American Plum Line Pattern Virus Research Articles

Virome analysis deciphered the infection of American plum line pattern virus, little cherry virus 1 and plum bark necrosis stem pitting-associated virus in plum from India

Virome analysis deciphered the infection of American plum line pattern virus, little cherry virus 1 and plum bark necrosis stem pitting-associated virus in plum from India

American plum line pattern virus (American line pattern of plum)

American plum line pattern virus (American line pattern of plum)

Plum Pox Virus Strain C Isolates Can Reduce Sour Cherry Productivity.

The impact of plum pox virus (PPV) on sour cherry (Prunus cerasus L.) productivity has been studied by comparing the yield of PPV-infected and PPV-free fruit-bearing trees. A total of 152 16- to 17-year-old trees of nine cultivars and hybrids were surveyed in the production orchards (cultivar collection and hybrid testing plots) in the Republic of Tatarstan, Russia. Sixty trees tested positive for PPV using ELISA and RT-PCR. Among them, 58 PPV isolates belonged to the strain C and the other 2 isolates to the strain CV. For the cultivars Sevastyanovskaya, Shakirovskaya, hybrids 88-2 and 80-8, the average (2012 to 2019) productivity of infected trees was 38% to 45% lower than for PPV-free trees of the same cultivar or hybrid. No ilarviruses (prunus necrotic ringspot virus, prune dwarf virus, apple mosaic virus, American plum line pattern virus) were detected in PPV-infected trees, suggesting that reduced cherry productivity was attributed to the PPV infection. Thus, it was shown for the first time that PPV can reduce the productivity of at least some sour cherry cultivars and hybrids, and strain C isolates are responsible for crop losses.

Pest categorisation of non-EU viruses and viroids of Prunus L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Prunus L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of Ilarvirus S1 and Ilarvirus S2, for which very limited information exists, the pest categorisation was completed for 26 viruses and 1 viroid having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative plant propagation techniques, with plants for planting representing the major pathway for long‐distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Prunus seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Apple scar skin viroid, American plum line pattern virus, cherry mottle leaf virus, cherry rasp leaf virus, cherry rosette virus, cherry rusty mottle‐associated virus, cherry twisted leaf‐associated virus, peach enation virus, peach mosaic virus, peach rosette mosaic virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, apricot vein clearing virus, Asian prunus virus 1, Asian prunus virus 2, Asian prunus virus 3, Caucasus prunus virus, cherry virus B, Mume virus A, nectarine stem pitting‐associated virus, nectarine virus M, peach chlorotic mottle virus, peach leaf pitting‐associated virus, peach virus D, prunus virus F and prunus virus T satisfy all the other criteria to be considered as potential Union QPs. Prunus geminivirus A does not meet the criterion of having negative impact in the EU. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non‐EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non‐quarantine pests.

Discovery of Plant Viruses From Tea Plant (Camellia sinensis (L.) O. Kuntze) by Metagenomic Sequencing.

The tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important woody species. In this study, we collected 26 tea plant samples with typical discoloration symptoms from different tea gardens and performed metagenomic analysis based on next-generation sequencing. Homology annotation and PCR sequencing validation finally identified seven kinds of plant viruses from tea plant. Based on abundance distribution analysis, the two most abundant plant viruses were highlighted. Genetic characterization suggested that they are two novel virus species with relatively high homology to Blueberry necrotic ring blotch virus and American plum line pattern virus. We named the newly discovered viruses tea plant necrotic ring blotch virus (TPNRBV) and tea plant line pattern virus (TPLPV). Evolutionary relationship analysis indicated that TPNRBV and TPLPV should be grouped into the Blunervirus and the Ilarvirus genera, respectively. TPLPV might have same genome activation process with known ilarviruses based on sequence analysis. Moreover, specific primers for both viruses detection were designed and validated. The symptoms and ultrastructure of TPNRBV infected leaves were first recorded. Virus detections in the symptomatic and asymptomatic tissues from field plants showing tea plant necrotic ring blotch disease suggest that TPNRBV has a systemic movement feature. In summary, we first identified seven kinds of putative plant viruses by metagenomic analysis and report two novel viruses being latent pathogens to tea plant. The results will advance our understanding of tea plant virology and have significance for the genetic breeding of tea plants in the future.

