Field-based polymerase chain reaction detection of key sweetpotato viruses

Abstract

Globally, Sweetpotato feathery mottle virus (SPFMV) and Sweetpotato chlorotic stunt virus (SPCSV) occur frequently and in combination cause sweetpotato virus disease (SPVD). In SPVD infections, plants are severely stunted with severe chlorosis, thus farmers and seed inspectors easily select against such plants as source of planting material. However, in single virus infections, symptoms are not visible and so, the sweetpotato vines appear healthy. Farmers can easily pick them for seed while at the same time they can easily pass the plant inspection and certification checks since seed inspectors are currently using visual diagnosis. Virus diagnosis is very expensive and requires the inspectors to refer samples to some central laboratory at the Ministry of Agriculture, Animal Industry and Fisheries and using fixed and expensive equipment. The available private laboratories that can do laboratory tests are also based around Kampala yet sweetpotato vine multipliers are located upcountry and close to the farming communities. This slows down the speed of inspections and certification, hence limiting timely access to certified seed hence potential degradation of samples, culminating in limited access to quality seed. Thus the main objective of this study was to contribute to management of sweetpotato viruses in the field through development of a modified field-based nucleic acid extraction protocol and on-site virus detection method for key sweetpotato viruses. This involved (i) modification of the Lodhi et al. (1994) total plant CTAB RNA extraction procedure, by omitting some of the laboratory specific steps and varying the incubation time in liquid nitrogen, instead of the freezer, (ii) use of a portable miniature miniPCR thermocycler to run reverse transcriptase (RT) and the resulting cDNA amplification instead of a stationed and expensive conventional PCR thermocycler, and (iii) the amplicons stained with an environmentally safe GelGreen® dye viewed using a portable Bluegel® imaging system. The modified RNA extraction procedure reduced the RNA extraction time from overnight (960 minutes) to only about 2 hours and 45 minutes. Incubation of samples in liquid nitrogen for only 90 minutes yielded RNA of comparable quality and quantity (P > 0.05) to the RNA yield obtained with the original protocol, when incubation was done at 4 °C overnight in a freezer. The miniPCR efficiently amplified the viral cDNA and yielded amplicons that were similar (100 %) to those obtained with original extraction protocol and subsequent amplification by a stationed conventional RT-PCR. Hence, a method that allows for quick diagnosis of sweetpotato virus infections in the field has been developed. This has an advantage of avoiding delays in availing quality certified sweetpotato seed and circumvents any risks associated with sample quality decline during transportation. Adoption of this method by the crop inspection and certification department of MAAIF will contribute greatly towards sustainable quality sweetpotato production through making timely recommendations.