[1]任胜林,梅慧玲,李映萱,等.百合枯萎病病原菌的分离鉴定及其接种浓度对发病程度的影响[J].南京农业大学学报,2020,43(6):1097-1106.[doi:10.7685/jnau.202001038]
 REN Shenglin,MEI Huiling,LI Yingxuan,et al.Isolation and identification of lily Fusarium wilt pathogen and the effect of spore suspension concentration on the extent of disease[J].Journal of Nanjing Agricultural University,2020,43(6):1097-1106.[doi:10.7685/jnau.202001038]
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百合枯萎病病原菌的分离鉴定及其接种浓度对发病程度的影响()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
43卷
期数:
2020年6期
页码:
1097-1106
栏目:
生物与环境
出版日期:
2020-11-10

文章信息/Info

Title:
Isolation and identification of lily Fusarium wilt pathogen and the effect of spore suspension concentration on the extent of disease
作者:
任胜林 梅慧玲 李映萱 杜彭涛 文涛 乔亦铸 张赫 麦远愉 董银霜 李静 黄启为
南京农业大学资源与环境科学学院/江苏省固体有机废弃物资源化高技术研究重点实验室/江苏省有机固体废弃物资源化协同创新中心/国家有机类肥料工程技术研究中心, 江苏 南京 210095
Author(s):
REN Shenglin MEI Huiling LI Yingxuan DU Pengtao WEN Tao QIAO Yizhu ZHANG He MAI Yuanyu DONG Yinshuang LI Jing HUANG Qiwei
College of Resources and Environmental Sciences/Jiangsu Provincial Key Laboratory for Organic Solid Waste Utilization/Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization/National Engineering Research Center for Organic-Based Fertilizers, Nanjing Agricultural University, Nanjing 210095, China
关键词:
百合枯萎病病原菌分离孢子悬液浓度病情指数发病率
Keywords:
lilyFusarium wiltpathogen isolationspore suspension concentrationdisease indexincidence of a disease
分类号:
S432.4
DOI:
10.7685/jnau.202001038
摘要:
[目的] 江苏宜兴是我国百合生产的传统优势产区,百合枯萎病是严重阻碍百合产业化健康发展的一个关键因子。通过分离鉴定百合枯萎病的致病菌株,接种不同浓度的病原菌孢子悬液,研究百合的发病程度,为江苏宜兴百合枯萎病的生物防治提供科学基础。[方法] 采集宜兴百合种植区典型枯萎病发病严重的植株及鳞茎分离致病菌株,通过柯赫氏法则回接试验初步确定致病性,对致病性强的菌株进行DNA提取、PCR扩增和测序,基于病原菌rDNA-ITS基因序列和GenBank中已有相关尖孢镰刀菌序列,应用MEGA 7.0软件构建系统发育树;采用刺伤百合鳞片接种不同浓度的病原菌孢子悬液与在土壤中接种不同浓度病原菌孢子悬液的方法,评估不同浓度孢子悬液对百合枯萎病发病程度的影响。[结果] 从典型枯萎病严重发病的植株上共分离得到4株病原菌株:P-OF、P-PF、P-RF和P-BF,分离率分别为25%、20%、10%和5%。柯赫氏法则回接试验表明:接种菌株P-OF、P-PF百合发病率为100%,鳞片病情指数分别为92.50、48.25、15.00、30.00;盆栽试验表明:接种菌株P-OF的百合植株最先发病,初步确定菌株P-OF的致病性最强;通过形态学和分子生物学对菌株P-OF的鉴定结果表明,从典型枯萎病严重发病百合植株中所分离的病原菌为尖孢镰刀菌(Fusarium oxysporum)。百合枯萎病的病情指数与接种病原菌孢子悬液浓度相关,百合鳞片接种试验和盆栽试验结果表明,土壤病原菌浓度为103 CFU·g-1,是导致百合植株发病的临界浓度,当土壤病原菌浓度低于105 CFU·g-1时,病情指数随接种浓度增加而增加,当土壤病原菌浓度高于105 CFU·g-1时,病情指数不再显著增加。[结论] 从江苏宜兴百合枯萎病的典型发病植株中分离到4株病原菌株,其中菌株P-OF的致病性最强,经鉴定为尖孢镰刀菌百合专化型(Fusarium oxysporum sp.f.lilii),导致百合植株发病的临界土壤病原菌浓度为103 CFU·g-1
Abstract:
[Objectives] Lily(Lilium spp.) is one of the dominant traditional production areas in Yixing City,Jiangsu Province,China. Fusarium wilt is a key stunting factor for the healthy development of lily production. The isolation and identification of the pathogenic strains of lily Fusarium wilt in Yixing City,inoculation of spore suspensions of pathogens of different concentrations and the relationship between occurrence degree and the concentration of spore suspensions of pathogens were carried out for providing scientific basis in biological control technology of lily Fusarium wilt in Yixing.