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We conducted a survey of the phytosanitary status of the Prunus germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. The virome of plants displaying virus-like symptoms was studied using Illumina MiSeq high-throughput sequencing. Reads related to prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and ourmia-like virus 1 (OuLV1) were generated in a number of samples. Near complete genomes of two divergent PNRSV isolates, PDV isolate, and a contig partly covered OuLV1 genome were assembled de novo using the metaSPAdes program. The structure of the genomic RNA1, RNA2, and RNA3 of the new ilarvirus isolates was shown to be typical of PNRSV and PDV. This is the first report and characterization of the PNRSV and PDV full-length genomes from Russia, expanding the information on their geographical distribution and genetic diversity. An open reading frames (ORF)-based phylogeny of all full-length PNRSV and PDV genomes available in GenBank divided each ORF into two or three main clusters. A number of isolates migrated from one cluster to another cluster, depending on the analyzed genome segment. The different branching order may indicate reassortment in the evolutionary history of some PDV and PNRSV isolates. [ABSTRACT FROM AUTHOR]

We conducted a survey of the phytosanitary status of the Prunus germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. The virome of plants displaying virus-like symptoms was studied using Illumina MiSeq high-throughput sequencing. Reads related to prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and ourmia-like virus 1 (OuLV1) were generated in a number of samples. Near complete genomes of two divergent PNRSV isolates, PDV isolate, and a contig partly covered OuLV1 genome were assembled de novo using the metaSPAdes program. The structure of the genomic RNA1, RNA2, and RNA3 of the new ilarvirus isolates was shown to be typical of PNRSV and PDV. This is the first report and characterization of the PNRSV and PDV full-length genomes from Russia, expanding the information on their geographical distribution and genetic diversity. An open reading frames (ORF)-based phylogeny of all full-length PNRSV and PDV genomes available in GenBank divided each ORF into two or three main clusters. A number of isolates migrated from one cluster to another cluster, depending on the analyzed genome segment. The different branching order may indicate reassortment in the evolutionary history of some PDV and PNRSV isolates.

In order to fill in a decade-long information gap regarding the biological, serological and molecular data for plum tree viruses in Bosnia and Herzegovina, a three-phase study combining symptom evaluation, and serological and molecular assays with high-throughput sequencing (HTS) technology was conducted. The most frequently observed symptoms were discolorations in the form of ring patterns, bands and irregular shapes, as well as vein banding. Sharka-associated symptoms in the form of ring patterns and semicircles were prevalent. A total of 468 plum tree samples were tested by ELISA for the presence of PPV, ApMV, PDV, PNRSV, PBNSPaV, ACLSV and MLRSV. An overall infection incidence of 51.9% was detected, with PPV being the most prevalent (48.7%), followed by PDV (2.99%), PNRSV (0.21%) and mixed infections of PPV+PDV (1.71%). RT-PCR-assisted strain typing in 45 samples revealed PPV-D as the most common strain (22.22%), followed by PPV-REC (6.66%). Mixed infections of PPV-D+PPV-REC were detected (6.66%). HTS enabled the recovery of a 9743 nts long sequence of PPV-D (PPV_O7/80, MW412433), which shared the highest nucleotide and amino acid identities with isolates S13 (LC375131) from Serbia, SVN1 (LC375132) from Slovenia and N9 (LC375129) from Bulgaria. The phylogenetic analysis of the whole genome placed the isolate of the D strain in a distinctive group with the Slovenian isolate SVN1 (LC375132). In addition, the (Cter)NIb/(Nter)CP fragment of a PPV-REC isolate (MW412434) obtained in this survey formed a separate group with previously known isolates from Bosnia and Herzegovina (BOS64Pl and BOS257Pl). [ABSTRACT FROM AUTHOR]

Biological and molecular properties of five Prune dwarf virus cherry isolates were studied. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) proved PDV infection in the respective source trees and absence of six other viruses infecting the cherry. Two of infected source trees were symptomless, and the other three trees showed deformed and elongated leaves with chlorotic lines and spots. The reaction of greenhouse-grown seedlings of 22 herbaceous test plant species was investigated. The woody indicators Prunus tomentosa and Prunus cerasifera were chip-budded. Cucumis sativus cv. Amelia, C. sativus cv. Levina, C. sativus cv. Tessa and Prunus tomentosa appeared as the most suitable test plants for bioassay of studied PDV isolates. The nucleotide sequences corresponding to the full-length coat protein (CP) of 657 bp were obtained. The identity among studied isolates in CP was 95.5–100% and 97.4–100% at nucleotide and amino acid levels, respectively. The movement protein (MP) sequences were 753 nucleotides long, translating 251 amino acids and shared 94.1–98.4% nucleotide and 96.8–99.2% amino acid identity. Constructed trees with the nucleotide sequences of both MP and CP genes of studied isolates and referent PDV cherry variants showed the formation of two phylogroups in the two genomic regions. All studied isolates separated into two clusters in group I. No correlation between the phylogenetic grouping and the biological properties, as well the geographical origin, was observed. [ABSTRACT FROM AUTHOR]

