Lack of Evidence for Seed Transmission of 'Candidatus Liberibacter africanus' Associated with Greening (Huanglongbing) in Citrus in South Africa.

'Candidatus Liberibacter africanus' is associated with citrus greening (huanglongbing [HLB]) in South Africa. Various unpublished reports have suggested that the related bacterium 'Ca. L. asiaticus' associated with HLB in citrus might be seed transmissible based on real-time PCR results. Seed transmission poses a risk of long distance disease spread, especially with the dissemination of rootstock seed. Therefore, it was essential to determine whether 'Ca. L. africanus' is seed transmitted in citrus. Fruit from 26 'Ca. L. africanus'-infected branches of six citrus cultivars showing greening symptoms were collected and the seed was harvested. Cultivars included were Minneola tangelo (Citrus reticulata × C. paradisi); sweet oranges (C. sinensis) Premier midseason, Clanor midseason, and Olinda Valencia; Eureka lemon (C. limon) and Troyer citrange (Poncirus trifoliata × C. sinensis) rootstock variety. Branches bearing each fruit were collected and confirmed to contain 'Ca. L. africanus' by real-time PCR testing using Taqman probe HLBp and HLBaf and HLBr primers as described by Li et al. (3). The seed of each sample was sorted into five categories ranging from healthy looking to totally aborted based on their appearance before planting. Germination was done in seed trays under vector-free conditions at 24 to 28°C. Thereafter the seedlings were planted in small, plastic bags and monitored for greening-like symptoms or other abnormalities for up to 2 years. A slow-release, balanced fertilizer was applied and supplemented with micro-nutrient sprays for plant maintenance. Plants showing abnormal symptoms were potted into larger pots and closely monitored. These samples and a number of other seedlings showing growth abnormalities were tested for 'Ca. L. africanus' by real-time PCR as described above. In total, 1,570 seedlings were obtained. Some abnormal symptoms such as small chlorotic leaves, interveinal chlorosis, yellow veins, and stunting were seen in some seedlings. Most symptoms resembled deficiencies, and no blotchy mottle typical of 'Ca. L. africanus' infection was noted on any of the seedlings. Abnormal seedlings arose from normal and abortive seed. One hundred and eighteen of these seedlings (8 Minneola tangelo; 24 Premier midseason, 42 Clanor midseason, 33 Olinda Valencia, and 11 Troyer citrange seedlings) were individually tested using real-time PCR for 'Ca. L. africanus' detection. These seedlings had germinated from essentially healthy-looking seed (category 1) to seeds with severe abnormalities (category 5) and 33, 24, 23, 30, and 8 seedlings, respectively, were tested from each of the five seed categories. No samples tested positive with real-time PCR based on a positive/negative threshold Cq value of 35. Buds of some seedlings that yielded the lowest Cq values above 35 were grafted onto healthy 'Madam vinous' sweet orange (C. sinensis) seedlings and monitored for symptom development for 3 months. No symptoms developed and all these indicators also tested negative for 'Ca. L. africanus', indicating the absence of a transmissible agent. Just as other researchers (1,2) have recently indicated a lack of evidence for seed transmission of 'Ca. L. asiaticus', no seed transmission of 'Ca. L. africanus' could be demonstrated in this experiment either. References: (1) U. Albrecht and K. D. Bowman. HortScience 44:1967, 2009. (2) J. S. Hartung et al. Plant Dis. 94:1200, 2010. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006.

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5'UTR.

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.

Lynch syndrome: the influence of environmental factors on extracolonic cancer risk in hMLH1 c.C1528T mutation carriers and their mutation-negative sisters.

