Establishment and Application of Visual LAMP Method for Rapid Detection of Sphaerotheca aphanis Causing Strawberry Powdery Mildew

doi:10.7668/hbnxb.20193522

Abstract

Abstract:

Strawberry powdery mildew caused by Sphaerotheca aphanis is one of the strawberry diseases with large incidence area and high incidence frequency in the protected cultivation areas, which usually occurs from seedling stage to fruiting stage. Therefore, the establishment of a rapid and efficient detection method of S. aphanis is quite important for the diagnosis and control of strawberry powdery mildew.A set of LAMP amplification primers including F3/B3、FIP/BIP and LF/LB were designed for the conserved sequence of ITS gene of Sphaerotheca aphanis as the target gene. Through the double judgment method of Real-time fluorescence curve and fluorescence color change, the main factors in the LAMP reaction system including the final concentrations of dNTPs, MgSO₄, BstDNA polymerase and betaine and amplification temperature were selected and optimized to increased etections ensitivity and specificity. Finally, the effects of field samples detection by optimized LAMP were evaluated.The results showed that the LAMP detection system of strawberry powdery mildew was established successfully. The optimized reaction conditions included dNTPs 1.6 mmol/L, MgSO₄ 8 mmol/L, BstDNA polymerase 320 U/moL and betaine 1.2 mmol/L and the amplification temperature was 65 ℃. Under such conditions, the minimum detection sensitivity was 3.2×10^-4 ng/μL within 60 min, and the efficiency of which was 100 times higher than the result of PCR amplification.The visual LAMP method could effectively detect the samples with no obvious symptoms infected by S. aphanis in the early stage. This method has the advantages of short detection time, direct observation with eyes, low requirements for operators and low detection cost. It has important guiding significance for the early diagnosis, monitoring, early prevention and determination of the best control time of strawberry powdery mildew.

Key words: Strawberry powdery mildew, Sphaerotheca aphanis, Loop-mediateddisothermal amplification (LAMP), Detection

BEN Haiyan, HAO Yongjuan, HUO Jianfei, YAO Yurong, GAO Wei, WANG Wanli. 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. doi: 10.7668/hbnxb.20193522.

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Figures/Tables 11

Tab.1 Designed primer set for LAMP detection of strawberry powdery mildew

引物名称 Prime name	引物序列(5'—3') Prime sequence(5'—3')	引物组碱基长度/bp Prime set length of base
F3-1	CGGTATTCCGAGGGGCAT	18
B3-1	CCTGATCCGAGGTCATCCAA	20
FIP-1	CTGTTTAGGGGACGCCGAGCTGTTCGAGCGTCAGAACATC	40
BIP-1	GGCGGTACCGTTGTGCTCTCTAGGTGGGTTTTGGCAGGT	38
LF-1	ACCAAGCCAGGCTTGAGAG	19
LB-1	GTAGTCACGTATCTCGCGACAGAG	24

Fig.1 Optimized results of LAMP detection system for final concentration of dNTPs A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—6 were the final dNTP concentrations of 0.8,1.0,1.2,1.4,1.6,1.8 mmol/L,respectively;N was the negative control).

Fig.2 Optimized results of LAMP detection system for final concentration of Mg²⁺ A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—5 were the final Mg²⁺ concentrations of 4,6,8,10,12 mmol/L,respectively;N was the negative control).

Fig.3 Optimized results of LAMP detection system for final concentration of betaine A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—7 were the final betaine concentrations of 0,0.2,0.4,0.6,0.8,1.0,1.2 mmol/L,respectively,N was the negative control).

Fig.4 Optimized results of LAMP detection system for final concentration of BstDNA polymerase A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—5 were the final BstDNA polymerase concentrations of 40,80,160,320,340 U/mL,respectively;N was the negative control).

Fig.5 Optimized results of LAMP detection system for final concentration of temperature A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution (Numbers 1—3 were temperatures of 63,65,67 ℃,respectively;N was the negative control).