First Report of American plum line pattern virus Infecting Flowering Cherry (Prunus serrulata) in Japan

First report of [i]American plum line pattern virus[/i] infecting flowering cherry ([i]Prunus serrulata Lindl.[/i]) in Japan

Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus.

The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus, occurring in 48 of the 61 Ilarvirus-positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus-like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus-like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the need for a standardized approach to accurately determine what constitutes an active, viable virus infection after detection by molecular based methods.

American plum line pattern virus. [Distribution map

Abstract A new distribution map is provided for American plum line pattern virus. Bromoviridae: Ilarvirus. Hosts: Prunus spp. Information is given on the geographical distribution in Europe (Albania, Italy, Mainland Italy and Sicily), Asia (Israel, Korea Republic and Lebanon), Africa (Tunisia), North America (Canada, Nova Scotia, Ontario, USA, California, Kentucky, Michigan, Ohio, Oregon and Washington), South America (Argentina) and Oceania (New Zealand).

SURVEY OF STONE FRUIT VIRUSES AND VIROIDS IN CHILE

Stone fruits orchards from three Chilean regions were visited to collect leaf samples which were tested for the presence of the most important viruses and viroids. Molecular nonradioactive hybridization (MH) testing of 2,456 samples and confirmatory RT-PCR of some MH-negative samples gave the following infection rates: 31.5% for Prunus necrotic ringspot virus (PNRSV); 31.3% for Prune dwarf virus (PDV); 25.7% for Peach latent mosaic viroid (PLMVd); 3.1% for Tomato ringspot virus (ToRSV) only in rootstocks; 1.8% for Plum pox virus (PPV); 1.2% for Hop stunt viroid (HSVd); 1.1% for Apple mosaic virus (ApMV); and 0.9% for Apple chlorotic leaf spot virus (ACLSV). American plum line pattern virus (APLPV) was not detected. The overall infection rate in the surveyed orchards was 29.6%, specifically 28.2% (Region Metropolitana), 23.4% (Valparaiso) and 33% (Bernardo O’Higgins) in the three regions surveyed. ApMV and HSVd, detected only in peach and nectarine, are new records in stone fruits in Chile.

IMPROVED DETECTION OF ILARVIRUSES AND NEPOVIRUSES AFFECTING FRUIT TREES USING QUANTITATIVE RT-qPCR

Reverse transcription quantitative PCR (RT-qPCR) assays were developed for the detection of the ilarviruses Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), and American plum line pattern virus (APLPV), and the nepoviruses Tomato ringspot virus (ToRSV) and Cherry leafroll virus (CLRV). These viruses affect various stone fruits such as apricots, cherries, peaches, plums, and almonds. The goal of this work was to improve the RT-qPCR detection of PNRSV, PDV, and ApMV in addition to developing three new RT-qPCR assays for the detection of APLPV, ToRSV and CLRV. Primers for conventional RT-PCR as well as primers and probes for RT-qPCR assays were designed after aligning coat protein (CP) gene sequences of geographically diverse isolates with the corresponding CP gene sequences from GenBank, targeting regions with 100% sequence identity. The efficiency of each RT-qPCR assay, as well as the intra- and inter-assay variability, were determined. These conventional RT-PCR and RT-qPCR assays were validated using purified total RNAs from 221 trees from the USDA Clonal Germplasm Repository orchards. The data showed that more isolates were detected by RT-qPCR than by RT-PCR.

First Report of Infection of Cherry Rusty Mottle Associated Virus in Portuguese Laurel (Prunus lusitanica) in Washington State.

