• 百种中国杰出学术期刊
  • 中国精品科技期刊
  • 中国高校百佳科技期刊
  • 中国高校精品科技期刊
  • 中国国际影响力优秀学术期刊
  • 中国科技核心期刊

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

百日草炭疽病病原菌的分离鉴定及防治药剂筛选

李文 何月秋 王佳莹 王国良 王志龙

李文, 何月秋, 王佳莹, 王国良, 王志龙. 百日草炭疽病病原菌的分离鉴定及防治药剂筛选[J]. 农药学学报, 2021, 23(2): 341-347. doi: 10.16801/j.issn.1008-7303.2021.0017
引用本文: 李文, 何月秋, 王佳莹, 王国良, 王志龙. 百日草炭疽病病原菌的分离鉴定及防治药剂筛选[J]. 农药学学报, 2021, 23(2): 341-347. doi: 10.16801/j.issn.1008-7303.2021.0017
Wen LI, Yueqiu HE, Jiaying WANG, Guoliang WANG, Zhilong WANG. Isolation and identification of pathogen causing anthracnose on Zinnia elegans Jacq. and fungicides screening[J]. Chinese Journal of Pesticide Science, 2021, 23(2): 341-347. doi: 10.16801/j.issn.1008-7303.2021.0017
Citation: Wen LI, Yueqiu HE, Jiaying WANG, Guoliang WANG, Zhilong WANG. Isolation and identification of pathogen causing anthracnose on Zinnia elegans Jacq. and fungicides screening[J]. Chinese Journal of Pesticide Science, 2021, 23(2): 341-347. doi: 10.16801/j.issn.1008-7303.2021.0017

百日草炭疽病病原菌的分离鉴定及防治药剂筛选

doi: 10.16801/j.issn.1008-7303.2021.0017
基金项目: 宁波市自然科学基金(2018A610215);宁波城市职业技术学院科研项目(ZZX16047);宁波市公益计划(2019C10087)
详细信息
    作者简介:

    李文,女,博士,讲师,主要从事植物病虫害防治研究,E­mail:liwen547249317@126.com

    通讯作者:

    王志龙,通信作者 (Author for correspondence),男,教授,从事植物病虫害防治研究,E­mail:wangzhl01@163.com

  • 中图分类号: S436.8; S482.2; TQ455

Isolation and identification of pathogen causing anthracnose on Zinnia elegans Jacq. and fungicides screening

  • 摘要: 为明确百日草炭疽病的病原菌,依据柯赫氏法则,对采集的病叶进行病原物分离并进行分离菌株致病性试验。确定菌株的致病性后,结合菌落形态,初步判断其为炭疽菌。运用分子生物学方法,扩增获得肌动蛋白基因 (actin gene, ACT)、几丁质合酶基因 (chitin synthase A gene, CHS)、磷酸甘油醛脱氢酶基因 (glyceraldehyde-3-phosphate dehydrogenase, GAPDH)、核糖体内部转录间隔区 (ribosomal internal transcribed spacer, ITS)、锰超氧化物歧化酶 (manganese-superoxide dismutase, SOD2)、谷氨酰胺合酶 (glutamine synthatase, GS)、微管蛋白 (beta-tubulin-TUB2) 以及钙调蛋白 (calmodulin, CaM ) 8个基因序列,进行联合系统发育分析,确定该菌为暹罗刺盘孢菌Colletotrichum siamense。离体抑菌活性测定结果表明,9种杀菌剂制剂75%肟菌 • 戊唑醇水分散粒剂、35%氟菌 • 戊唑醇悬浮剂、75%百菌清可湿性粉剂、250 g/L嘧菌酯悬浮剂、50%咪鲜胺可湿性粉剂、10%苯醚甲环唑水分散粒剂、10%丙硫菌唑悬浮剂、500 g/L氟啶胺悬浮剂和25%溴菌晴可湿性粉剂对供试病原菌的EC50值分别为0.152、0.407、2.48、252、0.0342、0.556、317、0.00291和27.4 mg/L,其中500 g/L氟啶胺悬浮剂和50%咪鲜胺可湿性粉剂抑菌活性最强。
  • 图  1  百日草田间发病症状

    Figure  1.  Disease symptoms of Zinnia elegans in the field

    图  2  分离物致病性测定

    a: 对照,喷洒无菌水;b: 喷洒1 × 106 个/mL分生孢子处理,10 d后拍照记录;c: 喷洒分生孢子处理,15 d后拍照记录。

    Figure  2.  Pathogenicity test of the isolate with sterilized water

    Pathogenicity test of the isolate with sterilized water (a) and 1 × 106 conidia/mL suspension (b and c). Pictures were taken at 10 d (b) and 15 d (c) after the inoculation.

