Dilemmas caused by endogenous pararetroviruses regarding the taxonomy and diagnosis of yam (Dioscorea spp.) badnaviruses: analyses to support safe germplasm movement.

The discovery of endogenous pararetroviral sequences (EPRVs) has had a deep impact on the approaches needed for diagnosis, taxonomy, safe movement of germplasm and management of diseases caused by pararetroviruses. In this article, we illustrate this through the example of yam (Dioscorea spp.) badnaviruses. To enable progress, it is first necessary to clarify the taxonomical status of yam badnavirus sequences. Phylogeny and pairwise sequence comparison of 121 yam partial reverse transcriptase sequences provided strong support for the identification of 12 yam badnavirus species, of which ten have not been previously named. Virus prevalence data were obtained, and they support the presence of EPRVs in D. rotundata, but not in D. praehensilis, D. abyssinica, D. alata or D. trifida. Five yam badnavirus species characterised by a wide host range seem to be of African origin. Seven other yam badnavirus species with a limited host range are probably of Asian-Pacific origin. Recombination under natural circumstances appears to be rare. Average values of nucleotide intra-species genetic distances are comparable to data obtained for other RNA and DNA virus families. The dispersion scenarios proposed here, combined with the fact that host-switching events appear common for some yam badnaviruses, suggest that the risks linked to introduction via international plant material exchanges are high.

Strategies for the Construction of Cassava Brown Streak Disease Viral Infectious Clones.

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.

16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia.

The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage-within the gamma-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species.

Rapid single-tube immunocapture RT-PCR for the detection of two yam potyviruses.

An immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay has been developed for the detection of yam-infecting potyviruses. Based upon the same format two distinct simple tests have been developed, which allow the reliable diagnosis of yam mosaic virus and the tentatively named yam mild mosaic virus. By using immunocapture and a single-buffer RT-PCR reaction, the test can be performed in a single tube. The tests described have been shown to exhibit a thousand-fold increase in detection sensitivity compared to existing ELISA assays.

First Report of the Bemisia tabaci B Biotype in India and an Associated Tomato leaf curl virus Disease Epidemic.

In May 1999, in the Kolar district of Karnataka State, Bemisia tabaci numbers on tomato increased by approximately 1,000-fold that observed previously (3). This was associated with an epidemic of severe tomato leaf curl disease that caused complete crop failure. DNAs extracted from 35 symptomatic tomato leaf samples collected within the epidemic region all gave the expected 500 to 600 bp amplicon with begomovirus-specific primers A/B (1). These primers amplify from the conserved nonanucleotide TAATATTAC in the common region of DNA-A to the conserved amino acid sequence CEGPCKYG within the coat protein gene. AluI and TaqI restriction patterns of all 35 polymerase chain reaction (PCR) products were identical. One PCR product from an epidemic (GenBank no. AF321929) and a non-epidemic (AF321930) site (Bangalore) were cloned and sequenced. The two 531-bp inserts showed 96% nucleotide identity to each other and 94% nucleotide identity to the equivalent region of Tomato leaf curl Bangalore virus (ToLCBV-Ban-4) (AF165098), suggesting that the epidemic was caused by an indigenous ToLCBV strain. Adult B. tabaci were collected from tomato plants at nine sites within the epidemic. DNA was extracted from 9 to 13 individuals per site and analyzed by RAPD-PCR using primers OpB20 and OpB11. Eighty to 100% of individuals per site had identical patterns to those of B biotype individuals from Israel and Florida, which were different to the patterns produced by the indigenous Indian B. tabaci. Adult B. tabaci from the epidemic and nonepidemic (Bangalore) regions were cultured separately on zucchini plants (n = 20) vars. Fordhook and Ambassador. Distinct silverleaf symptoms appeared in all plants fed on by the epidemic B. tabaci, but not on those fed on by the nonepidemic whiteflies. Irregular ripening of tomatoes was also a widespread problem in the epidemic area. Cytochrome oxidase I (COI) (720 bp) gene sequences were obtained for epidemic (AF321927) and nonepidemic (AF321928) B. tabaci, which had only 80% nucleotide identity to each other. A GenBank BLAST search showed that the former were most similar to B biotype whitefly from Israel (AF164667; 97%) and Texas (AF164675; 99%). The B biotype transmits Indian ToLCBV (2) and its introduction into India is of great concern as it is already associated with a devastating plant-disease epidemic. References: (1) D. Deng et al. Ann. App. Biol. 125:327, 1994. (2) P. F. McGrath and B. D. Harrison. Ann. App. Biol. 126:307, 1995. (3) H. K. Ramappa et al. Ann. App. Biol. 133:187, 1998.

Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus.

Sampling procedures and diagnostic protocols were optimized for accurate diagnosis of Cassava brown streak virus (CBSV) (genus Ipomovirus, family Potyviridae). A cetyl trimethyl ammonium bromide (CTAB) method was optimized for sample preparation from infected cassava plants and compared with the RNeasy plant mini kit (Qiagen) for sensitivity, reproducibility and costs. CBSV was detectable readily in total RNAs extracted using either method. The major difference between the two methods was in the cost of consumables, with the CTAB 10x cheaper (0.53 pounds sterling=US$0.80 per sample) than the RNeasy method (5.91 pounds sterling=US$8.86 per sample). A two-step RT-PCR (1.34 pounds sterling=US$2.01 per sample), although less sensitive, was at least 3-times cheaper than a one-step RT-PCR (4.48 pounds sterling=US$6.72). The two RT-PCR tests revealed consistently the presence of CBSV both in symptomatic and asymptomatic leaves and indicated that asymptomatic leaves can be used reliably for virus diagnosis. Depending on the accuracy required, sampling 100-400 plants per field is an appropriate recommendation for CBSD diagnosis, giving a 99.9% probability of detecting a disease incidence of 6.7-1.7%, respectively. CBSV was detected at 10(-4)-fold dilutions in composite sampling, indicating that the most efficient way to index many samples for CBSV will be to screen pooled samples. The diagnostic protocols described below are reliable and the most cost-effective methods available currently for detecting CBSV.

Yams (Dioscorea spp.) from the South Pacific Islands contain many novel badnaviruses: implications for international movement of yam germplasm.

Yam (Dioscorea spp.) samples (n = 690) from seven South Pacific Islands were screened for badnavirus infection by ELISA using two antisera to African badnaviruses. Positive readings were obtained for 26.4-34.6% of samples representing both known (D. bulbifera, D. nummularia and D. pentaphylla) and unreported host species (D. alata, D. esculenta, D. rotundata and D. trifida) in this region. Total DNAs were extracted from 25 ELISA-positive plants and 4 ELISA-negative controls and subjected to PCR amplification with badnavirus-specific primers targeting the reverse transcriptase (RT)-RNaseH genes. All 29 samples yielded the expected size PCR-product for badnaviruses, which were cloned and sequenced. Phylogenetic analyses of the resulting 45 partial (500-527 bp) RT-RNaseH sequences revealed 11 new sequence groups with <79% nucleotide identity to each other or any EMBL sequence. Three sequences (two groups) were highly divergent to the other nine new South Pacific yam badnavirus groups (47.9-57.2% identity) and probably represent either new Caulimoviridae genera or endogenous pararetrovirus sequences. Some sequence groups appeared specific to particular Dioscorea host species. Four 99.9% identical RT-RNaseH sequences possessing nine amino acid deletions from D. esculenta from three islands represent a putative integrated sequence group. The distribution of sequence groups across the islands indicates that badnaviruses have spread extensively between islands and continents through infected germplasm.

Taxonomy, molecular phylogeny and evolution of plant reverse transcribing viruses (family Caulimoviridae) inferred from full-length genome and reverse transcriptase sequences.

