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地锦叶斑病菌鉴定及LAMP快速检测体系的建立

黄成玉, 陈健鑫, 郑星月, 吴峰婧琳, 马焕成, 伍建榕

黄成玉, 陈健鑫, 郑星月, 等. 地锦叶斑病菌鉴定及LAMP快速检测体系的建立[J]. 华南农业大学学报, 2025, 46(1): 89-96. DOI: 10.7671/j.issn.1001-411X.202401012
引用本文: 黄成玉, 陈健鑫, 郑星月, 等. 地锦叶斑病菌鉴定及LAMP快速检测体系的建立[J]. 华南农业大学学报, 2025, 46(1): 89-96. DOI: 10.7671/j.issn.1001-411X.202401012
HUANG Chengyu, CHEN Jianxin, ZHENG Xingyue, et al. Identification of pathogen causing leaf spot disease of Parthenocissus tricuspidata and establishment of LAMP rapid detection system[J]. Journal of South China Agricultural University, 2025, 46(1): 89-96. DOI: 10.7671/j.issn.1001-411X.202401012
Citation: HUANG Chengyu, CHEN Jianxin, ZHENG Xingyue, et al. Identification of pathogen causing leaf spot disease of Parthenocissus tricuspidata and establishment of LAMP rapid detection system[J]. Journal of South China Agricultural University, 2025, 46(1): 89-96. DOI: 10.7671/j.issn.1001-411X.202401012

地锦叶斑病菌鉴定及LAMP快速检测体系的建立

基金项目: 国家自然科学基金(31860208);国家重点研发计划(2019YFD100200X)
详细信息
    作者简介:

    黄成玉,硕士研究生,主要从事森林病理学研究,E-mail: 468588408@qq.com

    陈健鑫,博士研究生,主要从事森林病理学研究,E-mail: 535975299@qq.com,†表示同等贡献

    通讯作者:

    伍建榕,教授,博士,主要从事森林病理学及资源微生物利用研究,E-mail: 1176279044@qq.com

  • 中图分类号: S432.1

Identification of pathogen causing leaf spot disease of Parthenocissus tricuspidata and establishment of LAMP rapid detection system

  • 摘要:
    目的 

    明确引起云南玉龙县地锦叶斑病的病原种类,并建立快速检测方法。

    方法 

    采用组织分离法分离病原菌,并依据科赫氏法则确定地锦叶斑病病原,通过形态学特征及多基因联合构建系统发育树,明确病原菌的分类地位;以病原菌的Alt a1基因序列为靶标设计特异性引物建立环介导等温扩增(Loop-mediated isothermal amplification,LAMP)检测方法。

    结果 

    地锦叶斑病病原菌鉴定为链格孢菌Alternaria alternata。建立的LAMP反应体系可特异有效地检测出地锦叶斑病菌,该体系最佳反应温度为65 ℃、最佳反应时间为50 min、最低检测灵敏度为1 pg/μL。使用该体系对人工接种病原菌不同时间的地锦叶片进行链格孢LAMP检测,检出时间为接种12 h及以上。

    结论 

    本研究可为地锦叶斑病的早期检测和科学防控提供理论依据。

    Abstract:
    Objective 

    To identify the pathogens causing leaf spot disease of Parthenocissus tricuspidata in Yunnan Yulong County, and establish a rapid detection method.

    Method 

    The pathogens were isolated by tissue separation method and verified by Koch’s postulate. Through morphological characteristics and multi-gene joint construction of phylogenetic tree, the taxonomic status of pathogenic bacteria was clarified. A loop-mediated isothermal amplification (LAMP) assay was established by designing specific primers based on the Alt a1 gene sequence of the pathogen.

    Result 

    The pathogen causing P. tricuspidata leaf spot was identified as Alternaria alternata. The established LAMP reaction system could specifically and effectively detect A. alternata. The optimal reaction temperature of the system was 65 ℃, the optimal reaction time was 50 min, and the minimum detection sensitivity was 1 pg/μL. The system was used to detect A. alternata in the leaves of the P. tricuspidata artificially inoculated with pathogens at different time, and the detection time was 12 h or more.

    Conclusion 

    This study provides a theoretical basis for early detection, scientific prevention and control of the leaf spot of P. tricuspidata.

  • 图  1   地锦叶斑病的田间症状

    Figure  1.   Field symptoms on leaves of Parthenocissu tricuspidata

    图  2   地锦叶斑病菌的形态学特征

    A:菌落正反面;B:分生孢子链;C:分生孢子梗;D:分生孢子。

    Figure  2.   Morphological characters of the pathogen causing leaf spot of Parthenocissu tricuspidata

    A: Surface of colony; B: Chains of conidia; C: Conidiophore; D: Conidia.

