RESUMO
Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.
Assuntos
Primers do DNA/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Phytoplasma/genética , Phytoplasma/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Xylella/genética , Xylella/isolamento & purificação , Misturas Complexas , Sondas de Oligonucleotídeos/genética , Vitis/microbiologiaRESUMO
Deep-sequencing analysis of nucleic acids extracted from leaf tissue of an American persimmon (Diospyros virginiana L.) and subsequent-sequencing analyses uncovered at least four distinct closterovirus-like molecules. Two complete genomes of 18,569 and 18,030 nucleotides (nt) and partial genomes of 4,899 and 9,019 nt were determined. The two complete genomes encoded 11 potential open reading frames and the characteristic organization of closteroviruses. Among the four genomes, the putative heat shock protein 70 homolog (HSP70h), RNA-dependent RNA polymerase, and coat protein showed 82-85, 72-91, and 84-87 % amino acid sequence identities, respectively. These results suggested that the four identified viruses could be divergent variants in a single host plant. The phylogenetic tree based on HSP70h showed that their closest relative, although distant, is Olive leaf yellowing-associated virus, a putative unassigned member of the family Closteroviridae. The name Persimmon virus B was proposed for this new virus, representing another unassigned member of the family.
Assuntos
Closterovirus/classificação , Closterovirus/isolamento & purificação , Diospyros/virologia , Genoma Viral , RNA Viral/genética , Análise de Sequência de DNA , Closterovirus/genética , Análise por Conglomerados , Dados de Sequência Molecular , Filogenia , Folhas de Planta/virologia , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
A unique circular molecule of 358 nucleotides was detected in American persimmon (Diospyros virginiana L.). The molecule was graft-transmissible and had genetic characteristics of members of the genus Apscaviroid. It had the highest sequence similarity (72-73 %) to citrus viroid VI (CVd-VI) and formed a clade with CVd-VI, citrus dwarfing viroid, and apple dimple fruit viroid in a phylogenetic tree. The molecule was not detected in citrus, unlike CVd-VI, which infects citrus and persimmon, and it was genetically distant from persimmon latent viroid, which infects persimmon only. The genetic and biological features indicated that the molecule may be a member of a new apscaviroid species.
Assuntos
Diospyros/virologia , RNA/genética , Análise de Sequência de DNA , Viroides/genética , Análise por Conglomerados , Dados de Sequência Molecular , Filogenia , RNA Circular , RNA Viral/genética , Homologia de Sequência do Ácido Nucleico , Viroides/isolamento & purificaçãoRESUMO
Deep-sequencing analysis of nucleic acids from leaf tissue of Japanese persimmon trees exhibiting fruit apex disorder in some fruits detected two molecules that were graft transmitted to healthy seedlings. One of the complete genomes consisted of 13â467 nt and encoded six genes similar to those of plant rhabdoviruses. The virus formed a distinct cluster in the genus Cytorhabdovirus with lettuce necrotic yellows virus, lettuce yellow mottle virus and strawberry crinkle virus in a phylogenetic tree based on the L protein (RNA-dependent RNA polymerase, RdRp). The other consisted of 7475 nt and shared a genome organization similar to those of some insect and fungal viruses having dsRNA genomes. In a phylogenetic tree using the RdRp sequence of several unassigned dsRNA viruses, the virus formed a possible new genus cluster with two insect viruses, Circulifer tenellus virus 1 and Spissistilus festinus virus 1, and one plant virus, cucurbit yellows-associated virus.
