福建省一种蓝莓枝枯病病原鉴定

张小艳; 陈细红; 谢丽雪; 张立杰; 郑姗; 李韬

doi:10.19303/j.issn.1008-0384.2023.10.010

福建省一种蓝莓枝枯病病原鉴定

doi: 10.19303/j.issn.1008-0384.2023.10.010

1.
福建省农业科学院果树研究所，福建　福州　350013
2.
福州海关技术中心，福建　福州　350001

基金项目: 福建省科技计划公益类专项（2020R10280011）

详细信息

作者简介:
张小艳（1982 —），女，硕士，助理研究员，主要从事果树病虫害综合治理研究，E-mail：zxyan1204@163.com

通讯作者:
李韬（1969 —），男，学士，副研究员，主要从事果树遗传育种与病虫害研究，E-mail: leetao06@163.com

中图分类号: S436.63
计量
- 文章访问数: 284
- HTML全文浏览量: 141
- PDF下载量: 54
- 被引次数: 0
出版历程
- 收稿日期: 2023-06-30
- 修回日期: 2023-07-26
- 网络出版日期: 2023-10-25
- 刊出日期: 2023-10-28

Pathogen of Branch Blight on Blueberry Plants in Fujian

1.
Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
Fujian Key Laboratory for Technology Research of Inspection and Quarantine, Technology Center of Fuzhou Customs District, Fuzhou, Fujian　350001, China

摘要

摘要: 目的明确福建省三明地区蓝莓枝枯病的病原菌，为该病害的有效防治提供理论依据。方法采集受害蓝莓枝条样品，通过常规组织分离法进行病原菌分离纯化，采用针刺法进行致病性测定完成柯赫氏法则验证，利用形态学特征并联合分子生物学，明确致病菌的分类地位。结果从病组织中分离获得菌株KW1-4，将其回接至健康蓝莓枝条后，接种部位出现干枯病斑，再次分离获得的菌株与接种菌株一致，表明该菌株是引起蓝莓枝枯病的病原菌。菌株KW1-4菌落圆形，白色至灰色，有2种类型分生孢子，α型分生孢子长椭圆形至梭形，具2个明显油球，（4.77～7.63）µm×（1.55～2.71）µm，β型分生孢子线形，无油球，（11.95～19.65）µm×（1.05～1.94）µm。采用ITS，TEF1-ɑ，β-tubulin3段基因联合构建系统发育树，供试菌株KW1-4与Diaporthe australiana聚在同一分支。对比形态学特征，确定菌株KW1-4为间座壳属真菌Diaporthe australiana。结论引起福建省三明市蓝莓枝枯病的病原菌是Diaporthe australiana，本研究首次报道Diaporthe australiana能够侵染蓝莓枝条。
- 蓝莓 /
- 枝枯病 /
- 间座壳菌 /
- 鉴定
Abstract: Objective The pathogen that caused the blueberry branch blight in Sanming, Fujian was identified. Method Microbes were isolated and purified from the blueberry branches showing anthracnose symptoms collected from the infected area. Pathogenicity of the isolates was tested by needle injection on the plant and verified by the Koch's method. Pathogen taxonomy was determined by morphological observations and molecular technology. Result Strain KW1-4 was isolated from the diseased tissue and inoculated onto healthy blueberry branches to show on the inoculation site dry spots same as what produced by the strain from the infected plants. The white to gray, round colonies of KW1-4 had two types of conidia. The alpha conidia were elliptical or spindle in shape and 4.77-7.63 μm × 1.55-2.71 μm in size with two distinct oil globules, while the linear beta conidia sized 11.95-19.65 μm × 1.05-1.94 μm with no oil globule. The 3 fragments of the genes, ITS, TEF1-ɑ, and β-tubulin, were used to construct the phylogenetic tree that showed KW1-4 to cluster with Diaporthe australiana in a same branch, which agreed with the morphological characteristics. Conclusion It was determinedD. australiana to be the pathogen that caused the blight on blueberry bushes in Sanming. The identification paved the way for the disease control.
- Blueberry /
- branch blight /
- Diaporthe australiana /
- identification

HTML全文

图 1 蓝莓枝枯病田间症状

Figure 1. Symptoms of blueberry branch blight in the field

下载: 全尺寸图片幻灯片

图 2 KW1-4的接种症状

Figure 2. Pathogenicity of KW1-4

下载: 全尺寸图片幻灯片

图 3 KW1-4 菌落形态及其分生孢子

A：3 d ；B：9 d ；C：25 d ；D：α型分生孢子； E：β型分生孢子。

Figure 3. Colony morphology and conidia of KW1-4

A: 3 d; B: 9 d; C: 25 d; D: alpha conidia; E: beta conidia.

