BackgroundPlant viruses in the genus Begomovirus, family Geminiviridae often cause substantial crop losses. These viruses have been emerging in many locations throughout the tropics and subtropics. Like many plant viruses, they are often not recognized by plant diagnostic clinics due in large part to the lack of rapid and cost effective assays. An isothermal amplification assay, Recombinase polymerase amplification (RPA), was evaluated for its ability to detect three begomoviruses and for its suitability for use in plant diagnostic clinics. Methods for DNA extraction and separation of amplicons from proteins used in the assay were modified and compared to RPA manufacturer’s protocols. The modified RPA assays were compared to PCR assays for sensitivity, use in downstream applications, cost, and speed.ResultsRecombinase polymerase amplification (RPA) assays for the detection of Bean golden yellow mosaic virus, Tomato mottle virus and Tomato yellow leaf curl virus (TYLCV) were specific, only amplifying the target viruses in three different host species. RPA was able to detect the target virus when the template was in a crude extract generated using a simple inexpensive extraction method, while PCR was not. Separation of RPA-generated amplicons from DNA-binding proteins could be accomplished by several methods, all of which were faster and less expensive than that recommended by the manufacturer. Use of these modifications resulted in an RPA assay that was faster than PCR but with a similar reagent cost. This modified RPA was the more cost effective assay when labor is added to the cost since RPA can be performed much faster than PCR. RPA had a sensitivity approximate to that of ELISA when crude extract was used as template. RPA-generated amplicons could be used in downstream applications (TA cloning, digestion with a restriction endonuclease, direct sequencing) similar to PCR but unlike some other isothermal reactions.ConclusionsRPA could prove useful for the cost effective detection of plant viruses by plant diagnostic clinics. It can be performed in one hour or less with a reagent cost similar to that of PCR but with a lower labor cost, and with an acceptable level of sensitivity and specificity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0504-8) contains supplementary material, which is available to authorized users.

Full-length sequences of a bipartite begomovirus were obtained from a plant of Jatropha multifida in Florida showing symptoms of foliar mosaic, distortion and necrosis. Sequences of four clones each of a DNA-A and DNA-B were obtained, which showed very low sequence diversity among themselves. The clones were infectious when biolistically inoculated to J. multifida, Phaseolus vulgaris and Nicotiana tabacum, but not to J. curcas. The DNA-A sequences had less than 89 % pairwise identity scores with the DNA-A of other begomoviruses. The DNA-A appeared to be a recombinant in that 18 % of the DNA-A (470 nt) had a pairwise identity score of 91.98 % with RhRGMV, indicating that this portion most likely originated from a virus closely related to RhRGMV. The remaining 82 % of the DNA-A had lower identity scores with TbMoLCV (87.84 %) and RhRGMV (87.46 %), which suggests that this part of the component originated from an undescribed virus. There was no evidence for recombination in the DNA-B. Equivalent sequences of the DNA-A had the highest identity score (94.18 %) with a 533-nt sequence obtained from J. multifida from Puerto Rico in 2001 (GenBank accession no. AF058025). Pairwise comparison, recombination and phylogenetic analysis, and biology suggest that these clones are those of jatropha mosaic virus first reported from Puerto Rico. This is the first report of the complete genome sequence of jatropha mosaic virus.

The genome sequence of Southern tomato virus in asymptomatic Solanum lycopersicum ‘Sweet Hearts’ (STV-Florida) in Florida was assembled from small RNAs sequenced by Illumina RNA-seq. The STV-Florida genome shared 99.0 to 99.9% similarity with full genome sequences from Bangladesh, China, Mexico, and the United States (Mississippi and North Carolina).

Euphorbia mosaic virus (EuMV) was found in a symptomatic passionfruit (Passiflora edulis) plant from Homestead, Florida, USA, as well as in the symptomatic weed Euphorbia heterophylla. This is the first identification of EuMV in Florida and the United States and the first report of a natural infection of passionfruit by EuMV.