    图  3  百日草炭疽病菌形态学观察

    a:菌落形态;b:分生孢子形态,比例尺 = 10 μm。

    Figure  3.  Morphological characteristics of colonies of anthracnose pathogen on Z. elegans

    a: Colony; b: Spores, scale bar = 10 μm.

    图  4  系统发育进化树

    基于8个基因的进化树,分离得到的菌株F3与Colletotrichum siamense聚在一起。

    Figure  4.  Phylogenetic tree based on the concatenated sequences

    (ACT, CHS, GAPDH, ITS, SOD2, GS, CaM and TUB2) placed the isolate F3 within the Colletotrichum siamense cluster.

    表  1  PCR扩增引物

    Table  1.   PCR primers used in this study

    基因
    Gene
    引物序列
    Primer sequence
    参考文献
    Reference
    ACT ATGTGCAAGGCCGGTTTCGC [6]
    TACGAGTCCTTCTGGCCCAT
    CHS TGGGGCAAGGATGCTTGGAAGAAG [6]
    TGGAAGAACCATCTGTGAGAGTTG
    GAPDH GCCGTCAACGACCCCTTCATTGA [7]
    GGGTGGAGTCGTACTTGAGCATGT
    ITS CTTGGTCATTTAGAGGAAGTAA [8]
    TCCTCCGCTTATTGATATGC
    SOD2 CAGATCATGGAGCTGCACCA [9]
    TAGTACGCGTGCTCGGACAT
    GS ATGGCCGAGTACATCTGG [9]
    GAACCGTCGAAGTTCCAC
    TUB2 AACATGCGTGAGATTGTAAGT [10]
    TAGTGACCCTTGGCCCAGTTG
    CaM GAATTCAAGGAGGCCTTCTC [10]
    CTTCTGCATCATGAGCTGGAC
    下载: 导出CSV

    表  2  本研究所用菌株信息

    Table  2.   Bacterial strains used in this study

    菌株名称
    Complex strains
    GenBank 序列编号
    GenBank accession number
    ITSGAPDHACTCHSSOD2TUB2CaMGS
    C. siamense F3 MN972439 MN972438 MN972436 MN972437 MN972440 MT266560 MT266561 MT266559
    C. aenigma ICMP 18608 JX010244 JX010044 JX009443 JX009774 JX010311 JX010389 JX009683 JX010078
    C. aeschynomenes ICMP 17673 JX010176 JX009930 JX009483 JX009799 JX010314 JX010392 JX009721 JX010081
    C. alatae CBS 304.67 JX010190 JX009990 JX009471 JX009837 JX010305 JX010383 JX009738 JX010065
    C. alienum ICMP 12071 JX010251 JX010028 JX009572 JX009882 JX010333 JX010411 JX009654 JX010101
    C. aotearoa ICMP 18537 JX010205 JX010005 JX009564 JX009853 JX010345 JX010420 JX009611 JX010113
    C. asianum ICMP 18580 FJ972612 JX010053 JX009584 JX009867 JX010328 JX010406 FJ917506 JX010096
    C. clidemiae ICMP 18658 JX010265 JX009989 JX009537 JX009877 JX010356 JX010438 JX009645 JX010129
    C. cordylinicola MFLUCC 090551 JX010226 JX009975 HM470235 JX009864 JX010361 JX010440 HM470238 JX010122
    C. fructicola ICMP 18581 JX010165 JX010033 FJ907426 JX009866 JX010327 JX010405 FJ917508 JX010095
    C. gloeosporioides IMI 356878 JX010152 JX010056 JX009531 JX009818 JX010365 JX010445 JX009731 JX010085
    C. horii NBRC 7478 GQ329690 GQ329681 JX009438 JX009752 JX010370 JX010450 JX009604 JX010137
    C. kahawae subsp. ciggaro ICMP 18539 JX010230 JX009966 JX009523 JX009800 JX010346 JX010434 JX009635 JX010132
    C. musae CBS 116870 JX010146 JX010050 JX009433 JX009896 JX010335 HQ596280 JX009742 JX010103
    C. nupharicola CBS 470.96 JX010187 JX009972 JX009437 JX009835 JX010320 JX010398 JX009663 JX010088
    C. psidii CBS 145.29 JX010219 JX009967 JX009515 JX009901 JX010366 JX010443 JX009743 JX010133
    C. queenslandicum ICMP 1778 JX010276 JX009934 JX009447 JX009899 JX010336 JX010414 JX009691 JX010104
    C. salsolae ICMP 19051 JX010242 JX009916 JX009562 JX009863 JX010325 JX010403 JX009696 JX010093
    C. siamense ICMP 18578 JX010171 JX009924 FJ907423 JX009865 JX010326 JX010404 FJ917505 JX010094
    C. theobromicola CBS 124945 JX010294 JX010006 JX009444 JX009869 JX010372 JX010447 JX009591 JX010139
    C. ti ICMP 4832 JX010269 JX009952 JX009520 JX009898 JX010362 JX010442 JX009649 JX010123
    C. tropicale CBS 124949 JX010264 JX010007 JX009489 JX009870 JX010329 JX010407 JX009719 JX010097
    C. xanthorrhoeae BRIP 45094 JX010261 JX009927 JX009478 JX009823 JX010369 JX010448 JX009653 JX010138
    C.kahawae subsp. Kahawae IMI 319418 JX010231 JX010012 JX009452 JX009813 JX010350 JX010444 JX009642 JX010130
    下载: 导出CSV

