Identification of the pathogen of Prunus davidiana canker in Xinjiang

doi:10.6048/j.issn.1001-4330.2024.12.024

Abstract

Abstract:

【Objective】 There is a typical symptom about canker disease in Prunus davidiana from Kuitun City (44°26'35″N, 84°54'24″ E) and Aral City (40°32'30″N, 81°17'35″E), Xinjiang.With this in mind, this research aim to identify the real cause.【Methods】 Pathogens (KTST and TDST) were isolated from the susceptible Prunus davidiana branches.【Results】 Based on morphological characteristics, combined with ITS, RPB2 and Tef1-α multi-genesequence analysis, Cytospora leucostoma and Cytospora chrysosperma were isolates from KTST and TDST.【Conclusion】 The Koch postulates was verified by inoculating the pathogen of canker disease on the healthy branches in vitro to cause canker disease symptoms.

Key words: Valsa canker; morphological characteristics; pathogenicity determination; multigene analysis

摘要：

【目的】 鉴定在新疆奎屯市(44°26'35″N,84°54'24″E)和阿拉尔市(40°32'30″N,81°17'35″E)的山桃腐烂枝条病症。【方法】 从山桃腐烂枝条上分离出2株山桃腐烂病病原菌(KTST和TDST),测定分析田间症状、无性形态、致病性,并结合ITS、RPB2、Tef1-α多基因序列联合鉴定分析病原菌。【结果】 KTST和TDST的子座结构、孔口的数量、腔室形状及孢子类型均符合壳囊孢属(Cytospora)特征,KTST与TDST分离株与GenBank中已有的Cytospora leucostoma、Cytospora chrysosperma菌株聚为一个独立的分支。将腐烂病病原菌接种在离体的健康山桃枝上,引起腐烂病症状,验证其致病性。【结论】 新疆奎屯市和阿拉尔市的山桃腐烂病致病菌为Cytospora chrysosperma。

关键词: 山桃腐烂病, 形态特征, 致病性测定, 多基因联合分析

CLC Number:

S436.629

TANG Li, LI Chunyan, JIA Wenhao, LIU Zhenya, LI Yapeng, DAN Hongxia, ZHANG Wangbin. Identification of the pathogen of Prunus davidiana canker in Xinjiang[J]. Xinjiang Agricultural Sciences, 2024, 61(12): 3089-3096.

唐丽, 李春艳, 贾文浩, 刘振亚, 李亚鹏, 但红侠, 张王斌. 新疆山桃腐烂病病原菌鉴定[J]. 新疆农业科学, 2024, 61(12): 3089-3096.

