苹果树腐烂病病原种类及其亲缘关系分析

doi:10.6048/j.issn.1001-4330.2022.03.020

摘要/Abstract

摘要：

【目的】研究苹果树腐烂病菌的种类及其亲缘关系,为有效防治苹果树腐烂病提供理论依据。【方法】以阿克苏地区不同区域苹果树腐烂病病样为材料,采用常规组织分离法和枝条烫伤接种法分离和鉴定菌株,记录形态学特征和发病情况;利用分子生物学方法获得β-tubulin与EF1-α序列,在NCBI网站对比分析Blast同源性,采用邻接法构建系统发育树分析同源性。【结果】菌丝生长最适温度 20~30℃,最适pH值为5~6,最适碳氮源分别是葡萄糖和蛋白胨;共分离出11个菌株,将菌株接种到健康苹果枝条上均可发病且能分离出相同的菌株,各菌株分别与Valsa mali和Valsa ceratosperma具有较近的亲缘关系。【结论】阿克苏地区苹果树腐烂病菌为黑腐皮壳属V. mali(无性型C. mali)和V. ceratosperma(无性型C. sacculus),其中V. mali为阿克苏地区的主要致病种,在PDA上存在不同类型的培养性状。

关键词: 苹果树; 腐烂病菌; β-tubulin; EF1-α; 培养性状

Abstract:

【Objective】 Based on the observation of the characteristics of pathogen culture, combined with molecular biology technology, to determine the species and related relationship of apple tree rot fungus in Aksu area in the hope of providing theoretical basis for effective control of apple canker. 【Methods】 The pure culture of Apple Tree Valsa Canker was obtained by Koch's Rule. The morphology and culture characters were observed and recorded. The DNA of the strain was extracted by improved CTAB method and then sequenced. After sequencing, the blast homology was compared and analyzed on NCBI website. Then the β - tubulin and EF1 - α sequences were analyzed by adjacency method and after that, phylogenetic tree was constructed. 【Results】 The optimum temperature for mycelium growth was 20-30℃, the optimum PH value was 5-6, and the optimum carbon and nitrogen sources were glucose and peptone; A total of 11 strains were isolated. The strain could be inoculated on healthy apple branches and the same strains could be isolated. It turned out that each strain was closely related to Valsa Mali, Valsa ceratosperma. 【Conclusion】 The apple tree rotting bacteria in Aksu area are V. Mali (asexual C. Mali) and V. ceratosperma (asexual C. sacculus), among which, V. Mali is the main pathogenic species. There are different types of culture characters on PDA.

Key words: apple tree; valsa canker; β-tubulin; EF1-α; culture traits

中图分类号:

S436.611

李亚鹏, 张王斌, 仪子博. 苹果树腐烂病病原种类及其亲缘关系分析[J]. 新疆农业科学, 2022, 59(3): 700-706.

LI Yapeng, ZHANG Wangbin, YI Zibo. Analysis of Pathogen Species and Their Genetic Relationship of Apple Tree canker[J]. Xinjiang Agricultural Sciences, 2022, 59(3): 700-706.

