Establishment and Optimization of a Dual Loop-Mediated Isothermal Amplification (LAMP) Rapid Detection System for Sclerotinia sclerotiorum and Alternaria alternata — Oak Academic Publishing
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Establishment and Optimization of a Dual Loop-Mediated Isothermal Amplification (LAMP) Rapid Detection System for Sclerotinia sclerotiorum and Alternaria alternata
School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
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School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
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School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
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School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
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School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
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School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
1 School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
2 School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
3 School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
4 School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
5 School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
6 School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, China
In order to effectively address the problem of the decline in quality and yield of C. morifolium cv. Chuju caused by the simultaneous infection of two pathogenic fungi, the existing LAMP technology (a detection method that can be achieved without expensive equipment) provides a window period for disease prevention. Developing a LAMP detection method that can simultaneously detect different pathogens and has high sensitivity has become the key to solving this problem. In this study, we first designed specific primers based on the conserved sequences of the two common pathogens of C. morifolium cv. Chuju, namely Sclerotinia sclerotiorum and Alternaria alternata , for LAMP amplification. Subsequently, we gradually optimized each aspect of the LAMP method to enhance the detection efficiency and sensitivity. The LAMP assay showed high specificity for both pathogens and a detection limit of 1.43 × 10 − 6 ng/µL, which is about 100-fold higher than that of conventional PCR. These improvements aid in the effective monitoring and early diagnosis of disease during the cultivation of C. morifolium cv. Chuju, which will contribute to improved disease management and reduced economic losses.
Wang, J., Li, X., Xing, S., Ma, Z., Hu, S. and Tu, C. (2017) Bio-Organic Fertilizer Promotes Plant Growth and Yield and Improves Soil Microbial Community in Continuous Monoculture System of Chrysanthemum morifolium cv. Chuju. International Journal of Agriculture and Biology , 19, 563-568. https://doi.org/10.17957/ijab/15.0339
Jin, M., Zhu, Z., Guo, Q., Shen, H. and Wang, Y. (2012) Growth and Accumulation of Bioactive Compounds in Medicinal Chrysanthemum morifolium Ramat. cv. ‘Chuju’ under Different Colored Shade Polyethylene. Journal of Medicinal Plants Research , 6, 398-404. https://doi.org/10.5897/jmpr11.1026
Yuan, H., Jiang, S., Liu, Y., Daniyal, M., Jian, Y., Peng, C., et al . (2020) The Flower Head of Chrysanthemum morifolium Ramat. (Juhua): A Paradigm of Flowers Serving as Chinese Dietary Herbal Medicine. Journal of Ethnopharmacology , 261, Article 113043. https://doi.org/10.1016/j.jep.2020.113043