Figures/Tables 9

References 29

[1]	刘佳, 刘雅琴, 李靖, 等. 碱胁迫对山桃叶片形态结构及光合特性的影响[J]. 西南农业学报, 2017, 30(2): 327-332.
	LIU Jia, LIU Yaqin, LI Jing, et al. Effects of alkali stress on Prunus davidiana (carr.) leaf morphological structure and photosynthetic characteristics[J]. Southwest China Journal of Agricultural Sciences, 2017, 30(2): 327-332.
[2]	安海军. 山桃在城市园林绿化中的应用[J]. 现代农村科技, 2021, (6): 67-68, 44.
	AN Haijun. The application of mountain peach in urban landscaping[J]. Modern Rural Science and Technology, 2021, (6): 67-68, 44.
[3]	Xu D D, Li H Y, Lin L J, et al. Effects of carboxymethyl chitosan on the growth and nutrient uptake in Prunus davidiana seedlings[J]. Physiology and Molecular Biology of Plants, 2020, 26(4): 661-668.
[4]	刘永刚. 山桃育苗及梅花嫁接培育技术[J]. 江西农业, 2019, (4): 11-12.
	LIU Yonggang. Cultivation Technology of mountain peach seedling and plum blossom grafting[J]. Jiangxi Agriculture, 2019, (4): 11-12.
[5]	程瑞春. 林木嫁接砧穗平衡理论及其应用[J]. 内蒙古林业调查设计, 2021, 44(1): 14-17, 55.
	CHENG Ruichun. The rootstock-scion balance theory and its application in tree grafting[J]. Inner Mongolia Forestry Investigation and Design, 2021, 44(1): 14-17, 55.
[6]	Fotouhifar K B, Hedjaroude G A, Leuchtmann A. ITS rDNA phylogeny of Iranian strains of Cytospora and associated teleomorphs[J]. Mycologia, 2010, 102(6): 1369-1382.
[7]	Stewart J E, Miller S T, Zink F A, et al. Genetic and phenotypic characterization of the fungal pathogen Cytospora plurivora from western Colorado peach orchards and the development of a ddPCR assay for detection and quantification[J]. Phytopathology, 2022, 112(4): 917-928.
[8]	杨跃文, 范明浩, 季蒙, 等. 干旱胁迫对5个种源山桃生长特性的影响研究[J]. 内蒙古林业科技, 2020, 46(4): 33-36.
	YANG Yuewen, FAN Minghao, JI Meng, et al. Effects of drought stress on growth characteristics of Prunus davidiana from five provenances[J]. Journal of Inner Mongolia Forestry Science and Technology, 2020, 46(4): 33-36.
[9]	尚建力. 山桃树种育苗造林技术分析[J]. 种子科技, 2020, 38(12): 59, 62.
	SHANG Jianli. Analysis of seedling cultivation and afforestation technology of mountain peach[J]. Seed Science & Technology, 2020, 38(12): 59, 62.
[10]	牛茹萱. 甘肃地方桃资源抗寒性评价及其对低温胁迫的响应机制[D]. 兰州: 甘肃农业大学, 2020.
	NIU Ruxuan. Cold resistance evaluation and response mechanism of local peach resources to low temperature stress in Gansu[D]. Lanzhou: Gansu Agricultural University, 2020.
[11]	方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社, 1998.
	FANG Zhongda. Research methods of plant diseases[M]. Beijing: China Agriculture Press, 1998.
[12]	唐俊煜, 王瑜, 李春艳, 等. 新疆南疆9种不同林果树木腐烂病菌无性形态比较[J]. 塔里木大学学报, 2015, 27(2): 23-27.
	TANG Junyu, WANG Yu, LI Chunyan, et al. Comparison of clonal morphology of rot pathogen from 9 kinds of trees in South Xinjiang[J]. Journal of Tarim University, 2015, 27(2): 23-27.
[13]	桂腾茸, 孔宝华, 马学林, 等. 云南苹果树腐烂病菌分离株生物学特性和致病性研究[J]. 西南农业学报, 2015, 28(5): 2096-2102.
	GUI Tengrong, KONG Baohua, MA Xuelin, et al. Studies on biological characters and pathogencity of isolates of Cytospora from apple tree in Yunnan[J]. Southwest China Journal of Agricultural Sciences, 2015, 28(5): 2096-2102.
[14]	冯宝珍, 李培谦. 月季黑斑病病原菌鉴定及室内药剂初步筛选[J]. 植物保护学报, 2019, 46(5): 1147-1154.