Fig.6 Detection results of lamp primer specificity of strawberry powdery mildew A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution (Number 1 was strawberry powdery mildew(Sphaerotheca aphanis),2—7 were the control strains,followed by powdery mildew of pepper(Oidiopsis taurica),cucumber powdery mildew(Sphaerotheca cucurbitae),strawberry gray mold(Botrytis cinerea),strawberry root rot(Fusarium sp.),strawberry anthracnose(Colletotrichum gloeosporioides),white leaf spot of strawberry(Mycosphaerella fragariae),respectively;N was the negative control).

Fig.7 Detection results of sensitivity of strawberry powdery mildew by using lamp A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(The bold DNA concentrations of strawberry pathogens numbered 1—7 were 3.2,3.2×10^-1,3.2×10^-2,3.2×10^-3,3.2×10^-4,3.2×10^-5,3.2×10^-6ng/μL,respectively;N was the negative control).

Fig.8 Detection results of sensitivity of strawberry powdery mildew by using PCR amplification

Fig.9 Repeatability test results of lamp optimized system for strawberry powdery mildew 1.Strawberry blade (mild disease);2.Strawberry fruit (mild disease);3.Strawberry stalk(slightly affected);4.Strawberry blade (serious disease);5.Strawberry fruit (serious disease);6.Strawberry stalk(serious disease);7.Strawberry healthy fruit.

Fig.10 Field detection results of strawberry powdery mildew by using LAMP 1—7.Strawberry leaf samples:1.Leaves with lighter disease,2,3.Leaves with more heavy disease,4,5.Leaves with serious disease,6,7.Healthy leaves;8—14.Strawberry fruit samples:8,9.Mild disease,10.More severe disease,11,12.Serious disease,13,14.Healthy fruits;15—21.Strawberry stalk samples:15.Stalk with light disease,16,17.Stalks with heavy disease,18,19.Stalks with serious disease,20,21.Healthy leaves.

