Early and Rapid Detection of Tomato Gray Mold Based on LAMP Technology

doi:10.15933/j.cnki.1004-3268.2025.06.009

Abstract

Abstract: Gray mold caused by Botrytis cinerea is one of the main diseases of tomatoes，which severely affects the yield and quality of tomatoes. In order to achieve the early and rapid detection of tomato gray mold，by using the ACTIN gene of Botrytis cinerea as the target gene，and based on the loop‐mediated isothermal amplification（LAMP）technology，a set of LAMP specific primers was designed and screened，and the reaction system and reaction conditions were optimized to realize the rapid isothermal amplification of Botrytis cinerea.Through agarose gel electrophoresis and SYBR Green Ⅰ visualization analysis，the optimal dosages of Bst Ⅱ DNA polymerase and dNTPs，as well as the optimal ratio of inner and outer primers，were determined to be 0.6 U/μL，1.25 mmol/L，and 2∶1 respectively.The specific detection of Botrytis cinerea could be achieved at 61 ℃ for 40 min，and its sensitivity could reach 100 ag/μL，which was 106 times the sensitivity of ordinary PCR detection. When this method was applied to the detection of tomato diseases，the spores detection limit for Botrytis cinerea could reach 20 spores/mL，and the pathogen could be detected in tomato leaves that had been infected for 4 days without obvious phenotypic symptoms of gray mold，making it applicable for the early，rapid，sensitive and visual detection of tomato gray mold.

Key words: Tomato, Botrytis cinerea, Gray mold, Loop‐mediated isothermal amplification, ACTIN gene

摘要： 由灰葡萄孢菌引起的番茄灰霉病是番茄主要病害之一，对番茄的产量和品质造成严重影响。为了对番茄灰霉病进行早期快速检测，以灰葡萄孢菌的ACTIN基因为靶基因，基于环介导等温扩增技术（Loop‐mediated isothermal amplification，LAMP），设计并筛选出一组LAMP特异性引物，优化其反应体系和反应条件，实现对灰葡萄孢菌的快速等温扩增。通过琼脂糖凝胶电泳和SYBR Green Ⅰ可视化分析，确定Bst Ⅱ DNA 聚合酶、dNTPs 的最佳用量和内外引物最佳比例分别为0.6 U/μL、1.25 mmol/L、2∶1，在61 ℃反应40 min即可特异性检测出灰葡萄孢菌，其灵敏度可达100 ag/μL，是普通PCR检测灵敏度的106倍。将该方法应用于番茄病害检测时，其对灰葡萄孢菌的孢子检出限达到20个/mL，且可在番茄被侵染4 d、尚无明显灰霉病感病表型的番茄叶片中检测出来，可用于番茄灰霉病的早期快速、灵敏、可视化检测。

关键词: 番茄, 灰葡萄孢菌, 灰霉病, 环介导等温扩增, ACTIN基因

CLC Number:

S436.412

ZHAO Qian, LI Wen, LI Xiliu, JIA Zhenhua, FENG Xiaojuan, SONG Shuishan. Early and Rapid Detection of Tomato Gray Mold Based on LAMP Technology[J]. Journal of Henan Agricultural Sciences, 2025, 54(6): 84-91.

赵芊, 李文, 李西柳, 贾振华, 封晓娟, 宋水山. 基于LAMP 技术的番茄灰霉病早期快速检测[J]. 河南农业科学, 2025, 54(6): 84-91.

Figures/Tables 8

References

［1］杨向云，刘方，赵芊，等. N-3-羰基十四烷酰基高丝氨酸内酯诱导番茄对灰霉病抗性机制初探［J］.华北农学报，2020，35（4）：169‐176.
YANG X Y，LIU F，ZHAO Q，et al. The exploration of the effect of N‐3‐oxo‐tetradecanoyl homoserine lactone on the resistance to Botrytis cinerea on tomato［J］. Acta Agriculturae Boreali‐Sinica，2020，35（4）：169‐176.

［2］ROMANAZZI G，SMILANICK J L，FELIZIANI E，et al.Integrated management of postharvest gray mold on fruit crops［J］. Postharvest Biology and Technology，2016，113：69‐76.

