Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.

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Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5' ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5'UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.

Three genetic variants of Grapevine leafrollassociated virus 3 (GLRaV-3) were identified from vineyards of the Western Cape, South Africa. In a previous study, three full genome sequences of isolates representing each of the variant groups were determined. The three variant groups were represented by accessions 621, 623 and PL-20, of variant groups I, II and III respectively. A specific single strand conformation polymorphism (SSCP) profile was assigned to each variant which was used as a quick, reliable detection and differentiation method. In this study we analysed the occurrence of these three GLRaV-3 variants in mother blocks in different cultivars and from different vine growing regions using SSCP. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of three GLRaV-3 variants within a spatiotemporally recorded cluster of diseased plants was studied by means of SSCP profile analysis of ORF5 amplified PCR products. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows.

Tomato yellow leaf curl virus (TYLCV) causes a serious disease of tomato in many countries throughout the world. Preliminary reports suggested that TYLC disease was present in 1997 in South Africa. In 1998 140 ha of tomato fields in the Onderberg area were assessed for possible presence of TYLCV. Symptoms like those caused by TYLCV isolates in Israel were observed in most fields, and disease incidence ranged from <1 to 50%. Yield losses in individual plants ranged from negligible to 100% and appeared related to the age of the plants at time of infection. Two isolates of the suspect virus were experimentally transmitted from symptomatic tomato to virus-free, glasshouse-grown tomato seedlings by colony. Field and colony whiteflies were identified as the Bemisia tabaci based on mt COI sequence analysis (1). Attempts to transmit the suspect begomovirus by sap inoculation between tomato plants were unsuccessful. Polymerase chain reaction (PCR) amplification with degenerate PCR primers (2) that permit detection of the coat protein gene (AV1) and the common region (CR) of other begomoviruses yielded an amplicon of the expected size (2,100 bp), suggesting begomovirus association with diseased tomato plants. Nucleotide (nt) sequence analysis of AV1 for both tomato isolate AF261885 indicated that they were indistinguishable and shared less than 78% sequence identity with other well-studied begomoviruses, indicating a distinct, previously undescribed begomovirus species. AV1 sequence comparisons also revealed that its closest relatives were members of the TYLCV cluster, which includes South African cassava mosaic virus (77.4%) (AF11785), East African cassava mosaic virus (77.3%) (AJ006459), and TYLCV-IS (76.2%) (X15656). The theoretical Rep binding element in the CR, TCGGT, was identical to TYLCV-IS and Cotton leaf curl virus-Pakistan (AJ002448) (AJ002449). Here, we provisionally designate this new tomato-infecting begomoviral species, Tomato curly stunt virus from South Africa (ToCSV-SA). References: (1) D. R. Frohlich et al. Mol. Ecol. 8:1683, 1999. (2) A. M. Idris and J. K. Brown. Phytopathology 88:648, 1998.

Four different mealybug species (Dysmicoccus brevipes, Planococcus citri, P. ficus, and Pseudococcus longispinus) were evaluated for their ability to transmit putative activated-episomal Banana streak OL (badna)virus (BSOLV) to banana cv. Williams (Cavendish subgroup, AAA). Expressible endogenous sequences of banana streak viruses (BSVs) have been reported to be present in the DNA of various Musa hybrids, including FHIA-21 (AAAB). To obtain activated episomal BSOLV for this experimental transmission study, intentional stress by tissue culture propagation was applied to indexed FHIA-21 which, while free of other viruses, can contain activated episomal BSOLV. Immunocapture polymerase chain reaction and triple-antibody sandwich enzyme-linked immunosorbent assay results revealed that 13.4% of the derived progeny of the mother plants were infected with episomal BSOLV. Four of these BSOLV-infected progeny were used as sources of episomal virus for transmission studies. D. brevipes, Planococcus citri, and P. ficus mealybugs were able to transmit the putative activated episomal BSOLV. Control plants for the transmission experiments included FHIA-21 corms with no background history of tissue culture, as well as virus-free Williams plants. Episomal Banana streak GF (badna)virus (BSGFV) was transmitted from asymptomatic corm-derived FHIA-21 plants by P. citri and P. ficus. This is the first report of P. ficus as a vector of BSVs.

Greening disease of citrus is a serious disease known in South Africa since the late 1920s. In South Africa, it is associated with infection by ‘Candidatus Liberibacter africanus’, a heat sensitive, phloem-limited, noncultured alpha-proteobacterium. Huanglongbing (HLB), a similar, but more devastating disease that was described initially from China but which now occurs in several citrus producing countries, is associated with a different Liberibacter species, ‘Ca. L. asiaticus’. A ‘Ca. L. africanus’ subspecies, ‘Ca. L. africanus subsp. capensis’, has been found only in South Africa infecting an indigenous Rutaceous species, Calodendrum capense (Cape Chestnut), in the Western Cape in 1995. The discovery of a new Liberibacter species in Brazil, ‘Ca. L. americanus’, and the spread of ‘Ca. L. asiaticus’ to a number of additional countries over the last few years prompted us to assess whether only ‘Ca. L. africanus’ is present in commercial citrus orchards in South Africa. Samples displaying greening or similar symptoms were collected from 249 citrus trees from 57 orchards distributed throughout the greening affected citrus production areas of South Africa. Multiplex polymerase chain reaction (PCR) was performed on DNA extracts to detect the known citrus Liberibacters. Amplicons were obtained from 197 samples. None of the samples yielded a 1,027-bp amplicon indicative of ‘Ca. L. americanus’ infection. The amplicons of 84 samples were sequenced, and all were identical to the cognate ‘Ca. L. africanus’ Nelspruit sequence in GenBank. No instance of ‘Ca. L. asiaticus’ or ‘Ca. L. africanus subsp. capensis’ sequence was found. Geographically representative samples that tested negative for Liberibacter also tested negative for phytoplasmas based on real-time PCR results. Based on the results of this survey, it is concluded that to date only ‘Ca. L. africanus’ is associated with citrus greening in commercial citrus in South Africa.

The biological and molecular characterization of a virus recognized as a distinct begomovirus species, Tomato curly stunt virus (ToCSV), first observed in South Africa in 1997, is reported here. Whitefly-transmission and host-range studies were carried out using a Bemisia tabaci colony identified as the B-biotype. The experimental host range of ToCSV spanned primarily species in the Solanaceae and Fabaceae. The complete ToCSV genome (2·766 kb) was amplified by PCR, cloned, and the DNA sequence determined. Phylogenetic analysis revealed that ToCSV was most closely related to Tobacco leaf curl Zimbabwe virus (TbLCZV), at 84% nucleotide identity, indicating that ToCSV is a new species in the genus Begomovirus that is probably endemic to southern Africa. The ToCSV genome sequence contained all of the hallmark coding and non-coding features characteristic of other previously recognized monopartite begomoviruses. ToCSV is only the second begomovirus described from southern Africa that infects solanaceous species. Neither a begomoviral DNA-B component nor a satellite-like DNA molecule was detected by PCR in extracts of ToCSV-infected plants.