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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (6): 692-698    DOI: 10.3785/j.issn.1008-9209.2019.03.061
植物保护     
云南地区月季灰霉病病原菌的分离鉴定
张双艳(),孟静,韩洋琳,吴红芝()
云南农业大学园林园艺学院，昆明 650201
Isolation and identification of gray mold pathogens on roses in Yunnan area
Shuangyan ZHANG(),Jing MENG,Yanglin HAN,Hongzhi WU()
College of Horticulture and Landscape, Yunnan Agricultural University, Kunming 650201, China
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摘要：

为了明确引起月季灰霉病的病原菌，为防治月季灰霉病提供理论依据，本研究从云南18个切花月季产区采集月季灰霉病样本，采用浇注平板法对病原菌进行分离纯化，用传统形态学鉴定的方法对病原菌进行菌落形态特征观察和显微特征观察，并结合ITS和PRB2基因联合分析来构建系统发育树，进行分子生物学鉴定。结果表明：引起云南地区月季灰霉病的病原菌主要是灰葡萄孢菌（Botrytis cinerea），但种内变异非常丰富。根据菌落形态特征、分生孢子梗及分生孢子形态，从206个月季灰霉病样本中分离到124株灰霉病病原菌。根据菌落形态特征将病原菌分为菌丝型、孢子型和菌核型，所占比例分别为39%、28%和33%。41株菌核型菌株中只获得1株黄色菌核菌株，不产生孢子或产生少量孢子。在由88株代表菌株构建的系统发育树中，87株与B. cinerea聚为一支，1株与蚕豆葡萄孢菌（B. fabae）聚为一支，同源性达到99%以上。综上表明，侵染云南地区月季的灰霉菌主要是B. cinerea，且除了B. cinerea外的另一个侵染月季的葡萄孢属真菌是B. fabae，这为进一步推进月季灰霉病菌特性的研究及其防治提供了重要的理论支持。
关键词： 月季;  灰霉病;  灰葡萄孢菌;  蚕豆葡萄孢菌    
Abstract:

In order to clarify the pathogens causing gray mold on roses and provide theoretical basis for its prevention and control, samples of rose gray mold were collected from 18 production bases of cut flowers in Yunnan area. The pathogens were isolated and purified by pouring plate method. The morphological and microscopic characteristics observation of colonies of pathogens were carried out by traditional morphological identification method, and ITS and PRB2 gene analysis was combined to construct a phylogenetic tree for molecular biological identification. The results showed that 124 strains of gray mold pathogens were isolated from 206 samples of rose according to the morphological characteristics of colonies, the morphology of conidial pedicels and conidia. Pathogenic fungi were classified into mycelial, conidial and sclerotial types according to their colony morphological characteristics, with the proportions of 39%, 28% and 33%, respectively. Only one strain with yellow sclerotia was obtained from 41 sclerotia-type strains, and no spores or a few spores were produced. Of the phylogenetic trees constructed by 88 representative strains, 87 strains were clustered with B. cinerea, and only one strain was clustered with B. fabae, and their homology was more than 99%. The above results show that B. cinerea is the main pathogen causing gray mold on roses in Yunnan area, but the intraspecific variation in B. cinerea is rich. This study lays a foundation for further exploring the happening pattern of rose gray mold and control techniques on it in the future.
Key words: rose (Rosa hybrida L.)    gray mold    Botrytis cinerea    Botrytis fabae
收稿日期: 2019-03-06 出版日期: 2020-01-20
CLC:  S 436.8  
基金资助: 国家重点研发计划(2018YFD1000407);云南省科技厅院士专家工作站项目(2018IC098);云南省高水平大学创新人才培养基地创新训练项目(2017CX015)
通讯作者: 吴红芝     E-mail: 465682882@qq.com;hwu1128@163.com
作者简介: 张双艳（https://orcid.org/0000-0003-2667-4997），E-mail：465682882@qq.com
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张双艳
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引用本文:

张双艳,孟静,韩洋琳,吴红芝. 云南地区月季灰霉病病原菌的分离鉴定[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(6): 692-698.

Shuangyan ZHANG,Jing MENG,Yanglin HAN,Hongzhi WU. Isolation and identification of gray mold pathogens on roses in Yunnan area. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(6): 692-698.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.03.061    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I6/692

基因名称

Gene name

引物名称

Primer name

引物序列（5′→3′）

Primer sequence (5′→3′)

来源

Source

ITSITS1TCCGTAGGTGAACCTGCGGWHITE等[10]
ITS4TCCTCCGCTTATTGATATGC
RPB2RPB2forGATGATCGTGATCATTTCGGSTAATS等[11]
RPB2revCCCATAGCTTGCTTACCCAT
表1  引物序列信息
图1  月季灰霉病症状
图2  云南地区不同月季灰霉菌培养3、15 d的菌落特征A～B.菌丝型；C～D.孢子型；E～F.菌核型；G～H.黄色菌核菌株。
图3  云南地区月季灰霉病病原菌的显微特征A～C.分生孢子梗；D～F.分生孢子。
图4  ITS、RPB2引物对云南地区月季灰霉病病原菌的PCR扩增结果A. ITS序列的PCR扩增结果（M： DL2000 DNA标志物；1～3：样本）。B. PRB2序列的PCR扩增结果（M： DL2000 DNA标志物；1～8：样本）。
图5  云南地区月季灰霉病病原分离物的系统发育树
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