［3］LI L L，GUO P，JIN H，et al. Different proteomics of Ca2+on SA‐induced resistance to Botrytis cinerea in tomato［J］. Horticultural Plant Journal，2016，2（3）：154‐162.
［4］NOTOMI T，OKAYAMA H，MASUBUCHI H，et al.Loop‐mediated isothermal amplification of DNA［J］.Nucleic Acids Research，2000，28（12）：E63.
［5］刘旺，靳晶豪，陈孝仁. 环介导等温扩增技术的应用进展［J］. 生物技术进展，2021，11（2）：128‐135.
LIU W，JIN J H，CHEN X R. Application progress of loop‐mediated isothermal amplification technique［J］.Current Biotechnology，2021，11（2）：128‐135.
［6］罗阳，田唯嘉，何芳，等. 奶牛乳房炎源肺炎克雷伯菌PCR和LAMP检测方法的建立［J］.河南农业科学，2023，52（2）：145‐150.
LUO Y，TIAN W J，HE F，et al. Establishment of PCR and LAMP methods for detection of Klebsiella pneumoniae from dairy mastitis［J］. Journal of Henan Agricultural Sciences，2023，52（2）：145‐150.
［7］张民秀，谢芝勋，张艳芳，等. 鸡圆圈病毒3型可视化LAMP检测方法的建立及应用［J］. 河南农业科学，2024，53（4）：128‐136.
ZHANG M X，XIE Z X，ZHANG Y F，et al. A visual LAMP assay for detection of chicken gyrovirus 3［J］.Journal of Henan Agricultural Sciences，2024，53（4）：128‐136.
［8］蔡树东，吴双，赵维芯，等. 环介导等温扩增技术的改良及其在检测中的应用进展［J］.河南农业科学，2016，45
（8）：7‐11.
CAI S D，WU S，ZHAO W X，et al. Progress on improvement and development of loop‐mediated isothermal amplification method［J］. Journal of Henan Agricultural Sciences，2016，45（8）：7‐11.
［9］史芳芳，王雷. 草莓枯萎病和炭疽病的双重LAMP快速病原鉴定［J］. 农业生物技术学报，2021，29（6）：1215‐1221.

SHI F F，WANG L. Rapid pathogen identification of Fusarium wilt and anthracnose in strawberry（Fragaria ananassa）by double LAMP［J］. Journal of Agricultural Biotechnology，2021，29（6）：1215‐1221.

［10］王一波，祝山，郭文通，等. 猕猴桃细菌性溃疡病可视化LAMP检测方法的建立与应用［J］. 西北农业学报，2021，30（5）：761‐766.
WANG Y B，ZHU S，GUO W T，et al. Establishment and application of visual LAMP method for rapid detection of Pseudomonas syringae pv.actinidiae［J］.Acta Agriculturae Boreali‐Occidentalis Sinica，2021，30（5）：761‐766.

［11］兰成忠，阮宏春，甘林，等. 苜蓿根腐病菌（Fusarium solani）的LAMP快速检测方法［J］. 江西农业学报，2020，32（1）：27‐32.
LAN C Z，RUAN H C，GAN L，et al. Rapid detection method for Fusarium solani by loop‐mediated isothermal amplification（LAMP）［J］.Acta Agriculturae Jiangxi，2020，32（1）：27‐32.

［12］汪少丽，曲恒华，王英姿，等. 苹果轮纹病菌LAMP快速检测方法的建立［J］.植物保护学报，2020，47（1）：127‐133.
WANG S L，QU H H，WANG Y Z，et al. Development of a loop‐mediated isothermal amplification assay for rapid detection of apple ring rot pathogen Botryosphaeria dothidea［J］. Journal of Plant Protection，2020，47（1）：127‐133.

［13］闫思远，王小利，杜娟，等. 葡萄霜霉病菌环介导等温扩增检测体系的建立［J］. 河南农业大学学报，2020，54（6）：995‐1001.
YAN S Y，WANG X L，DU J，et al. Development of LAMP detection system for Plasmopara viticola［J］.Journal of Henan Agricultural University，2020，54（6）：995‐1001.

［14］杨帆，胡章薇，严浩浩，等. 青稞黑穗病菌LAMP检测体系的建立［J］. 西北农林科技大学学报（自然科学版），2021，49（9）：67‐76.
YANG F，HU Z W，YAN H H，et al. Establishment of LAMP detection system for barley smut［J］.Journal of Northwest A & F University（Natural Science Edition），2021，49（9）：67‐76.