Sweet cherry is an important fruit crop in Bulgaria. One of the most common viruses of sweet cherry and other stone fruits in the world is Prune dwarf virus (PDV). Field surveys were conducted in 43 commercial and 3 collection sweet and sour cherry orchards at 10 locations in six regions of Bulgaria. Leaf samples from 2090 trees showing and not showing virus-like symptoms were tested by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for PDV infection. The average PDV infection level in sweet and sour cherry was 14.4%. In 92.4% of the trees, PDV caused single infection, whereas in 7.6%, it caused mixed infection with Prunus necrotic ringspot virus (PNRSV). Visual observations were made to connect the symptoms with PDV infection. There were PDV symptoms in 56.2% of infected trees whereas, 43.8% of the trees were asymptomatic. The coat protein (CP) sequences of 14 isolates from four regions were determined. The obtained sequences were 654 nucleotides (nts) in length, translated to 218 amino acids. Sequence comparisons showed 87%–100% to 93%–100% identity at nucleotide and amino acid level, respectively. Phylogenetic analyses using nucleotide and amino acid sequences of the CP genes of PDV isolates from Bulgaria and 38 cherry isolates retrieved from the GenBank database indicated the existence of three and two phylogenetic groups, respectively. No association between the phylogenetic grouping of the studied isolates and the symptoms of infected trees and/or the sampling-region was observed.

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Economically important viruses infect plums and other stone fruits cause lower yields, fruit size and quality and also affect its chemical composition. Fruits of the genetically modified (GM) plum 'HoneySweet' growing on trees deliberately infected with Plum pox virus (PPV), Prune dwarf virus (PDV) and Apple chlorotic leaf-spot virus (ACLSV) were analysed in detail to determine what effects these virus infections have on selected pomological traits, dissolved solids and titratable acids. Assessments of the fruits were made in the years from 2011 to 2014. The GM plum 'HoneySweet' was chosen for this experiment, and uninfected trees of the same variety in the same orchard were used as control. It was shown that there was no effect on dissolved solids resulting from PPV infections, either alone or in combination with the other viruses, but that there was a significant effect on levels of titratable acids where trees had been inoculated with all three viruses (treatment I). Regarding pomological traits, in most cases there were no significant effects seen to affect internal characteristics, only that in the assessments were lower although still of an acceptable quality for commercial fruit.

Prune dwarf virus (PDV) is a plant RNA viral pathogen in many orchard trees worldwide. Our knowledge about resistance genes or resistant reactions of plant hosts to PDV is scant. To fill in part of this gap, an aim of this study was to investigate reactions to PDV infection in a model host, Chenopodium quinoa. Our investigations concentrated on morphological and ultrastructural changes after inoculation with PDV strain 0599. It turned out that PDV infection can cause deformations in host cells but also induce changes in the organelles, such as chloroplasts in inoculated leaves. Moreover, we also demonstrated specific reactions/changes, which could be associated with both types of vascular tissue capable of effectively blocking the systemic spread of PDV to upper leaves. Furthermore, the relative amount of virus, P1 protein deposition, and movement protein (MP) gene expression consequently decreased in PDV-inoculated leaves. [ABSTRACT FROM AUTHOR]

