Necrosis Stem Pitting-associated Virus Research Articles

Analysis of Subcellular Localization and Pathogenicity of Plum Bark Necrosis Stem-Pitting Associated Virus Protein P6

Infection of <i>plum bark necrosis stem pitting associated virus</i> (PBNSPaV) has been reported in many <i>Prunus</i> species in several countries, causing significant economic losses. The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance. However, numerous studies have indicated that they might play important roles in the pathogenesis of virus infection. The role of small hydrophobic protein P6, encoded by the open reading frame 2 of PBNSPaV, has not been well explored. In this study, we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain. Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane. To further clarify the pathogenicity of P6 proteins, we constructed a PVX-P6 expression vector by inserting the p6 fragment into a <i>potato virus X</i> (PVX)-based vector and transformed it into <i>Agrobacterium tumefaciens GV3101</i>. Infiltration of <i>Nicotiana benthamiana</i> (<i>N. benthamiana)</i> with the PVX vector-transformed <i>A. tumefaciens</i> led to slight mosaic symptoms at 14 days of post-inoculation. Meanwhile, infiltration with the PVX-P6 vector-transformed <i>A. tumefaciens</i> resulted in no significant symptoms. These results demonstrated that heterologous expression of P6 in <i>N. benthamiana</i> could not enhance the pathogenicity of PVX. Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms, and the mode of action of PBNSPaV-P6 protein remains to be further studied.

Updating the Quarantine Status of Prunus Infecting Viruses in Australia.

One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus.

Complete Genomic Characterization of Plum bark necrosis stem pitting-associated virus Infecting Sweet Cherry in China.

Plum bark necrosis stem pitting–associated virus (PBNSPaV) causes the plum bark necrosis stem pitting–associated disease. We obtained the complete genome of a PBNSPaV isolate (PBNSPaV-TA) using small RNA deep sequencing followed by overlapping RT-PCR. To our knowledge, this is the first report of a completed genome of PBNSPaV identified from cherry trees.

Development of a multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic viroid

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.

Partial characterisation of a cherry isolate of a newly emerging stone fruit virus: Plum bark necrosis stem pitting associated virus

Plum bark necrosis stem pitting associated virus (PBNSPaV) has occurred globally over the last two decades and is becoming one of the major agricultural issues of stone fruits. The virus was detected for the first time in plums and has been reported in the other stone fruit species. The agent, whose dissemination mode is still unknown, has been first reported in cherries in Turkey grown in Malatya province. In this study, the K1 isolate of PBNSPaV, identified in a sweet cherry plant with severe stem pitting and gumming symptoms on its trunk, was partially characterised. A fragment of Hsp70h gene located on ORF3 of viral genome has been cloned, sequenced and analysed phylogenetically (Accession number. FJ231498). The PBNSPaV-K1 isolate showed 93–96% nucleotide sequence identity to sequences of Italian and American isolates in databases. An RNA probe has been raised for fast and reliable detection of the agent by molecular hybridisation. The studies on development of genome specific primers for the detection of the isolate by one-tube RT-PCR have failed.

Use of dried high-phenolic laden host leaves for virus and viroid preservation and detection by PCR methods

The efficiency of RNA extraction for Apricot latent virus (ApLV), Plum bark necrosis stem pitting associated virus (PBNSPaV), Prunus necrotic ring spot virus (PNRSV), Potato virus Y (PVY), and Apple scar skin viroid (ASSVd) from infected hosts is of great importance for molecular diagnosis by the polymerase chain reaction (PCR). A method is described for drying tissue to overcome phenolic inhibitors of viral RNA. This study showed that the infected host leaves, dried at 65 °C for 2 days and conserved at 4 °C in air proof conditions, serve as good sources for detection of viral and viroid pathogens by PCR methods. Preliminary results suggest that ApLV, PNRSV, PVY, and ASSVd were detected easily by reverse transcriptase-polymerase chain reaction (RT-PCR) and PBNSPaV by nested-RT-PCR with high amplification yields. No significant difference was observed between ethidium bromide-stained band profiles of dried compared to fresh leaves of infected samples. The RNA derived from dry leaf samples was suitable for detection studies. This simple and inexpensive method has proved very effective for long term conservation of virus and viroid isolates.

Simultaneous detection and identification of eight stone fruit viruses by one-step RT-PCR

A sensitive and reliable one step RT-PCR reaction with an internal control has been developed to detect and differentiate eight important viruses that affect stone fruit tress: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV) and Plum bark necrosis stem pitting associated virus (PBNSPaV). In addition, we investigated the detection limit and the efficiency of three different nucleic acid extraction methods that avoid the use of organic solvents, for both multiplex RT-PCR and dot-blot hybridisation assays. The primer cocktail was used to analyse 38 stone fruits originating from nine different countries and six species. A large number of virus combinations was detected and up to three different viruses were observed in five samples. A decrease in sensitivity was observed when the primer cocktail contained more than five different pair primers. However, comparative analyses showed that the multiplex RT-PCR containing the eight virus pair primers was even more sensitive than the ELISA or molecular hybridisation assays. The use of the multiplex RT-PCR technology in routine diagnosis of stone fruit tree viruses is discussed.