芍药黑斑病病原菌鉴定及其对杀菌剂敏感性分析

doi:10.16420/j.issn.0513-353x.2020-0236

摘要/Abstract

摘要：

对在江苏省扬州市芍药（Paeonia lactiflora）叶片上发现的一种黑斑病,采集其病叶,分离和纯化病原物,根据柯赫氏法则明确其致病性。基于病原物形态特征和多基因（rDNA-ITS、endoPG、OPA2-1、Alt a 1）序列联合分析,鉴定该病原物为链格孢（Alternaria alternata）。该病原菌的最适生长条件是:温度为28 ℃,pH 7.0,最适碳源为可溶性淀粉,最适氮源为硝酸钾。室内毒力测定发现,在测试的4种杀菌剂中,嘧菌酯和吡唑醚菌酯乳油对病原菌离体生长的抑制作用较强,而戊唑醇和异菌脲对其生长的抑制作用较差。

关键词: 芍药, 黑斑病, 链格孢, 生长条件, 杀菌剂

Abstract:

A black spot disease was discovered on peony（Paeonia lactiflora）leaves in Yangzhou City,Jiangsu Province in 2017. To determine the causing agent,the fungus was isolated and purified from the diseased leaves,and was tested for its pathogenicity following Koch’s postulation. The fungus was identified as Alternaria alternata based on its morphological characteristics and the combined sequence analyses of rDNA-ITS,endoPG,OPA2-1 and Alt a 1. Analysis of its growth conditions showed that the optimal growth conditions of A. alternata on Potato Dextrose Agar（PDA）include the temperature at 28 ℃ and pH 7.0. On defined Czapek media,soluble starch and KNO₃ are the optimal carbon source and nitrogen source,respectively. What’s more,among four fungicides tested in toxicity assays,both Azoxystrobin and Pyraclostrobin exhibited more stronger inhibition effect on the mycelial growth of the pathogen on PDA,compared with Tebuconazole and Iprodione.

Key words: peony, leaf black spot disease, Alternaria, growth conditions, fungicide

中图分类号:

S682.1

陶航, 扎依娜·玛合巴提, 张烨, 孙李瞳, 黄沈鑫, 张子辉, 刘旺, 施宁雪, 陈孝仁. 芍药黑斑病病原菌鉴定及其对杀菌剂敏感性分析[J]. 园艺学报, 2021, 48(1): 173-182.

TAO Hang, ZAYINA Mahabat, ZHANG Ye, SUN Litong, HUANG Shenxin, ZHANG Zihui, LIU Wang, SHI Ningxue, CHEN Xiaoren. Identification,Growth Conditions and Fungicide Sensitivity of the Pathogen Causing Black Spot Disease on Peony Leaves[J]. Acta Horticulturae Sinica, 2021, 48(1): 173-182.

