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Two large contigs with sequence similarities to different carlaviruses were identified by high-throughput sequencing in samples from a cactus plant. The complete genomes of the two viruses, tentatively named "cactus carlavirus 1" (CCV-1) and "cactus carlavirus 2" (CCV-2), were determined to be 8,441 and 8,396 nucleotides long, respectively, excluding the poly(A) tail. These viruses have the typical genomic organization of members of the genus Carlavirus. CCV-1 appears to be a cactus isolate of the carlavirus HSO-2016a, with 90.1% nucleotide sequence identity between the two virus genomes, whereas CCV-2 may be classified as a member of a new species. The sequences of CCV-2 and other carlaviruses are 48.9-60.0% identical at the whole-genome level.

Asian prunus viruses (APV 1, APV 2 and APV 3), Plum bark necrosis stem pitting associated virus (PBNSPaV) and Peach latent mosaic viroid (PLMVd) are pathogens that infect Prunus species. A single-tube multiplex, TaqMan real-time RT-PCR assay was developed for the simultaneous detection and identification of these pathogens. The protocol includes amplification and detection of a fluorogenic cytochrome oxidase gene (COX) as an internal control. The results of the multiplex TaqMan RT-PCR assay correlated with those from conventional RT-PCR, with a 10-fold increase in sensitivity in the multiplex real-time format. The efficiency and accuracy of the assay was evaluated by testing stone fruit trees from positive control collections and several orchard locations. Several mixed infections of target pathogens were detected in peach orchard samples. This assay is simple, rapid and cost-effective and can be used by quarantine and certification programs where numerous stone fruit trees need to be tested for these pathogens.

Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Apple fruit crinkle viroid (AFCVd), and Pear blister canker viroid (PBCVd) infect pome fruit trees. These viroids are important quarantine pathogens for the international movement of pome germplasm. A single-step multiplex reverse transcription polymerase chain reaction assay (mRT-PCR) was developed for the simultaneous detection of these viroids. Four pairs of primers specific for each of the four viroids were used to amplify PCR products of different sizes that can be resolved by agarose gel electrophoresis. Amplification of a plant 18S rRNA was included in the assay as an internal control. Amplicons of 371 bp (AFCVd), 270 bp (ADFVd), 186 bp (ASSVd), 120 bp (PBCVd), and 844 bp (18S rRNA) were obtained in both uniplex and mRT-PCR assays. The identities of the amplification products were confirmed by sequencing. The specificities and limits of detection for all four viroids by uniplex and mRT-PCR assays were comparable. The assay was further validated using samples from pome trees inoculated with all four viroids, as well as field samples from commercial orchards in Colorado. All four viroids were detected from inoculated pear trees and up to three viroids were detected from inoculated apple trees. This is a simple, rapid and cost-effective technique to detect these four viroids in fruit trees. The procedure is especially applicable to certification and quarantine programs, where numerous samples must be tested for all four viroids.

A simple and sensitive dot blot hybridization assay using a digoxigenin-labeled cRNA polyprobe was developed for the simultaneous detection of six viroids that infect pome and stone fruit trees. The polyprobe was constructed by cloning sequentially partial sequences of each viroid into a single vector, with run-off transcription driven by the T7 promoter. All six viroids were detectable within a dilution range of 5−3 to 5−4 in total nucleic acid extracts from infected trees. Individual trees were co-inoculated to create mixed infections and all four pome fruit viroids and both stone fruit viroids could be detected in pear and peach trees, respectively, using the polyprobe. The results of the assays using the polyprobe were comparable to those using single probes. The methods were validated by testing geographically diverse isolates of viroids, as well as field samples from several collections in the US. The assay offers a rapid, reliable and cost-effective approach to the simultaneous detection of six fruit trees viroids and has the potential for routine use in quarantine, certification, and plant genebank programs where many samples are tested and distributed worldwide.

A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.

The complete genomic sequences of two isolates of pokeweed mosaic virus (PkMV) were determined to be 9512 nucleotides long, excluding the poly(A) tail. Their genomic organization is typical of potyviruses and contains conserved motifs found in members of the genus Potyvirus. Pairwise comparisons showed that PkMV and other members of the genus Potyvirus share 51.0-57.5 % sequence identity at the genome sequence level and 39.8-53.0 % at the polyprotein sequence level. Phylogenetic analysis indicated that PkMV is most closely related to several viruses in the PVY group of the genus Potyvirus. The genomic information obtained for PkMV suggests that this virus is a distinct potyvirus.

The presence of Gooseberry vein banding associated virus (GVBaV), a badnavirus in the family Caulimoviridae, is strongly correlated with gooseberry vein banding disease in Ribes spp. In this study, full-length genomic sequences of four GVBaV isolates from different hosts and geographic regions were determined to be 7649-7663 nucleotides. These isolates share identities of 96.4-97.3% for the complete genomic sequence, indicating low genetic diversity among them. The GVBaV genome contains three open reading frames (ORFs) on the plus strand that potentially encode proteins of 26, 16, and 216 kDa. The size and organization of GVBaV ORFs 1-3 are similar to those of most other badnaviruses. The putative amino acid sequence of GVBaV ORF 3 contained motifs that are conserved among badnavirus proteins including aspartic protease, reverse transcriptase, and ribonuclease H. The highly conserved putative plant tRNA(met)-binding site is also present in the 935-bp intergenic region of GVBaV. The identities of the genomic sequences of GVBaV and other badnaviruses range from 49.1% (Sugarcane bacilliform Mor virus) to 51.7% (Pelargonium vein banding virus, PVBV). Phylogenetic analysis using the amino acid sequence of the ORF 3 putative protein shows that GVBaV groups most closely to Dioscorea bacilliform virus, PVBV, and Taro bacilliform virus. These results confirm that GVBaV is a pararetrovirus of the genus Badnavirus in the family Caulimoviridae.