Lynch Syndrome (LS) is a cancer susceptibility syndrome caused mostly by mutations in the mismatch repair genes, hMLH1, hMSH2 and hMSH6. Mutation carriers are at risk of colorectal and endometrial cancer and, less frequently, cancer of the ovaries, stomach, small bowel, hepatobiliary tract, ureter, renal pelvis and brain. The influence of environmental factors on extracolonic cancer risk in LS patients has not been investigated thus far. The aim of this study was to investigate some of these factors in South African females carrying the hMLH1 c.C1528T mutation and their mutation-negative relatives. Data were collected from 87 mutation-positive females and 121 mutation-negative female relatives regarding age, cancer history, hormonal contraceptive use, parity, duration of breast feeding, height, weight and age at first birth, last birth, menarche and menopause. Influence of these factors on cancer risk was analysed by mixed-effects generalised linear models. Extracolonic cancer occurred in 14% (12/87) of mutation-positive females versus 7% (8/121) of mutation-negative females, (P = 0.0279, adjusted for age and relatedness between women). Breast cancer was the most common extracolonic cancer. An association was found for oral contraceptive use and extracolonic cancer risk in mutation-negative females only. No association was found for any of the other risk factors investigated, when adjusted for age. This might be due to the scarcity of extracolonic cancers in our data. Future knowledge on the influence of additional environmental factors on cancer risk in LS females can lead to evidence-based lifestyle advice for mutation carriers, thereby complementing the prevention strategies available today. In addition, it can contribute to an integrated model of cancer aetiology. Therefore, this study should be taken as a thrust for further research.

Soybean blotchy mosaic virus, a New Cytorhabdovirus Found in South Africa.

A previously unidentified plant Rhabdovirus sp. associated with a blotchy mosaic symptom of soybean (Glycine max), prevalent in the lower-lying, warmer soybean production areas of South Africa, was isolated and partially characterized. The virus was shown to be transmitted by mechanical inoculation and at least one species of leafhopper (Peragallia caboverdensis Lindberg (Cicadellidae, Agalliinae)). To determine the morphology and virion size, as well as intercellular accumulation, negative-stained preparations or embedded ultrathin sections of infected plant samples were observed under a transmission electron microscope. The distribution of the virions within the cytoplasm and its bullet-shaped morphology and size (338 to 371 nm by 93 nm) suggested that it is a putative member of the genus Cytorhabdovirus. Degenerate primers designed to a conserved region of the polymerase gene of a number of Rhabdovirus spp. were used in reverse-transcriptase polymerase chain reaction with total RNA from symptomatic plants as template. Amplicons were sequenced and compared with related sequences available on GenBank. The analysis confirmed that the virus was related to Cytorhabdovirus spp., with the highest nucleotide similarity being 60.7% with Northern cereal mosaic virus. The particle morphology, typical virion accumulation in the cytoplasm of infected cells, nucleotide sequence similarity with that of other plant Rhabdovirus spp., and unique symptoms on soybean suggest that the virus is a previously unknown Cytorhabdovirus sp., for which we propose the name Soybean blotchy mosaic virus (SbBMV).

A Survey for 'Candidatus Liberibacter' Species in South Africa Confirms the Presence of Only 'Ca. L. africanus' in Commercial Citrus.

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.

A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae).

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.

Transmission of Activated-Episomal Banana streak OL (badna)virus (BSOLV) to cv. Williams Banana (Musa sp.) by Three Mealybug Species.

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.

The extracolonic cancer spectrum in females with the common 'South African' hMLH1 c.C1528T mutation.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease, characterized by the occurrence of predominantly colon and endometrial cancer and, less frequently, cancer of the small bowel, stomach, hepatobiliary tract, ureter, renal pelvis, ovaries and brain. The phenotypic diversity may partially be explained by allelic heterogeneity. The aim of this study was to investigate the frequency of extracolonic cancers in a cohort of females sharing the same c.C1528T disease-predisposing mutation in the hMLH1 gene. Data on cancer history were obtained from 87 mutation-positive females and 121 mutation-negative sisters, as a control group. Testing for microsatellite instability (MSI) and expression of the wild-type hMLH1 allele was performed on extra-colonic tumour tissue blocks of mutation-positive individuals. Extracolonic cancer occurred in 14% (12/87) of mutation-positive females vs. 7% (8/121) of mutation-negative females (P = 0.10). Multiple primary cancers occurred at a significantly higher incidence in the first group. Breast cancer, which was the most frequent extra-colonic cancer in mutation positive females (53%), occurred at a young age, and occurred bilaterally in two out of seven cases. Involvement of the hMLH1 gene was confirmed in five out of seven cases of breast cancer, two cases of endometrial cancer, one case of ovarian cancer and one case of renal cell carcinoma, by detecting immunohistochemical compromise of the gene product. Although the study might not have been adequately statistically powered (to provide a significant P value), the noteworthy findings in this study include the confirmation of a range of Lynch II type cancers in a cohort we previously thought was wholly predisposed to Lynch I features, and a confirmation of breast cancer as part of the spectrum of Lynch syndrome cancers affecting women.

