. 农业科技与信息, 2016(10): 101.
	YANG Y. Common diseases and insect pests of Camellia and their control countermeasures[J]. Journal of Landscape Science, 2016(10): 101. (in Chinese)
[7]	RAABE R D, MCCAIN A H, PAULUS A O. Diseases and pests[M]// FEATHERS D L,BROWN M H. The Camellia, its history, culture, genetics and a look into its future development. Columbia: Southern Californian Camellia Society Inc, 1978: 279-285.
[8]	TOOR R F V. Development of biocontrol methods for Camellia flower blight caused by Ciborinia camelliae[J]. American Camellia Yearbook, 2003:29-33.
[9]	KONDRATEV N, DENTON-GILES M, BRADSHAW R E, et al. Camellia plant resistance and susceptibility to petal blight disease are defined by the timing of defense responses[J]. Mol Plant Microbe Interact, 2020, 33(7): 982-995. DOI URL
[10]	陈秀虹, 伍建榕. 观赏植物病害诊断与治理[M]. 北京: 中国建筑工业出版社, 2009.
[11]	PU M Y, WU Z Q, ZHANG S W, et al. First report of Chaetomium pseudocochliodes causing petal blight disease of Camellia reticulata in Yunnan, China[J]. Plant Disease, 2022, 106(11): 3001.
[12]	方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社, 1998.
[13]	任纬恒. 高山杜鹃病害的病原菌分离鉴定与防治基础研究[D]. 贵阳: 贵州师范大学, 2019: 1-68.
	REN W H. Pathogenic identification and basic researches on diseases control of Rhododendron[D]. Guiyang: Guizhou Normal University, 2019: 1-68. (in Chinese with English abstract)
[14]	朱轶慧, 刘玉军, 毕飞虎, 等. 红叶石楠叶斑病病原鉴定及其生物学特性研究[J]. 植物保护, 2021, 47(5): 210-215.
	ZHU Y H, LIU Y J, BI F H, et al. Identification and biological characteristics of the pathogen causing leaf spot disease on Photinia×fraseri[J]. Plant Protection, 2021, 47(5): 210-215. (in Chinese with English abstract)
[15]	张小彦, 何静, 侯彩霞, 等. 枸杞根腐病菌拮抗菌株的筛选与鉴定[J]. 浙江农业学报, 2020, 32(5): 858-865. DOI
	ZHANG X Y, HE J, HOU C X, et al. Screening and identification of antagonistic strains of wolfberry root rot[J]. Acta Agriculturae Zhejiangensis, 2020, 32(5): 858-865. (in Chinese with English abstract) DOI
[16]	李戌清, 严建立, 阮松林. 三叶青炭疽病病原菌的鉴定与生物学特性[J]. 浙江农业学报, 2020, 32(11): 2009-2019. DOI
	LI X Q, YAN J L, RUAN S L. Identification and biological characteristics of anthracnose pathogen on Tetrastigma hemsleyanum[J]. Acta Agriculturae Zhejiangensis, 2020, 32(11): 2009-2019. (in Chinese with English abstract)
[17]	HENSON J M, FRENCH R. The polymerase chain reaction and plant disease diagnosis[J]. Annual Review of Phytopathology, 1993, 31: 81-109. PMID
[18]	刘彬, 张祥林, 王羽中, 等. 向日葵白锈病菌巢式PCR检测方法的研究[J]. 新疆农业科学, 2011, 48(5): 859-863.
	LIU B, ZHANG X L, WANG Y Z, et al. Study on the nest-PCR to detect the Albugo tragopogi (Persoon) schruter var helianthi novotlenova[J]. Xinjiang Agricultural Sciences, 2011, 48(5): 859-863. (in Chinese with English abstract)
[19]	GLASS N L, DONALDSON G C. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes[J]. Nano Research, 1995, 61(4): 1323-1330.
[20]	O’DONNELL K, CIGELNIK E. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous[J]. Molecular Phylogenetics and Evolution, 1997, 7(1): 103-116. DOI URL
[21]	MAHARACHCHIKUMBURA S S N, HYDE K D, GROENEWALD J Z, et al. Pestalotiopsis revisited[J]. Studies in Mycology, 2014, 79: 121-186. DOI URL
