Detection Of Plum Pox Virus Research Articles

BLUE LAMP: A FAST AND RELIABLE NUCLEIC ACID AMPLIFICATION TECHNIQUE FOR THE DETECTION OF PLUM POX VIRUS

ISHS II EUFRIN Plum and Prune Working Group Meeting on Present Constraints of Plum Growing in Europe BLUE LAMP: A FAST AND RELIABLE NUCLEIC ACID AMPLIFICATION TECHNIQUE FOR THE DETECTION OF PLUM POX VIRUS

Production and in vitro assessment of transgenic plums for resistance to Plum pox virus: a feasible, environmental risk‐free, cost‐effective approach

AbstractDuring the production and assessment of transgenic plants resistant to quarantine viruses, the need to contain genetically modified plants (GMPs) and pathogens severely limits working options. Moreover, in the case of fruit trees, acclimatisation and viral inoculation are very time‐consuming, thus a quick and safe method to assess the resistance to quarantine viruses, such as Plum pox virus (PPV), is desirable. This article focuses on the production of transgenic plums together with a contained and rapid evaluation in vitro for PPV resistance. The plum ‘Stanley’ was transformed by Rhizobium radiobacter (syn. Agrobacterium tumefaciens) using a PPV‐M derived hairpin construct (h‐UTR/P1) that had previously been shown to confer high and broad‐spectrum PPV resistance in model plant. Two transgenic clones, St24 and St28, were obtained. To assess their ability to resist PPV infection, micropropagated shoots of the transgenic clones were micrografted in vitro onto PPV‐D infected ‘GF305’ rootstock. Following successful grafting, the transgenic scions were analysed by immunocapture‐reverse transcription‐polymerase chain reaction (IC‐RT‐PCR) for PPV detection. A total of 97% (47/48) of St24 and 73% (17/23) of St28 tested plants were resistant to the heterologous strain of PPV. In line with an RNA silencing mediated resistance mechanism, the St24 clone was shown to accumulate higher concentration of PPV UTR/P1‐specific small interfering RNAs (siRNAs) than St28 one. The results are of practical interest not only developing plum clones that are highly resistant to PPV, but also for setting up quick and contained inoculation test procedure.

Fast and reliable detection of Plum pox virus in woody host plants using the Blue LAMP protocol

Up to now, the polymerase chain reaction is the most widely used method for the amplification of nucleic acids in vitro, especially for pathogen detection because of its high sensitivity. In the recent years, however, numerous isothermal amplification methods were developed to avoid the need for thermal cycling. The most frequently applied approach seems to be loop-mediated isothermal amplification (LAMP). The great advantage of LAMP is its enormous rate of amplification paired with a very high specificity and low artefact susceptibility. This study presents a straightforward procedure for Plum pox virus (PPV) detection. A modified one-step reverse transcription loop-mediated isothermal amplification protocol of Varga and James is applied to virus suspensions from plant extracts obtained by a simplified and standardised procedure. Gel electrophoresis is substituted by a homogenous colour test upon nucleic acid amplification. This procedure takes only 2.5 h from sampling to result and requires minimal technical equipment. With amplification and visualisation homogenously taking place in non-opened tubes the risk of cross-contamination of subsequent samples by former amplification products via facilities and equipment is strongly minimised. Hence, the Blue LAMP provides a fast and reliable detection of PPV both for single samples and for large-scale surveys.

Impedimetric Immunosensor for Detection of Plum Pox Virus in Plant Extracts

AbstractThis paper concerns the development of an immunosensor for detection of Plum pox virus in plant extracts. The sensor was based on gold electrodes modified with: 1,6‐hexanedithiol, gold nanoparticles, anti‐PPV IgG polyclonal antibody and BSA. It was used for determination of the virus in extracts from plum (Prunus domestica) and tobacco (Nicotiana benthamiana) leaves. The proposed electrochemical impedance spectroscopy (EIS) immunosensor displayed a very good detection limit of 10 pg/mL and a wide dynamic range from 10 pg PPV/ mL to 200 pg/mL. The presence of extracts from plant materials has no influence on the immunosensor’s response. The immunosensor was capable of discriminating between samples from healthy plants and samples containing 0.01 % of extract from infected plant material. The conditions for immunosensor regeneration are also presented.