	FENG Baozhen, LI Peiqian. Identification of the pathogen causing black spot of Chinese rose and fungicide screening for the disease control[J]. Journal of Plant Protection, 2019, 46(5): 1147-1154.
[15]	Liu Y J, Whelen S, Hall B D. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit[J]. Molecular Biology and Evolution, 1999, 16(12): 1799-1808. DOI PMID
[16]	Carbone I, Kohn L M. A method for designing primer sets for speciation studies in filamentous ascomycetes[J]. Mycologia, 1999, 91(3): 553-556.
[17]	Kumar S, Stecher G, Li M, et al. MEGA X: molecular evolutionary genetics analysis across computing platforms[J]. Molecular Biology and Evolution, 2018, 35(6): 1547-1549. DOI PMID
[18]	Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap[J]. Evolution, 1985, 39(4): 783-791. DOI PMID
[19]	Fan X L, Bezerra J D P, Tian C M, et al. Cytospora (diaporthales) in China[J]. Persoonia, 2020, 45: 1-45. DOI PMID
[20]	郭众仲, 张王斌, 李亚鹏, 等. 不同寄主来源腐烂病菌对库尔勒香梨树致病性研究[J]. 新疆农业科学, 2014, 51(12): 2245-2250.
	GUO Zhongzhong, ZHANG Wangbin, LI Yapeng, et al. Study on the Pathogenicity of Canker Pathogen from Different Hosts to Korla fragrant pear Tree[J]. Xinjiang Agricultural Sciences, 2014, 51(12): 2245-2250.
[21]	李春艳, 张王斌, 苟巧, 等. 不同寄主来源腐烂病菌对枣树致病性研究[J]. 新疆农业科学, 2016, 53(1): 51-58.
	LI Chunyan, ZHANG Wangbin, GOU Qiao, et al. Study on the pathogenicity of Cytospora spp. from different hosts to jujube[J]. Xinjiang Agricultural Sciences, 2016, 53(1): 51-58.
[22]	张星耀, 陈海燕, 梁军, 等. 金黄壳囊孢菌(Cytospora chrysosperma)的培养性状和营养体亲和性[J]. 西北农林科技大学学报(自然科学版), 2007, 35(3): 99-105.
	ZHANG Xingyao, CHEN Haiyan, LIANG Jun, et al. Cultural morphology and vegetative compatibility of Cytospora chrysosperma isolates[J]. Journal of Northwest A & F University (Natural Science Edition), 2007, 35(3): 99-105.
[23]	张俊娥, 范鑫磊, 梁英梅, 等. 杨树腐烂病菌表观特征及遗传多样性分析[J]. 北京林业大学学报, 2017, 39(7): 76-86.
	ZHANG June, FAN Xinlei, LIANG Yingmei, et al. Analysis of appearance features and genetic diversity of Cytospora chrysosperma causing poplar canker[J]. Journal of Beijing Forestry University, 2017, 39(7): 76-86.
[24]	孙祥瑞, 朱杰华, 刘蕊, 等. 几种树皮腐烂病菌的分子鉴定[J]. 河北农业大学学报, 2010, 33(6): 36-42.
	SUN Xiangrui, ZHU Jiehua, LIU Rui, et al. Molecular identification of pathogens of several trees canker[J]. Journal of Agricultural University of Hebei, 2010, 33(6): 36-42.
[25]	冀瑞卿, 李玉, 宋瑞清, 等. 金黄壳囊孢菌rDNA ITS序列测定及生理学特性[J]. 吉林农业大学学报, 2010, 32(5): 483-487.
	JI Ruiqing, LI Yu, SONG Ruiqing, et al. Cultural characteristics and rDNA its sequence analysis of Cytospora chrysosperma[J]. Journal of Jilin Agricultural University, 2010, 32(5): 483-487.
[26]	HAN Yuli, WANG Di, ZHOU Jie, et al. Intragenomic polymorphisms of the ITS, EF1-α and RPB2 sequences of Ganoderma australe[J]. Mycosystema, 2019, 38(5): 679-685. DOI
[27]	陆家云. 植物病原真菌学[M]. 北京: 中国农业出版社, 2001.
	LU Jiayun. Plant pathogeny mycology[M]. Beijing: China Agriculture Press, 2001.
[28]	范鑫磊. 中国黄河流域壳囊孢属的分类和系统学研究[D]. 北京: 北京林业大学, 2016.
	FAN Xinlei. Taxonomy and systematics of the genus Cytospora in the Yellow River Basin of China[D]. Beijing: Beijing Forestry University, 2016.
[29]	马荣. 新疆壳囊孢属真菌的分类及系统学研究[D]. 北京: 北京林业大学, 2017.
	MA Rong. Study on taxonomy and systematics of the fungi of the genus Cystispora in Xinjiang[D]. Beijing: Beijing Forestry University, 2017.

