A Precise TaqMan-Based Real-Time qPCR Assay for Detecting and Quantifying Blackberry Chlorotic Ringspot Virus, Blueberry Shock Virus, and Plum Pox Virus in Fruit Tree Seedlings

A new procedure is described for the detection of Plum pox virus (PPV) in samples from large-scale field tests. The new direct real-time polymerase chain reaction (drtPCR) procedure was based on crude supernatants collected from peach (Prunus persica) leaves macerated in a buffer that was specially developed for this purpose and named “direct pathogen extract buffer.” Specific TaqMan primers and probes were designed for PPV detection: two sets specific to PPV D and M strains, respectively, and one for universal detection of PPV strains D, M, C, EA, and W. These primer and probe sets can be used singly or for duplex differentiation of D and M strains. Using Prunus spp. tissue infected with 30 known fruit tree viruses, the universal primer and probe set correctly identified PPV-infected and PPV-free samples, with no nonspecific cross-reactions. Based on endpoint analysis of the drtPCR reaction, a threshold cyle value of 36 was suggested as the maximum threshold to declare a sample positive for PPV. A comparative analysis comparing drtPCR and ELISA using 12 200 field samples revealed that drtPCR was approximately 100- to 1000-fold more sensitive than ELISA and was able to detect PPV at an earlier stage of infection than ELISA. The drtPCR is a valuable tool for PPV diagnosis and may also be applicable to other studies, including pathogen population dynamics and vector transmission efficiency.

Sharka, caused by Plum pox virus (PPV) is considered the most detrimental disease of plum. Although PPV is widespread in all plum growing areas from Romania and causes serious yield losses, limited information about the occurrence of PPV strains are available in some regions like Moldavia plum area. To supplement this information, fifty PPV isolates collected from five different commercial plum orchards from Moldavia were investigated. PPV detection was made by DAS-ELISA and by IC-RT-PCR. PPV strains were serologically determined by TAS-ELISA using PPV-D and PPV-M specific monoclonal antibodies. Molecular strain typing was done by RT-PCR targeting three genomic regions corresponding to (Cter)CP, (Cter)NIb/(Nter)CP and CI. RFLP analysis at the C-ter of CP cistron was used to distinguish the two major strains, D and M based on a RsaI polymorphism located in this genomic section. All PCR products targeting (Cter)CP and one PCR product spanning the (Cter)NIb/(Nter)CP were sequenced. All PPV isolates typed as PPV-M by serological analysis and by molecular differentiation in the genomic region corresponding to (C-ter)CP proved to be PPV recombinant (PPV-Rec) when the molecular analysis were performed in the region corresponding to NIb/CP. The sequencing results confirmed a high similarity with different sequences of PPV-Rec previously reported. Overall results provided that in Moldavia the predominant strain is PPV-D, followed with a much lower frequency by PPV-Rec which shares the CP gene with M strain. The mixed infections (PPV-D+PPV-Rec), were sporadically recorded in this plum growing area.

Serological and Molecular Typing of Plum pox Virus Isolates in the Transylvania, Romania

Apricot (Prunus armeniaca L.) production was 2.8 million tons in 2001 (FAO, 2001), making it the third most important stone fruit species worldwide. However, the spread of Plum Pox Virus (PPV), also known as sharka, is the most important limiting factor to the apricot industry in Europe. During the 1980s and 1990s, apricot production was seriously affected by PPV in Spain, France, and Italy (Roy and Smith, 1994). Apricot is very sensitive to all strains of PPV, resulting in serious damage to the fruits (deformation, necrotic spots on pits and flesh, and dry flesh). PPV is spread by aphids in a nonpersistent way: acquisition time by the aphids lasts seconds, retention lasts minutes and the virus is transmitted via the stylet (Nault, 1997). This fact makes chemical control of the aphid vector inefficient. Removal of infected trees was ineffective in eradicating the disease in Spain (Llácer and Cambra, 1998). The only reliable control of the PPV is to use resistant cultivars. However, there are no resistant cultivars adapted to the mild winters in southern Europe.