    图  3   基于ITS、Alt a1gpd序列构建的地锦叶斑病菌及相关菌株的系统发育树

    Figure  3.   Phylogenetic tree based on ITS, Alt a1 and gpd sequences of the pathogen causing leaf spot of Parthenocissu tricuspidata and other related strains

    图  4   地锦叶斑病菌致病性验证

    A~E:接种12、24、48、72和96 h;F:CK。

    Figure  4.   Pathogenicity assay of pathogen causing leaf spot of Parthenocissu tricuspidata

    A−E: 12, 24, 48, 72 and 96 h after inoculation; F: CK.

    图  5   LAMP反应体系的优化

    A、B:LAMP反应温度优化;C、D: LAMP反应时间优化;A、C:以SYBR Green I为显色剂的LAMP反应;B、D: LAMP检测的电泳结果。在图A、B中,M:DL2000 DNA Marker;1~7:分别为60、61、62、63、64、65、66 ℃;8:阴性对照;在图C、D中,M:DL2000 DNA Marker;1~5:分别为20、30、40、50、60 min,6:阴性对照。

    Figure  5.   Optimization of LAMP reaction system

    A, B: Optimization of LAMP reaction temperature; C, D: Optimization of LAMP reaction time; A, C: LAMP reaction with SYBR Green I as color developer; B,D: Electrophoresis results detected by LAMP. In figure A and B, M: DL2000 DNA Marker; 1−7 represent 60, 61, 62, 63, 64, 65, 66 ℃ respectively; 8: Negative control; In figure C and D, M: DL2000 DNA Marker; 1−5 represent 20, 30, 40, 50, 60 min respectively, 6: Negative control.

    图  6   LAMP反应的特异性

    M:DL2000 DNA Marker;1:链格孢菌Alternaria alternaria;2:小孢拟盘多毛孢Pestalotiopsis microspora;3:高粱附球菌Epicoccum sorghinum;4:暹罗炭疽菌Colletotrichum siamense;5:尖孢镰刀菌Fusarium oxysporum;6:葡萄座腔菌Botryosphaeria dothidea;7:平脐蠕孢Bipolaris sp.;8:草茎点霉Phoma herbarum;9:越橘间座壳Diaporthe vaccinii;10:木贼镰刀菌Fusarium equisetum;11:阴性对照Negative control。

    Figure  6.   Specificity of LAMP reaction

    图  7   LAMP反应的灵敏性

    M:DL2000 DNA Marker;1:1 ng/μL;2~4:分别为100、10和1 pg/μL;5~7:分别为100、10和1 fg/μL;8:100 ag/μL;9:阴性对照。

    Figure  7.   Sensitivity of LAMP reaction

    M: DL2000 DNA Marker, 1: 1 ng/μL; 2−4 represent 100, 10 and 1 pg/μL respectively; 5−7 represent 100, 10 and 1 fg/μL, respectively; 8: 100 ag/μL; 9: Negative control.

    图  8   LAMP反应体系对人工接种链格孢地锦叶片的检测

    M:DL2000 DNA Marker;1:阳性对照Positive control;2~9: 0、3、6、12、24、48、72、96 h。

    Figure  8.   LAMP detection of Alternaria alternata in artificially inoculated leaves of Parthenocissu tricuspidata

    表  1   本研究PCR采用的引物信息

    Table  1   Primers for PCR used in this study

    基因/序列
    Gene/Sequence
    引物名称
    Primer name
    引物序列(5′→3′)
    Primer sequence
    ITS ITS1
    ITS4
    TCCGTAGGTGAACCTGCGG
    TCCTCCGCTTATTGATATGC
    gpd gpd1
    gpd2
    CAACGGCTTCGGTCGCATTG
    GCCAAGGAGTTGGTTGTGC
    Alt a1 Alt-for
    Alt-rev
    ATGCAGTTCACCACCATCGC
    ACGAGGGTGAYGTAGGCGTC
    下载: 导出CSV
  • [1] 高国平, 邓秋越, 王红, 等. 地锦叶斑病病原菌的鉴定及其生物学特性[J]. 东北林业大学学报, 2015, 43(3): 112-116.
    [2] 柴军红, 何婷婷, 赵楠, 等. 五叶地锦果实多糖、花色苷抗氧化、抗肿瘤活性研究[J]. 湖北农业科学, 2019, 58(15): 97-101.
    [3]

    ZHOU C H, SONG J, XU B. First report of Neophysopella vitis causing leaf rust on Parthenocissus semicordata in Guangdong Province, China[J/OL]. Plant Disease, 2024, 108(1): 211. doi: 10.1094/PDIS-06-23-1104-PDN.

