新疆发现甜菜新病害甜菜黄萎病

doi:10.6048/j.issn.1001-4330.2022.02.020

摘要/Abstract

摘要：

【目的】 研究甜菜黄萎病病原及病名,为有效防治该病害奠定基础。【方法】 采用常规组织分离法对病株叶柄组织进行分离、纯化;通过牙签接种法对纯培养菌株进行致病性测定;结合形态学特征与多基因序列分析方法对病原菌进行鉴定。【结果】 分离菌株的菌落形态、分生孢子梗、分生孢子以及厚垣孢子的形态与变黑轮枝菌(Gibellulopsis nigrescens)基本一致; 基于ITS、LSU及ACT基因构建的系统进化树显示,菌株BV4均与经G. nigrescens 聚为一支,且同源性分别为99%、99%、100%。【结论】 G. nigrescens可侵染甜菜并引起甜菜黄萎症状,所致病害可定名为甜菜黄萎病。

关键词: 甜菜; 黄萎病; 病原鉴定; 变黑轮枝菌

Abstract:

【Objective】 To identify the pathogenic species for this disease and determine the disease name can lay a foundation for the prevention and control of disease. 【Method】 A conventional tissue isolation method was employed to isolate and purify the tissues of the sugar beet plants with the disease; the pathogenicity of pure-culture strains of the pathogen was determined with a toothpick inoculation method; the pathogen was identified according to the morphological characteristics and the results of multiple-gene sequence analysis. 【Result】 The isolated strain had the similar characteristic on cultural, conidiophores, conidia and chlamydospores to the reported Gibellulopsis nigrescens; Construction of phylogenetic based on ITS, LSU and ACT sequences showed that the strain BV4 and G. nigrescens were grouped into one branch and the homology were 99%, 99% and 100% with G. nigrescens, respectively. 【Conclusion】 In brief, G. nigrescensis identified as the pathogen causing the wilt of sugar beet in the Xinjiang region, and the disease can be named Gibellulopsis yellow wilt disease of sugar beet.

Key words: sugar beet; yellow wilt; pathogen identification; Gibellulopsis nigrescens

中图分类号:

S566.3

赵志强, 张卢慧, 赵全新, 郭庆元. 新疆发现甜菜新病害甜菜黄萎病[J]. 新疆农业科学, 2022, 59(2): 425-432.

ZHAO Zhiqiang, ZHANG Luhui, ZHAO Quanxin, GUO Qingyuan. Identification of Pathogen Causing Sugar Beet Gibellulopsis Yellow Wilt Based on Multiple Gene Sequence Analysis[J]. Xinjiang Agricultural Sciences, 2022, 59(2): 425-432.

图/表 8

表 1 扩增片段及其引物

Table 1 Primer sequence for PCR amplification

位点名称 Clone name	引物名称 Primer name	引物类型 Primer type	引物序列(5'-3') Primer Sequence	引物长度 The length of primer (bp)	文献来源 Reference
ITS	ITS1	Forward	TCCGTAGGTGAACCTGCGG	19	[9]
ITS	ITS4	Reverse	TCCTCCGCTTATTGATATGC	20	[9]
LSU	LROR	Forward	ACCCGCTGAACTTAAGC	17	[10]
LSU	LR5	Reverse	TCCTGAGGGAAACTTCG	17	[10]
ACT	ACT 512F	Forward	ATGTGCAAGGCCGGTTTCGC	20	[11]
ACT	ACT 783R	Reverse	TACGAGTCCTTCTGGCCCAT	20	[11]

图1 甜菜黄萎病田间症状

Fig.1 Sugar beet yellow wilt Symptoms on naturally infected leaf

图2 甜菜黄萎病室内接种症状注:A,B.发病甜菜植株;C.健康甜菜植株

Fig.2 Symptom after inoculation with Gibellulopsis nigrescens strain BV4 indoor Note:A,B.Diseased plant; C.Healthy plant

图3 病原菌形态特征注:A.PDA培养基上的菌落;B.分生孢子梗;C.分生孢子;D.厚垣孢子标尺:B,C,D=20 μm

Fig.3 Morphology characteristics of the pathogen causing Gibellulopsis yellow wilt on sugar beet Note:A.Colony on PDA; B.Conidiophore ; C.Condium; D.Chlamydospore.Scale bars:B,C,D=20 μm