［15］胡章薇，杨帆，张学飞，等. 青稞条纹病LAMP检测体系的建立［J］.西北农林科技大学学报（自然科学版），2021，49（11）：105‐114.
HU Z W，YANG F，ZHANG X F，et al. Establishment of LAMP detection system of Pyrenophora graminea［J］. Journal of Northwest A & F University（Natural Science Edition），2021，49（11）：105‐114.

［16］杜然，张静，杨龙，等. 油菜黑胫病菌和茎基溃疡病菌的LAMP检测方法的建立［J］.植物病理学报，2021，51（1）：123‐134.
DU R，ZHANG J，YANG L，et al. Development of a LAMP method for detection of Leptosphaeria biglobosa and L.maculans in oilseed rape ［J］. Acta Phytopathologica Sinica，2021，51（1）：123‐134.

［17］吴娜，李淑君，康业斌. 烟草疫霉菌LAMP检测方法的建立及验证［J］. 中国烟草科学，2020，41（1）：74‐80.
WU N，LI S J，KANG Y B. Establishment of the loop‐mediated isothermal amplification assay for detection of Phytophthora parasitica［J］. Chinese Tobacco Science，2020，41（1）：74‐80.
［18］刘树森，郭宁，孙华，等. 玉米根腐病菌麦根腐平脐蠕孢的LAMP检测方法［J］.华北农学报，2023，38（1）：162‐167.

LIU S S，GUO N，SUN H，et al. Loop‐mediated isothermal amplification assay for detection of Bipolaris sorokiniana causing maize root rot［J］. Acta Agriculturae Boreali‐Sinica，2023，38（1）：162‐167.
［19］贲海燕，郝永娟，霍建飞，等. 草莓白粉病菌LAMP可视化快速检测方法的建立及初步应用［J］.华北农学报，2022，37（6）：182‐190.
BEN H Y，HAO Y J，HUO J F，et al. Establishment and application of visual LAMP method for rapid detection of Sphaerotheca aphanis causing strawberry powdery mildew［J］.Acta Agriculturae Boreali‐Sinica，2022，37（6）：182‐190.
［20］FAN F，YIN W X，LI G Q，et al. Development of a LAMP method for detecting SDHI fungicide resistance in Botrytis cinerea［J］. Plant Disease，2018，102（8）：1612‐1618.
［21］FAN F，HAHN M，LI G Q，et al. Rapid detection of benzimidazole resistance in Botrytis cinerea by loop‐mediated isothermal amplification［J］.Phytopathology Research，2019，1（1）：10.
［22］蒋荷，李旭，郑慧慧，等. 黄瓜种传镰刀菌种类的鉴定及其致病性研究［J］.中国农业大学学报，2013，18（2）：86‐92.
JIANG H，LI X，ZHENG H H，et al. Study on identification of cucumber seedborne Fusarium species and testing of their pathogenicity［J］.Journal of China Agricultural University，2013，18（2）：86‐92.
［23］戴婷婷，陆辰晨，郑小波. 环介导等温扩增技术在病原物检测上的应用研究进展［J］.南京农业大学学报，2015，38（5）：695‐703.
DAI T T，LU C C，ZHENG X B. Application research progress of Loop‐mediated isothermal amplification in the pathogenic microorganism［J］.Journal of Nanjing Agricultural University，2015，38（5）：695‐703.
［24］梁伊菲，王春伟.黄芪根腐病尖孢镰刀菌LAMP可视化快速检测方法的建立［J］. 山西农业科学，2023，51（4）：420‐426.
LIANG Y F，WANG C W. Establishment of LAMP visual rapid detection for Fusarium oxysporum of root rot of Astragalus membranaceus［J］.Journal of Shanxi Agricultural Sciences，2023，51（4）：420‐426.

