Nested PCR multiplex assay for detection of phytoplasma and begomoviruses identified in soybean in Cuba

Main Article Content

Robert M. Leyva Martínez
Madelaine L. Quiñones Pantoja
Karel Acosta Peréz
Berta Piñol Pérez
S. Pérez-Álvarez

Abstract

The objective of the work was to develop a nested multiplex PCR assay for the simultaneous detection of phytoplasmas and the begomoviruses identified in soybean in Cuba. Two pairs of specific primers to the begomoviruses Tobacco yellow crinkle virus (TbYCV) and Rhynchosia golden mosaic Yucatan virus (RhGMYuV) were designed, and they were used in conjunction with two pairs of universal primers for the generic detection of phytoplasms under established conditions. The evaluation of the optimal temperature for alignment of the primers to the template, the analytical sensitivity, and the specificity of the primers, critical parameters for the nested PCR assay for begomoviruses, were optimized. Critical parameters for the nested multiplex PCR assay, such as the evaluation of different combinations of concentrations of the primers designed with the conditions established for phytoplasm nPCR, the analytical sensitivity, and the intra and inter-assay repeatability, were also optimized. Performance parameters of the multiplex nPCR assay were determined. In the multiple assay, the fragments expected for TbYCV (988 kb), RhGMYuV (968 kb), and phytoplasms (1250 kb) were successfully amplified, confirming the simultaneous, sensitive and specific detection of these pathogens. The analytical parameters of the trial performance were over 90%. The multiple nested PCR assay here developed allows a sensitive, fast and low-cost detection of phytoplasmas sp. and begomoviruses that affect soybean in Cuba. These results are the first of this type for the country and provide new methodological elements for the validation of plant pathogen detection assays.

Article Details

How to Cite
Leyva Martínez, R. M., Quiñones Pantoja, M. L., Acosta Peréz, K., Piñol Pérez, B., & Pérez-Álvarez, S. (2019). Nested PCR multiplex assay for detection of phytoplasma and begomoviruses identified in soybean in Cuba. Revista De Protección Vegetal, 34(2). Retrieved from https://revistas.censa.edu.cu/index.php/RPV/article/view/1026
Section
ORIGINAL ARTICLES

References

Reveles-Torres LR, Velásquez-Valle VR, Castillo-Mauricio JA. Fitoplasmas: Otros agentes fitopatógenos. Campo experimental Zacatecas. CIRNOC-INIFAP. Folleto técnico. 2014; 56: 41.

Jamil N, Rehman A, Hamza M, Hafeez A, Ismail H, Zubair M, et al. First Report of Tomato leaf curl New Delhi virus, a Bipartite Begomovirus, Infecting Soybean (Glycine max). Plant Disease. 2017; 101 (5): 845.

Ramesh SV, Chouhan BS, Gupta GK, Husain SM, Chand S. Genomic sequence characterization of Begomovirus infecting soybean and molecular evolutionary genomics of Legume yellow mosaic viruses (LYMVs). Plants Omics Journal. POJ. 2017; 10 (02): 88-96.

Acosta K, Silva FN, Zamora L, Quiñones M, Martínez R, Piñol B, et al. Detection and molecular characterization of an aster yellows group phytoplasma associated with soybean and evidence of two new 16SrI subgroups in Cuba. Journal of Plant Pathology. 2015; 97(2): 339-344.

Quiñones ML, Acosta KI, Castillo Urquiza G, Zamora L, Murilo Zerbini F. Soya y rábano como nuevos hospedantes de Begomovirus en Cuba. Rev. Protección Veg. 2014; 29(3): 235-235.

Chang-Sidorchuk L, González H, Martínez-Zubiaur Y, Navas-Castillo J, Fiallo-Olivé E. First report of Rhynchosia golden mosaic Yucatan virus infecting soybean in Cuba. Journal of Plant Pathology. 2016; 98: 174.

Lau HY, Botella JR. Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection. Front. Plant Sci. 2017; 8.

Bertaccini A. Phytoplasma research between past and future: what directions? Phytopathogenic Mollicutes. 2015; 5 (1): S1-S4.

Khan J, Srivastava P, Singh K. Efficacy of nested-PCR for the detection of phytoplasma causing spike disease of sandal. Current Science. 2004; 86:1530-1533.