Sweet cherry is an important fruit crop in Bulgaria. One of the most common viruses of sweet cherry and other stone fruits in the world is Prune dwarf virus (PDV). Field surveys were conducted in 43 commercial and 3 collection sweet and sour cherry orchards at 10 locations in six regions of Bulgaria. Leaf samples from 2090 trees showing and not showing virus-like symptoms were tested by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for PDV infection. The average PDV infection level in sweet and sour cherry was 14.4%. In 92.4% of the trees, PDV caused single infection, whereas in 7.6%, it caused mixed infection with Prunus necrotic ringspot virus (PNRSV). Visual observations were made to connect the symptoms with PDV infection. There were PDV symptoms in 56.2% of infected trees whereas, 43.8% of the trees were asymptomatic. The coat protein (CP) sequences of 14 isolates from four regions were determined. The obtained sequences were 654 nucleotides (nts) in length, translated to 218 amino acids. Sequence comparisons showed 87%–100% to 93%–100% identity at nucleotide and amino acid level, respectively. Phylogenetic analyses using nucleotide and amino acid sequences of the CP genes of PDV isolates from Bulgaria and 38 cherry isolates retrieved from the GenBank database indicated the existence of three and two phylogenetic groups, respectively. No association between the phylogenetic grouping of the studied isolates and the symptoms of infected trees and/or the sampling-region was observed. [ABSTRACT FROM AUTHOR]

Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea.

Prune dwarf virus (PDV) is one of the members of Bromoviridae family, genus Ilarvirus. Host components that participate in the regulation of viral replication or cell-to-cell movement via plasmodesmata are still unknown. In contrast, viral infections caused by some other Bromoviridae members are well characterized. Bromoviridae can be distinguished based on localization of their replication process in infected cells, cell-to-cell movement mechanisms, and plant-specific response reactions. Depending upon the genus, "genome activation" and viral replication are linked to various membranous structures ranging from endoplasmic reticulum, to tonoplast. In the case of PDV, there is still no evidence of natural resistance sources in the host plants susceptible to virus infection. Apparently, PDV has a great ability to overcome the natural defense responses in a wide spectrum of plant hosts. The first manifestations of PDV infection are specific cell membrane alterations, and the formation of replicase complexes that support PDV RNA replication inside the spherules. During each stage of its life cycle, the virus uses cell components to replicate and to spread in whole plants, within the largely suppressed cellular immunity environment. This work presents the above stages of the PDV life cycle in the context of current knowledge about other Bromoviridae members. [ABSTRACT FROM AUTHOR]

Prune dwarf virus (PDV) is one of the most dangerous pathogens of fruit trees worldwide. One of the most important proteins required for PDV infection is replicase. (P1 protein) which anchored viral RNA and builds replication complex along with RNA depended polymerase. Despite the importance of PDV as a pathogen, our knowledge regarding tissue/cellular localization and structure of PDV P1 protein is still incomplete. The aim of this work was to localize replicase distribution in leaf tissues and cells by immunofluorescent and immunogold labeling of Nicotiana tobaccum cv Samsun and development of a 3D model of PDV replicase. In this paper we demonstrate that PDV replication, is similar to that of Alfalfa mosaic virus and is strongly connected with tonoplasts. In addition, PDV replicase and coat protein (CP) were also found to be strongly associated with membranes of endoplasmic reticulum and, indicating the potential involvement of these membrane structures in the processes related to viral infection. Bioinformatic analyzes based on 3D modeling and structure prediction revealed that P1 protein has a potential transmembrane domain which enables protein anchoring to tonoplast during replication complex assembly. [ABSTRACT FROM AUTHOR]

Economically important viruses infect plums and other stone fruits cause lower yields, fruit size and quality and also affect its chemical composition. Fruits of the genetically modified (GM) plum 'HoneySweet' growing on trees deliberately infected with Plum pox virus (PPV), Prune dwarf virus (PDV) and Apple chlorotic leaf-spot virus (ACLSV) were analysed in detail to determine what effects these virus infections have on selected pomological traits, dissolved solids and titratable acids. Assessments of the fruits were made in the years from 2011 to 2014. The GM plum 'HoneySweet' was chosen for this experiment, and uninfected trees of the same variety in the same orchard were used as control. It was shown that there was no effect on dissolved solids resulting from PPV infections, either alone or in combination with the other viruses, but that there was a significant effect on levels of titratable acids where trees had been inoculated with all three viruses (treatment I). Regarding pomological traits, in most cases there were no significant effects seen to affect internal characteristics, only that in the assessments were lower although still of an acceptable quality for commercial fruit. [ABSTRACT FROM AUTHOR]