[1]	MA Li, GUO Xueliang, YAO Hongyu, LIU Haobin, XING Xiaolong, ZHU Chunhua, QI Hongzhi, LI Xianwei, YU Dongdong, ZHANG Li, LI Chengwei, PEI Dongli, WANG Fang. Isolation and Identification of Antagonistic Endophytic Bacteria against Strawberry Gray Mold and Their Antibacterial Effects [J]. Journal of Henan Agricultural Sciences, 2025, 54(6): 100-109.
[2]	ZENG Wanning, WANG Yanxuan, WANG Fanzhen, CHEN Zhaorong, LIU Huiqin, JIANG Yongcheng, Azmat·Turhorn, LI Baotong. Biocontrol Effect of Bacillus siamensis Y‐54 against Tomato Leaf Mold [J]. Journal of Henan Agricultural Sciences, 2024, 53(3): 103-109.
[3]	HUANG Jiaqing, LIU Cenwei, YE Jing, WANG Yixiang, REN Lihua. Physicochemical Properties and Microstructure Changes of Tomato Straw Hydrothermal Biochar under Different Reaction Conditions [J]. Journal of Henan Agricultural Sciences, 2023, 52(5): 170-180.
[4]	SHAO Zeqiang, BU Xintong, GOU Qiandong, GUO Xiaoxiao, ZHOU Linyuan, WANG Jingbo, LI Yinghua. Effects of Alcoholic Waste Mash Liquid Organic Carbon Fertilizer on Root Growth，Yield and Quality of Small Tomato [J]. Journal of Henan Agricultural Sciences, 2023, 52(3): 109-117.
[5]	XU Mengzhu, YANG Xiaohui, LIU Yan, SHI Yu, SONG Hongxia, ZHANG Yi. Effects of Combined Application of Calcium and Zinc on Fruit Quality of Different Tomato Varieties [J]. Journal of Henan Agricultural Sciences, 2023, 52(2): 124-135.
[6]	LI Jianxin, GE Guimin, HUANG Wen, SHEN Aimin. Application Effect of Mushroom Residue Organic Fertilizer on Taste Tomato Cultivation [J]. Journal of Henan Agricultural Sciences, 2022, 51(5): 117-125.
[7]	DONG Xiaoxing, YU Luming, NIU Xuxu, ZHANG Zengguang, LI Shengli. Temperature Suitability Evaluation and Run Analysis of Tomato Cultivation in Wide⁃span Plastic Greenhouse with External Thermal Insulation [J]. Journal of Henan Agricultural Sciences, 2022, 51(3): 127-138.
[8]	YAN Zongshan, YANG Shimei, LI Yanrong, WANG Lei, ZHANG Xiangping, CHEN Liang, . Effects of Different Yellow Sand Cultivation Patterns on the Rhizosphere Temperature of Tomato in Greenhouse [J]. Journal of Henan Agricultural Sciences, 2021, 50(5): 107-114.
[9]	YANG Dongyan, SANG Ting, FENG Haiping, WANG Rong, MA Ling. Effects of Planting Density on Yield Composition and Light Environment of East-West Ridge of Tomato in Greenhouse [J]. Journal of Henan Agricultural Sciences, 2021, 50(5): 99-106.
[10]	CHEN Yuehao, HE Guangxiong, LI Jie, SHI Liangtao, FANG Haidong, SHI Zhengtao. Tomato Recognition in Yuanmou Hot Area Based on Object‐Oriented GF‐2 Remote Sensing Data [J]. Journal of Henan Agricultural Sciences, 2021, 50(12): 170-180.
[11]	QIN Tao, LIU Xinshe. Effects of Different Tomato Rootstocks on Growth and Physiological Characteristics of Eggplant Grafted Seedlings under Heat Shock Treatment [J]. Journal of Henan Agricultural Sciences, 2021, 50(1): 129-133.
[12]	GAO Zhenfeng, LI Na, ZHANG Xiaoyu, HAN Jucai. Screening and Colonization Characteristics of Efficient Endophytic Antifungal Bacteria against Botrytis cinerea [J]. Journal of Henan Agricultural Sciences, 2020, 49(3): 88-100.
[13]	XI Beibei , YE Jianren. Screening and Identification of High-efficiency Potassium-solubilizing Bacteria and Their Growth-promoting Effects on Plants [J]. Journal of Henan Agricultural Sciences, 2020, 49(2): 81-88.
[14]	CHEN Yan. The Final Residues and Degradation Dynamics of 1.5% Matrine·Osthol AS in Tomato and Soil [J]. Journal of Henan Agricultural Sciences, 2020, 49(2): 74-80.
[15]	GUO Chenxi, ZHOU Guifang, CHEN Bihua, GUO Weili, CAI Zucong, HUANG Xinqi, WANG Guangyin. Effect of Reductive Soil Disinfestation on Growth,Yield,Diseases and Insect Pests of Three Continuous Cultivation Vegetables in Plastic Greenhouse#br# [J]. Journal of Henan Agricultural Sciences, 2020, 49(11): 98-109.