图/表 8

参考文献 23

[1]	Chen Xiao-hong, Ye Hua-zhi, Yan Ji-ming, Qin Yun, Wu Guang-qing. 2006. Investigation on the diseases of medicinal plants in Sichuan Province and pathogen identification I. A list of diseases of main cultivated medicinal plants. Southwest China Journal of Agricultural Sciences, 19(1):58-62. (in Chinese)
	陈小红, 叶华智, 严吉明, 秦芸, 伍光庆. 2006. 四川药用植物病害调查与病原鉴定Ⅰ. 主要栽培药用植物病害. 西南农业学报, 19(1):58-62.
[2]	Chen X R, Liu B B, Xing Y P, Cheng B P, Liu M L, Tong Y H, Xu J Y. 2016. Identification and characterization of Phytopythium helicoides causing stem rot of Shatangju mandarin seedlings in China. European Journal of Plant Pathology, 146:715-727. doi: 10.1007/s10658-016-0952-4 URL
[3]	Fang Zhong-da. 1998. Methods used in plant pathology. 3rd ed. Beijing: China Agriculture Press. (in Chinese)
	方中达. 1998. 植病研究法. 3版. 北京: 中国农业出版社.
[4]	Huang Zhang-xin. 2001. Experiment guidance for plant chemical protection. Beijing: China Agriculture Press. (in Chinese)
	黄彰欣. 2001. 植物化学保护实验指导. 北京: 中国农业出版社.
[5]	Hong S G, Cramer R A, Lawrence C B, Pryor B M. 2005. Alt a 1 allergen homologs from Alternaria and related taxa:analysis of phylogenetic content and secondary structure. Fungal Genetics and Biology, 42(2):119-129. doi: 10.1016/j.fgb.2004.10.009 URL
[6]	Jiang Yao, Ge Jin-tao, Ning Chuan-long, Tao Jun. 2013. Disease investigation of herbaceous peony and disease susceptibility of cultivars. Jiangsu Agricultural Science, 41(1):125-127. (in Chinese)
	姜瑶, 葛金涛, 宁传龙, 陶俊. 2013. 芍药病害种类及其品种感病性调查. 江苏农业科学, 41(1):125-127.
[7]	Lan Ying, Zhao Gui-hua, Zheng Peng-peng. 1984. Studies on Cladosporium red spot of peony. Journal of Nanjing Institute of Forestry,(1):16-29. (in Chinese)
	蓝莹, 赵桂华, 郑彭彭. 1984. 芍药红斑病的研究. 南京林业院学报,(1):16-29.
[8]	Li Gui-fan, Wang Qi-yan, Hu Guo-yan. 2001. Common disease and control in herbaceous peony in cold district. Northern Horticulture,(5):56. (in Chinese)
	李桂凡, 王启燕, 胡国岩. 2001. 高寒地区芍药露地栽培常见病害及其防冶. 北方园艺,(5):56.
[9]	Li Li, Song Shu-xiang, Liu Hui-xiang, Guo Xian-feng. 2016. Identification of red spot pathogens on peony in Shandong Province. Acta Horticulturae Sinica, 43(2):365-372. (in Chinese)
	李丽, 宋淑香, 刘会香, 郭先锋. 2016. 山东省芍药红斑病病原菌鉴定. 园艺学报, 43(2):365-372.
[10]	Liu Chu-rong, Dong Yue, Lü Qing-yang, Li Ying-bin, Jiang Na, Luo Lai-xin, Zhu Tian-sheng, Li Jian-qiang. 2019. Screening of fungicides and their cocktail mixture for controlling black spot of jujube. Plant Protection, 45(5):263-268. (in Chinese)
	刘础荣, 董玥, 吕青阳, 李迎宾, 蒋娜, 罗来鑫, 朱天生, 李健强. 2019. 防治红枣黑斑病的杀菌剂筛选和复配研究. 植物保护, 45(5):263-268.
[11]	Liu Xia, Yang Ke-qiang, Jiang Xing-yin, An Hai-shan, Wang Can-can. 2013. Sensitivity of walnut-associated Alternaria alternata to four fungicides. Agrochemicals, 52(1):67-70. (in Chinese)
	刘霞, 杨克强, 姜兴印, 安海山, 王灿灿. 2013. 危害核桃的链格孢(Alternaria alternata)对4种杀菌剂的敏感性. 农药, 52(1):67-70.
[12]	Peever T L, Carpenter-Boggs L, Timmer L W, Carris L M, Bhatia A. 2005. Citrus black rot is caused by phylogenetically distinct lineages of Alternaria alternata. Phytopathology, 95:512-518. doi: 10.1094/PHYTO-95-0512 pmid: 18943316
[13]	Shi Liang-hong, Li Ling, Shen Hong-wei, Sun Feng-yi, Zhao Lan-yong. 2014. The identification and analysis of ITS sequence on tree peony red spot. Journal of Agriculture, 4(10):32-35. (in Chinese)
	石良红, 李玲, 申宏伟, 孙逢毅, 赵兰勇. 2014. 牡丹红斑病病原鉴定与ITS序列分析. 农学学报, 4(10):32-35.
[14]	Shi Yan-tong, Zhang Xiu-xin, Xue Jing-qi, Wu Rui, Shi Feng-rui. 2014. Diseases investigation of herbaceous peony and selection of resistant varieties. Southwest China Journal of Agricultural Sciences, 27(5):1979-1983. (in Chinese)
	石颜通, 张秀新, 薛璟祺, 吴蕊, 石丰瑞. 2014. 芍药病害调查及抗性品种筛选. 西南农业学报, 27(5):1979-1983.
[15]	White T J, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics//Innis M A,Gelfand D H,Sninsky J J,White T J. PCR protocols:a guide to methods and applications. PCR protocols: a guide to methods and applications. San Diego:Academic Press.
[16]	Wu Yu-zhu, Ji Yan-ping, Liu Yu, Zhao Gui-hua, Niu Ying-fu, Wang Hai-ming, Zhao Hai-jun. 2005. Study on red spot disease of peony tree. Forest Research, 15(6):711-716. (in Chinese)
	吴玉柱, 季延平, 刘愚, 赵桂华, 牛迎福, 王海明, 赵海军. 2005. 牡丹红斑病的研究. 林业科学研究, 15(6):711-716.
[17]	Xiao Chun-fang, Tian Heng-lin, Shen Yan-fen, Gao Jian-hua, Zhang Yuan-xue, Chen Jia-ji. 2015. Toxicity and control efficacy of azoxystrobin against potato early blight Alternaria solani. Hunan Agricultural Sciences,(8):46-48. (in Chinese)
	肖春芳, 田恒林, 沈艳芬, 高剑华, 张远学, 陈家吉. 2015. 嘧菌酯对马铃薯早疫病菌Alternaria solani的毒力及防效. 湖南农业科学,(8):46-48.
[18]	Yang Rui-xian, Wang Zu-hua, Ye Wen-yu. 2010. Identification of four kinds of fungal diseases on tree peony and herbaceous peony in Luoyang. Subtropical Agriculture Research, 6(2):102-105. (in Chinese)
	杨瑞先, 王祖华, 叶文雨. 2010. 洛阳牡丹、芍药4种真菌性病害鉴定. 亚热带农业研究, 6(2):102-105.
[19]	Yang Ting, Yang Kuan, He Chi, Wang Hui-ling, Wang Wen-peng, Jin Xin, Zhu Shu-sheng, Zhu You-yong, He Xia-hong. 2018. Biological characteristics of Alternaria alternata causing dark speckle disease of Panax notoginseng. Journal of Chinese Medicinal Materials, 41(4):763-770. (in Chinese)
	杨婷, 杨宽, 何迟, 王慧玲, 王文鹏, 金鑫, 朱书生, 朱有勇, 何霞红. 2018. 三七黑斑病病原菌Alternaria alternata Keissl.生物学特性研究. 中药材, 41(4):763-770.
[20]	Yu Si-jia, Zhang Zhou-shuang, Lei Zeng-pu, Fei Yu-zhen. 1993. The preliminary study of integrated management of major diseases of tree peony and peony in Beijing area. Journal of Beijing Forestry University, 15(2):103-108. (in Chinese)
	俞思佳, 张佐双, 雷增普, 费玉珍. 1993. 北京地区牡丹和芍药主要病害的综合防治. 北京林业大学学报, 15(2):103-108.
[21]	Zhang D, Gao F, Li W X, Jakovlić I, Zou H, Zhang J, Wang G T. 2020. PhyloSuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources, 20:348-355. doi: 10.1111/1755-0998.13096 pmid: 31599058
[22]	Zhang Jun-hua, Li Yun-peng, Han Yu-tong, Yang Ming-xiu, Song Shuang, Zhong Qing-yan, Fan Lin. 2018. Study on pathogen identification and biological characteristics of rice brown panicle disease in Heilongjiang Province. Journal of Northeast Agricultural University, 49(1):27-38. (in Chinese)
	张俊华, 李云鹏, 韩雨桐, 杨明秀, 宋爽, 钟庆艳, 范琳. 2018. 黑龙江省水稻褐变穗病病原鉴定及生物学特性研究. 东北农业大学学报, 49(1):27-38.
[23]	Zhang Tian-yu. 2003. Flora Fungorum sinicorum. Vol 16. Alternaria. Beijing: Science Press. (in Chinese)
	张天宇. 2003. 中国真菌志. 16卷. 链格孢属. 北京: 科学出版社.

