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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2019, Vol. 45 Issue (6): 692-698    DOI: 10.3785/j.issn.1008-9209.2019.03.061
Plant protection     
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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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     
Received: 06 March 2019      Published: 20 January 2020
CLC:  S 436.8  
Corresponding Authors: Hongzhi WU     E-mail: 465682882@qq.com;hwu1128@163.com
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Shuangyan ZHANG
Jing MENG
Yanglin HAN
Hongzhi WU
Cite this article:

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.

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


云南地区月季灰霉病病原菌的分离鉴定

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 

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
Table1 Information of primer sequences
Fig. 1 Symptoms of rose gray mold
Fig. 2 Colony characteristics of different pathogenic fungi of rose gray mold cultured for 3 and 15 days in Yunnan areaA-B. Mycelial type; C-D. Conidial type; E-F. Sclerotial type; G-H. Yellow sclerotia strains.
Fig. 3 Microscopic characteristics of rose gray mold pathogens in Yunnan areaA-C. Conidiophores; D-F. Conidia.
Fig. 4 PCR amplification results of rose gray mold pathogens in Yunnan area by ITS and RPB2 primersA. PCR amplification results of ITS sequences (M: DL2000 DNA marker; 1-3: Samples). B: PCR amplification results of PRB2 sequences (M: DL2000 DNA marker; 1-8: Samples).
Fig. 5 Phylogenetic tree of gray mold pathogens isolated from roses in Yunnan area
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