图4 基于ITS、LSU、ACT基因扩增的电泳图

Fig 4. The PCR amplification product of ITS、LSU and ACT sequences

图5 甜菜黄萎病病原菌菌株BV4的ITS序列系统进化树

Fig.5 Phylogenetic tree of the pathogenic strain BV4 based on rDNA-ITS sequence

图6 甜菜黄萎病病原菌菌株BV4的LSU序列系统进化树

Fig.6 Phylogenetic tree of the pathogenic strain BV4 based on rDNA-LSU sequence

图7 甜菜黄萎病病原菌菌株BV4的ACT序列系统进化树

Fig.7 Phylogenetic tree of the pathogenic strain BV4 based on ACT sequence

参考文献 30

[1]	张冰. 新疆甜菜产业发展研究[D]. 北京: 中国农业科学院, 2010.
	ZHANG Bing. Study on the development of sugar beet industry in Xinjiang[D]. Beijing: Chinese Academy of Agricultural Sciences, 2010.
[2]	国家统计局. 中国统计年鉴[J]. 北京: 中国统计出版社, 2019.
	Nation Bureau of Statistics of China. China Statistical Yearbook 2019[J]. Beijing: China Statistics Press, 2019.
[3]	董金皋, 李洪连, 王建明, 等. 农业植物病理学[M].(第2版.) 北京: 中国农业出版社, 2009: 226.
	DONG Jingao, LI Honglian, WANG Jianming, et al. Agricultural Plant pathology [M].(2rd Ed.) Beijing: China Agricultural Press, 2009: 226.
[4]	魏良民, 冯建忠. 新疆甜菜黄萎病发生的调查研究[J]. 中国糖料, 2001, 23(4):31-33.
	WEI Liangmin, FENG Jianzhong. Investigation on sugar beet yellow wilt disease happened in Xinjiang[J]. Sugar Crops of China, 2001, 23(4):31-33.
[5]	刘正简, 王瑞英, 沈菊英, 等. 甜菜黄萎病[J]. 植物保护学报, 1985, 12(2):118,114.
	LIU Zhengjian, WANG Ruiying, SHENG Juying, et al. Yellow wilt of sugar beet[J]. Journal of Plant Protection, 1985, 12(2):118,114.
[6]	Brantner J R, Windels C E, Omer M A. Verticillium dahliae Causes Wilt on Sugar Beet Following Potato in Eastern North Dakota[J]. Plant Health Progress, 2008.
[7]	Zare R, Gams W, Starink-Willemse M, et al. Gibellulopsis, a suitable genus for Verticillium nigrescens, and Musicillium, a new genus for V.theobromae[J]. Nova Hedwigia, 2007, 85(3-4):463-489. DOI URL
[8]	黄小琴, 刘勇, 张蕾, 等. 四川盆地主栽油菜品种菌核病抗性评价及稳定性分析[J]. 中国油料作物学报, 2012, 34(5):514-517.
	HUANG Xiaoqin, LIU Yong, ZHANG Lei, et al. Resistance evaluation and stability of rapeseed cultivars to Sclerotinia sclerotiorum in Sichuan Province[J]. Chinese Journal of Oil Crop Sciences, 2012, 34(5):514-517.
[9]	White T J, Bruns T, Lee S. Analysis of phytogenetic relationships by amplification and direct sequencing of ribosomal RNA genes [M] New York: Academic Press, 1990, 315-322.
[10]	Vilgalys R, Hester M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species[J]. The Journal of Bacteriology, 1990, 172(8):4238-4246. DOI URL
[11]	Shimomoto Y, Sato T, Hojo H, et al. Pathogenic and genetic variation among isolates of Corynespora cassiicola in Japan[J]. Plant Pathology, 2010, 60(2):253-260. DOI URL
[12]	Pethybridge G H. Notes on some saprophytic species of fungi, associated with diseased potato plants and tubers[J]. Transactions of the British Mycological Society, 1917, 6:104-120. DOI URL
[13]	云雷. 向日葵变黑轮枝菌的鉴定与生物学特性的研究[D]. 呼和浩特:内蒙古农业大学, 2014.
	YUN Lei. Identification of Gibellulopsis nigrescens from Sunflower and Study on its Biological Characteristics[D]. Hohhot: Inner Mongolia Agricultural University, 2014.
[14]	Inderbitzin P, Bostock R M, Davis R M, et al. Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species[J]. PLos One, 2011, 6(12):e28341. DOI URL
[15]	王丽丽, 蔡超, 罗明, 等. 马铃薯黄萎病研究现状[J]. 生物安全学报, 2017, 26(1):30-38.
