Genetic identification of two sweet-potato-infecting begomoviruses in South Africa.

The complete genome sequences of two monopartite begomovirus isolates (genus Begomovirus, family Geminiviridae) that occurred either alone or in mixed infection in sweet potato (Ipomoea batatas) plants collected in Waterpoort, South Africa, are presented. One of the isolates corresponds to sweet potato mosaic-associated virus (SPMaV; SPMaV-[ZA:WP:2011]), with which it shared 98.5 % nucleotide identity, whereas the second isolate corresponds to a new variant of sweet potato leaf curl Sao Paulo virus (SPLCSPV; SPLCSPV-[ZA:WP:2011]), with which it shared 91.4 % nucleotide identity. The phylogenetic and recombination relationships of these isolates to other monopartite Ipomoea-infecting begomoviruses were also investigated. SPLCSPV-[ZA:WP:2011] was found to be a natural recombinant of swepoviruses consisting of two distinct parental genomic sequences from SPLCSPV and sweet potato leaf curl Georgia virus (SPLCGV).

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.

Relative Pathogenicity of Cryphonectria cubensis on Eucalyptus Clones Differing in Their Resistance to C. cubensis.

Cryphonectria cubensis causes a destructive canker disease of Eucalyptus species. Management of this disease is primarily through breeding and selection of disease resistant trees. One means of selecting such trees is by artificial inoculation with the pathogen. In routine screening trials in South Africa, an isolate of C. cubensis, considered to be highly pathogenic, has been used for such inoculations. Although the most resistant clones under natural conditions are the same as those detected in inoculation trials, a question has arisen whether all clones respond similarly to different C. cubensis isolates. Thus, a trial consisting of five clones, known to differ in susceptibility to infection by C. cubensis, was established. These trees were inoculated with nine South African C. cubensis isolates previously shown to differ in pathogenicity. Inoculations showed a significant isolate × clone interaction as well as an "apparent immunity" for one clone × isolate interaction, providing evidence highly suggestive of a vertical resistance component in the pathosystem. Disease screening in this pathosystem has traditionally relied on a single pathogen isolate; however, considering data presented here, future reliance on a single isolate may be inadequate.

Characterization of South African Cryphonectria cubensis Isolates Infected with a C. parasitica Hypovirus.

ABSTRACT Cryphonectria cubensis is the causal agent of a serious canker disease of Eucalyptus spp. in tropical and subtropical parts of the world. In this study, a South African C. cubensis isolate was transfected by electroporation with a synthetic RNA transcript corresponding to the full-length coding strand of the C. parasitica hypovirus (CHV1-EP713). Hypovirus infection resulted in pronounced morphological changes that included a striking increase in bright yellow-orange pigment production, a reduction in mycelial growth rate, and reduced sporulation. Greenhouse studies revealed that the virus-containing strain was significantly less virulent than the original virulent C. cubensis isolate. Although the hypovirus was not transmitted through conidia produced by infected C. cubensis, the virus was readily transmitted via hyphal anastomosis to C. cubensis isolates representing a broad range of vegetative compatibility groups. These results suggest that vegetative incompatibility may not pose a strong barrier against virus transmission in South African isolates of C. cubensis and that hypovirus-mediated biological control could provide opportunities to reduce the impact of Cryphonectria canker in South Africa.