During late spring of 2012 in Snohomish County of Washington State, chlorotic yellow leaf blotch symptoms suggestive of a virus infection were observed on Portuguese laurel (Prunus lusitanica) planted in a hedge row. Leaf samples from representative trees were initially tested for the presence of Cherry leaf roll virus (CLRV) and Plum pox virus (PPV) by ELISA with antibodies specific to CLRV and general potyvirus, respectively (Agdia, Inc., Elkhart, IN). The ELISA test yielded negative results for both viruses. Reverse transcription (RT)-PCR was pursued to detect other viruses known to infect Prunus spp., namely American plum line pattern virus (APLPV), Apple chlorotic leafspot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry raspleaf virus (CRLV), Cherry virus A (CVA), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV), as well as CLRV and PPV. None of these viruses were detected. However, RT-PCR with a generic primer pair Fovea2/AdPr (3) that amplifies the coat protein (CP) coding sequence and 3'-untranslated regions (3'-UTR) of several members of the family Betaflexiviridae yielded a 1.4-kb amplicon that was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. KF356396). The sequences from three clones were 99.8% identical to each other at the nucleotide level. Comparison of the consensus CP coding region with the nucleotide sequence database revealed 86 to 93% identity to Cherry rusty mottle associated virus (CRMaV), and only 73 to 75% identities to Cherry necrotic rusty mottle virus (CNRMV) (1) and 71 to 76% identities to Cherry green ring mottle virus (CGRMV) (4) isolates from sweet cherry (P. avium). This result suggested that the cloned fragment represents a strain of CRMaV. Prunus avium 'Bing' and 'Sam,' and P. serrulata 'Kwanzan' were grafted with bark patches from the symptomatic tree and observed for induction of cherry rusty mottle disease (CRMD) symptoms. Ninety days after grafting, symptoms typical of CRMD consisting of chlorotic yellow mottle appeared on 'Bing,' 'Sam,' and 'Kwanzan' indicators. Small necrotic spots also appeared on the leaves of the latter. Angular necrotic lesions on 'Sam' and epinasty of 'Kwanzan' that are diagnostic symptoms of cherry necrotic rusty mottle disease (CNRMD) and cherry green ring mottle disease (CGRMD), respectively, were absent from graft inoculated indicators. Further RT-PCR tests on the indicators using primers specific to CNRMV, CGRMV, and CRMaV (2) yielded negative results for CNRMV and CGRMV but showed positive amplification for CRMaV. The results of the woody indexing corroborate the presence of CRMaV but the absence of CNRMV and CGRMV in the symptomatic Portuguese laurel. To our knowledge, this is the first report of CRMaV in Portuguese laurel in the United States and the first description of symptoms associated with CRMaV in this host. As a potential reservoir of CRMaV, Portuguese laurel could play an important component in management of CRMD in cherry production areas where this ornamental cherry is also present.

Relationship of viruses and viroids with apricot “viruela” disease

In this study, 34 apricot orchards in south-eastern Spain planted with the Spanish cultivar ‘Bulida’ and showing “viruela” symptoms were studied for 2 years. Leaf and fruit samples from four trees in each orchard, either with or without “viruela” symptoms, were collected and analysed by multiplex RT-PCR for the detection of American plum line pattern virus (APLPV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Apricot latent virus (ApLV), Plum bark necrosis and stem pitting-associated virus (PBNSPaV), Plum pox virus (PPV), Prune dwarf virus (PDV), and Prunus necrotic ring spot virus (PNRSV). In addition, molecular hybridization assays were performed for the detection of Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). All fruits showing “viruela” symptoms were infected with ACLSV and HSVd, suggesting that these pathogens may be responsible for “viruela” disease. Other viruses including PNRSV, PBNSPaV, ApLV, PDV, ApMV and PPV, were detected to a lesser degree. Detection of ACLSV and HSVd in samples without symptoms could be explained by the influence of environmental conditions and/or the physiological stage of fruits on the expression of symptoms.