[Methods] The pathogenic strains were isolated from the plants with severe blight disease and bulbs in the lily field of Yixing. The pathogenicity was preliminarily determined by the Koch’s rule backhaul experiment. The highly pathogenic strains were subjected to DNA extraction,PCR amplification and sequencing. The phylogenetic dendrogram was constructed based on the rDNA-ITS gene sequence of the pathogens and the related Fusarium oxysporum sequence in GenBank by using MEGA 7.0 software. The incidence of lily Fusarium wilt was evaluated by inoculating experiments both in inoculating pathogen spore suspension to the soil and in wounded inoculating pathogen spore suspension on the plants.[Results] A total of 4 pathogenic strains of P-OF,P-PF,P-RF and P-BF were isolated from severely blighted plants,with isolation rates of 25%,20%,10% and 5%,respectively. The back-to-back experiments of the law showed that the incidence of P-OF and P-PF was 100%,and the scale index was 92.50,48.25,15.00 and 30.00,respectively. The results of pot experiment showed that the treatment of lily plants inoculated with P-OF was the first to develop the disease. This hinted that the pathogenicity of P-OF might be the strongest. The identification of P-OF by morphology and molecular biology showed that the pathogen isolated from lily plants with severe wilt was Fusarium oxysporum. The results of lily scale inoculation and pot experiment showed that the concentration of soil pathogen at 103 CFU·g-1 was the critical concentration of lily Fusarium wilt. The disease index increased with increasing inoculation concentration within 105 CFU·g-1 and no longer increased significantly if the pathogen concentration in soil was higher than 105 CFU·g-1. The disease index of lily Fusarium wilt was related to the concentration of spore suspension of inoculated pathogens.[Conclusions] Four pathogenic strains identified as Fusarium oxysporum were isolated from Fusarium wilt plants in Yixing lily field. The P-OF was the most pathogenic strain,identified as Fusarium oxysporum sp. f. lilii. The critical concentration of soil pathogen was 103 CFU·g-1.

参考文献/References:

[1] Cao X,Shi S,Zhang Z. First report of botrytis leaf blight on lily(Lilium longiflorum)caused by botrytis cinerea in Beijing,China[J]. Plant Disease,2018,102(5):1033.
[2] 赵祥云,王文和. 拓展市场强强联手合作共赢——我国百合产业现状、存在问题和发展前景[J]. 中国花卉园艺,2017(13):10-13. Zhao X Y,Wang W H. Expanding the market,strong and strong cooperation and win-win cooperation:the current situation,problems and development prospects of China’s lily industry[J]. China Flowers and Horticulture,2017(13):10-13(in Chinese).
[3] 吕书年,赵鹂. 百合属植物资源的保护与利用[J]. 丽水学院学报,2001,23(5):35-36,38. Lü S N,Zhao L. Protection and utilization of Lilium plant resources[J]. Journal of Lishui University,2001,23(5):35-36(in Chinese).
[4] 朱茂山,关天舒. 百合主要病害及其防治关键技术[J]. 辽宁农业科学,2007(6):41-43. Zhu M S,Guan T S. The main diseases of lily and the key technologies for its control[J]. Liaoning Agricultural Sciences,2007(6):41-43(in Chinese with English abstract).
[5] van Heusden A W,Jongerius M C,van Tuyl J M,et al. Molecular assisted breeding for disease resistance in lily[J]. Acta Horticulturae,2002,572:131-138.
[6] Prados-Ligero A M,Basallote-Ureba M J,Melero-Vara J M. First report of Fusarium oxysporum f. sp. lilii and F.proliferatum affecting Lilium crops in Spain[J]. Tropical Plant Pathology,2008,33(3):235-236.
