ABSTRACT
The genus Tospovirus was thought to be composed only of the tomato spotted wilt virus (TSWV), but now at least four Tospovirus species have been proposed based on serological and molecular data. A classification of tospoviruses has been proposed taking into account global similarities of the N gene and N protein sequences of 7 isolates of Tospovirus. Because phylogenetic analyses based on global similarities can lead to classifications which do not mirror the genealogy of the group, we have employed a cladistic analysis using parsimony of this genus with RNA sequences of 450 nucleotides of the N gene from 14 new Argentinean isolates and 4 previously described isolates. Representatives of the Bunyaviridae family, Rift Valley Fever Virus (Phlebovirus) and Bunyamwera (Bunyavirus), were used as the outgroup in separate analyses.
Subject(s)
Nucleocapsid/genetics , Phylogeny , Tospovirus/genetics , Argentina , Base Sequence , Bunyaviridae/genetics , Genetic Variation , Molecular Sequence Data , Nucleocapsid Proteins , RNA, Viral/genetics , Rift Valley fever virus/genetics , Selection, Genetic , Sequence AlignmentABSTRACT
Tomato spotted wilt is a serious disease that affects several economically important crops. From the epidemiological point of view and for the development of a successful plan for transgenic resistance plants, the four known Tospovirus species must be discriminated at the molecular level. A RT-PCR assay using primers complementary to the N gene was used to detect and differentiate fourteen Argentinian isolates of Tospovirus from different crops and geographical areas. Extracts were reverse transcribed using a thermo-resistant reverse transcriptase and PCR reactions were performed for 30 min in a capillar thermo-cycler. The products were digested with restriction enzymes and three of the four described species were identified. Additionally, the results were confirmed by DAS-ELISA. The method described here is rapid and reliable.
Subject(s)
DNA Restriction Enzymes , Polymerase Chain Reaction/methods , Tospovirus/isolation & purification , Base Sequence , DNA Primers , Deoxyribonucleases, Type II Site-Specific , Enzyme-Linked Immunosorbent Assay , Molecular Sequence Data , Tospovirus/genetics , Transcription, GeneticABSTRACT
The restriction map of rDNA from South American camelids and the Bactrian camel was analyzed by digestion of high-molecular-weight DNA with endonucleases EcoRI,BamHI and the two combined followed by Southern blot hybridization with probes for the 18S and 28S rDNA sequences. We scored a total of 17 restriction sites, six of which were mapped conserved in all the species. The other eleven corresponded to spacer regions and revealed variations between these taxa. The study showed that the two groups differ in the length of the internal transcribed spacer. Also they showed the existence of two regions of fast evolution on the opposite termini of the external spacer. A restriction site present at low frequency in the non-transcribed spacer of guanaco and llama was the only difference encountered within the South American group.
