Rapid Nucleic Acid Extraction and PCR Detection of Three Significant Banana Viruses in the Philippines
Paul D. Kohnen, Maura Luisa S. Gabriel, Marissa I. Atis, and Miriam E. Pascua
College of Agriculture, Food and Sustainable Development,
Mariano Marcos State University, Ilocos Norte, Philippines
Whole nucleic acid was rapidly isolated from leaf tissue of banana cultivars ‘Cardaba’ and ‘Lacatan’ (Musa spp.) for use in polymerase chain reactions (PCR) designed to detect three viruses commonly found in the Philippines; banana bunchy top nanovirus (BBTV), banana streak badnavirus (BSV), and banana bract mosaic potyvirus (BBrMV). Reliable sequence-specific amplification (~1100 bp, 220 bp, and 324 bp PCR products for BBTV, BSV, and BBrMV, respectively) was observed. Furthermore, BBTV and BSV could be detected simultaneously using two primer sets in the same PCR reaction. Use of these modified techniques could aid in future banana virus detection endeavors.
Bananas (Musa spp.) are the number one export commodity in the Philippines today, making the banana industry the nation’s top dollar earner. The country ranks number five in the world in terms of production volume, in part because the country is blessed with a favorable climate well-suited for year-round banana cultivation. In addition, various cultivars including ‘Cavendish’, ‘Saba’, ‘Cardaba’, ‘Lacatan’, ‘Bungulan’, ‘Latundan’, and ‘Senorita’, are accepted in both the domestic and export markets, either as fresh or processed products (Faylon et al. 2004).
In 2002, however, the alarming spread of banana bunchy top virus (BBTV) and fusarium wilt in the northern part of Luzon culminated in an epidemic that resulted in substantial losses to growers. The research and development efforts that followed focused on the management of BBTV as well as industry rehabilitation. Virus disease surveillance was subsequently expanded to include not only BBTV, but also banana bract mosaic virus (BBrMV) and banana streak virus (BSV), especially in materials selected for micropropagation. Ongoing efforts to reduce disease incidence continue but are sometimes hampered by the use of less reliable diagnostic methods such as symptomatology and some serological assays. . . .
DALE JL, LOCKHAR BE, PLOETZ RC. 1994. Banana diseases caused by viruses. In: Compendium of Tropical Fruit Diseases. Ploetz RC, Zentmyer GA, Nishijima WT, Rohrbach KG, Ohr HD eds. Minnesota, United Sates: American Phytopathological Society. p. 17-20.
FAYLON PS, EUSEBIO JE, ANIT EA. 2004. Banana R&D in the Philippines: updates and highlights. Proceedings of the 2nd BAPNET Steering Committee Meeting. 6-19 October 2003; Jakarta, Indonesia: Bioversity International. p. 81-94.
GEERING ADW, McMICHAEL LA, DIETZGEN RG, THOMAS JE. 2000. Genetic diversity among banana streak virus isolates from Australia. Phytopathology 90: 921-927.
HARDING RM, BURNS TM, HAFNER G, DIETZGEN RG, DALE JL. 1993. Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene. Journal of General Virology 74: 323-328.
HARPER G, OSUJI J, HESLOP-HARRISON JS, HULL R. 1999. Integration of banana streak badnavirus into the Musa genome: Molecular and cytogenetic evidence. Virology 255: 207-213.
JAMES AP, GEIJSKES RJ, DALE JL, HARDING RM. 2011. Development of a novel rolling-circle amplification technique to detect banana streak virus that also discriminates between integrated and episomal virus sequences. Plant Disease 95: 57-62.
LE PROVOST G, ISKRA-CARUANA ML, ACINA I, TEYCHENEY PY. 2006. Improved detection of episomal banana streak viruses by multiplex immunocapture PCR. Journal of Virological Methods 137: 7-13.
NDOWORA T, DAHAL G, LaFLEUR D, HARPER G, HULL R, OLSZEWSKI NE, LOCKHART B. 1999. Evidence that badnavirus infection in Musa can originate from integrated pararetroviral sequences. Virology 255: 214-220.
SU HJ. 1998. First occurrence of banana streak badnavirus and studies on its vectorship in Taiwan. Proceedings of ProMusa Virology workshop. 19-21 January 1998; Montpellier, France. International Network for the Improvement of Banana and Plantain. p. 20-25.
SU HJ, HWANG AS, LEE SY, CHAO CP. 2007. Conservation, disease indexing and utilization of pathogen-free citrus and banana genetic resources in Taiwan. International Workshop on the Conservation and Utilization of Tropical/Subtropical Plant Genetic Resources. 9-11 January 2007; Tien Giang, Vietnam: Taiwan Agricultural Research Institute. p. 1-24.
SU HJ, TSAO LY, WU ML, HUNG TH. 2003. Biological and molecular categorization of strains of banana bunchy top virus. Journal of Phytopathology 151: 290-296.