[7] 王祥会,王军,蒋小龙,等. 云南进境百合种球病虫害调查研究[J]. 江西农业学报,2005,17(3):42-45. Wang X H,Wang J,Jiang X L,et al. Investigation on diseases and insect pests of imported Lilium bulb in Yunnan Province[J]. Acta Agriculturae Jiangxi,2005,17(3):42-45(in Chinese with English abstract).
[8] 刘新月,李凡,陈海如,等. 致病性尖孢镰刀菌生物防治研究进展[J]. 云南大学学报(自然科学版),2008,30(增刊1):89-93. Liu X Y,Li F,Chen H R,et al. Research progress on biological control of pathogenic Fusarium oxysporum[J]. Journal of Yunnan University(Natural Science Edition),2008,30(S1):89-93(in Chinese with English abstract).
[9] 张丽丽,刘冬云,赵婵璞. 百合枯萎病抗性鉴定及发病因素的研究[J]. 河北农业大学学报,2012,35(6):27-32. Zhang L L,Liu D Y,Zhao C P. Identification and pathogenesis of lily Fusarium wilt resistance[J]. Journal of Hebei Agricultural University,2012,35(6):27-32(in Chinese with English abstract).
[10] Chung W C,Wu R S,Hsu C P,et al. Application of antagonistic rhizobacteria for control of Fusarium seedling blight and basal rot of lily[J]. Australasian Plant Pathology,2011,40(3):269-276.
[11] 何欣,黄启为,杨兴明,等. 香蕉枯萎病致病菌筛选及致病菌浓度对香蕉枯萎病的影响[J]. 中国农业科学,2010,43(18):3809-3816. He X,Huang Q W,Yang X M,et al. Screening and identification of pathogen causing banana Fusarium wilt and the relationship between spore suspension concentration and the incidence rate[J]. Scientia Agricultura Sinica,2010,43(18):3809-3816(in Chinese with English abstract).
[12] 王拱辰,陈辉珍. 促进镰刀菌产孢的培养基[J]. 植物病理学报,1995,25(2):165-166. Wang G C,Chen H Z. Media for promoting Fusarium sporulation[J]. Chinese Journal of Plant Pathology,1995,25(2):165-166(in Chinese with English abstract).
[13] 方中达. 植病研究方[M]. 3版. 北京:中国农业出版社,1998:122-137. Fang Z D. Plant Disease Research Party[M]. 3rd ed. Beijing:China Agriculture Press,1998:122-137(in Chinese).
[14] 梁巧兰,徐秉良,刘艳梅. 观赏百合根腐病病原鉴定及药剂筛选[J]. 甘肃农业大学学报,2004,39(1):25-28. Liang Q L,Xu B L,Liu Y M. Identification and screening of root rot of ornamental lily[J]. Journal of Gansu Agricultural University,2004,39(1):25-28(in Chinese with English abstract).
[15] 张晨智,张旭,沈其荣,等. 健康与发病香蕉植株内生细菌菌群差异[J]. 南京农业大学学报,2019,42(2):284-291. DOI:10.7685/jnau.201804036. Zhang C Z,Zhang X,Shen Q R,et al. Differences in endophytic bacterial flora of healthy and diseased banana plant[J]. Journal of Nanjing Agricultural University,2019,42(2):284-291(in Chinese with English abstract).
[16] Booth C.The Genus Fusarium[M]. Surrey:CMI,1971:14-19.
[17] Geiser D M,del mar Jiménez-Gasco M,Kang S,et al. FUSARIUM-ID v. 1.0:a DNA sequence database for identifying Fusarium[J]. European Journal of Plant Pathology,2004,110(5/6):473-479.
[18] Simonetti E,Roberts I N,Montecchia M S,et al. A novel Burkholderia ambifaria strain able to degrade the mycotoxin fusaric acid and to inhibit Fusarium spp. growth[J]. Microbiological Research,2018,206:50-59.
[19] Shen W S,Lin X G,Gao N,et al. Land use intensification affects soil microbial populations,functional diversity and related suppressiveness of cucumber Fusarium wilt in China’s Yangtze River delta[J]. Plant and Soil,2008,306(1/2):117-127.
[20] Michielse C B,Rep M. Pathogen profile update:Fusarium oxysporum[J]. Molecular Plant Pathology,2009,10(3):311-324.