Liu, Y., Xin, J., Liu, L., Song, A., Guan, Z., Fang, W., et al . (2020) A Temporal Gene Expression Map of Chrysanthemum Leaves Infected with Alternaria alternata Reveals Different Stages of Defense Mechanisms. Horticulture Research , 7, Article No. 23. https://doi.org/10.1038/s41438-020-0245-0
Liu, B., Li, Z., Du, J., Zhang, W., Che, X., Zhang, Z., et al . (2022) 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. Pathogens , 11, Article 1221. https://doi.org/10.3390/pathogens11111221
Zhang, S., Liu, L., Li, W., Yin, M., Hu, Q., Chen, S., et al . (2025) Alternaria alternata Effector Aaalta1 Targets CmWD40 and Participates in Regulating Disease Resistance in Chrysanthemum morifolium . PLOS Pathogens , 21, e1012942. https://doi.org/10.1371/journal.ppat.1012942
Seliem, M.K., Taha, N.A., El-Feky, N.I., Abdelaal, K., El-Ramady, H., El-Mahrouk, M.E., et al . (2024) Evaluation of Five Chrysanthemum morifolium Cultivars against Leaf Blight Disease Caused by Alternaria alternata at Rooting and Seedling Growth Stages. Plants , 13, Article 252. https://doi.org/10.3390/plants13020252
Prova, A., Akanda, A.M., Islam, S. and Hossain, M.M. (2018) Characterization of Sclerotinia sclerotiorum , an Emerging Fungal Pathogen Causing Blight in Hyacinth Bean ( Lablab purpureus ). The Plant Pathology Journal , 34, 367-380. https://doi.org/10.5423/ppj.oa.02.2018.0028
Mekapogu, M., Jung, J., Kwon, O., Ahn, M., Song, H. and Jang, S. (2021) Recent Progress in Enhancing Fungal Disease Resistance in Ornamental Plants. International Journal of Molecular Sciences , 22, Article 7956. https://doi.org/10.3390/ijms22157956
Duan, Y., Ge, C., Zhang, X., Wang, J. and Zhou, M. (2013) A Rapid Detection Method for the Plant Pathogen Sclerotinia sclerotiorum Based on Loop-Mediated Isothermal Amplification (LAMP). Australasian Plant Pathology , 43, 61-66. https://doi.org/10.1007/s13313-013-0239-6
Zhang, X., Xu, G., Tang, H., Li, Y. and Liu, C. (2017) Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Alternaria alternata . Journal of AOAC International , 100, 99-103. https://doi.org/10.5740/jaoacint.16-0196
Nath, V.S., Hegde, V.M., Jeeva, M.L., Misra, R.S., Veena, S.S., Raj, M., et al . (2014) Rapid and Sensitive Detection of Phytophthora colocasiae Responsible for the Taro Leaf Blight Using Conventional and Real-Time PCR Assay. FEMS Microbiology Letters , 352, 174-183. https://doi.org/10.1111/1574-6968.12395
Ioos, R., Fabre, B., Saurat, C., Fourrier, C., Frey, P. and Marçais, B. (2010) Development, Comparison, and Validation of Real-Time and Conventional PCR Tools for the Detection of the Fungal Pathogens Causing Brown Spot and Red Band Needle Blights of Pine. Phytopathology® , 100, 105-114. https://doi.org/10.1094/phyto-100-1-0105
Thomson, D. and Dietzgen, R.G. (1995) Detection of DNA and RNA Plant Viruses by PCR and RT-PCR Using a Rapid Virus Release Protocol without Tissue Homogenization. Journal of Virological Methods , 54, 85-95. https://doi.org/10.1016/0166-0934(95)00022-m
Green, S.J., Venkatramanan, R. and Naqib, A. (2015) Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches. PLOS ONE , 10, e0128122. https://doi.org/10.1371/journal.pone.0128122
Orpana, A.K., Ho, T.H., Alagrund, K., Ridanpää, M., Aittomäki, K. and Stenman, J. (2013) Novel Heat Pulse Extension-PCR-Based Method for Detection of Large CTG-Repeat Expansions in Myotonic Dystrophy Type 1. The Journal of Molecular Diagnostics , 15, 110-115. https://doi.org/10.1016/j.jmoldx.2012.07.004