图/表 7

参考文献 20

[1]	陈学森, 韩明玉, 苏桂林, 等. 当今世界苹果产业发展趋势及我国苹果产业优质高效发展意见[J]. 果树学报, 2010,(4): 598-604.
	CHEN Xuesen, HAN Mingyu, SU Guilin, et al. Current development trend of apple industry in the world and opinions on high quality and efficient development of apple industry in China[J]. Acta Pomology Sinica, 2010, 27(4): 598-604.
[2]	罗睿. “冰糖心苹果”试题设计[J]. 中学地理教学参考, 2018,(1): 9-10.
	LUO Rui. Test design of "bingtangxin apple"[J]. Teaching Reference of Middle School Geography, 2018,(1): 9-10.
[3]	刘淑玉, 祁东文. 浅析阿克苏苹果产业发展[J]. 山西果树, 2018,(1): 17-19.
	LIU Shuyu, QI Dongwen. Analysis on the development of apple industry in Aksu[J]. Shanxi Fruits, 2018(01): 17-19.
[4]	苟巧, 张王斌, 但红侠, 等. 不同寄主来源腐烂病菌对苹果树致病性研究[J]. 新疆农业科学, 2015, 52(11): 2085-2092.
	GOU Qiao, ZHANG Wangbin, DAN Hongxia, et al. Pathogenicity of Fusarium canker from different host sources on apple trees[J]. Xinjiang Agricultural Sciences, 2015, 52(11): 2085-2092.
[5]	曾士迈. 宏观植物病理学[M]. 北京: 中国农业出版社, 2005.
	ZENG Shimai. Macro plant pathology [M]. Beijing: China Agricultural Press, 2005.
[6]	王卫雄. 甘肃省苹果树腐烂病菌种类、遗传多样性及生防细菌培养条件优化研究[D]. 兰州:甘肃农业大学, 2015.
	WANG Weixiong. Study on species, genetic diversity and optimization of culture conditions of biocontrol bacteria of Apple Tree Canker in Gansu Province[D]. Lanzhou: Gansu Agricultural University, 2015.
[7]	刘伟. 苹果树腐烂病病斑扩展及分生孢子器发育研究[D]. 保定:河北农业大学, 2014.
	LIU Wei. Study on the expansion of apple canker and the development of conidia[D]. Baoding: Hebei Agricultural University, 2014.
[8]	李宏. 新疆特色林果主要有害生物[M]. 乌鲁木齐: 新疆生产建设兵团出版社, 2009.
	LI Hong. Main Pests of Characteristic Fruits in Xinjiang [M]. Urumqi: Xinjiang Production and Construction Corps Press, 2009.
[9]	董金皋. 农业植物病理学(第二版)[M]. 北京: 中国农业出版社. 2008: 247-248.
	DONG Jingao. Agricultural Plant Pathology (2nd Ed.)[M]. Beijing: China Agricultural Press, 2008: 247-248.
[10]	王卫雄, 徐秉良, 薛应钰, 韩健, 陈臻. 甘肃省苹果树腐烂病菌Valsa mali培养性状及致病力研究[J]. 果树学报, 2015, 32(4): 666-671, 520.
	WANG Weixiong, XU Bingliang, XUE Yingyu, et al. Culture characteristics and pathogenicity of Valsa Mali of Apple Tree Canker in Gansu Province[J]. Acta Pomologica Sinica, 2015, 32(4): 666-671, 520.
[11]	陈策. 国外对于苹果树腐烂病、桃树腐烂病以及其他同类病害的研究近况[J]. 中国果树, 1980,(S1): 68-77.
	CHEN Ce. Recent studies on apple canker, peach canker and other similar diseases abroad[J]. China Fruit Tree, 1980,(S1): 68-77.
[12]	臧睿. 中国苹果树腐烂病菌的种群组成、分子检测及其ISSR遗传分析[D]. 杨凌:西北农林科技大学, 2012.
	ZANG Rui. Population composition, molecular detection and ISSR Genetic Analysis of apple canker in China[D]. Yangling: Northwest agricultural and Forestry University, 2012.
[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. Biological characteristics and pathogenicity of apple canker pathogen isolated from Yunnan[J]. Southwest Agricultural Journal, 2015, 28(5): 2096-2102.
[14]	杜琴. 新疆主要林木腐烂病菌种类鉴定及其防治方法研究[D]. 石河子:石河子大学, 2013.
	DU Qin. Identification and control methods of main forest canker pathogens in Xinjiang[D]. Shihezi: Shihezi University, 2013
[15]	刘应敏, 尹永香, 张艳, 等. 新疆苹果树腐烂病病原菌(Cytospora schulzeri)的分离、鉴定及其生物学特性的研究[J]. 新疆农业大学学报, 2016, 39(6): 447-452.
	LIU Yingmin, YIN Yongxiang, ZHANG Yan, et al. Isolation, identification and biological characteristics of the pathogen of apple canker in Xinjiang[J]. Journal of Xinjiang Agricultural University, 2016, 39(6): 447-452.
[16]	陈玮君. 酸性PDA培养基的制作[J]. 河南农业, 1996,(1): 28.
	CHEN Weijun. Preparation of acid PDA medium[J]. Henan Agriculture, 1996, (1): 28.
[17]	臧睿, 黄丽丽, 康振生, 等. 陕西苹果树腐烂病菌(Cytospora spp.)不同分离株的生物学特性与致病性研究[J]. 植物病理, 2007,(4): 343-351.
	ZANG Rui, HUANG Lili, KANG Zhensheng, et al. Study on biological characteristics and pathogenicity of different isolates of Apple Tree Canker (Cytospora spp.) in Shaanxi[J]. Plant Pathology, 2007,(4): 343-351.
[18]	唐俊煜. 新疆绿洲生态枣园林木腐烂病菌种类鉴定及交互侵染机制研究[D]. 阿拉尔:塔里木大学, 2016.
	TANG Junyu. Species identification and cross infection mechanism of Tree Canker pathogens in Xinjiang Oasis Ecological jujube garden[D]. Aral: Tarim University, 2016.
[19]	Saitou N,. and Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, (4): 406-425. DOI PMID
[20]	郭开发. 新疆林木腐烂病菌种类鉴定及其主要种Valsa mali遗传分化和初侵染源快速检测研究[D]. 石河子:石河子大学, 2016.
	GUO Kaifa. Identification of Tree Canker pathogens in Xinjiang and rapid detection of its main species, Valsa Mali, genetic differentiation and primary infection source[D]. Shihezi: Shihezi University, 2016.