First reported in Bulgaria in 1933, Plum pox virus (PPV), the causal agent of sharka disease, has spread throughout Europe, despite its classification as a quarantine pest. In recent years, PPV has reached North Africa, America, and Japan. Sharka disease causes severe yield losses to the stone-fruit industry. As is typical for plant virus diseases, there is neither cure nor effective treatment of infected trees. Detection and quarantine measures are relied upon to delay or halt virus spread. PPV detection has led to the harmonization of preventive measures such as spring season surveys of nurseries and orchards, the use of certified virus-free plants, removal of infected trees, and creation of quarantine zones. In summer, while a majority of susceptible cultivars exhibit PPV symptoms, some tolerant cultivars are symptomless, and infected fruits become marketable. This situation led us to analyze fruits through RT-PCR to evaluate levels of PPV in these symptomless marketed fruits. Because evidence of PPV infection was previously found in some fruit samples, this study may have implications for the export and marketing of fruits from PPV-tolerant plum cultivars.

Plum pox virus (PPV) is the causal agent of sharka disease of stone fruit trees which is considered to be one of the most serious plant diseases. Sensitive techniques are needed for its diagnosis. Immunosorbent assays based on the use of monoclonal antibodies (1) and different PCR techniques have been described with or without previous immunocapture (2,3). In all instances plant extracts are needed, even though the sample grinding operation is time consuming and entails risks of contamination and of release of PCR inhibitors. Several reports have demonstrated the potential of using immobilized targets in PCR (4,5) or tissue of printing techniques for the detection of viral agents by immunological (6) or nucleic acid-based techniques (7). In this article we describe a simple direct tissue blotting PCR assay called print-capture PCR (PC-PCR) that allows the rapid and sensitive detection of PPV from infected plants without the need for grinding the samples. We demonstrate that a number of proteins may be used for the capture phase of PC-PCR, thus obviating the need for virus-specific immunoglobulins. For tissue printing, the fresh sections of leaves or stems from infected or healthy control plants (GF305 peach seedlings, apricots, Nicotiana benthamiana) were pressed onto Whatman 3MM paper. The prints can be processed directly or stored at room temperature for up to 1 month without any detrimental effects on amplification. Plant extracts used for immunocapture-PCR (IC-PCR) (3) or for spot-capture PCR (SC-PCR) were prepared by grinding (1/30, w/v) the samples used for tissue printing in PBS (3) supplemented with 2% polyvinylpyrrolidone and 0.2% sodium diethyl dithiocarbamate. For SC-PCR, 4 μl of the plant extracts were spotted on small squares of Whatman 3MM paper; the paper dried and processed as the tissue prints. Similar 0.5 ml microtubes (Sarstedt) were used for all capture techniques. For IC-PCR, the plant extracts were submitted to an immunocapture performed directly in the tubes used for the reverse transcriptase–PCR (RT–PCR) as described previously (3). The coating of the immunocapture tubes was performed using either a specific monoclonal antibody against PPV, immunoglobulins purified from an antiserum against PPV or skimmed milk as described below for SC-PCR and PC-PCR tubes. For PC-PCR and SC-PCR, the squares of paper harboring either the tissue prints or the spotted extracts were introduced in an Eppendorf tube and 120 μl 0.5% Triton X-100 was added, vortexed and incubated for 2 min at room temperature. Triton extracts (100 μl) were then recovered and transferred to tubes previously coated in carbonate buffer (3) with one of the following proteins: anti-PPV monoclonal antibodies 5B (1) (1 μg/ml), antiserum against PPV (2 μg/ml), anti-citrus tristeza virus (CTV) monoclonal antibodies [3DF1+ 3CA5 (8), 1 μg/ml], skimmed milk (Sveltesse, Nestle, 5%), bovine serum albumin (BSA fraction V, Boehringer, 5%) or Triptone (Oxoid, 5%). The Triton extracts were incubated in the coated tubes for 2 h at 37 C and washed twice with PBS-Tween (3). The RT–PCR one step protocol (3) was used for amplification purposes. Ten microliters of PCR products were analyzed by electrophoresis in 3% agarose gels and stained by ethidium bromide. Comparison of IC-PCR (3), SC-PCR and PC-PCR indicates that all three techniques allow the successful amplification and detection of PPV from infected herbaceous and woody plant hosts (Fig. 1). Evaluation of various substrates for the preparation of tissue prints or the spotting of extracts in SC-PCR indicated that both paper such as Whatman 3MM and nylon membranes (Immobilon-N, Millipore) are suitable but that no amplification products could be obtained from printed or spotted nitrocellulose membranes (HA45, Millipore) (results not shown). For obvious cost reasons, Whatman paper was therefore selected for the rest of this study. Attempts at PCR amplification by directly including the pieces of paper in the RT–PCR mix resulted in non-specific amplifications and were therefore not pursued. Attempts to amplify material released from the prints or spotted extracts in the absence of Triton X-100 proved unsuccessful, indicating the requirement for Triton in order to release amplifiable targets, possibly by disruption of the viral particles (9). Similarly, attempts to directly amplify the Triton extracts prepared from the printed or spotted papers failed, demonstrating the need for the capture step. Direct comparison, using the same plant extracts of IC-PCR and SC-PCR indicated that both techniques have sensitivities in the same range, with positive amplification still observed with crude infected Nicotiana extracts diluted 106-fold in healthy control extract (results not shown). One surprising observation made during the course of this work is that while specific anti-PPV immunoglobulins are necessary for a successful capture in IC-PCR (Fig. 1, compare tracks 1 and 3), the capture

Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR. Validation and practice parameters

Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR.

Specific detection and quantification of Plum pox virus by real-time fluorescent reverse transcription-PCR

Plum pox virus detection in dormant plum trees by PCR and ELISA

Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B‐IVIA, used in DASI‐ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV‐D, AL for PPV‐M, EA24 for PPV‐EA, and TUV and AC for PPV‐C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co‐operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization.

Plum pox virus (Potyvirus plumpoxi-PPV), one of the most significant diseases affecting stone fruit trees, has been extensively studied worldwide since its initial detection in 1917, with various diagnostic approaches explored. This study investigates the performance of colorimetric and real-time diagnostic kits developed by DOSA Information and Communication Technologies Ltd. for detecting PPV under isothermal conditions. These isothermal diagnostic kits, developed and optimized with domestic resources, stand out as highly practical diagnostic tools, leveraging an advantage of containing proprietary primer sets that enable direct testing of RNA from infected plants, independent of existing isothermal methods. In this study, the isothermal diagnostic kits enabled successful amplification of PPV at 65°C within 60 minutes, without cross-reaction with potential host genomes or other viruses that could co-infect. The colorimetric and real-time diagnostic kits demonstrated detection sensitivities of 10-4 and 10-5 ng/µL of PPV RNA, respectively, meeting the desired standards for PPV detection. These kits also facilitated effective amplification of PPV-M, D, and T strains, commonly found in Türkiye. Colorimetric and real-time isothermal diagnostic kits can be utilized as rapid and cost-effective tools in PPV screening programs. The application of these kits, especially in field settings and areas with limited laboratory infrastructure, should be encouraged. This successfully developed and optimized isothermal diagnostic platform could also be adapted for the detection of other significant plant viruses, offering a strategic advantage in managing viral diseases in agricultural regions such as Türkiye.

Plum pox virus (PPV) is the etiological agent of sharka, the most important viral disease of stone fruit worldwide. In this study, a one-step reverse transcription real-time PCR test (RT-qPCR) was modified and translated as a one-step RT-droplet digital PCR (RT-ddPCR) for sensitive, direct, and accurate detection and quantification of PPV. The modified RT-qPCR and RT-ddPCR PPV detection tests were validated using both plant purified total RNA (TRNA) and crude extract as templates. The proposed tests were sensitive, specific, selective, repeatable, and reproducible in detecting PPV from fresh, lyophilized, and in vitro plant samples. RT-ddPCR was more sensitive than RT-qPCR in detecting PPV using purified TRNA while showing the same sensitivity using crude extract. This work highlights the robustness, time-saving, and cost-effective nature of the proposed one-step RT-ddPCR test, offering a potential reduction in resources for PPV detection and quantification even with raw extracts.

Oligonucleotide microarray-based detection and genotyping of Plum pox virus

Transgenic plum Prunus domestica L., clone C5 (cv. HoneySweet) for protection against sharka disease