种名 Species	菌株 Isolate	NCBI基因数据库登录号 GenBank accession
种名 Species	菌株 Isolate	rDNA-ITS	endoPG	OPA2-1	Alt a 1
链格孢菌Alternaria alternata	EGS 34-016	AF347031	AY295024	AY295065	KP275691
	EGS 34-039	―	AY295025	AY295064	―
	39-190	―	AY629218	AY295060	―
	S2	MT269274	MT505726	MT505728	MT505725
橘树链格孢Alternaria citriarbusti	SH-MIL-15s	―	AY295026	AY295057	―
橘斑链格孢Alternaria citrimacularis	BC2-RLR-32s	JX397903	JX397906	AY295069	―
草莓链格孢Alternaria fragaria	0-187	―	AY629222	AY631462	―
梨黑斑链格孢Alternaria gaisen	EGS 90-0512	―	AY295033	AY631460	KP275694
粗柠檬链格孢Alternaria limoniasperae	PR325	―	AY629233	AY295062	―
蜜柚链格孢Alternaria tangelonis	EV-MIL-2s	―	AY295031	AY295066	JQ646392
细极链格孢Alternaria tenuissima	EGS 34-015	AF347032	JQ811977	AY631463	―
土耳其链格孢Alternaria turkisafria	EGS 44-159	―	AY295022	AY295054	JQ646404