Prunus necrotic ring spot virus (PNRSV) y Prune dwarf virus (PDV) constituyen los virus más ampliamente distribuidos en todas las regiones del mundo productoras de frutales de carozo. Apple chlorotic leaf spot virus (ACLSV) también ha sido detectado en estos frutales en otras regiones y Plum pox virus (PPV), agente causal de Sharka, constituye una amenaza permanente para los cultivos de la zona. Considerando que estos virus son responsables de pérdidas significativas en la producción de los Prunus infectados, se programó analizar su presencia en plantaciones comerciales de Río Negro, Argentina, con el propósito de conocer su estado sanitario. Se empleó la técnica inmunoenzimática DAS-ELISA y se analizaron hojas y pétalos en primavera y yemas en reposo durante el invierno, de las siguientes especies: duraznero, cerezo, ciruelo y guindo. PNRSV resultó el virus de mayor difusión en las plantaciones comerciales analizadas. Sobre un total de 390 plantas, el 39.26% resultó infectado con el patógeno. PDV estuvo presente solamente en el 5.88% de las muestras estudiadas. A diferencia de lo que ocurre en otras zonas frutícolas, según los resultados obtenidos en el presente estudio, en ningún caso se encontraron PNRSV y PDV en la misma planta. ACLSV fue detectado solamente en dos muestras de ciruelo (0.47% del total de muestras analizadas). No se evidenció la presencia de PPV, agente causal de Sharka. Al analizar los resultados en las diferentes especies frutícolas, se observó que cerezos y durazneros presentaron mayor porcentaje de plantas enfermas, 58.59 y 41 %, respectivamente. En ciruelo el 26% de las plantas presentó alguno de los virus analizados y en guindo el 7.84%

The effect of infection with Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV) on vegetative and reproductive growth of sweet cherry trees (Prunus avium L.) was investigated. Infected trees were smaller and the growth reduction was more severe in Lapins than in Sam; when trees were infected in the nursery (early) compared to an infection two years later in the orchard; and when trees were grafted on the dwarfing Inmil rootstock compared to the more vigorous Damil. Tree mortality after establishment in the orchard was not a problem and only one virus infected tree died during the 5-year observation period. Reduced vigour was accompanied by increased blind wood formation. While the infected trees had a higher generative spur density the total number of generative spurs per tree was less than in the virus free controls, thus reducing yield. In addition the germination of pollen from infected trees was reduced. In general PNRSV has little or no significant influence on vegetative or reproductive behaviour, while PDV significantly reduced both characteristics. The combined infection had dramatic effects, especially on the dwarfing Inmil rootstock with the susceptible cv. Lapins. After four years in the orchard 10% of the initially virus free trees were infected by PDV and 48% by PNRSV.

During 2006-2008 field surveys were carried out in the important cherry growing areas of the Czech Republic to assess the incidence of Prune dwarf virus and Prunus necrotic ring spot virus in commercial orchards and nurseries. A total of 1,438 samples from 1,198 sweet cherry trees and from 240 sour cherry trees were tested by ELISA for the presence of Prune dwarf virus and Prunus necrotic ring spot virus. The overall average infection level was 17.7%. The most infected species were sour cherry trees (22.5%). The most frequently detected virus was Prune dwarf virus (10.9%). Prunus necrotic ring spot virus occurred in 6.3% of samples. Our study provided an indication of a sanitary status of sweet and sour cherry in commercial orchards and nurseries in the Czech Republic.

Leaves were collected from a total of 62 trees displaying virus-like disease symptoms such as rugose mosaic in leaf, irregular shaped leaf blades or small leaf blades in a survey of sweet cherry (Prunus avium L. cv Red Lamp) in Shandong Province, the largest cherry production area in China. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus- 2 (LChV-2), Cherry virus A (CVA) and Cherry green ring mottle virus (CGRMV) were present and that most of the samples (70%) were infected by at least two viruses. A case study in this survey of two selected trees revealed that a mixed infection with these five viruses simultaneously caused two types of field symptoms: late-maturity in fruits or fruit blast. Real-time quantitative RT-PCR analysis revealed that the relative proportion of the virus titers was distinct in the two sampled trees. The PNRSV titer was significantly higher than the four other viruses in the trees that exhibited fruit blast. Phylogenetic analysis of the PNRSV coat protein (CP) gene showed that the isolates HSY-4-1 and X-3, obtained from the two sampled trees separately, were classified into different subgroups of Group I isolates, which is characterized by a six-nucleotide insertion. A non-synonymous substitution was identified in the six-nucleotide insertion in the PNRSV CP gene in one of the two isolates. This is the first detailed study of the incidence of multiple sweet cherry viruses in China. [ABSTRACT FROM AUTHOR]

