Detection of coffee ringspot virus (CoRSV) in the mite vector by RT-qPCR.

The mite Brevipalpus phoenicis (Geijskes, 1939) (Acari: Tenuipalpidae) is responsible for the transmission of coffee ringspot. There is a great interest in the study of Brevipalpus transmitted viruses (BrTVs), and the interaction between them, their vectors and hosts. Electron microscopy analyses suggest that CoRSV replicates in the mite tissue, what would characterize a persistent propagative virus-vector relationship. This study aimed to establish a transcriptase quantitative polymerase chain reaction (RT-qPCR) method to evaluate and compare the replication capacity of the virus in the vector. Total RNA was extracted from a sample of 100 mites reared in symptomatic plants for CoRSV using two different methods: CTAB and Nucleo Spin RNA XS Kit (Macherey-Nagel) and quantified in a NanoDrop 8000 (Thermo Scientific).Abstract 10

Citrus bright spot virus: a new dichorhavirus, transmitted by Brevipalpus azores, Causing Citrus Leprosis Disease in Brazil.

Citrus leprosis (CL) is the main viral disease affecting the Brazilian citriculture. Sweet orange (Citrus sinensis L. Osbeck) trees affected by CL were identified in small orchards in Southern Brazil. Rod-like particles of 40 × 100 nm and electron lucent viroplasm were observed in the nucleus of infected cells in symptomatic tissues. RNA extracts from three plants, which proved negative by RT-PCR for known CL-causing viruses, were analyzed by high throughput sequencing and Sanger sequencing after RT-PCR. The genomes of bi-segmented ss(?)RNA viruses, with ORFs in a typical organization of members of the genus Dichorhavirus, were recovered. These genomes shared 98?99% nt sequence identity among them but <73% with those of known dichorhavirids, a value below the threshold for new species demarcation within that genus. Phylogenetically, the three haplotypes of the new virus called citrus bright spot virus (CiBSV) are clustered with citrus leprosis virus N, which is a dichorhavirus transmitted by Brevipalpus phoenicis sensu stricto. In CiBSV-infected citrus plants, B. papayensis and B. azores were found, but the virus could only be transmitted to Arabidopsis plants by B. azores. The study provides the first evidence of the role of B. azores as a viral vector and supports the assignment of CiBSV to the tentative new species Dichorhavirus australis

Diversity of “Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “Ca. Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “Ca. Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating “Ca. Liberibacter asiaticus” isolates