Ilarviruses ofPrunusspp.: A Continued Concern for Fruit Trees

Prunus spp. are affected by a large number of viruses, causing significant economic losses through either direct or indirect damage, which results in reduced yield and fruit quality. Among these viruses, members of the genus Ilarvirus (isometric labile ringspot viruses) occupy a significant position due to their distribution worldwide. Although symptoms caused by these types of viruses were reported early in the last century, their molecular characterization was not achieved until the 1990s, much later than for other agronomically relevant viruses. This was mainly due to the characteristic liability of virus particles in tissue extracts. In addition, ilarviruses, together with Alfalfa mosaic virus, are unique among plant viruses in that they require a few molecules of the coat protein in the inoculum in order to be infectious, a phenomenon known as genome activation. Another factor that has made the study of this group of viruses difficult is that infectious clones have been obtained only for the type member of the genus, Tobacco streak virus. Four ilarviruses, Prunus necrotic ringspot virus, Prune dwarf virus, Apple mosaic virus, and American plum line pattern virus, are pathogens of the main cultivated fruit trees. As stated in the 9th Report of the International Committee on Taxonomy of Viruses, virions of this genus are "unpromising subjects for the raising of good antisera." With the advent of molecular approaches for their detection and characterization, it has been possible to get a more precise view of their prevalence and genome organization. This review updates our knowledge on the incidence, genome organization and expression, genetic diversity, modes of transmission, and diagnosis, as well as control of this peculiar group of viruses affecting fruit trees.

Evaluation of the phytosanitary status of the Prunus species in the National Clonal Germplasm Repository in California: survey of viruses and viroids.

The USDA National Clonal Germplasm Repository (NGCR) at the University of California, Davis is recognized as one of the richest sources of Prunus species material in the US. The repository maintains more than 2450 trees representing 96 taxa collected from around the world. However, the phytosanitary status of the NCGR Prunus collection has not been thoroughly evaluated. In this study, a comprehensive screening of a selected subset of the collection for virus and virus-like diseases affecting Prunus trees has been completed. Two factors were considered for selecting the trees: the country of origin and the observation of suspicious disease symptoms. A total of 221 trees representing 185 different cultivars of cherry, almond, peach, apricot and plum were sampled. Conventional RT-PCR detection was used to test for 13 different viruses and two viroids. The pathogens included in the survey were Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Plum bark necrosis stem pitting-associated virus (PBNSPaV), American plum line pattern virus (APLPV), Cherry virus A (CVA), Cherry leafroll virus (CLRV), Cherry rasp leaf virus (CRLV), Cherry green ring mottle virus (CGRMV), Cherry necrotic rusty mottle virus (CNRMV), Apple chlorotic leafspot virus (ACLSV) Tomato ringspot virus (ToRSV), Little cherry virus 1 (LChV-1), Little cherry virus 2 (LChV-2), Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). Though the majority of these trees were asymptomatic, all tested pathogens were detected except for ToRSV and CRLV. Two of the viruses detected (ACLSV and LChV-2) had never been reported from California. Incidence of PNRSV in tested trees was the highest (55 trees) followed by the two viroids (PLMVd and HSVd), with 33 and 32 infected trees, respectively. The incidence for the rest of the pathogens ranged between 0 to 19 trees. The infection rate of all tested samples ranged from 0.5% to 24.9%.

First Report of Cherry virus A in Sweet Cherry Trees in China.