References 21

[1]	杨俊誉, 魏世杰, 苏代发, 陈杉艳, 罗志伟, 沈雪梅, 赖泳红, Arslan Jamil, 童江云, 崔晓龙. 草莓白粉病病原菌及分子防御机制的研究进展[J]. 云南大学学报(自然科学版), 2019, 41, 47(4):842-850.doi:10.7540/j.ynu.20180733. doi: 10.7540/j.ynu.20180733
	Yang J Y, Wei S J, Su D F, Chen S Y, Luo Z W, Shen X M, Lai Y H, Arslan J, Tong J Y, Cui X L. Research advances on powdery mildew pathogens and molecular defense mechanism of strawberries[J]. Journal of Yunnan University(Natural Sciences Edition), 2019, 41, 47(4):842-850.
[2]	杨书珍, 柳丽梅, 彭丽桃, 周厚成, 潘思轶, 张丽华. 草莓采后真菌病害控制研究进展[J]. 华中农业大学学报, 2012, 31 (1):127-132.doi:10.3969/j.issn.1000-2421.2012.01.023. doi: 10.3969/j.issn.1000-2421.2012.01.023
	Yang S Z, Liu L M, Peng L T, Zhou H C, Pan S Y, Zhang L H. Advances in controlling of pathogenic fungi infections in post-harvest strawberry[J]. Journal of Huazhong Agricultural University, 2012, 31 (1):127-132.
[3]	关玲, 赵密珍, 王庆莲, 于红梅, 袁华招. 草莓品种 (系) 白粉病田间抗性鉴定[J]. 吉林农业大学学报, 2018, 40 (3):276-284.doi:10.13327/j.jjlau.2017.3787. doi: 10.13327/j.jjlau.2017.3787
	Guan L, Zhao M Z, Wang Q L, Yu H M, Yuan H Z. Field natural evaluation of powdery mildew resistance in different cultivars and progenies of strawberry[J]. Journal of Jilin Agricultural University, 2018, 40 (3):276-284.
[4]	董华芳, 姚智, 许延波, 张旭东, 杨军, 刘永碧. 十种药剂对草莓白粉病的田间防效[J]. 北方园艺, 2020, 40 (5):28-33.doi:doi:10.11937/bfyy.20192813. doi: doi:10.11937/bfyy.20192813
	Dong H F, Yao Z, Xu Y B, Zhang X D, Yang J, Liu Y B. Control effect of ten medicines on strawberry powdery mildew[J]. Northern Horticulture, 2020, 40(5):28-33.
[5]	刘永春, 张红玲. 腈苯唑与氟啶胺混剂防治草莓白粉病和灰霉病[J]. 农药, 2017, 56 (5):374-376.doi:10.16820/j.cnki.1006-0413.2017.05.018. doi: 10.16820/j.cnki.1006-0413.2017.05.018
	Liu Y C, Zhang H L. The mixture of fenbuconazole and fluazinam against strawberry powdery mildew and strawberry gray mould[J]. Agrochemicals, 2017, 56 (5):374-376.
[6]	杨瑞, 王建立, 于同泉, 沈元月, 房克凤, 王绍辉. 运用扫描电镜技术观察草莓白粉病菌[J]. 电子显微学报, 2013, 32 (3):260-265.doi:10.3969/j.1000-6281.2013.03.013. doi: 10.3969/j.1000-6281.2013.03.013
	Yang R, Wang J L, Yu T Q, Shen Y Y, Fang K F, Wang S H. SEM studies on strawberry powdery-mildew[J]. Journal of Chinese Electron Microscopy Society, 2013, 32 (3):260-265.
[7]	杨俊誉, 魏世杰, 苏代发, 张振荣, 陈杉艳, 罗志伟, 沈雪梅, 赖泳红, Arslan Jamil, 童江云, 崔晓龙. 温室中患白粉病与健康草莓植株根际原核生物群落的比较研究[J]. 西南农业学报, 2020, 33 (1):85-91.doi:10.16213/j.cnki.scjas.2020.1.014. doi: 10.16213/j.cnki.scjas.2020.1.014
	Yang J Y, Wei S J, Su D F, Zhang Z R, Chen S Y, Luo Z W, Shen X M, Lai Y H, Arslan J, Tong J Y, Cui X L. Study on prokaryotic communities in rhizospheres of powdery mildew-infected and non-infected strawberry in greenhouse by high-through sequencing technology[J]. Southwest China Journal of Agricultural Sciences, 2020, 33 (1):85-91.
[8]	Notomi T. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28 (12):63. pmid: 10871386
[9]	Goto M, Honda E, Ogura A, Nomoto A. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue[J]. Biotechniques, 2009, 46 (3):167-172.doi:10.2144/000113072. doi: 10.2144/000113072 pmid: 19317660
[10]	王一波, 祝山, 郭文通, 秦虎强, 刘巍, 徐亮胜, 黄丽丽. 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.doi:10.7606/j.issn.1004-1389.2021.05.016. doi: 10.7606/j.issn.1004-1389.2021.05.016
	Wang Y B, Zhu S, Guo W T, Qin H Q, Liu W, Xu L S, Huang L L. 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]	谷莉, 郑玉东, 张翔, 白昌明, 刘金兰, 辛鲁生, 李晨, 王崇明. 鲍疱疹病毒原位LAMP检测方法的建立与初步应用[J]. 水产学报, 2022, 46 (5):885-894.doi:10.11964/jfc.20211013104. doi: 10.11964/jfc.20211013104