    表  3  不同杀菌剂制剂在培养基中的浓度梯度

    Table  3.   Concentration of fungicide in the medium

    杀菌剂
    Fungicide
    杀菌剂质量浓度
    Concentration/(mg/L)
    75% 肟菌 • 戊唑醇水分散粒剂
    trifloxystrobin + tebuconazole 75% WG
    0.5, 1, 2, 4, 8
    35% 氟菌 • 戊唑醇悬浮剂
    triflumizole + tebuconazole 35% SC
    75% 百菌清可湿性粉剂
    chlorothalonil 75% WP
    1, 2, 4, 8, 16
    250 g/L 嘧菌酯悬浮剂
    azoxystrobin 250 g/L SC
    50% 咪鲜胺可湿性粉剂
    prochloraz 50% WP
    0.0156, 0.03125, 0.0625, 0.125, 0.25
    10% 苯醚甲环唑水分散粒剂
    difenoconazole 10% WG
    0.125, 0.25, 0.5, 1, 2
    10% 丙硫菌唑悬浮剂
    prothioconazole 10% SC
    500 g/L 氟啶胺悬浮剂
    fluazinam 500 g/L SC
    25% 溴菌晴可湿性粉剂
    bromothalonil 25% WP
    下载: 导出CSV

    表  4  9种杀菌剂制剂对暹罗刺盘孢菌的杀菌活性

    Table  4.   Antibiotic activities of 9 fungicides to C. siamense

    杀菌剂
    Fungicide
    毒力回归方程
    Toxicity regression equation
    EC50/(mg/L)相关系数
    r
    75% 肟菌 • 戊唑醇水分散粒剂 trifloxystrobin+tebuconazole 75% WG y = 5.4972 + 0.5910x 0.152 0.9927
    35% 氟菌 • 戊唑醇悬浮剂 triflumizole+tebuconazole 35% SC y = 5.3649 + 0.9240x 0.407 0.9893
    75% 百菌清可湿性粉剂 chlorothalonil 75% WP y = 4.6483 + 0.9137x 2.48 0.9809
    250 g/L 嘧菌酯悬浮剂 azoxystrobin 250 g/L SC y = 4.5407 + 0.1934x 252 0.9263
    50% 咪鲜胺可湿性粉剂 prochloraz 50% WP y = 6.5097 + 1.0149x 0.0342 0.9965
    10% 苯醚甲环唑水分散粒剂 difenoconazole 10% WG y = 5.2999 + 1.0991x 0.556 0.9925
    10% 丙硫菌唑悬浮剂 prothioconazole 10% SC y = 3.9007 + 0.4397x 317 0.9869
    500 g/L 氟啶胺悬浮剂 fluazinam 500 g/L SC y = 6.3497 + 0.5290x 0.00291 0.9936
    25% 溴菌晴可湿性粉剂 bromothalonil 25% WP y = 4.2447 + 0.5403x 27.4 0.9198
    下载: 导出CSV
  • [1] 闫凯. 百日菊播种育苗初探[J]. 河南林业科技, 2003, 3: 27. doi: 10.3969/j.issn.1003-2630.2003.02.014