Natural Occurrence of Groundnut ringspot virus on Soybean in South Africa.

Mechanically transmissible viruses were isolated from two soybean (Glycine max Merr.) plants from Rustenburg, Northwest Province and Greytown, KwaZulu-Natal, South Africa, respectively (2). Viruses were isolated by two serial local-lesion transmissions on Chenopodium quinoa. Ringspot symptoms on Nicotiana benthamiana suggested the presence of tospoviruses. This was supported by the detection of typical tospoviruslike particles in ultrathin sections of infected plants. Serological analysis of samples using various tospovirus antisera in a number of enzymelinked immunosorbent assay formats suggested the two isolates had epitopes in common with Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), and Tomato chlorotic spot virus (TCSV). Nucleotide sequences were determined using reverse transcriptionpolymerase chain reaction (RT-PCR) for 857 bp of the nucleoprotein gene of the two isolates (GenBank Accession Nos. AF487516 and AF 487517). These revealed a 99% nucleotide identity with each other. Sequence comparison with cognate regions of TSWV (GenBank Accession No. D00645), GRSV-SA-05 (GenBank Accession No. S54327), and TCSV (GenBank Accession No. S54325) revealed that both isolates share 97% nucleotide sequence identity with GRSV (SA-05) from peanut also originally from South Africa (1). Both isolates are therefore considered GRSV. To our knowledge, this is the first report of the natural occurrence of GRSV on soybean worldwide. References: (1) A. C. de Avila et al. J. Gen. Virol. 74:153, 1993. (2) G. Pietersen et al. Afr. Plant Prot. 4:65, 1998.

Tomato curly stunt virus, a New Begomovirus of Tomato Within the Tomato yellow leaf curl virus-IS Cluster in South Africa.

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.

First Report of Cucumber Mosaic Cucumovirus Subgroup 1 in South Africa, from Banana with Infectious Chlorosis.

Cucumber mosaic cucumovirus (CMV) is the etiological agent of infectious chlorosis disease in bananas (Musa spp.). In South Africa, diagnosis of CMV on banana has been based only on symptoms (1). A Grande Naine (Cavendish, AAA) plant with typical infectious chlorosis disease was obtained from Letsitele. Sap from this plant was inoculated to indicator plants. Virus was isolated by two serial local lesion transfers on Chenopodium quinoa and maintained on Nicotiana benthamiana. It was identified as a subgroup I CMV based on its reaction to CMV DTL-, but not CMV ToRS- monoclonal antibodies in double antibody sandwich (DAS)-enzyme-linked immunosorbent assay (ELISA). The identity was further confirmed by the polyhedral particle morphology under the electron microscope by negatively staining, and decoration in immunoelectron microscopy with antiserum to a local tomato isolate of CMV. Infectious chlorosis disease symptoms were induced by sap inoculation to healthy Grande Naine plants, and the virus detected by ELISA. This is the first confirmed diagnosis of CMV on bananas in South Africa. The isolate was deposited in the PPRI Virus Collection. Reference: (1) B. Q. Manicom. 1993. Pages 102-103 in: Handbook of banana growing in South Africa. J. C. Robinson, ed. Agric. Res. Counc., Inst. Trop. Subtrop.Crops, Nelspruit, South Africa.

Molecular characterization of a subgroup I geminivirus from a legume in South Africa.

A South African geminivirus for which we propose the name bean yellow dwarf virus (BeYDV) has been isolated from French bean (Phaseolus vulgaris cv. Bonus) showing stunting, chlorosis and leaf curl symptoms. A full-length cloned copy of the viral genome produced characteristic symptoms of the disease when reintroduced into French bean by agroinoculation, and was systemically infectious in Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum, Datura stramonium and Arabidopsis thaliana. BeYDV resembles subgroup I geminiviruses which infect monocotyledonous plants in having a single DNA component, two non-overlapping virion-sense (V1 and V2) and two overlapping complementary-sense (C1 and C2) coding regions, and an intron within the complementary-sense coding regions that is excised to produce a C1C2 fusion protein. It is most closely related to tobacco yellow dwarf virus from Australia, the only subgroup I geminivirus previously known to infect dicotyledonous plants, although it is sufficiently dissimilar (65% nucleotide sequence identity) to be considered a distinct virus.