	WANG Lili, CAI Chao, LUO Ming, et al. Research advances of Verticillium wilt of potato[J]. Journal of Biosafety, 2017, 26(1):30-38.
[16]	王明霞, 杨继娟, 白应文, 等. 苜蓿来源的变黑轮枝菌及其致病性[J]. 西北农业学报, 2011, 20(1):165-169.
	WANG Mingxia, YANG Jijuan, BAI Yingwen. Verticillium nigrescens from alfalfa and its pathogenicity[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2011, 20(1):165-169.
[17]	Barbara D J, Clewes E. Plant pathogenic Verticillium species: how many of them are there?[J]. Molecular Plant Pathology, 2003, 4(4):297-305. DOI PMID
[18]	商鸿生, 杨家荣, 赵小明. 苜蓿黄萎病菌与轮枝孢属近似种的比较研究[J]. 草业学报, 1998, 7(4):32-37.
	SHANG Hongsheng, YANG Jiarong, ZHAO Xiaoming. A Comparative Study on the Pathogen of Verticillium Wilt of Alfalfa and Other Verticillium Species[J]. Acta Prataculturae Sinica, 1998, 7(4):32-37.
[19]	张传博, 苏晓庆. 几种基于基因组DNA的真菌分类技术研究进展[J]. 贵州师范大学学报(自然科学版), 2006, 24(1):113-118.
	ZHANG Chuanbo, SU Xiaoqing. Advanced of research on several technologies for fungi identification based on genome DNA[J]. Journal of Guizhou Normal University (Natural science Ed.), 2006, 24(1):113-118.
[20]	李艳春, 吴刚, 杨祝良. 我国云南食用牛肝菌的DNA条形码研究[J]. 植物分类与资源学报, 2013, 35(6):725-732.
	LI Yanchun, WU Gang, YANG Zhuliang. DNA Barcoding of Edible Boletes (Boletaceae) from Yunnan, China[J]. Plant Diversity and Resources, 2013, 35(6):725-732.
[21]	Pfister D H, Slater C, Hansen K. Chorioactidaceae: a new family in the Pezizales (Ascomycota) with four genera[J]. Mycological Research, 2008, 112(5):513-527. DOI URL
[22]	Vu D, Groenewald M, Vries M D, et al. Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom Fungi and reveals thresholds for fungal species and higher tax on delimitation[J]. Studies in Mycology, 2019, 92:135-154. DOI PMID
[23]	Karadimos D A, Karaoglanidis G S, Klonari K. First Report of Verticillium Wilt of Sugar Beet, Caused by Verticillium dahliae, in Greece[J]. Plant Disease, 2000, 84(5):593. DOI PMID
[24]	Strausbaugh C A, Eujayl I A, Martin F N. Pathogenicity, vegetative compatibility and genetic diversity of Verticillium dahliae isolates from sugar beet[J]. Canadian Journal of Plant Pathology, 2016, 38(4):492-505. DOI URL
[25]	Gaskill J O, Krentzer W A. Verticillium wilt of sugar beet[J]. Phytopathology, 1940, 30(9):769-774.
[26]	Isaac I. A comparative study of pathogenic isolates of Verticillium[J]. Transactions of the British Mycological Society, 1949, 32(2):137-157. DOI URL
[27]	陈吉棣. 变黑轮枝菌—我国几种植物上首次报道的一种轮枝菌[J]. 植物病理学报, 1982, 12(4): 35-39,67-68.
	CHEN Jidi. Verticillium nigrescens Pethybr.A newly recorded species from several plants in China[J]. Acta Phytopathologica Sinica, 1982, 12(4): 35-39,67-68.
[28]	王克荣, 陆家云. 变黑轮枝菌种的鉴定及其致病力的测定[J]. 南京农业大学学报, 1987, 10(2):59-62.
	WANG Kerong, LU Jiayun. Identification and pathogenicity determination of isolates of Verticillium nigrescens Pethybra[J]. Journal of Nanjing Agricultural University, 1987, 10(2):59-62.
[29]	段维军, 郭立新, 张慧丽, 等. 进境台湾芹菜上一株变黑轮枝菌的鉴定[J]. 植物检疫, 2012, 26(4):30-34.
	DUAN Weijun, GUO Lixing, ZHANG Huili, et al. Identification of Verticillium nigrescens isolated in celery From Taiwan[J]. Plant Quarantine, 2012, 26(4):30-34.
[30]	Hu X P, Wang M X, Hu D F, et al. First Report of Wilt on Alfalfa in China Caused by Verticillium nigrescens[J]. Plant Disease, 2011, 95(12):159.