Abstract
Coffee leaf rust (CLR) caused by Hemileia vastatrix is a devastating worldwide disease. Early monitoring is crucial for controlling CLR quickly and efficiently. However, accurately identifying CLR in its early stages via the naked eye is challenging. Moreover, detecting H. vastatrix using PCR-based methods is time-consuming, labour-intensive, and occasionally exhibits low sensitivity. Loop-Mediated Isothermal Amplification (LAMP) technology is known for its speed, specificity, and sensitivity to identifying many pathogens accurately. Therefore, in this study, we conducted a comparative analysis of ITS sequences from H. vastatrix and other H. vastatrix and Uredinales strains available in the National Center for Biotechnology Information (NCBI) database using the BLASTn tool. Based on this analysis, we designed specific primers that target the unique region and its flanking regions within the ITS sequences of H. vastatrix. Using SYBR Green I dye, we established a LAMP technique for rapid and sensitive detection of H. vastatrix. Moreover, we optimised the LAMP protocol to enhance sensitivity and specificity for H. vastatrix detection. Under the optimised conditions, the established LAMP protocol detected as little as 1pg/μL of H. vastatrix DNA within 60min at 63°C. This sensitivity is approximately 100 times higher than that achieved using conventional PCR. Our method proved effective in detecting H. vastatrix at the early stages of CLR symptom development on the coffee leaves in field conditions.
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Data availability
DNA sequences are available in the GenBank database, with the accession numbers listed in the Results. All other relevant data are within the paper.
References
Badela JL, Zambolimb L (2019) Coffee bacterial diseases: a plethora of scientific opportu- nities. Plant Pathology 68:411–425
Bebber DP, Castillo ÁD, Gurr SJ (2016) Modelling coffee leaf rust risk in Colombia with climate reanalysis data. Philosophical Transactions of The Royal Society B-Biological Sciences 371:20150458
Beirn LA, Clarke BB, Crouch JA (2014) Influence of host and geographic locale on the distribution of Colletotrichum cereale lineages. PLoS One 9:e97706
Belan LL, de Jesus WC, de Souza AF, Zambolim L, Tomaz MA, Alves FR, Ferrao MAG, do Amaral JFT (2015) Monitoring of leaf rust in conilon coffee clones to improve fungicide use. Australasian Plant Pathology 44: 5-12.
Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H (2010) ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases. BMC Microbiology 10:189
Bertacca S, Caruso AG, Trippa D, Marchese A, Giovino A, Matic S, Noris E, San Ambrosio MIF, Alfaro A, Panno S, Davino S (2022) Development of a real-time loopmediated isothermal amplification assay for the rapid detection of Olea Europaea geminivirus. Plants (Basel) 11:660
Capucho AS, Zambolim L, Lopes UN, Milagres NS (2013) Chemical control of coffee leaf rust in Coffea canephora cv. Conilon. Australasian Plant Pathology 42:667–673
Chen W, Djama ZR, Coffey MD, Martin FN, Bilodeau GJ, Radmer L, Denton G, Lévesque CA (2013) Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species. Phytopathology 103:43–54
Chen X, Ma L, Qiang S, Ma D (2016) Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas. Scientific Reports 6:25688
Crouch JA, Tredway LP, Clarke BB, Hillman BI (2009) Phylogenetic and population genetic divergence correspond with habitat for the pathogen Colletotrichum cereale and allied taxa across diverse grass communities. Molecular Ecology 18:123–135
de Resende MLV, Pozza EA, Reichel T, Botelho DMS (2021) Strategies for coffee leaf rust management in organic crop systems. Agronomy 11:1865
Feng W, Ishiguro Y, Hotta K, Watanabe H, Suga H, Kageyama K (2015) Simple detection of Pythium irregulare using loop-mediated isothermal amplification assay. FEMS Fems Microbiology Letters 362:174