[22]	吴晓鹏. 杭州茶内生拟盘多毛孢资源、多样性及分类研究[D]. 杭州: 浙江大学, 2006.
	WU X P. Resource, diversity and taxonomy of endophytic Pestalotiopsis on Camellia sinensis in Hangzhou[D]. Hangzhou: Zhejiang University, 2006. (in Chinese with English abstract)
[23]	ISMAIL A M, CIRVILLERI G, POLIZZI G. Characterisation and pathogenicity of Pestalotiopsis uvicola and Pestalotiopsis clavispora causing grey leaf spot of mango (Mangifera indica L.) in Italy[J]. European Journal of Plant Pathology, 2013, 135(4): 619-625. DOI URL
[24]	REN H Y, LI G, QI X J, et al. Identification and characterization of Pestalotiopsis spp. causing twig blight disease of bayberry (Myrica rubra Sieb. & Zucc) in China[J]. European Journal of Plant Pathology, 2013, 137(3): 451-461. DOI URL
[25]	赵洪海, 岳清华, 梁晨. 蓝莓拟盘多毛孢枝枯病的病原菌[J]. 菌物学报, 2014, 33(3): 577-583.
	ZHAO H H, YUE Q H, LIANG C. The pathogen causing Pestalotiopsis twig dieback of blueberry[J]. Mycosystema, 2014, 33(3): 577-583. (in Chinese with English abstract)
[26]	韦继光, 徐同, 潘秀湖, 等. 拟盘多毛孢属的分类学研究进展[J]. 广西农业生物科学, 2006, 25(1): 78-85.
	WEI J G, XU T, PAN X H, et al. Progress of research on taxonomy of Pestalotiopsis[J]. Journal of Guangxi Agricultural and Biological Science, 2006, 25(1): 78-85. (in Chinese with English abstract)
[27]	韦继光, 徐同, 黄伟华, 等. 从分子系统发育探讨内生拟盘多毛孢与病原拟盘多毛孢的关系[J]. 浙江大学学报(农业与生命科学版), 2008, 34(4): 367-373.
	WEI J G, XU T, HUANG W H, et al. Molecular phylogenetic investigation on relationship of endophytic and pathogenic Pestalotiopsis species[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2008, 34(4): 367-373. (in Chinese with English abstract)
[28]	张凡, 冯婷, 韩正敏, 等. 落羽杉赤枯病病原菌的分离鉴定及生物学特性分析[J]. 植物保护学报, 2018, 45(4): 900-907.
	ZHANG F, FENG T, HAN Z M, et al. Biological characteristics and molecular identification the pathogen of Taxodium red blight[J]. Journal of Plant Protection, 2018, 45(4): 900-907. (in Chinese with English abstract)
[29]	陈立杰, 宋玉, 陈通政, 等. 一个分离自贵州省茶叶上的拟盘多毛孢属新种[J]. 种子, 2019, 38(8): 70-73.
	CHEN L J, SONG Y, CHEN T Z, et al. One new Pestalotiopsis species isolated from Camellia sinensis of Guizhou Province[J]. Seed, 2019, 38(8): 70-73. (in Chinese)
[30]	何翔, 李翱, 李楚, 等. 滇重楼稻曲拟盘多毛孢叶斑病病原鉴定及其生物学特性测定[J]. 植物保护, 2020, 46(6): 47-54, 64.
	HE X, LI A, LI C, et al. Pathogen identification and biological characteristics of Paris Polyphylla var. Yunnanensis leaf spot disease caused by Pestalotiopsis oryzae[J]. Plant Protection, 2020, 46(6): 47-54, 64. (in Chinese with English abstract)
[31]	秦绍钊, 王建伟, 张柱亭. 贵州油茶叶枯病病原鉴定研究[J]. 南方农业, 2019, 13(26): 134-135.
	QIN S Z, WANG J W, ZHANG Z T. Identification of pathogen of Camellia oleifera blight in Guizhou Province[J]. South China Agriculture, 2019, 13(26): 134-135. (in Chinese)
[32]	张晓勇, 李树江, 王亮, 等. 山茶灰斑病病原菌鉴定及防治药剂初步筛选[J]. 植物保护, 2019, 45(4): 209-215, 242.
	ZHANG X Y, LI S J, WANG L, et al. Pathogen identification of gray leaf spot on Camellia japonica and screening of the fungicides[J]. Plant Protection, 2019, 45(4): 209-215, 242. (in Chinese with English abstract)

华东山茶花腐病病原菌分离鉴定

Isolation and identification of petal blight disease of Camellia japonica

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