Differential disease phenotype of begomoviruses associated with tobacco leaf curl disease in Comoros.

In the 2000s, tobacco plantations on the Comoros Islands were afflicted with a previously unobserved tobacco leaf curl disease characterised by symptoms of severe leaf curling and deformation. Previous molecular characterization of potential viral pathogens revealed a complex of African monopartite tobacco leaf curl begomovirus (TbLCVs). Our molecular investigation allowed the characterization of a new monopartite virus involved in the disease: tomato leaf curl Namakely virus (ToLCNamV). Agroinoculation experiments indicated that TbLCVs and tomato leaf curl viruses (ToLCVs) can infect both tomato and tobacco but that infectivity and symptom expression fluctuate depending on the virus and the plant cultivar combination.

First Report of Tomato chlorosis virus in Tomato on Mauritius Island.

In February of 2007, a virus disease survey on tomato plants (Solanum lycopersicum) in greenhouses and open fields was conducted on the island of Mauritius at the request of the Agricultural Research and Extension Unit (AREU), sponsored by the European Union, and funded by the Programme Régional de Protection des Végétaux (PRPV). Yellowing symptoms on the lower and middle leaves of tomato plants and whiteflies (Bemisia tabaci) were observed in greenhouses in Pailles, located in the north region of the island. The interveinal chlorosis pattern of the discolored leaves was similar to symptoms described for Tomato chlorosis virus (ToCV; genus Crinivirus) detected on tomato in 2004 on Reunion Island (1), suggesting the possible involvement of the same virus. Six symptomatic tomato leaf samples were collected from separate plants in the Pailles greenhouses. Total RNA was extracted from these samples with the Qiagen (Courtaboeuf, France) RNeasy Plant Mini Kit. Reverse transcription-PCR was used for molecular diagnosis, independently using two sets of specific ToCV primers. The first set of primers, ToCV-172 and ToCV-610, was designed to amplify the highly conserved region of the heat shock protein 70 (HSP70) gene (2). The second set of primers was designed to amplify the coat protein (CP) gene (forward-CP-ToCV-4384: 5'-ATCCTCTGGTTAGACCGTTAG-3' and reverse as in Segev et al. [3]). PCR products of the expected size (439 and 725 bp, respectively) were observed for the six samples from the greenhouse from Pailles. For each set of primers, two PCR products obtained from two different samples were cloned using the pGEM-T Easy Vector system (Promega, Madison, WI) and sequenced (Macrogen, Seoul, Korea). The two HSP70 sequences (GenBank-EMBL-DDBJ Accession Nos. AM884013 and AM884014) and the two CP sequences (FM206381 and FM206382) had 100% nucleotide identities (DNAMAN; Lynnon BioSoft, Quebec City, Canada). The highest nucleotide identities of the 439-bp fragment of HSP70 gene (NCBI, BLASTn) were 97% with ToCV isolates from France (DQ355214, DQ355215, and DQ355216), Florida (AY903448), Italy (AM231038 and AY048854), Mayotte Island (AM748818), Portugal (AF234029), and Reunion Island (AM748816). Similarly, the highest nucleotide identities (98%) were obtained with ToCV isolates from France (EU625350) and Spain (DQ136146), with the 725-bp fragments of CP gene. Interestingly, ToCV isolates from Mauritius and Reunion are as divergent as isolates from the rest of the world, which suggests the possibility of different introductions. In conclusion, observed symptoms and laboratory results based on two different regions of the genome confirm the presence of ToCV in symptomatic tomatoes on the island of Mauritius, for the first time to our knowledge. The visual survey carried out in June of 2008 confirmed the presence of typical interveinal chlorosis symptoms in other greenhouses, requiring further studies to assess the incidence of ToCV on tomato crops. References: (1) H. Delatte et al. Plant Pathol. 55:289, 2006. (2) D. Louro et al. Eur. J. Plant Pathol. 106:589, 2000. (3) L. Segev et al. Plant Dis. 88:1160, 2004.

Begomovirus 'melting pot' in the south-west Indian Ocean islands: molecular diversity and evolution through recombination.

During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.

A multiplex PCR method discriminating between the TYLCV and TYLCV-Mld clades of tomato yellow leaf curl virus.

Tomato yellow leaf curl virus (TYLCV) is one of the causal agents of tomato yellow leaf curl disease (TYLCD) and can cause up to 100% yield losses in tomato fields. As TYLCV continues to spread, many isolates have been described in different parts of the world. Recently two closely related but distinct TYLCV clades, called TYLCV and TYLCV-Mld, have been identified. Isolates from those two clades differ mainly in the nucleotide sequences of their replication associated protein genes but do not display significantly different symptomatology. In order to improve monitoring of the rapidly expanding worldwide TYLCD epidemic, a multiplex polymerase chain reaction assay (mPCR) was developed. A set of three primers were designed to detect and characterize the TYLCV and TYLCV-Mld clade isolates. The specificity and sensitivity of the mPCR were validated on TYLCV infected tomato plants and Bemisia tabaci whiteflies. Being cheap, fast and highly sensitive this new diagnostic tool should greatly simplify efforts to trace the global spread of TYLCV.

Another use of silica gel and aqueous eluent for HPLC analysis of clozapine and desmethylclozapine.

A procedure that involves a high-performance liquid chromatographic system with silica-bonded columns and reversed-phase eluents was fitted from a previously described method to measure clozapine and desmethylclozapine plasma levels. Clozapine and its demethylated metabolite were extracted from alkalinized serum by a liquid-liquid extraction, separated in 10 min, then quantitated at 254 nm at a minimum concentration of 20 ng/mL. The standard curves were linear over the range of 50-3000 ng/mL (r > 0.99) both for clozapine and desmethylclozapine and the assay had good sensitivity and recovery. Intra- and interday coefficients of variation for 200 and 800 ng/mL controls were less than 11.5% for clozapine and desmethylclozapine. This simple and efficient assay was used to monitor clozapine and desmethylclozapine levels from some treatment-refractory schizophrenic patients.