Lugo MOY, Guzmán UR, García ERS, León FJ. Geminivirus transmitidos por mosca blanca (Bemisia tabaci) en tomate del Valle Agrícola de Culiacán, Sinaloa. Revista Mexicana de Fitopatología. 2011; 29:109-118.

Kwon JY, Hong JS, Kim MJ, et al. Simultaneous multiplex PCR detection of seven cucurbit infecting viruses. J Virol Methods. 2014; 206:133-139.

Venkataravanappa V, Prasanna HC, Lakshminarayana-Reddy CN, Krishna-Reddy M. Molecular detection and characterization of phytoplasma in association with begomovirus in eggplant. Acta virológica. 2018; 62: 246-258.

Gundersen D, Lee IM. Ultrasensitive detection of phytoplasmas by nested PCR assay using two universal primer pairs. Phytopathology Mediterranean. 1996; 35: 144-51.

Rychlik W. Selection of primers for Polimerase Chain Reaction. Molecular Biotechnology. 1995; 3:129-134.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution. 2013; 30 (12): 2725-2729.

Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. Segunda Edición. Eds by: Cold Spring Harbor Laboratory Press. 1989.

Massart S, Brostaux Y, Barbarossa L, Battle A, Cesar V, Dutercq O, et al. Inter-laboratory evaluation of two Reverse-transcriptase Polymeric Chain Reaction-based methods for detection of four fruit tree viruses. Ann. Appl. Biol. 2009; 154: 133-141.

Majumder S, Baranwal VK, Joshi S. Simultaneous detection of Onion yellow dwarf virus and Shallot latent virus in infected leaves and cloves of garlic by duplex RT-PCR. J. Plant Pathol. 2008; 90 (2): 371-374.

Firrao G, Garcia-Chapa M, Marzachí C. Phytoplasmas: genetics, diagnosis and relationships with the plants and insects host. Frontiers in Bioscience. 2007; 12: 1353-1375.

OIE Terrestrial Manual. Principles and methods of validation of diagnostic assays for infectious diseases. Version adopted by the World Assembly of Delegates of the OIE in May 2013. Chapter 1.1.5.

Díaz A, Quiñones M, Arana F, Soto M, Hernández A. Potyvirus: características generales, situación de su diagnóstico y determinación de su presencia en cultivo de pimiento en cuba. Rev. Protección Veg. 2010; 25(2): 69-79.

Acosta K, Martínez Y, Zamora L, Fernández A, Santos-Cervantes ME, Leyva-López NE. PCR múltiple anidada para detección de fitoplasmas y rickettsia asociados con los síntomas del cogollo arrepollado (Bts) en papayo. Rev. Protección Veg. 2011; 26 (3): 156-163.

Manual OIE. Principles of validation of diagnostic assays for infectious diseases. Chapter 1.1.3, version adopted, May. 2006.

Galetto L, Marzachi C. Real-time PCR Diagnosis and Quantification of Phytoplasmas. In: Weintraub P.G. and Jones P. (ed.). Phytoplasmas: Genomes, Plant Hosts and Vectors. United Kingdom: CAB International. 2010: 1-18.

Arana F, Pacheco R, Díaz De la Osa A, Piñol B, Quiñones M. Desarrollo de un método para el diagnóstico específico del PepMoV basado en la RT-PCR. Rev. Protección Veg. 2016; 31(1): 20-28.

Naranjo FE, Martínez ZY. Avances en el diagnóstico de la marchitez bacteriana (Ralstonia solanacearum): situación actual y perspectivas en Cuba. Rev. Protección Veg. 2013; 28 (3): 160-170.

Tao Y, Man J, Wu Y. Development of a multiplex polymerase chain reaction for simultaneous detection of wheat viruses and a phytoplasma in China. Arch Virol. 2012; 157: 1261-1267.

Swarnalatha P, Krishna-Reddy M. Duplex PCR for simultaneous detection of Begomovirus and Phytoplasma from naturally infected tomato. Pest Management in Horticultural Ecosystems. 2014; 20 (1): 59-68.

Biswas C, Dey P, Sat pathy S. A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.). Lett. Appl. Microbiol. 2013; 56: 373-378.

Most read articles by the same author(s)