Stevens, A.J., Appleby, S. and Kennedy, M.A. (2016) [Letter to the Editor] Many Commercial Hot-Start Polymerases Demonstrate Activity Prior to Thermal Activation. BioTechniques , 61, 293-296. https://doi.org/10.2144/000114481
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. (2000) Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Research , 28, E63. https://doi.org/10.1093/nar/28.12.e63
Drais, M.I., Maheshwari, Y., Selvaraj, V., Varvaro, L., Yokomi, R. and Djelouah, K. (2019) Development and Validation of a Loop-Mediated Isothermal Amplification Technique (LAMP) for the Detection of Spiroplasma Citri, the Causal Agent of Citrus Stubborn Disease. European Journal of Plant Pathology , 155, 125-134. https://doi.org/10.1007/s10658-019-01755-6
Winkworth, R.C., Nelson, B.C.W., Bellgard, S.E., Probst, C.M., McLenachan, P.A. and Lockhart, P.J. (2020) A LAMP at the End of the Tunnel: A Rapid, Field Deployable Assay for the Kauri Dieback Pathogen, Phytophthora Agathidicida. PLOS ONE , 15, e0224007. https://doi.org/10.1371/journal.pone.0224007
Ravindran, A., Lévy, J., Pierson, E. and Gross, D.C. (2015) Loop-Mediated Isothermal Amplification Procedure (LAMP) for Detection of the Potato Zebra Chip Pathogen “Candidatus Liberibacter Solanacearum”. In: Lacomme, C., Ed., Methods in Molec ular Biology , Springer, 85-97. https://doi.org/10.1007/978-1-4939-2620-6_7
Yin, Y.B., Li, D.L., Yang, B.H., Zhao, Z., Meng, S.Y., Liu, B.S. and Chen, Z.L. (2022) Establishment of Loop-Mediated Isothermal Amplification for Brucella Detection Using a Warmer Pad as a Heating Source. BioTechniques , 73, 142-150. https://doi.org/10.2144/btn-2021-0043
Parida, M., Posadas, G., Inoue, S., Hasebe, F. and Morita, K. (2004) Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus. Journal of Clinical Microbiology , 42, 257-263. https://doi.org/10.1128/jcm.42.1.257-263.2004
Niessen, L. and Vogel, R.F. (2010) Detection of Fusarium Graminearum DNA Using a Loop-Mediated Isothermal Amplification (LAMP) Assay. International Journal of Food Microbiology , 140, 183-191. https://doi.org/10.1016/j.ijfoodmicro.2010.03.036
Grabicoski, E.M.G., Jaccoud-Filho, D.d.S., Lee, D., Henneberg, L. and Pileggi, M. (2020) Real-time Quantitative and Ion-Metal Indicator Lamp-Based Assays for Rapid Detection of Sclerotinia sclerotiorum . Plant Disease , 104, 1514-1526. https://doi.org/10.1094/pdis-07-19-1455-re
Pecchia, S., Caggiano, B., Da Lio, D., Cafà, G., Le Floch, G. and Baroncelli, R. (2019) Molecular Detection of the Seed-Borne Pathogen Colletotrichum Lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster. Plants , 8, Article 222. https://doi.org/10.3390/plants8070222
Chand, P. and Rai, D.C. (2008) In Vitro Evaluation of Different Organic Amendments against Sclerotinia sclerotiorum (Lib) de Bary. Plant Disease Research , 23, 84-85.
Kim, E., Lee, H.M. and Kim, Y.H. (2017) Morphogenetic Alterations of Alternaria alternata Exposed to Dicarboximide Fungicide, Iprodione. The Plant Pathology Journal , 33, 95-100. https://doi.org/10.5423/ppj.nt.06.2016.0145
Bustin, S.A., Ruijter, J.M., van den Hoff, M.J.B., Kubista, M., Pfaffl, M.W., Shipley, G.L., et al . (2025) MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines. Clinical Chemistry , 71, 634-651. https://doi.org/10.1093/clinchem/hvaf043
Shu, R., Yin, X., Long, Y., Yuan, J. and Zhou, H. (2022) Detection and Control of Pantoea Agglomerans Causing Plum Bacterial Shot-Hole Disease by Loop-Mediated Isothermal Amplification Technique. Frontiers in Microbiology , 13, Article ID: 896567. https://doi.org/10.3389/fmicb.2022.896567