Estimation of the accuracy of two diagnostic methods for the detection of Plum pox virus in nursery blocks by latent class models

The control of Plum pox virus (PPV), the most important viral disease that affects stone fruit trees, requires the use of reliable detection methods. The effectiveness of spot real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) for the detection of PPV in samples collected from nursery blocks was compared with a validated PPV detection technique, the double antibody sandwich indirect enzyme‐linked immunosorbent assay (DASI‐ELISA) using the PPV‐specific monoclonal antibody 5B‐IVIA/AMR. In total, 5047 nursery plants were analysed by both techniques. The agreement between the techniques was almost perfect (Cohen’s kappa index of 0·88 ± 0·01). The diagnostic parameters (sensitivity, specificity and likelihood ratios) of both techniques were simultaneously evaluated in 2473 nursery plants by latent class models using maximum likelihood functions and a Bayesian approach. The sensitivity and specificity of both techniques did not vary according to the latent model applied. Spot real‐time RT‐PCR was more sensitive while DASI‐ELISA was more specific for PPV detection. In addition, the findings demonstrate that latent class models are a flexible and potent statistical method to estimate the accuracy of diagnostic tests for plant pathology.

GENETIC DIVERSITY OF CANADIAN ISOLATES OF PLUM POX VIRUS AND ITS SIGNIFICANCE

Since the initial detection of Plum pox virus (PPV) in Canada in 2000, and as a result of subsequent intensive surveys, isolates of three strains of PPV were detected; PPV-D, PPV-W, and PPV-Rec. Only isolates of PPV-D were found in trees in commercial orchards; while isolates of PPV-D, PPV-W, and PPV-Rec were found in trees on homeowner/residential properties. Phylogenetic analysis of D isolates from commercial orchards revealed two main clusters or subgroups (I and II, Theilmann et al., 2006). Some D isolates from homeowner properties cluster with subgroup I or II, but most appear to be distinct from either subgroup. PPV-W and PPV-Rec were each found in plum trees on individual homeowner properties. The complete genomes of 2 Canadian isolates of PPV Rec were sequenced and were 99% identical. In phylogenetic analysis based on the N-terminus of the CP, they were most similar to the Rec isolates J4c (Poland) and MYV-3 (Slovakia). The Canadian Rec isolates are the first isolates of PPV Rec found outside of Europe and sequence analysis reveals a close relationship to European Rec isolates. Isolates from homeowner properties form the major source of genetic diversity of PPV found in Canada.

Interaction of Plum Pox Virus with Specific Colloidal Gold-Labeled Antibodies and Development of Immunochromatographic Assay of the Virus

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in samples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by "sandwich"-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.

Preliminary results for obtaining nuclear-stock plum material in Romania.

A certification program focused on plum was established at Fruit Research & Development Station Bistrita, Romania started in 2008. The objective is to obtain in the first step the prebasic material and, subsequently, to establish plum mother plantations with basic material satisfying the EPPO recommended certification standards. Sixteen plum (Prunus domestica) cultivars (Iulia, Matilda, Geta, Zamfira, Ivan, Dani, Doina, Romaner, Elena, Jubileu 50, Delia, Flora, Renclod d’Althan, Stanley, Anna Spath and Carpatin) were subjected to this work. Selected plum trees from experimental orchards were monitored and tested. One shoot was collected from each candidate and tested before grafting onto virus-free Myrobolan 29C rootstock. Selected clones were also tested on woody indicators GF 305. Propagated material was individually tested one year later. All tests were done according to the EPPO recommendations. DAS-ELISA was used for Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Myrobalan latent ringspot virus (MLRSV). To increase the sensitivity of PPV and PDV detection, IC-RT-PCR was performed. Spot-Real Time RT-PCR was also used to detect PPV. Nested-PCR method was made to detect Candidatus Phytoplasma prunorum. Delia cultivar was found infected by PPV and eliminated from propagation by grafting. This cultivar is subjected to pathogen elimination by using in vitro chemo-thermotherapy. The other fifteen cultivars were found virus-free both before and after propagation. The certification of this material as ‘Prebasic category’ is in progress.