	Gu L, Zheng Y D, Zhang X, Bai C M, Liu J L, Xin L S, Li C, Wang C M. Establishment and application of in situ LAMP for detection of Haliotid herpesvirus 1 (HaHV-1)[J]. Journal of Fisheries of China, 2022, 46 (5):885-894.
[12]	Yu L Z, Song S Y, Yu C, Jiao B B, Tian Y M, Li Y J. Rapid detection of Anguina agrostis by loop-mediated isothermal amplification[J]. European Journal of Plant Pathology, 2020, 156 (3):819-825.doi:10.1007/s10658-020-01932-y. doi: 10.1007/s10658-020-01932-y URL
[13]	张振荣, 田云霞, 董琼娥, 谭海燕, 张丽芳, 冯德党, 赖泳红, 杨俊誉, 苏代发, 崔晓龙, 童江云. 基于高通量测序的草莓白粉病病原菌分析[J]. 西南农业学报, 2021, 34 (7):1439-1443.doi:10.16213/j.cnki.scjas.2021.7.011. doi: 10.16213/j.cnki.scjas.2021.7.011
	Zhang Z R, Tian Y X, Dong Q E, Tan H Y, Zhang L F, Feng D D, Lai Y H, Yang J Y, Su D F, Cui X L, Tong J Y. Analysis of pathogen of strawberry powdery mildew based on High-throughput sequencing[J]. Southwest China Journal of Agricultural Sciences, 2021, 34 (7):1439-1443.
[14]	曹学仁, 周益林. 基于PCR技术的植物病原菌分子定量检测技术研究进展[J]. 植物保护, 2020, 46 (4):7-11.doi:10.16688/j.zwbh.2019463. doi: 10.16688/j.zwbh.2019463
	Cao X R, Zhou Y L. Research progress in quantitative detection of plant pathogens using PCR technique[J]. Plant protection, 2020, 46 (4):7-11.
[15]	Rigotti S, Gindro K, Richter H, Viret O. Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.:Fr.in strawberry (Fragaria × ananassa Duch.) using PCR. Fems Microbiology Letters, 2002, 209 (2):169-174.doi:10.1016/S0378-1097(02)00491-3. doi: 10.1016/S0378-1097(02)00491-3
[16]	王楠, 王剑, 尹丹韩, 高观朋, 王伟. 三重PCR检测草莓灰霉病菌、炭疽病菌和黄萎病菌[J]. 中国农业科学, 2010, 43 (21):4392-4400.doi:10.3864/j.issn.0578-1752.2010.21.008. doi: 10.3864/j.issn.0578-1752.2010.21.008
	Wang N, Wang J, Yin D H, Gao G P, Wang W. Triplex PCR detection of Botrytis cinerea,Colletotrichum gloeosporioides and Verticillium dahliae in infected strawberry plant tissues[J]. Scientia Agricultura Sinica, 2010, 43 (21):4392-4400.
[17]	刘雅, 杨静, 张丽勍, 李斌, 周艳孔, 周洁, 高清华, 段可. 上海地区四种草莓病毒的检测分析[J]. 上海农业学报, 2021, 37 (4):63-67.doi:10.15955/j.issn1000-3924.2021.04.12 doi: 10.15955/j.issn1000-3924.2021.04.12
	Liu Y, Yang J, Zhang L Q, Li B, Zhou Y K, Zhou J, Gao Q H, Duan K. Detection of four viruses infecting strawberry in Shanghai[J]. Acta Agriculturae Shanghai, 2021, 37 (4):63-67.
[18]	陈柳, 尚巧霞, 陈笑瑜, 邢冬梅, 冉策, 魏艳敏, 赵晓燕, 刘正坪. 草莓轻型黄边病毒 RT-LAMP 检测方法的建立[J]. 中国农业科学, 2015, 48 (3):613-620.doi:10.3864/j.issn.0578-1752.2015.03.20. doi: 10.3864/j.issn.0578-1752.2015.03.20
	Chen L, Shang Q X, Chen X Y, Xing D M, Ran C, Wei Y M, Zhao X Y, Liu Z P. Detection of Strawberry mild yellow edge virus by RT-LAMP[J]. Scientia Agricultura Sinica, 2015, 48 (3):613-620.
[19]	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.doi:10.3969/j.issn.1674-7968.2021.06.019. doi: 10.3969/j.issn.1674-7968.2021.06.019
	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.
[20]	Kreitlow A, Becker A, Schotte U, Malorny B, Plötz M, Abdulmawjood A. Evaluation of different target genes for the detection of Salmonella spp. by loop-mediated isothermal amplification (LAMP)[J]. Letters in Applied Microbiology, 2021, 72 (4):420-426.doi:10.1111/lam.13409. doi: 10.1111/lam.13409 pmid: 33030743
[21]	Kokane A, Kokane S, Gubyad M, Ghosh D K. A Rapid and Sensitive Reverse Transcription-loop-mediated isothermal amplication (RT-LAMP) assay for the detection of Indian citrus ringspot virus[J]. Plant Disease, 2021,(105):1346-1355.doi:10.1094/PDIS-06-20-1349-RE. doi: 10.1094/PDIS-06-20-1349-RE

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