下载: 全尺寸图片幻灯片

图 4 基于rDNA-ITS、TEF1-ɑ和β-tubulin基因序列构建的间座壳菌系统发育树

Figure 4. Phylogenetic tree of Diaporthe species constructed based on concatenated sequences of ITS, TEF1-ɑ, and β-tubulin

下载: 全尺寸图片幻灯片

表 1 PCR扩增ITS、TEF1-ɑ和β-tubulin片段的引物

Table 1. Primers for PCR amplification of fragments of ITS, TEF1-ɑ, and β-tubulin

基因 Gene	引物 Primer	序列（5′-3′） Sequence（5′-3′）	参考文献 Reference
ITS	ITS1	TCCGTAGGTGAACCTGCGG	[21]
ITS	ITS4	TCCTCCGCTTATTGATATGC	[21]
TEF1-ɑ	EF-728F	CATYGAGAAGTTCGAGAAGG	[22]
TEF1-ɑ	EF2	GGARGTACCAGTSATCATGTT	[23]
β-tubulin	Bt2a	GGTAACCAAATCGGTGCTGCTTTC	[24]
β-tubulin	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	[24]

下载: 导出CSV

表 2 ITS、TEF1-ɑ和β-tubulin引物的PCR反应条件

Table 2. PCR reaction conditions for fragments of ITS, TEF1-ɑ, and β-tubulin

扩增片段 Amplified fragment	预变性 Pre-denaturation	变性 Denaturation	退火 Annealing	延伸 Extension	循环次数 Cycle number	延伸 Extension
ITS	94 ℃，3 min	94 ℃，45 s	52 ℃，45 s	72 ℃，60 s	30	72 ℃，7 min
TEF1-ɑ	95 ℃，5 min	95 ℃，60 s	55 ℃，80 s	72 ℃，90 s	35	72 ℃，5 min
β-tubulin	95 ℃，5 min	95 ℃，60 s	45 ℃，80 s	72 ℃，90 s	35	72 ℃，5 min

下载: 导出CSV

参考文献(31)

