RESUMO
Symptoms of phytoplasma infection were observed in different weed species, Bidens subalternans, Conyza bonariensis, Heterosperma ovatifolium and Conium maculatum, collected from diverse geographical regions in Argentina. To confirm the association of phytoplasma infection with symptomatic plants, PCR, RFLP and phylogenetic analyses based on 16S rRNA-encoding sequences were performed. In this work, we report the presence of phytoplasmas from group 16SrVII (subgroup 16VII-B) infecting C. bonariensis and B. subalternans and from group 16SrIII (subgroup 16SrIII-X) B. subalternans, H. ovatifolium, and C. maculatum. Phytoplasmas from the aster yellows group were detected infecting C. bonariensis and B. subalternans. Analysis of 16S rRNA-encoding genes revealed the presence of two distinct operons, rrnB (16SrI-B) and newly described rrnA, which is different from the reference RFLP patterns of all previously established 16SrI-subgroups. A single rp operon sequence analysis reveals the presence of simple infection and confirms a description of a novel subgroup. On the basis of these results we propose a designation of new subgroup 16SrI-(B/AJ) AJ (rp-AJ). To our knowledge, this is the first report of phytoplasmas infecting Bidens subalternans¸ Heterosperma ovatifolium and Conium maculatum.
Assuntos
Filogenia , Phytoplasma/classificação , Doenças das Plantas/microbiologia , Plantas Daninhas/microbiologia , Argentina , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Óperon , Phytoplasma/isolamento & purificação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Strawberry plants showing symptoms of lethal redness disease were found in production fields located in Tucumán province, Argentina. The presence of phytoplasmas was confirmed by PCR of 16S rDNA gene using phytoplasma universal primers. According to the 16S rDNA gene sequence identity, the four isolates analysed are related to the X-disease group (16SrIII) (identity ~99 %). These results were confirmed by in silico RFLP, actual RFLP and also by phylogenetic analyses of the 16S rDNA gene. This new phytoplasma was named as Strawberry X-Redness (StrawXR). The results from virtual and actual RFLP analyses of 16S rDNA gene revealed the presence of subgroup 16SrIII-J and three new 16SrIII subgroups. This is the first record of phytoplasmas from X-disease group associated strawberry in Argentina. These results confirm the prevalence of X-disease group and also contribute to the knowledge of diversity of phytoplasmas in this region.
Assuntos
Fragaria/microbiologia , Filogenia , Phytoplasma/classificação , Doenças das Plantas/microbiologia , Argentina , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Phytoplasma/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Strawberry red leaf phytoplasma was found in strawberry plants from production fields in Lules (Tucumán province) and Bella Vista (Corrientes province), Argentina. Characteristic strawberry red leaf symptoms were stunting, young leaves with yellowing at the edges, mature leaves which curled and were reddish at the abaxial face, flower and fruit deformation and death. The pathogen was detected with phytoplasma-universal primer pairs P1/P7 followed by R16F2n/R16R2 as nested primers in 13 diseased plants. Based on RFLP and sequence analysis of the amplified 16S rRNA gene, the phytoplasma was related to the 16SrXIII group (Mexican periwinkle virescence). In silico the RFLP profile of all the samples analysed revealed the presence of a unique pattern, showing that the novel phytoplasma is different from all the phytoplasmas currently composing the 16SrXIII group. The phylogenetic analysis was consistent with RFLP analysis as the strawberry red leaf phytoplasma was grouped within the 16SrXIII group, but formed a particular cluster. On this basis, the Strawberry red leaf phytoplasma associated with strawberry red leaf disease was assigned to a new subgroup, 16SrXIII-F.
Assuntos
Fragaria/microbiologia , Filogenia , Phytoplasma/classificação , Doenças das Plantas/microbiologia , Argentina , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Dados de Sequência Molecular , Phytoplasma/genética , Phytoplasma/isolamento & purificação , Folhas de Planta/microbiologia , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Mal de Río Cuarto virus (MRCV), a member of the genus Fijivirus, family Reoviridae, has a genome consisting of 10 dsRNA segments. The segment 9 (S9) possesses two non-overlapping open reading frames (ORF-1 and ORF-2) encoding two putative proteins, MRCV P9-1 and MRCV P9-2, both of unknown function. The MRCV S9 ORF-1 was RT-PCR amplified, expressed in pET-15b vector, and the recombinant protein produced was used to raise an antiserum in rabbit. Western blot with the specific MRCV P9-1 antiserum detected a protein of about 39 kDa molecular weight present in crude protein extracts from infected plants and insects. However, no reaction was observed when this antiserum was tested against purified virus. In contrast, only virus particles were detected by a MRCV-coat antiserum used as a validation control. These results suggest that MRCV S9 ORF-1 encodes a non-structural protein of MRCV. Immunoelectron microscopy assays confirmed these results, and localized the MRCV P9-1 protein exclusively in electron-dense granular viroplasms within the cytoplasm of infected plants and insects cells. As viroplasms are believed to be the replication sites of reoviruses, the intracellular location of MRCV P9-1 protein suggests that it might be involved in the assembly process of MRCV particles.
Assuntos
Hemípteros/virologia , Doenças das Plantas/virologia , Infecções por Reoviridae/virologia , Reoviridae/fisiologia , Sorghum/virologia , Proteínas não Estruturais Virais/metabolismo , Animais , Feminino , Hemípteros/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Fases de Leitura Aberta , Reoviridae/genética , Sorghum/ultraestrutura , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/ultraestrutura , Vírion/imunologia , Vírion/metabolismoRESUMO
Mal de Río Cuarto virus (MRCV) is a newly described species of the genus Fijivirus, family Reoviridae. The nucleotide sequence of four MRCV genome segments was determined. MRCV S1, S2, S3 and S6 were predicted to encode proteins of 168.4, 134.4, 141.7 and 90 kDa, respectively. MRCV S1 encodes a basic protein that contains conserved RNA-dependent RNA polymerase motifs, and is homologous to Rice black streaked dwarf virus (RBSDV), Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV) polymerases as well as to corresponding proteins of members of other genera of the Reoviridae. MRCV S2 codes for a protein with intermediate homology to the ones coded by RBSDV S4 and FDV S3 'B' spike, which is presumably the B-spike protein. MRCV S3 most probably encodes the major core protein and is highly homologous to corresponding proteins of RBSDV S2 and FDV S3. MRCV S6-encoded protein has low homology to the proteins of unknown function coded by RBSDV S6 and FDV S6. The identity levels between all analyzed MRCV coded proteins and their RBSDV counterparts varied between 84.5 and 44.8%. The analysis of the reported sequences allowed a phylogenetic comparison of MRCV with other reovirus and supported its taxonomic status within the genus.