Fukuta S, Takahashi R, Kuroyanagi S, Ishiguro Y, Miyake N, Nagai H, Suzuki H, Tsuji T, Hashizume F, Watanabe H, Kageyama K (2014) Development of loop- mediated isothermal amplification assay for the detection of Pythium myriotylum. Letters in Applied Microbiology 59:49–57
Galvis CA, Leguizamon JE, Gaitan AL, Mejia JF, Alvarez E, Arroyave J (2007) Detection and identification of a group 16SrIII-related phytoplasma associated with coffee crispiness disease in Colombia. Plant Disease 91:248–252
Gehesquière B, D'Haeyer S, PhamK TK, Van Kuik AJ, Maes M, Höfte M, Heungens K (2013) qPCR assays for the detection of Cylindrocladium buxicola in plant, water, and air samples. Plant Disease 97:1082–1090
Gichuru E, Alwora G, Gimase J, Kathurima C (2021) Coffee leaf rust (Hemileia vasta- trix) in Kenya—a review. Agronomy 11:2590
Grisham MP, Pan Y-B (2007) A genetic shift in the virus strains that cause mosaic in Louisi- ana sugarcane. Plant Disease 91:453–458
Harder CB, Læssøe T, Frøslev TG, Ekelund F, Rosendahl S, Kjøller R (2013) A three-gene phylogeny of the Mycena pura complex reveals 11 phylogenetic species and shows ITS to be unreliable for species identification. Fungal Biology 117:764–775
Katoh H, Yamazaki S, Fukuda T, Sonoda S, Nishigawa H, Natsuaki T (2021) Detection of Fusarium oxysporum f. sp. fragariae by using loop-mediated isothermal amplification. Plant Disease 105:1072–1079
Khan M, Li B, Jiang Y, Weng Q, Chen Q (2017) Evaluation of different PCR-based assays and LAMP method for rapid detection of Phytophthora Infestans by targeting the Ypt1 gene. Frontiers in Microbiology 8:1920
Koo C, Malapi-Wight M, Kim HS, Cifci OS, Vaughn-Diaz VL, Ma B, Kim S, Abdel-Raziq H, Ong K, Jo YK, Gross DC, Shim WB, Han A, Abrams WR (2013) Development of a real-time microchip PCR system for portable plant disease diagnosis. PLoS One 8:e0082704
Lan C, Gan L, Dai Y, Liu X, Yang X (2022) Development of loop-mediated isothermal amplification (LAMP) assay for specific and sensitive detection of Mycocentrospora acerina (hart.) causing round leaf spot disease in sanqi (Panax notoginseng). Horticulturae 8:1060
Li L, Várzea VMP, Xia Q, Xiang W, Tang T, Zhu M, He C, Pereira AP, da Silva L, MdCM WW, Yi K (2021) First report of Hemileia vastatrix (coffee leaf rust) physiological races emergent in coffee germplasm collections in the coffee-cropping regions of China. Plant Disease 105:4162
Lu C, Song B, Zhang H, Wang Y, Zheng X (2015) Rapid diagnosis of soybean seedling blight caused by Rhizoctonia solani and soybean charcoal rot caused by Macrophomina phaseolina using LAMP assays. Phytopathology 105:1612–1617
Lu L, Tibpromma S, Karunarathna SC, Jayawardena RS, Lumyong S, Xu J, Hyde KD (2022) Comprehensive review of fungi on coffee. Pathogens 11:411
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28:e63
Ortega SF, Tomlinson J, Hodgetts J, Spadaro D, Gullino ML, Boonham N (2018) Development of loop-mediated isothermal amplification assays for the detection of seedborne fungal pathogens Fusarium fujikuroi and Magnaporthe oryzae in Rice seed. Plant Disease 102:1549–1558
Patel R, Mitra B, Vinchurkar M, Adami A, Patkar R, Giacomozzi F, Lorenzelli L, Baghini MS (2022) A review of recent advances in plant-pathogen detection systems. Heliyon 8:e11855
Ren W, Liu N, Li B (2021) Development and application of a LAMP method for rapid detection of apple blotch caused by Marssonina coronaria. Crop Protection 141:105452
Rizzo D, Moricca S, Bracalini M, Benigno A, Bernardo U, Luchi N, Da Lio D, Nugnes F, Cappellini G, Salemi C, Cacciola SO, Panzavolta T (2021) Rapid detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the loop-mediated isothermal amplification (LAMP) method. Plants(Basel) 10: 1048.