    YAN K. Study on sawing and seeding of Zinnia elegans Jacq.[J]. J Henan For Sci Tech, 2003, 3: 27. doi: 10.3969/j.issn.1003-2630.2003.02.014
    [2] 朱强, 周媛, 田丹青. 浙江省花海建设的现状、问题和对策[J]. 浙江农业科学, 2019, 60(9): 1617-1619.

    ZHU Q, ZHOU Y, TIAN D Q. The present situation, problems and countermeasures of flower sea construction in Zhejiang Province[J]. J Zhejiang Agric Sci, 2019, 60(9): 1617-1619.
    [3] 谢锐星, 陈庭. “仙湖花境”-亚热带地区花境营造探索[J]. 广东园林, 2018, 40(2): 88-94. doi: 10.3969/j.issn.1671-2641.2018.02.022

    XIE R X, CHEN T. To create and explore the flower border in the subtropical region of “Xianhu Flower Border”[J]. Guangdong Landscape Architec, 2018, 40(2): 88-94. doi: 10.3969/j.issn.1671-2641.2018.02.022
    [4] HOLCOMB G E. First report of bacterial leaf and flower spot of Zinnia in Louisiana[J]. Plant Dis, 1985, 69(4): 360.
    [5] STRIDER D L. Bacterial leaf and flower spot of Zinnia in North Carolina[J]. Plant Dis Reptr, 1973, 57(10): 1020.
    [6] STRIDER D L. Detection of Xanthomonas nigromaculans f. sp. zinniae in Zinnia seed[J]. Plant Dis Reptr, 1979, 63(10): 869-873.
    [7] WANG Y, QIN H Y, LIU Y X, et al. First report of anthracnose caused by Colletotrichum siamense on Actinidia arguta in China[J]. Plant Dis, 2019, 103: 372-373.
    [8] 杨敬辉, 陈宏州, 肖婷, 等. 草莓炭疽病病原鉴定及其对12种杀菌剂的毒力测定[J]. 西南农业学报, 2015, 28(6): 2527-2531.

    YANG J H, CHEN H Z, XIAO T, et al. Pathogen identification and toxicity determination of 12 different fungicides of strawberry anthracnose[J]. Southwest Chin J Agric Sci, 2015, 28(6): 2527-2531.
    [9] CARBONE I, KOHN L M. A method for designing primer sets for speciation studies in Filamentous ascomycetes[J]. Stud Mycol, 1999, 91(3): 553-556. doi: 10.1080/00275514.1999.12061051
    [10] TEMPLETON M D, RIKKERINK E H A, SOLON S L, et al. Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata[J]. Gene, 1992, 122(1): 225-230. doi: 10.1016/0378-1119(92)90055-T
    [11] GARDES M, BRUNS T D. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts[J]. Mol Ecol, 1993, 2(2): 113-118. doi: 10.1111/j.1365-294X.1993.tb00005.x
    [12] WHITE T J, BRUNS S, LEE S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J]. PCR Protocols: A Guide to Methods & Applications, 1990, 1: 315-322.
    [13] JONES D T, TAYLOR W R, THORNTON J M. The rapid generation of mutation data matrices from protein sequences[J]. Comput Appl Biosci, 1992, 8(3): 275-282.
    [14] O'DONNELL K, CIGELNIK E. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous[J]. Mol Phylogenetics Evol, 1997, 7(1): 103-116. doi: 10.1006/mpev.1996.0376
    [15] 邓洁, 莫飞旭, 石金巧, 等. 钩藤炭疽病病原鉴定、生物学特性及防治药剂筛选[J]. 中药材, 2020, 43(6): 1305-1309.