    [4]

    YIN X T, LI T G, WEI Y F, et al. First report of Coniella vitis causing white rot on Virginia creeper (Parthenocissus quinquefolia) in China[J]. Plant Disease, 2023, 107(4): 1244. doi: 10.1094/PDIS-09-22-2053-PDN

    [5]

    WANG K C, LI Z B, ZOU J, et al. First report of Septoria tormentillae causing leaf blotch of Parthenocissus tricuspidata in China[J]. Plant Disease, 2020, 104(2): 566. doi: 10.1094/PDIS-08-19-1666-PDN

    [6]

    HUANG C C, LIU H H, WU P H, et al. First report of leaf spot caused by Diaporthe tulliensis on Boston ivy (Parthenocissus tricuspidata) in Taiwan[J]. Plant Disease, 2021, 105(9): 2718. doi: 10.1094/PDIS-12-20-2652-PDN

    [7]

    ZHAO X Y, WU F, CHEN M, et al. First report of Colletotrichum siamense causing leaf spot on Parthenocissus tricuspidata in China[J]. Plant Disease, 2020, 104(8): 2290. doi: 10.1094/pdis-12-19-2535-pdn

    [8] 吴石平, 莫砚成, 饶岸. 地锦褐斑病病原菌鉴定及病害诊断[J]. 贵州农业科学, 2019, 47(1): 70-73.
    [9]

    SOROKA M, WASOWICZ B, RYMASZEWSKA A. Loop-mediated isothermal amplification (LAMP): The better sibling of PCR?[J]. Cells, 2021, 10(8): 1931. doi: 10.3390/cells10081931

    [10] 朱俊子, 邱泽澜, 高必达, 等. 黄花菜叶斑病菌鉴定、生物学特性分析、防治药剂筛选及LAMP检测体系的建立[J]. 植物保护学报, 2022, 49(6): 1631-1641.
    [11] 赖宝春, 姚锦爱. 蜜柚间座壳黑点病菌(Diaporthecitri) LAMP可视化检测技术的建立[J]. 福建农业学报, 2022, 37(11): 1470-1475.
    [12] 马骏. 山核桃真菌性叶斑病病原菌的分离鉴定及LAMP检测方法的建立[D]. 杭州: 浙江农林大学, 2022.
    [13] 崔林开, 和志华, 康业斌, 等. 环介导等温扩增技术快速检测麦根腐平脐蠕孢[J]. 植物病理学报, 2022, 52(2): 269-275.
    [14] 萨吉达木·艾则孜, 麦合木提江·米吉提, 赵志强, 等. 一种侵染红枣叶片和果实的病原真菌鉴定[J]. 新疆农业科学, 2018, 55(9): 1682-1688.
    [15] 吕则佳, 陈健鑫, 冯峻, 等. 楚雄华山松种子园无性系幼苗叶枯病病原菌鉴定[J]. 西北林学院学报, 2022, 37(4): 181-187.
    [16] 周莹, 周悦妍, 李永华, 等. 北京地区桃黑斑病新症状的发现与诊断[J]. 植物保护, 2023, 49(3): 214-218.
    [17] 周慧珍, 荣思文, 吴玉珠, 等. 青脆李果斑病病原鉴定及室内药剂筛选[J]. 中国南方果树, 2023, 52(4): 136-140.
    [18] 朱启寒, 何剑鹏, 张晓阳, 等. 莴笋链格孢叶斑病病原鉴定及室内生防菌剂筛选[J]. 江西农业大学学报, 2023, 45(2): 395-403.
    [19] 杨丽萍, 金梦军, 崔凌霄, 等. 甘肃省樱桃黑斑病病原菌的分离及鉴定[J]. 果树学报, 2020, 37(6): 891-899.
    [20] 刘俏, 宁楠楠, 马永强, 等. 青海省樱桃叶斑病病原菌的分离与鉴定[J]. 植物保护, 2020, 46(2): 48-55.
    [21]

    LIU B Y, LI Z W, DU J F, et al. Loop-mediated isothermal amplification (LAMP) for the rapid and sensitive detection of Alternaria alternata(Fr. ) Keissl in apple Alternaria blotch disease with aapg-1 encoding the endopolygalacturonase[J]. Pathogens, 2022, 11(11): 1221. doi: 10.3390/pathogens11111221

    [22]

    MOGHIMI H, MORADI A, HAMEDI J, et al. Development of a loop-mediated isothermal amplification assay for rapid and specific identification of ACT producing Alternaria alternata, the agent of brown spot disease in tangerine[J]. Applied Biochemistry and Biotechnology, 2016, 178(6): 1207-1219. doi: 10.1007/s12010-015-1939-x

    [23]

    ZHANG X Y, XU G J, TANG H Q, et al. Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Alternaria alternata[J]. Journal of AOAC International, 2017, 100(1): 99-103. doi: 10.5740/jaoacint.16-0196

    [24] 谢学文, 刘世程, 石延霞, 等. 茄匍柄霉LAMP快速检测体系的建立及应用[J]. 农业生物技术学报, 2022, 30(10): 2045-2052.
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出版历程
  • 收稿日期:  2024-01-09
  • 网络出版日期:  2024-03-26
  • 发布日期:  2024-03-19
  • 刊出日期:  2025-01-09

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