First Report of Pepino mosaic virus Infecting Tomato in South Africa.

Pepino mosaic virus (PepMV) (genus Potexvirus) is a highly infectious virus that is responsible for significant losses in yield of tomato fruit (Solanum lycopersicum) across Europe, Asia, and the Americas in the last decade (1). During the winter growing season of 2008, uneven discoloration of tomato fruit from farms in Limpopo Province, South Africa, was detected at the Pretoria fresh produce market. Twenty fruit were randomly selected from five different suppliers in this region and the 100 samples were batched into subsamples of five fruit. Leaves with suspect mosaic and bubbling symptoms were also detected from farms in Limpopo and were thus sampled. Leaf and fruit samples were tested by double antibody-sandwich (DAS)-ELISA (2) using polyclonal antibodies against PepMV (Agdia, Elkhart, IN) combined with appropriate positive and negative controls. Fruit samples from two of the suppliers, and all leaf samples tested, reacted strongly with PepMV antibodies. Inoculum was prepared from pooled DAS-ELISA-positive leaf samples and inoculated onto 10, 4-week-old, susceptible S. lycopersicum cv. Rooikhaki seedlings. After 3 weeks, all inoculated plants had developed characteristic PepMV symptoms (2) including leaf bubbling, distortion, and curled leaves. Older leaves developed yellow spots and light/dark green leaf mosaic while apical regions were stunted and branches were distorted to form 'nettle-head' symptoms. Fruit surfaces were marbled or displayed flaming and uneven discoloration. Leaves from symptomatic plants were sampled for confirmation of PepMV infection by DAS-ELISA and all samples reacted positively with PepMV antibodies. Total RNA was extracted from 500-μg replicates of pooled leaf samples from infected plants with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and amplified by conventional two-step reverse-transcription-PCR using a PepMV-specific primer set: Ker 1 (2) and PepCP-R (4) for a 986-bp region, including the coat protein, of the PepMV genome. PCR products were cloned into pTZ57R/T vector (Fermentas, Vilnius, Lithuania [UAB]) and six clones were purified and sequenced using universal M13 primers (3). Phylogenetic analysis clustered the sequence with EU (European), LP (Peruvian), US1 (United States)/CH1 (Chilean) and US2/CH2 PepMV isolates. The PepMV isolate accessions for US2/CH2 (AY509927, FJ612601, EF408821, FJ212288, and DQ000985) were identified as the closest relatives based on 98 to 99% nucleotide similarity obtained using BLASTN. The coat protein sequence of the South African isolate was submitted to GenBank (Accession No. HQ872607). To our knowledge, this is the first confirmed report of PepMV in South Africa. Further studies are necessary to determine its incidence and spread in this country. The presence of PepMV signals the urgent need for adoption of appropriate phytosanitary measures to restrict the spread and impact of this virus. References: (1) I. M. Hanssen and B. P. H. J. Thomma. Mol. Plant Pathol. 11:179, 2010. (2) I. M. Hanssen et al. Plant Pathol. 58:450, 2009. (3) J. Messing. Method Enzymol. 101:20, 1983. (4) I. Pagán et al. Phytopathology 96:274, 2006.