种名 Species	菌株 Isolate	NCBI基因数据库登录号 GenBank accession
种名 Species	菌株 Isolate	rDNA-ITS	endoPG	OPA2-1	Alt a 1
链格孢菌Alternaria alternata	EGS 34-016	AF347031	AY295024	AY295065	KP275691
	EGS 34-039	―	AY295025	AY295064	―
	39-190	―	AY629218	AY295060	―
	S2	MT269274	MT505726	MT505728	MT505725
橘树链格孢Alternaria citriarbusti	SH-MIL-15s	―	AY295026	AY295057	―
橘斑链格孢Alternaria citrimacularis	BC2-RLR-32s	JX397903	JX397906	AY295069	―
草莓链格孢Alternaria fragaria	0-187	―	AY629222	AY631462	―
梨黑斑链格孢Alternaria gaisen	EGS 90-0512	―	AY295033	AY631460	KP275694
粗柠檬链格孢Alternaria limoniasperae	PR325	―	AY629233	AY295062	―
蜜柚链格孢Alternaria tangelonis	EV-MIL-2s	―	AY295031	AY295066	JQ646392
细极链格孢Alternaria tenuissima	EGS 34-015	AF347032	JQ811977	AY631463	―
土耳其链格孢Alternaria turkisafria	EGS 44-159	―	AY295022	AY295054	JQ646404

碳源 Carbon source	菌落直径/mm Colony diameter	氮源 Nitrogen source	菌落直径/mm Colony diameter
蔗糖Sucrose（对照Control）	45.1 ± 3.9 cde	硝酸钾KNO₃（对照Control）	44.8 ± 0.8 a
葡萄糖Dextrose	43.4 ± 2.5 e	硝酸铵NH₄NO₃	34.5 ± 1.1 b
麦芽糖 Maltose	63.8 ± 4.1 a	硝酸钙Ca(NO₃)₂	42.0 ± 2.0 a
乳糖Lactose	59.7 ± 1.8 ab	氯化铵NH₄Cl	35.2 ± 2.7 ab
可溶性淀粉Soluble starch	67.1 ± 5.3 a	尿素Urea	26.2 ± 1.4 c
木糖Xylose	55.6 ± 1.7 abc	谷氨酸Glutamic acid	27.9 ± 2.2 c
半乳糖Galactose	49.4 ± 1.9 bcd

碳源 Carbon source	菌落直径/mm Colony diameter	氮源 Nitrogen source	菌落直径/mm Colony diameter
蔗糖Sucrose（对照Control）	45.1 ± 3.9 cde	硝酸钾KNO₃（对照Control）	44.8 ± 0.8 a
葡萄糖Dextrose	43.4 ± 2.5 e	硝酸铵NH₄NO₃	34.5 ± 1.1 b
麦芽糖 Maltose	63.8 ± 4.1 a	硝酸钙Ca(NO₃)₂	42.0 ± 2.0 a
乳糖Lactose	59.7 ± 1.8 ab	氯化铵NH₄Cl	35.2 ± 2.7 ab
可溶性淀粉Soluble starch	67.1 ± 5.3 a	尿素Urea	26.2 ± 1.4 c
木糖Xylose	55.6 ± 1.7 abc	谷氨酸Glutamic acid	27.9 ± 2.2 c
半乳糖Galactose	49.4 ± 1.9 bcd

药剂名称 Fungicide name	浓度/（mg · L^-1） Concentration					毒力回归方程 Toxicity regression equation	R²	EC₅₀/（mg · L^-1）
药剂名称 Fungicide name	1	0.5	0.1	0.05	0.01	毒力回归方程 Toxicity regression equation	R²	EC₅₀/（mg · L^-1）
嘧菌酯Azoxystrobin	39.6 ab	34.0 b	30.9 ab	20.2 b	6.2 b	y = 0.1595x + 0.4086	0.9301	3.7415 b
吡唑醚菌酯 Pyraclostrobin	45.1 a	40.7 a	33.4 a	31.8 a	20.6 a	y = 0.1165x + 0.4503	0.9843	2.6706 a
戊唑醇Tebuconazole	24.0 c	17.8 c	17.4 c	14.4 c	10.4 bc	y = 0.0592x + 0.2224	0.8870	4.8876 × 10⁴ c
异菌脲Iprodione	5.5 d	5.2 d	4.9 d	4.2 d	2.4 d	y = 0.0147x + 0.0577	0.8798	1.2260 × 10³⁰ d