编号 Number	寄主 Host	采集地点 Collection location	采集时间 Acquisition time
HQP₁	苹果	红旗坡一分厂	2018.5.16
HQP₂	苹果	红旗坡一分厂	2018.5.16
HQP₃	苹果	红旗坡一分厂	2018.5.16
YLC₁	苹果	第一师5团7连	2018.5.17
YLC₂	苹果	第一师5团7连	2018.5.17
YLC₃	苹果	第一师5团7连	2018.5.17

编号 Number	寄主 Host	采集地点 Collection location	采集时间 Acquisition time
HQP₁	苹果	红旗坡一分厂	2018.5.16
HQP₂	苹果	红旗坡一分厂	2018.5.16
HQP₃	苹果	红旗坡一分厂	2018.5.16
YLC₁	苹果	第一师5团7连	2018.5.17
YLC₂	苹果	第一师5团7连	2018.5.17
YLC₃	苹果	第一师5团7连	2018.5.17

菌株类别 Strain classification	菌落颜色 Colony color (3 d)	菌落颜色 Colony color (15 d)	菌落边缘 Edge of the colony	菌丝气生性 Mycelial aerogenesis	菌落直径 Diameter of the colony (2 d)(mm)	平均生长速率 Verage growth rate (mm/d)	菌落产孢情况 Sporulation of bacterial colonies
淡黄色菌株 Pale yellow strain	淡黄色	黄色、黄褐色	放射状	无	55.12±1.26	26.69±1.32	6株产孢第7~8 d
乳白色菌株 Milky white strain	乳白色	黄褐色	羽毛状	无	44.98±2.81	23.16±1.75	2株产孢第7~8 d

菌株类别 Strain classification	菌落颜色 Colony color (3 d)	菌落颜色 Colony color (15 d)	菌落边缘 Edge of the colony	菌丝气生性 Mycelial aerogenesis	菌落直径 Diameter of the colony (2 d)(mm)	平均生长速率 Verage growth rate (mm/d)	菌落产孢情况 Sporulation of bacterial colonies
淡黄色菌株 Pale yellow strain	淡黄色	黄色、黄褐色	放射状	无	55.12±1.26	26.69±1.32	6株产孢第7~8 d
乳白色菌株 Milky white strain	乳白色	黄褐色	羽毛状	无	44.98±2.81	23.16±1.75	2株产孢第7~8 d

编号 Number	寄主 Host	菌株类别 Strain classification	病原菌 Pathogenic bacteria
HQP2-1	苹果	淡黄色	V.ceratosperma
HQP2-2	苹果	淡黄色	V.ceratosperma
HQP2-3	苹果	淡黄色	V.ceratosperma
HQP2-4	苹果	淡黄色	V.ceratosperma
YLC3-1	苹果	淡黄色	V.mali
YLC3-2	苹果	淡黄色	V.mali
HQP3-1	苹果	乳白色	V.mali
YLC1-1	苹果	乳白色	V.mali
YLC1-2	苹果	乳白色	V.mali
YLC2-1	苹果	乳白色	V.mali