Salcedo AF, Purayannur S, Standish JR, Miles T, Thiessen L, Quesada-Ocampo LM (2021) Fantastic downy mildew pathogens and how to find them: advances in detection and diagnostics. Plants (Basel) 10:435
Shen DY, Li QL, Yu J, Zhao YY, Zhu Y, He X, Dou DL (2017) Development of a loop-mediated isothermal amplification method for the rapid detection of Pythium ultimum. Australasian Plant Pathology 46:571–576
Shen L, Huang M, Fang A, Yang Y, Yu Y, Bi C (2022) Loop-mediated isothermal amplification for the rapid detection of the mutation of carbendazim-resistant isolates in Didymella bryoniae. Agronomy(Basel) 12:2057
Shen W, Xu G, Sun L, Zhang L, Jiang Z (2016) Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Sporisorium scitamineum in sugarcane. Annals of Applied Biology 168:321–327
Shu R, Yin X, Long Y, Yuan J, Zhou H (2022) Detection and control of Pantoea agglomerans causing plum bacterial shot-hole disease by loop-mediated isothermal amplification technique. Frontiers in Microbiology 13:896567
Silva MC, Várzea V, Guerra-Guimarães L, Azinheira HG, Fernandez D, Petitot AS, Bertrand B, Lashermes P, Nicole M (2006) Coffee resistance to the main diseases: leaf rust and coffee berry disease. Brazilian Journal of Plant Physiology 18:119–147
Su Y, Yang Y, Peng Q, Zhou D, Chen Y, Wang Z, Xu L, Que Y (2016) Development and application of a rapid and visual loop-mediated isothermal amplification for the detection of Sporisorium scitamineum in sugarcane. Scientific Reports 6:23994
Sun J, Zheng HY, Zhong X, Ma CY (2019) Tropical crops industry development report. China Tropical Agricultural Science and Technology Press, Beijing, pp 147–156 (In Chi- nese)
Talhinhas P, Batista D, Diniz I, Vieira A, Silva DN, Loureiro A, Tavares S, Pereira AP, Azinheira HG, Guerra-Guimarães L (2017) The coffee leaf rust pathogen Hemileia vastatrix: one and a half centuries around the tropics. Molecular Plant Pathology 18:1039–1051
Tian Y, Liu D, Zhao Y, Wu J, Hu B, Walcott R (2017) Visual detection of Didymella bryoniae in cucurbit seeds using a loop-mediated isothermal amplification assay. European Journal of Plant Pathology 147:255–263
Venzon M (2021) Agro-ecological management of coffee pests in Brazil. Frontiers in Sustainable Food Systems 5:721117
Wang H, Wu WH, Li L, Liang YQ, Zheng JL, Xi JG, Li R, Huang X, He CP, Yi KX (2017) PCR-based molecular detection of Hemileia vastatrix in coffee. Chinese Journal of Tropical Crops 38:1677–1681
Wang T, Ji H, Yu Y, Wang X, Cheng Y, Li Z, Chen J, Guo L, Xu J, Gao C (2021) Development of a loop-mediated isothermal amplification method for the rapid detection of Phytopythium vexans. Frontiers in Microbiology 12:720485
Wellman FL (2021) Americans face up to the threat of coffee rust. Foreign Agriculture 17:1–7
White TJ, Bruns T, Lee S, Taylor J (1990) amplification and direct sequencing of fungal ribosomal RNA genes for phyloge-netics. In: PCR protocols: a guide to methods and applications. New York, Academic Press, Inc, pp 315–322
Wu WH, Liu BH, Wang QW, Lu PP, He CP, Liang YQ, Huang X, Yi KX (2022) Establishment and application of single tube nested PCR detection system for Hemileia vastatrix causing coffee leaf rust. Special Wild Economic Animal and Plant Research 164:1–8
Xiong Q, Zhang L, Zheng X, Qian Y, Zhang Y, Zhao L, Cheng Q (2021) Rapid and specific detection of the poplar black spot disease caused by Marssonina brunnea using loop-mediated isothermal amplification assay. Plants (Basel) 10:253
Yang X, Qi YJ, Al-Attala MN, Gao ZH, Yi XK, Zhang AF, Zang HY, Gu CY, Gao TC, Chen Y (2019) Rapid detection of alternaria species involved in pear black spot using loop-mediated isothermal amplification. Plant Disease 103:3002–3008
Zambolim L (2016) Current status and management of coffee leaf rust in Brazil. Tropical Plant Pathology 41:1–8
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Funding
This research was funded by the National Key R & D Program of China, grant number 2018YFD0201100; Special Funds for Efficient Tropical Agriculture Development of Hainan Province (UF37721); FAO/IAEA Collaborative Research Project, grant number No. 20380; Hainan Provincial Natural Science Foundation of China, grant number 320QN187; the Central Public-Interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences, grant number No. 1630042017021.
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Conceptualization, Kexian Yi; Methodology, Weihuai Wu; Validation, Han Wang; Formal analysis, Weihuai Wu, Han Wang, and Guihua Wang; Investigation, Thomas Gbokie, Jr., Yanqiong Liang, and Rui Li; Data curation, Weihuai Wu, Han Wang; Writing-original draft preparation, Weihuai Wu, Guihua Wang; Writing-review and editing, Guihua Wang, Weihuai Wu; Review, Kexian Yi; Supervision, Kexian Yi; and Project administration, Chunping He and Xing Huang. All authors have read and agreed to the published version of the manuscript.
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Wu, W., Wang, G., Wang, H. et al. Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Hemileia vastatrix in coffee plantations. Trop. plant pathol. (2024). https://doi.org/10.1007/s40858-023-00627-z
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DOI: https://doi.org/10.1007/s40858-023-00627-z