Lai, M.Y., Ooi, C.H. and Lau, Y.L. (2021) Validation of SYBR Green I Based Closed‐tube Loop‐Mediated Isothermal Amplification (LAMP) Assay for Diagnosis of Knowlesi Malaria. Malaria Journal , 20, Article No. 166. https://doi.org/10.1186/s12936-021-03707-0
Ghosh, R., Nagavardhini, A., Sengupta, A. and Sharma, M. (2015) Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Fusarium oxysporum F. Sp. Ciceris-Wilt Pathogen of Chickpea. BMC Research Note s , 8, Article No. 40. https://doi.org/10.1186/s13104-015-0997-z
Li, J., Wang, S., Yu, J., Wang, L. and Zhou, S. (2013) A Modified CTAB Protocol for Plant DNA Extraction. Chinese Bulletin of Botany , 48, 72-78. https://doi.org/10.3724/sp.j.1259.2013.00072
Porebski, S., Bailey, L.G. and Baum, B.R. (1997) Modification of a CTAB DNA Extraction Protocol for Plants Containing High Polysaccharide and Polyphenol Components. Plant Molecular Biology Reporter , 15, 8-15. https://doi.org/10.1007/bf02772108
He, Y., Tong, C., Chen, H., Zhao, W., Zhan, L., Wang, R., et al . (2025) A Rapid and Visual Detection Method for Alternaria alternata , the Causal Agent of Leaf Spot Disease on Yam, Based on RPA-CRISPR/Cas12a. Physiological and Molecular Plant Pathology , 137, Article 102612. https://doi.org/10.1016/j.pmpp.2025.102612
Yin, Y., Ding, L., Liu, X., Yang, J. and Ma, Z. (2009) Detection of Sclerotinia sclerotiorum in Planta by a Real‐Time PCR Assay. Journal of Phytopathology , 157, 465-469. https://doi.org/10.1111/j.1439-0434.2009.01543.x
Henke, W., Herdel, K., Jung, K., Schnorr, D. and Loening, S.A. (1997) Betaine Improves the PCR Amplification of GC-Rich DNA Sequences. Nucleic Acids Research , 25, 3957-3958. https://doi.org/10.1093/nar/25.19.3957
Banger, S., Pal, V., Tripathi, N.K. and Goel, A.K. (2021) Development of a Set of Three Real-Time Loop-Mediated Isothermal Amplification (LAMP) Assays for Detection of Bacillus Anthracis, the Causative Agent of Anthrax. Folia Microbiologica , 66, 587-596. https://doi.org/10.1007/s12223-021-00869-x
Trangoni, M.D., Gioffré, A.K., Cerón Cucchi, M.E., Caimi, K.C., Ruybal, P., Zumárraga, M.J., et al . (2015) LAMP Technology: Rapid Identification of Brucella and Mycobacterium Avium Subsp. Paratuberculosis. Brazilian Journal of Microbiology , 46, 619-626. https://doi.org/10.1590/s1517-838246220131206
Gunasegar, S. and Neela, V.K. (2021) Evaluation of Diagnostic Accuracy of Loop-Mediated Isothermal Amplification Method (LAMP) Compared with Polymerase Chain Reaction (PCR) for Leptospira spp. in Clinical Samples: A Systematic Review and Meta-Analysis. Diagnostic Microbiology and Infectious Disease , 100, Article 115369. https://doi.org/10.1016/j.diagmicrobio.2021.115369
Khan, M., Wang, R., Li, B., Liu, P., Weng, Q. and Chen, Q. (2018) Comparative Evaluation of the LAMP Assay and PCR-Based Assays for the Rapid Detection of Alternaria Solani. Frontiers in Microbiology , 9, Article ID: 2089. https://doi.org/10.3389/fmicb.2018.02089
Foo, P.C., Nurul Najian, A.B., Muhamad, N.A., Ahamad, M., Mohamed, M., Yean Yean, C., et al . (2020) Loop-Mediated Isothermal Amplification (LAMP) Reaction as Viable PCR Substitute for Diagnostic Applications: A Comparative Analysis Study of LAMP, Conventional PCR, Nested PCR (NPCR) and Real-Time PCR (qPCR) Based on Entamoeba hist olytica DNA Derived from Faecal Sample. BMC Biotechnology , 20, Article No. 34. https://doi.org/10.1186/s12896-020-00629-8
Hu, T. and Desai, J.P. (2004) Soft-Tissue Material Properties under Large De-Formation: Strain Rate Effect. Proceedings of the 26th Annual International Conference of the IEEE EMBS , San Francisco, 1-5 September 2004, 2758-2761.