    DENG J, MO F X, SHI J Q, et al. Pathogen identification, biological characteristics of anthracnose in Uncaris hirsute and screening of control fungicides[J]. J Chin Med Mater, 2020, 43(6): 1305-1309.
    [16] 杨晓琦, 周小军, 朱丽燕, 等. 金线莲炭疽病病原菌的分离鉴定及其对9 种杀菌剂的敏感性[J]. 农药学学报, 2020: 22. doi: 10.16801/j.issn.1008-7303.2020.0127

    YANG X Q, ZHOU X J, ZHU L Y, et al. Isolation and identification of pathogen causing anthracnose on Anoectochilus roxburghii and its sensitivity to nine fungicides[J]. Chin J Pestic Sci, 2020: 22. doi: 10.16801/j.issn.1008-7303.2020.0127
    [17] WEIR B S, JOHNSTON P R, DAMM U. The Colletotrichum gloeosporioides species complex[J]. Stud Mycol, 2012, 73: 115-180. doi: 10.3114/sim0011
    [18] UYSAL A, KURT S. First report of Colletotrichum siamense causing anthracnose on banana fruits in Turkey[J]. J Plant Pathol, 2020. doi: 10.1007/s42161-020-00534-1
    [19] PRASANNATH K, GALEA V J, AKINSANMI O A. Characterisation of leaf spots caused by Neopestalotiopsis clavispora and Colletotrichum siamense in macadamia in Australia[J]. Europ J Plant Pathol, 2020, 156: 1219-1225. doi: 10.1007/s10658-020-01962-6
    [20] 刘霞, 杨克强, 朱玉凤. 等8种杀菌剂对核桃炭疽病病原菌胶孢炭疽菌的室内毒力[J]. 农药学学报, 2013, 15(4): 412-420. doi: 10.3969/j.issn.1008-7303.2013.04.08

    LIU X, YANG K Q, ZHU Y F, et al. Laboratory toxicity of eight fungicides against Colletotrichum gloeosporioides causing walnut anthracnose[J]. Chin J Pestic Sci, 2013, 15(4): 412-420. doi: 10.3969/j.issn.1008-7303.2013.04.08
    [21] 胡晓颖, 赵杰. 5种杀菌剂对桃炭疽病菌的毒力测定[J]. 中国园艺文摘, 2018, 34(3): 61-62. doi: 10.3969/j.issn.1672-0873.2018.03.020

    HU X Y, ZHAO J. Toxicity of 5 fungicides to Colletotrichum gloeosporioides (Penz.) Sacc[J]. Chin Hortic Abstr, 2018, 34(3): 61-62. doi: 10.3969/j.issn.1672-0873.2018.03.020
    [22] 周小燕, 姜于兰. 不同杀菌剂对柑橘炭疽病的田间防治效果[J]. 湖北农业科学, 2011, 50(15): 3080-3082. doi: 10.3969/j.issn.0439-8114.2011.15.017

    ZHOU X Y, JIANG Y L. Field trials of several fungicides for control of Citrus anthracnose[J]. Hubei Agric Sci, 2011, 50(15): 3080-3082. doi: 10.3969/j.issn.0439-8114.2011.15.017
    [23] 韩永超, 曾祥国, 向发云, 等. 湖北省草莓炭疽病菌对咪鲜胺的敏感性[J]. 农药学学报, 2014, 16(5): 535-540. doi: 10.3969/j.issn.1008-7303.2014.05.07

    HAN Y C, ZENG X G, XIANG F Y, et al. Sensitivity of Colletotrichum spp. from strawberry to prochloraz in Hubei Province of China[J]. Chin J Pestic Sci, 2014, 16(5): 535-540. doi: 10.3969/j.issn.1008-7303.2014.05.07
  • 百日草炭疽病病原菌的分离鉴定及防治药剂筛选_附图1.docx
  • 加载中
图(4) / 表(4)
计量
  • 文章访问数:  707
  • HTML全文浏览量:  359
  • PDF下载量:  31
  • 被引次数: 0
出版历程
  • 收稿日期:  2020-08-11
  • 录用日期:  2020-10-16
  • 网络出版日期:  2021-04-08
  • 刊出日期:  2021-04-10

目录

    /

    返回文章
    返回