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

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 ± 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses. [Int Microbiol 2009; 12(1):1-6].

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

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses.

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

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

Plum pox virus (PPV) is considered the most destructive viral pathogen of stone fruits. Although PPV is widespread in all plum growing areas from Romania and causes serious yield losses, little is known about the virus variability. For this reason we investigated 100 PPV isolates collected from 13 different plum orchards in the Transylvania - Romania. 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 different genomic segments corresponding to (Cter)CP, (Cter)NIb/(Nter)CP and CI. RFLP analysis was used to distinguish the two major strains, D and M based on a RsaI polymorphism located in (Cter)CP. All PCR products targeting (Cter)CP and 10 PCR products 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. Sequencing confirmed a high similarity with different sequences of PPV-Rec previously reported. All these recombinant isolates share the same recombination breakpoint and conserve the DAG motif, which is considered essential for aphid transmission. Overall results provided that in Transylvania the predominant strain is PPV-D, followed by PPV-Rec which shares the CP gene with M strain and, therefore, it is serologically detected as PPV-M with M-specific monoclonal antibodies. In this big plum growing area, mixed infections (PPV-D+PPV-Rec), which might generate additional variation by recombination, are also frequent.

BEHAVIOUR OF TRANSGENIC PLUM POX VIRUS-RESISTANT PRUNUS DOMESTICA L. CLONE C5 GROWN IN THE OPEN FIELD UNDER A HIGH AND PERMANENT INFECTION PRESSURE OF THE PPV-REC STRAIN

Transgenic plum (Prunus domestica L.) clone C5 was grafted on virus-free St. Julien rootstocks and inoculated by bud grafting with the recombinant strain (PPVRec) of the Plum pox virus (PPV). Non inoculated C5 plums were used as controls. Transgenic plums infected with PPV-Rec and control plants were evaluated for six years in the open field. PPV symptoms, mild diffuse spots and rings, appeared on the basal leaves the second year after inoculation. The presence of PPV was confirmed by DAS-ELISA, ISEM, and RT-PCR. Severe PPV symptoms appeared in the leaves of shoots grown from non-transgenic inoculum buds. A reduction of symptoms and a decrease of the relative PPV concentration, as determined by DAS-ELISA in transgenic C5 plants, were observed from the third to the fifth year after virus inoculation. Very mild symptoms in older leaves of the basal branches of trees subsided by the end of the vegetative period, and were difficult to observe. The level of PPV detection by DAS-ELISA dropped from June to August together with a reduction of symptoms. PPV was undetectable by DAS-ELISA in more than half of the trees by the end of August. PPV-Rec was detected by RT-PCR in the basal and middle parts of the C5 scions. The upper leaves did not contain a detectable level of the virus. These results show the high level and stability of PPV resistance in graft-inoculated C5 trees.

Direct real-time PCR detection of Plum pox virus in field surveys in Ontario

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.

An Evidence-Based Approach to Plum Pox Virus Detection by DASI-ELISA and RT-PCR in Dormant Period

An evidence-based approach, such as those developed in clinical and veterinary medicine, was applied to the detection of Plum pox virus (PPV) during the dormant period. A standardized methodology was used for the calculation of parameters of the operational capacity of DASI-ELISA and RT-PCR in wintertime. These methods are routinely handled to test the sanitary status of plants in national or international trading and in those cases concerning export-import of plant materials. Diagnosis often has to be performed during the dormant period, when plant material is commercialized. Some guidelines to interpret diagnostic results of wintertime are provided in an attempt to minimize risks associated with the methods and over-reliance on the binary outcome of a single assay. In order to evaluate if a complementary test increased the confidence of PPV diagnosis when discordant results between DASI-ELISA and RT-PCR are obtained, NASBA-FH also was included. Likelihood ratios of each method were estimated based on the sensitivity and specificity obtained in wintertime. Subsequently, a Bayesian approach was performed to calculate post-test probability of PPV infection in spring. Results of evidence-based approach show that different PPV prevalences require different screening tests. Thus, at very low PPV prevalence levels DASI-ELISA should be used as the election method, whilst at the highest PPV prevalence levels RT-PCR should be performed. NASBA-FH could be used at medium prevalences to clarify discordances between DASI-ELISA and RT-PCR.