[21] 边小荣,师桂英,梁巧兰,等. 兰州百合枯萎病病原菌的分离鉴定与致病性测定[J]. 甘肃农业大学学报,2016,51(4):58-64. Bian X R,Shi G Y,Liang Q L,et al. Isolation and identification of wilt disease pathogen from Lanzhou lily and its pathogenicity[J]. Journal of Gansu Agricultural University,2016,51(4):58-64(in Chinese with English abstract).
[22] 边小荣. 兰州百合枯萎病病原鉴定及病原菌生物学特性研究[D]. 兰州:甘肃农业大学,2016. Bian X R. Study on identification and biological characters of Lanzhou lily wilt pathogen[D]. Lanzhou:Gansu Agricultural University,2016(in Chinese with English abstract).
[23] 冯昱人,武志江,张玉宝. 百合枯萎病病原菌的分离鉴定及致病性研究[J]. 兰州石化职业技术学院学报,2014,14(2):38-40. Feng Y R,Wu Z J,Zhang Y B. Isolation identification and pathogenicity study of pathogenic bacteria of lily blight[J]. Journal of Lanzhou Petrochemical College of Vocational Technology,2014,14(2):38-40(in Chinese with English abstract).
[24] 翟雅鑫,姚晨阳,薛丽芳,等. 一株百合枯萎病菌的鉴定及其生物学特性研究[J]. 山西农业大学学报(自然科学版),2018,38(5):1-6. Zhai Y X,Yao C Y,Xue L F,et al. Identification and biological characteristics of a fungal isolate causing lily Fusarium wilt[J]. Journal of Shanxi Agricultural University(Natural Science Edition),2018,38(5):1-6(in Chinese with English abstract).
[25] 王丽波,崔玥晗,岳玲,等. 百合枯萎病的研究进展[J]. 园艺与种苗,2018,38(12):50-53. Wang L B,Cui Y H,Yue L,et al. Research progress on Fusarium wilt of Lilium brownii var. viridulum[J]. Horticulture & Seed,2018,38(12):50-53(in Chinese with English abstract).
[26] 刘佳. 河北廊坊大豆枯萎病病原真菌的分离与鉴定[D]. 秦皇岛:河北科技师范学院,2016. Liu J. Isolation and identification of soybean Fusarium wilt pathogenic fungi in Langfang[D]. Qinhuangdao:Hebei Normal University of Science & Technology,2016(in Chinese with English abstract).
[27] 王小慧,朱震,马雪莲,等. 田间大棚西瓜枯萎病病原菌的分离与鉴定[J]. 南京农业大学学报,2012,35(6):61-67. DOI:10.7685/j.issn.1000-2030.2012.06.011. Wang X H,Zhu Z,Ma X L,et al. Identification and characterization of pathogens causing Fusarium wilt of watermelon from field greenhouses[J]. Journal of Nanjing Agricultural University,2012,35(6):61-67(in Chinese with English abstract).
[28] 潘汝浩,王继琛,王磊,等. 生姜枯萎病病原菌的分离鉴定及其接种浓度对生姜枯萎病发生程度的影响[J]. 南京农业大学学报,2014,37(1):94-100. DOI:10.7685/j.issn.1000-2030.2014.01.016 Pan R H,Wang J C,Wang L,et al. Isolation and identification of ginger Fusarium wilt pathogen and the effect of spore suspension concentration on the extent of disease[J]. Journal of Nanjing Agricultural University,2014,37(1):94-100(in Chinese with English abstract).
[29] González-Jartín J M,Alfonso A,Sainz M J,et al. First report of Fusarium foetens as a mycotoxin producer[J]. Mycotoxin Research,2019,35(2):177-186.
[30] Wu Z J,Yang L,Wang R Y,et al. In vitro study of the growth,development and pathogenicity responses of Fusarium oxysporum to phthalic acid,an autotoxin from Lanzhou lily[J]. World Journal of Microbiology and Biotechnology,2015,31(8):1227-1234.

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备注/Memo

备注/Memo:
收稿日期:2020-01-17。
基金项目:国家公益性行业(农业)科研专项(201503110);国家自然科学基金项目(31972506);江苏省重点研发计划(现代农业)项目(BE2018338);江苏省农业科技自主创新资金[CX(18)3095]
作者简介:任胜林,硕士研究生。
通信作者:黄启为,教授,研究方向为农业废弃物资源化与生物有机肥,E-mail:qwhuang@njau.edu.cn。
更新日期/Last Update: 1900-01-01