Prune dwarf virus (PDV) isolates have been investigated for genetic diversity. Full-length nucleotide and amino acid sequences of viral coat protein from 23 isolates collected from different stone fruit trees (sour and sweet cherry trees, wild cherry tree, plum tree, almond tree, peach tree) and different countries (Poland, Italy, Germany, USA, Israel) were analysed and compared to 57 others available in GenBank. Comparison of all sequenced virus isolates revealed diversity of 86-100 % at nucleotide level and 79-100 % at amino acid level. The ratio of non-synonymous to synonymous polymorphic sites indicated that purifying selection dominated in case of PDV. However, six codons showed to be under strong positive selection, including the codon located inside the structure involved in RNA-binding activity. [ABSTRACT FROM AUTHOR]

Prune dwarf virus (PDV) is one of the major positive RNA viruses which cause economical damages in stone fruit trees. The symptoms of PDV vary between different stone fruits namely sour and sweet cherry, almond, peach, apricot and plum including leaf narrowing, leaf chlorosis, vein clearing, mosaic, leaf whitening, leathery leaf, bushy branches and stunt trees. During the years 2011 and 2012, 251 leaf samples were collected for detection of PDV in stone fruit orchards of Charmahal-va- Bakhtiari province. DAS-ELISA test proved PDV presence serologically. Then, total RNA were extracted and tested by two-step RT-PCR which replicated partial and full coat protein sequence of PDV. One hundred and eighty one out of total samples (251 samples) showed PDV infection using serological and two-step RT-PCR assays, hence, incidence of PDV in Charmahal-va-Bakhtiari province was confirmed. This is the first report of PDV in stone fruit orchards of Charmahal-va-Bakhtiari province and in Iran. [ABSTRACT FROM AUTHOR]

Prune dwarf virus (PDV), a worldwide pathogen of stone fruit trees, has many isolates with different biological, serological and molecular properties. Monoclonal antibodies (MAbs) to the Prunus mahaleb isolate of PDV were used to investigate the serological variability of virus isolates, by TAS-ELISA (triple antibody sandwich enzyme-linked immunosorbent assay). The twenty-two PDV isolates from Germany (1), Italy (7), Poland (13) and the USA (1) were characterised against eight single MAbs. The virus showed high serological variability. Analysis of the MAbs reaction allowed for the identification of 13 serogroups. [ABSTRACT FROM AUTHOR]

A mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field-infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10−4 but not 10−3, after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at −15°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasi-isometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single-stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK. [ABSTRACT FROM AUTHOR]

Abstract: Fragaria chiloensis latent virus (FClLV), a member of the genus Ilarvirus was first identified in the early 1990s. Double-stranded RNA was extracted from FClLV infected plants and cloned. The complete nucleotide sequence of the virus has been elucidated. RNA 1 encodes a protein with methyltransferase and helicase enzymatic motifs while RNA 2 encodes the viral RNA dependent RNA polymerase and an ORF, that shares no homology with other Ilarvirus genes. RNA 3 codes for movement and coat proteins and an additional ORF, making FClLV possibly the first Ilarvirus encoding a third protein in RNA 3. Phylogenetic analysis reveals that FClLV is most closely related to Prune dwarf virus, the type member of subgroup 4 of the Ilarvirus genus. FClLV is also closely related to Alfalfa mosaic virus (AlMV), a virus that shares many properties with ilarviruses. We propose the reclassification of AlMV as a member of the Ilarvirus genus instead of being a member of a distinct genus. [Copyright &y& Elsevier]

Certification represents the first line of defense against fruit tree viruses. For certification or surveys dealing with large number of samples, ELISA is still considered the technique of choice and requires a continuous supply of good quality antibodies. Prune dwarf virus (PDV) is among the major viruses affecting stone fruits; it belongs to the genus Ilarvirus named so for its isometric labile particles. Recombinant DNA technology was investigated for production of PDV antiserum to avoid labile virus purification and virus maintenance problems. The PDV coat protein gene (CP) was cloned into a protein expression bacterial plasmid vector which allowed a good level of expression of up to 2 mg native protein/L culture. The recombinant PDV CP was injected into rabbits and the crude antiserum was successfully used in indirect ELISA at dilutions of up to 1:5000 to detect PDV in infected leaf samples. Similar results were obtained in dot blot immunoassays (DBIA). The antibodies were used in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and results were comparable to a reference commercial kit. The crude antiserum was efficiently used for coating ELISA plates, thereby reducing test costs. [Copyright &y& Elsevier]

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season. [Copyright &y& Elsevier]

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses. [ABSTRACT FROM AUTHOR]