[1]	李亚东. 越橘(蓝莓)栽培与加工利用[M]. 长春: 吉林科学技术出版社, 2001.
[2]	李亚东, 盖禹含, 王芳, 等. 2021年全球蓝莓产业数据报告 [J]. 吉林农业大学学报, 2022, 44(1):1−12. LI Y D, GAI Y H, WANG F, et al. Global blueberry industry report 2021 [J]. Journal of Jilin Agricultural University, 2022, 44(1): 1−12.(in Chinese)
[3]	肖秀珠, 刘佳明, 孙朝锋, 等. 福建省蓝莓种植气候适宜性区划 [J]. 中国农业气象, 2020, 41(8):520−528. XIAO X Z, LIU J M, SUN C F, et al. Climate suitability zoning of blueberry in Fujian Province [J]. Chinese Journal of Agrometeorology, 2020, 41(8): 520−528.(in Chinese)
[4]	张小艳, 郑姗, 谢丽雪, 等. 福建省蓝莓病虫害调查 [J]. 福建农业学报, 2019, 34(3):338−343. ZHANG X Y, ZHENG S, XIE L X, et al. A report on insect infestation and diseases on blueberry bushes in Fujian [J]. Fujian Journal of Agricultural Sciences, 2019, 34(3): 338−343.(in Chinese)
[5]	苏宝玲, 陈薇, 范业展, 等. 越橘病害概述 [J]. 北方园艺, 2010(6):218−223. SU B L, CHEN W, FAN Y Z, et al. A compendium of blueberry diseases [J]. Northern Horticulture, 2010(6): 218−223.(in Chinese)
[6]	徐成楠, 迟福梅, 冀志蕊, 等. 蓝莓葡萄座腔菌枝枯病研究进展 [J]. 中国果树, 2014(5):71−74. XU C N, CHI F M, JI Z R, et al. Research progress on branch blight of blueberry grape [J]. China Fruits, 2014(5): 71−74.(in Chinese)
[7]	余磊, 唐旭兵, 赵建荣, 等. 云南蓝莓枝干溃疡病病原菌鉴定及rDNA-ITS序列分析 [J]. 云南农业大学学报(自然科学), 2014, 29(1):27−31. YU L, TANG X B, ZHAO J R, et al. Identification and the internal transcribed spacer regions analysis of the pathogen causing stem canker on blueberry in Yunnan [J]. Journal of Yunnan Agricultural University (Natural Science), 2014, 29(1): 27−31.(in Chinese)
[8]	余磊, 赵建荣, Rarisara Impaprasert, 等. 蓝莓枝枯病病原菌鉴定 [J]. 植物病理学报, 2013, 43(4):421−425. YU L, ZHAO J R, IMPAPRASERT R, et al. Identification of the pathogen causing twigs and stem dieback in blueberry [J]. Acta Phytopathologica Sinica, 2013, 43(4): 421−425.(in Chinese)
[9]	赵洪海, 岳清华, 梁晨. 蓝莓拟盘多毛孢枝枯病的病原菌 [J]. 菌物学报, 2014, 33(3):577−583. ZHAO H H, YUE Q H, LIANG C. The pathogen causing Pestalotiopsis twig dieback of blueberry [J]. Mycosystema, 2014, 33(3): 577−583.(in Chinese)
[10]	岳清华, 赵洪海, 梁晨, 等. 蓝莓拟茎点枝枯病的病原 [J]. 菌物学报, 2013, 32(6):959−966. YUE Q H, ZHAO H H, LIANG C, et al. The pathogen causing Phomopsis twig blight of blueberry [J]. Mycosystema, 2013, 32(6): 959−966.(in Chinese)
[11]	WEINGARTNER D P. Etiology and symptomatology of canker and dieback diseases on highbush blueberries caused by Godronia (Fusicoccum) cassandrae and Diaporthe (Phomopsis) vaccinii [J]. Phytopathology, 1975, 65(2): 105. doi: 10.1094/Phyto-65-105
[12]	PARKER P E. Epidemiology and chemical control of Phomopsis canker of highbush blueberry [J]. Phytopathology, 1977, 77(12): 1481. doi: 10.1094/Phyto-67-1481
[13]	严雪瑞, 王旭, 胡梦琼, 等. 蓝莓间座壳芽枯病病原菌鉴定及其生物学特性 [J]. 植物病理学报, 2015, 45(5):556−560. YAN X R, WANG X, HU M Q, et al. Identification and biological characteristic of blueberry Diaporthe bud blight pathogen [J]. Acta Phytopathologica Sinica, 2015, 45(5): 556−560.(in Chinese)
[14]	ELFAR K, TORRES R, DÍAZ G A, et al. Characterization of Diaporthe australafricana and Diaporthe spp. associated with stem canker of blueberry in Chile [J]. Plant Disease, 2013, 97(8): 1042−1050. doi: 10.1094/PDIS-11-12-1030-RE
[15]	李媛, 石凌波, 费诺亚, 等. 蓝莓间座壳茎溃疡病病原鉴定及生物学特性研究 [J]. 植物保护, 2017, 43(1):89−94. doi: 10.3969/j.issn.0529-1542.2017.01.014 LI Y, SHI L B, FEI N Y, et al. Identification and biological characteristics of a blueberry Diaporthe stem canker pathogen [J]. Plant Protection, 2017, 43(1): 89−94.(in Chinese) doi: 10.3969/j.issn.0529-1542.2017.01.014