Plants in the genus Prunus of the family Rosaceae are important fruit and ornamental trees in China. In June of 2007, sweet cherry (Prunus avium) trees with mottling and mosaic symptoms were observed in a private garden near Kunming, Yunnan Province. Twenty-four samples, six each from sweet cherry, sour cherry (P. cerasus), flowering cherry (P. serrulata), and peach (P. persica) were collected from trees in private and community gardens in the area. The peach and sour and flowering cherry trees did not show any symptoms. Total nucleic acids were extracted using a cetyltrimethylammoniumbromide (CTAB) extraction method, and the extracts were tested for the following eight viruses by reverse transcription (RT)-PCR: American plum line pattern virus, Apple chlorotic leaf spot virus, Cherry green ring mottle virus, Cherry necrotic rusty mottle virus, Cherry virus A (CVA), Little cherry virus 1, Prune dwarf virus, and Prunus necrotic ringspot virus. Only CVA was detected in two symptomatic sweet cherry trees by RT-PCR with forward (5'-GTGGCATTCAACTAGCACCTAT-3') and reverse (5'-TCAGCTGCCTCAGCTTGGC-3') primers specific to an 873-bp fragment of the CVA replicase gene (2). The CVA infection of the two trees was confirmed by RT-PCR using primers CVA-7097U and CVA-7383L that amplified a 287-bp fragment from the 3'-untranslated region (UTR) of the virus (1). Amplicons from both amplifications were cloned and sequenced. Analysis of the predicted amino acid sequences of the 873-bp fragments (GenBank Accession Nos. EU862278 and EU862279) showed that they were 98% identical with each other and 97 to 98% with the type isolate of CVA from Germany (GenBank Accession No. NC_003689). The 286-bp sequences of the 3'-UTR (GenBank Accession Nos. FJ608982 and FJ608983) were 93% identical with each other and 93 to 98% with the type isolate. The sequence indicated that the three isolates were very similar and should be considered to be the same strain. CVA is a member of the genus Capillovirus in the family Flexiviridae and has been previously reported in Europe, North America, and Japan. The contribution of CVA to the symptoms observed and its distribution in China remain to be evaluated. To our knowledge, this is the first report of CVA in sweet cherry in China.

The promiscuous evolutionary history of the family Bromoviridae

Recombination and segment reassortment are important contributors to the standing genetic variation of RNA viruses and are often involved in the genesis of new, emerging viruses. This study explored the role played by these two processes in the evolutionary radiation of the plant virus family Bromoviridae. The evolutionary history of this family has been explored previously using standard molecular phylogenetic methods, but incongruences have been found among the trees inferred from different gene sequences. This would not be surprising if RNA exchange was a common event, as it is well known that recombination and reassortment of genomes are poorly described by standard phylogenetic methods. In an attempt to reconcile these discrepancies, this study first explored the extent of segment reassortment and found that it was common at the origin of the bromoviruses and cucumoviruses and at least at the origin of alfalfa mosaic virus, American plum line pattern virus and citrus leaf rugose virus. Secondly, recombination analyses were performed on each of the three genomic RNAs and it was found that recombination was very common in members of the genera Bromovirus, Cucumovirus and Ilarvirus. Several cases of recombination involving species from different genera were also identified. Finally, a phylogenetic network was constructed reflecting these genetic exchanges. The network confirmed the taxonomic status of the different genera within the family, despite the phylogenetic noise introduced by genetic exchange.

Low genetic variability in the coat and movement proteins of American plum line pattern virus isolates from different geographic origins

Until very recently isolates of American plum line pattern virus (APLPV) had not been reported from outside North America. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of eight APLPV isolates from five Mediterranean countries were determined. Sequence analysis showed that both MP and CP genes are highly conserved irrespective of geographic origin. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that these proteins are under the effect of negative purifying selection. The MP and CP of APLPV possess most of the functional motifs described for other members of the genus Ilarvirus.

Engineering Resistance to Multiple Prunus Fruit Viruses Through Expression of Chimeric Hairpins

Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

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.

Simultaneous detection of six stone fruit viruses by non-isotopic molecular hybridization using a unique riboprobe or ‘polyprobe’

A new strategy for the simultaneous detection of plant viruses by molecular hybridization has been developed. Two, four or six viral sequences were fused in tandem and transcribed to render unique riboprobes and designated as ‘polyprobes’. The ‘polyprobe four’ (poly 4) covered the four ilarviruses affecting stone fruit trees including apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and American plum line pattern virus (APLPV) whereas the ‘polyprobe two’ (poly 2) was designed to detect simultaneously, plum pox virus (PPV) and apple chlorotic leaf spot virus (ACLSV), the two more important viruses affecting these trees. Finally, a ‘polyprobe six’ (poly 6) was generated to detect any of the six viruses. The three polyprobes were comparable to the individual riboprobes in terms of end-point dilution limit and specificity. The validation of the new simultaneous detection strategy was confirmed by the analysis of 46 field samples from up to seven different hosts collected from 10 different geographical areas.