Oligonucleotide microarray-based detection and genotyping of Plum pox virus

Plum pox virus (PPV) is the most damaging viral pathogen of stone fruits. The detection and identification of its strains are therefore of critical importance to plant quarantine and certification programs. Existing methods to screen strains of PPV suffer from significant limitations such as the simultaneous detection and genotyping of several strains of PPV in samples infected with different isolates of the virus. A genomic strategy for PPV screening based on the viral nucleotide sequence was developed to enable the detection and genotyping of the virus from infected plant tissue or biological samples. The basis of this approach is a long 70-mer oligonucleotide DNA microarray capable of simultaneously detecting and genotyping PPV strains. Several 70-mer oligonucleotide probes were specific for the detection and genotyping of individual PPV isolates to their strains. Other probes were specific for the detection and identification of two or three PPV strains. One probe (universal), derived from the genome highly conserved 3′ non-translated region, detected all individual strains of PPV. This universal PPV probe, combined with probes specific for each known strain, could be used for new PPV strain discovery. Finally, indirect fluorescent labeling of cDNA with cyanine after cDNA synthesis enhanced the sensitivity of the virus detection without the use of the PCR amplification step. The PPV microarray detected and identified efficiently the PPV strains in PPV-infected peach, apricot and Nicotiana benthamiana leaves. This PPV detection method is versatile, and enables the simultaneous detection of plant pathogens.

British Columbia Regional Meeting, 2006 The Canadian Phytopathological Society

British Columbia Regional Meeting, 2006 The Canadian Phytopathological Society

Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus

A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV) . The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.

Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum pox virus

A one step, accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) procedure was developed for the detection of Plum pox virus (PPV). The six primers required for accelerated RT-LAMP were designed using a conserved region in the C-terminus of the coat protein coding region of PPV. RT-LAMP was used to detect isolates of five strains of PPV including the strains D, M, EA, C, and W. The virus was detected reliably in both infected herbaceous and woody hosts. RT-LAMP was compared to real-time RT-PCR with SYBR Green I and melting curve analysis, using serial dilutions of total RNA extracts. Similar sensitivities were observed, except that real-time RT-PCR was more consistent at lower template concentrations. The purity of the FIP and BIP primers affected the efficiency of the reaction, and incubation time and template concentration affected the ladder-like pattern observed after agarose gel electrophoresis. Although PPV could be detected after 30 min of incubation at 63 °C, a longer incubation time was required for lower concentrations of the target. RT-LAMP is a very sensitive, low cost diagnostic tool that should be of value in more accurate determination of the distribution of PPV. This should assist in preventing further spread of this devastating virus.

Detection and characterization of Plum pox virus: molecular methods

Since their initial description, molecular methods for the detection of Plum pox virus (PPV) have been increasingly used by diagnostic laboratories. Although they initially provided higher sensitivity than traditional serological (ELISA) techniques, molecular hybridization assays were rapidly superseded by RT‐PCR based systems for detecting and characterizing PPV isolates. These techniques have indeed surpassed all traditional methods in sensitivity and specificity. Continuous efforts are being made to improve the use of molecular techniques in routine tests, in particular to overcome specific problems caused by plant material with high contents of RT‐PCR inhibitors. Different systems for viral target preparation before the RT‐PCR reaction have been developed based on immunocapture, or, avoiding extract preparation, on print and squash capture. The use of immobilised targets on paper has allowed the detection of PPV in single aphids. Sequence data from a number of PPV isolates, often obtained through PCR‐based techniques has permitted the clustering of PPV strains into six groups: D, M, EA, C, W and Rec and the development of group‐ or strain‐specific detection assays. Other important advances have arisen with the determination of the viral load in a plant or aphid samples by quantitative, real‐time PCR, as automation and user‐friendly softwares have made this technology more available.