This study constitutes the first evaluation and application of quantitative taxonomy to the family Caulimoviridae and the first in-depth phylogenetic study of the family Caulimoviridae that integrates the common origin between LTR retrotransposons and caulimoviruses. The phylogenetic trees and PASC analyses derived from the full genome and from the corresponding partial RT concurred, providing strong support for the current genus classification based mainly on genome organisation and use of partial RT sequence as a molecular marker. The PASC distributions obtained are multimodal, making it possible to distinguish between genus, species and strain. The taxonomy of badnaviruses infecting banana (Musa spp.) was clarified, and the consequence of endogenous badnaviruses on the genetic diversity and evolution of caulimoviruses is discussed. The use of LTR retrotransposons as outgroups reveals a structured bipolar topology separating the genus Badnavirus from the other genera. Badnaviruses appear to be the most recent genus, with the genus Tungrovirus in an intermediary position. This structuring intersects the one established by genomic and biological properties and allows us to make a correlation between phylogeny and biogeography. The variability shown between members of the family Caulimoviridae is in a similar range to that reported within other DNA and RNA plant virus families.

Canna yellow streak virus: a new potyvirus associated with severe streaking symptoms in canna.

A new potyvirus has been found in canna. A 1700-nucleotide region at the 3' end of the genomic RNA has been sequenced from two isolates. The sequence reveals the virus to be a distinct member of the genus Potyvirus but most closely related to Johnsongrass mosaic virus. A specific primer pair was designed that enabled canna material to be screened specifically for this virus. The virus was consistently found in cannas showing severe virus symptoms. This virus has been found in different canna varieties from the UK, Belgium, Netherlands, France and Israel. The name Canna yellow streak virus (CaYSV) has been proposed for this new virus.

Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers.

Use of tRNA consensus primers to indicate subgroups of Pseudomonas solanacearum by polymerase chain reaction amplification.

Polymerase chain reaction amplification of DNA from 112 Pseudomonas solanacearum strains with the tRNA consensus primers T3A and T5A divided the species into three fingerprint groups. These groups correspond well with previous divisions made by restriction fragment length polymorphism analysis. This polymerase chain reaction test is a facile method for rapidly classifying P. solanacearum strains.

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria.

Mutants of a tomato strain of Xanthomonas campestris pv. vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kb EcoRI fragment of p6AD4.

Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction.

The sequence of a 292 bp segment of the DNA encoding 16S rRNA (corresponding to positions 44-337 of the Escherichia coli 16S rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16S rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16S rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

Identification and mapping of AFLP markers linked to peanut (Arachis hypogaea L.) resistance to the aphid vector of groundnut rosette disease.

Groundnut rosette disease is the most destructive viral disease of peanut in Africa and can cause serious yield losses under favourable conditions. The development of disease-resistant cultivars is the most effective control strategy. Resistance to the aphid vector, Aphis craccivora, was identified in the breeding line ICG 12991 and is controlled by a single recessive gene. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) analysis were employed to identify DNA markers linked to aphid resistance and for the development of a partial genetic linkage map. A F(2:3) population was developed from a cross using the aphid-resistant parent ICG 12991. Genotyping was carried out in the F2 generation and phenotyping in the F3 generation. Results were used to assign individual F2 lines as homozygous-resistant, homozygous-susceptible or segregating. A total of 308 AFLP (20 EcoRI+3/MseI+3, 144 MluI+3/MseI+3 and 144 PstI+3/MseI+3) primer combinations were used to identify markers associated with aphid resistance in the F(2:3) population. Twenty putative markers were identified, of which 12 mapped to five linkage groups covering a map distance of 139.4 cM. A single recessive gene was mapped on linkage group 1, 3.9 cM from a marker originating from the susceptible parent, that explained 76.1% of the phenotypic variation for aphid resistance. This study represents the first report on the identification of molecular markers closely linked to aphid resistance to groundnut rosette disease and the construction of the first partial genetic linkage map for cultivated peanut.