分离株 Strain	寄主 Host plant	寄主植物科 Host plant family	寄主植物属 Host plant genus	采集地点 Collection site	地理位置 Geographic position	海拔 Elevation(m)
KTST	山桃 Prunus davidiana	蔷薇科 Rosaceae Juss.	桃属 Amygdalus L.	新疆奎屯市	44°26'35″N,84°54'24″E	470
TDST	山桃 Prunus davidiana	蔷薇科 Rosaceae Juss.	桃属 Amygdalus L.	新疆阿拉尔市	40°32'30″N,81°17'35″E	1 010

分离株 Strain	寄主 Host plant	寄主植物科 Host plant family	寄主植物属 Host plant genus	采集地点 Collection site	地理位置 Geographic position	海拔 Elevation(m)
KTST	山桃 Prunus davidiana	蔷薇科 Rosaceae Juss.	桃属 Amygdalus L.	新疆奎屯市	44°26'35″N,84°54'24″E	470
TDST	山桃 Prunus davidiana	蔷薇科 Rosaceae Juss.	桃属 Amygdalus L.	新疆阿拉尔市	40°32'30″N,81°17'35″E	1 010

基因名称 Gene	引物名称 Primer	引物序列 Primer sequence	反应程序 Reaction program
核糖体基因转录间隔区 ITS	ITS1	5’-CCGTAGGTGAACCTGCGG-3’	95℃预变性5 min,95℃变性1 min,48℃退火1 min,72℃延伸1 min,72℃延伸10 min,35个循环
核糖体基因转录间隔区 ITS	ITS4	5’-TCCTCCGCTTATTGATATGC-3’
延伸因子 EF1-a	EF1-512F	5’-ATGTGCAAGGCCGGTTTCGC-3’	95℃预变性5 min,95℃变性1.5 min,56℃退火1 min,72℃延伸1.5 min,72℃延伸10 min,35个循环
延伸因子 EF1-a	EF1-783R	5’-TACGAGTCCTTCTGGCCCAT-3’
RBP2	RBP2-5F	5’-GATCGATCACGATGATCATCTTTCGG-3’	95℃预变性5 min,95℃变性1.5 min,56℃退火1 min,72℃延伸1.5 min,72℃延伸10 min,35个循环
RBP2	RBP2-7Cr	5’-CCCATAGGCTTGTCTTAGCCCAT-3’

基因名称 Gene	引物名称 Primer	引物序列 Primer sequence	反应程序 Reaction program
核糖体基因转录间隔区 ITS	ITS1	5’-CCGTAGGTGAACCTGCGG-3’	95℃预变性5 min,95℃变性1 min,48℃退火1 min,72℃延伸1 min,72℃延伸10 min,35个循环
核糖体基因转录间隔区 ITS	ITS4	5’-TCCTCCGCTTATTGATATGC-3’
延伸因子 EF1-a	EF1-512F	5’-ATGTGCAAGGCCGGTTTCGC-3’	95℃预变性5 min,95℃变性1.5 min,56℃退火1 min,72℃延伸1.5 min,72℃延伸10 min,35个循环
延伸因子 EF1-a	EF1-783R	5’-TACGAGTCCTTCTGGCCCAT-3’
RBP2	RBP2-5F	5’-GATCGATCACGATGATCATCTTTCGG-3’	95℃预变性5 min,95℃变性1.5 min,56℃退火1 min,72℃延伸1.5 min,72℃延伸10 min,35个循环
RBP2	RBP2-7Cr	5’-CCCATAGGCTTGTCTTAGCCCAT-3’

分离株 Strain	发病率 Morbidity (%)	显症时间 Disease time (h)	病斑扩展速度 Rate of lesion expansion (cm/d)	产孢时间 Sporulation time(d)	分生孢子角颜色 Conidia color	病斑颜色 Lesion color
KTST	100	48	0.56	13	橘黄色	褐色
TDST	100	48	0.79	15	橘黄色	褐色
CK	0	0	0	-	-	-