[16]	李媛, 石凌波, 费诺亚, 等. 蓝莓茎溃疡病病原菌鉴定及品种抗病性研究 [J]. 植物病理学报, 2017, 47(6):721−729. LI Y, SHI L B, FEI N Y, et al. Identification of causative agent of blueberry stem canker and screening of the disease resistant cultivars [J]. Acta Phytopathologica Sinica, 2017, 47(6): 721−729.(in Chinese)
[17]	林月莉, 黄丽丽, 索朗拉姆, 等. 苹果轮纹病室内快速评价体系的建立 [J]. 植物保护学报, 2011, 38(1):37−41. LIN Y L, HUANG L L, SUOLANGLAMU, et al. A rapid laboratory evaluation system for apple ring rot [J]. Journal of Plant Protection, 2011, 38(1): 37−41.(in Chinese)
[18]	魏景超. 真菌鉴定手册[M]. 上海: 上海科学技术出版社, 1979.
[19]	王先洪, 姜佳琦, 洪霓, 等. 贵州梨芽枯间座壳菌属Diaporthe的种类 [J]. 菌物学报, 2022, 41(8):1151−1164. WANG X H, JIANG J Q, HONG N, et al. Diaporthe species causing pear bud witherings in Guizhou, Southwest China [J]. Mycosystema, 2022, 41(8): 1151−1164.(in Chinese)
[20]	张颖慧, 魏东盛, 邢来君, 等. 一种改进的丝状真菌DNA提取方法 [J]. 微生物学通报, 2008, 35(3):466−469. ZHANG Y H, WEI D S, XING L J, et al. A modified method for isolating DNA from fungus [J]. Microbiology, 2008, 35(3): 466−469.(in Chinese)
[21]	BELLEMAIN E, CARLSEN T, BROCHMANN C, et al. ITS as an environmental DNA barcode for fungi: An in silico approach reveals potential PCR biases [J]. BMC Microbiology, 2010, 10: 189. doi: 10.1186/1471-2180-10-189
[22]	SAMUELS G J, ISMAIEL A, SOUZA J, et al. Trichoderma stromaticum and its overseas relatives [J]. Mycological Progress, 2012, 11(1): 215−254. doi: 10.1007/s11557-011-0743-4
[23]	VÁZQUEZ M B, BARRERA V, BIANCHINOTTI V. Molecular identification of three isolates of Trichoderma harzianum isolated from agricultural soils in Argentina, and their abilities to detoxify in vitro metsulfuron methyl [J]. Botany, 2015, 93(11): 793−800. doi: 10.1139/cjb-2015-0085
[24]	GLASS N L, DONALDSON G C. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes [J]. Applied and Environmental Microbiology, 1995, 61(4): 1323−1330. doi: 10.1128/aem.61.4.1323-1330.1995
[25]	杨琴. 中国间座壳属的分类和系统学研究[D]. 北京: 北京林业大学, 2019. YANG Q. Taxonomy and phylogeny of Diaporthe in China[D]. Beijing: Beijing Forestry University, 2019. (in Chinese)
[26]	龙慧. 西南地区间座壳属真菌(Diaporthe)的系统学研究[D]. 贵阳: 贵州大学, 2020. LONG H. The phylogenetic study of Diaporthe in southwest China[D]. Guiyang: Guizhou University, 2020. (in Chinese)
[27]	ERINCIK O, MADDEN L V, FERREE D C, et al. Effect of growth stage on susceptibility of grape berry and Rachis tissues to infection by Phomopsis viticola [J]. Plant Disease, 2001, 85(5): 517−520. doi: 10.1094/PDIS.2001.85.5.517
[28]	GOMES R R, GLIENKE C, VIDEIRA S I R, et al. Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi [J]. Persoonia, 2013, 31: 1−41. doi: 10.3767/003158513X666844
[29]	UDAYANGA D, CASTLEBURY L A, ROSSMAN A Y, et al. Insights into the genus Diaporthe: Phylogenetic species delimitation in the D. eres species complex [J]. Fungal Diversity, 2014, 67(1): 203−229. doi: 10.1007/s13225-014-0297-2
[30]	WRONA C J, MOHANKUMAR V, SCHOEMAN M H, et al. Phomopsis husk rot of macadamia in Australia and South Africa caused by novel Diaporthe species [J]. Plant Pathology, 2020, 69(5): 911−921. doi: 10.1111/ppa.13170
[31]	HUANG J Y, TANG H, GU B, et al. A Preliminary Investigation of Pathogenic Fungi from Lotus(Nelumbo nucifera Gaertn)in Nanchang City [J]. 生物灾害科学, 2021, 44(2):123−135. HUANG J Y, TANG H, GU B, et al. A preliminary investigation of pathogenic fungi from lotus(Nelumbo nucifera Gaertn)in Nanchang city [J]. Biological Disaster Science, 2021, 44(2): 123−135.(in Chinese)