Potato Leafroll Virus Isolates Research Articles

Molecular Determination of Some Important Viruses Causing Infection in Potato Fields in Turkey

Potato is one of the most important agricultural crops worldwide and known to be susceptible to more than 40 viruses in nature. In this research, 298 leaf samples collected from potato fields in Afyon, Nevşehir and Bolu provinces in the previous study were used to determine viruses affecting potato production in the region. The leaves of potato plants showing virus symptoms were subjected to RT-PCR using virus-specific primers, in order to detect the presence of Potato leafroll virus (PLRV), Potato virus A (PVA), Potato virus X (PVX) and Potato virus M (PVM). As a result of the study,one or more viruses were detected in 46 (15.43%) of the 298 leaf samples tested. A total of 43 samples infected with PLRV (14.42%) and 3 sample infected with PVX (1.%). It was determined that the most common virus in plant samples collected from Afyon, Nevşehir and Bolu Central regions was PLRV followed by PVX. PVA and PVM were not detected in the samples. The sequences of some positive isolates of PLRV were obtained and used along with the isolates registered in the GenBank to reveal phylogenetic relationships among them. PLRV isolates from Turkey were classified in Group 2 along with isolates from Serbia, Canada, and Pakistan based on phylogenetic analyses.

A new simple and effective method for PLRV infection to screen for virus resistance in potato

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

Global genetic diversity and evolutionary patterns among Potato leafroll virus populations.

Potato leafroll virus (PLRV) is a widespread and one of the most damaging viral pathogens causing significant quantitative and qualitative losses in potato worldwide. The current knowledge of the geographical distribution, standing genetic diversity and the evolutionary patterns existing among global PLRV populations is limited. Here, we employed several bioinformatics tools and comprehensively analyzed the diversity, genomic variability, and the dynamics of key evolutionary factors governing the global spread of this viral pathogen. To date, a total of 84 full-genomic sequences of PLRV isolates have been reported from 22 countries with most genomes documented from Kenya. Among all PLRV-encoded major proteins, RTD and P0 displayed the highest level of nucleotide variability. The highest percentage of mutations were associated with RTD (38.81%) and P1 (31.66%) in the coding sequences. We detected a total of 10 significantly supported recombination events while the most frequently detected ones were associated with PLRV genome sequences reported from Kenya. Notably, the distribution patterns of recombination breakpoints across different genomic regions of PLRV isolates remained variable. Further analysis revealed that with exception of a few positively selected codons, a major part of the PLRV genome is evolving under strong purifying selection. Protein disorder prediction analysis revealed that CP-RTD had the highest percentage (48%) of disordered amino acids and the majority (27%) of disordered residues were positioned at the C-terminus. These findings will extend our current knowledge of the PLRV geographical prevalence, genetic diversity, and evolutionary factors that are presumably shaping the global spread and successful adaptation of PLRV as a destructive potato pathogen to geographically isolated regions of the world.

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y

BackgroundCertification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine.ResultsWe developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay.ConclusionThis new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

Incidence and molecular characterization of potato leaf roll virus in seed potato production in Serbia

The distribution and frequency of potato leaf roll virus in the four most important potato growing regions in Serbia were studied during the seven years (2012–18). One hundred randomly collected potato tubers were sampled from each seed lot. The young leaves that developed in three weeks were sampled and tested to record infection rate. The presence of potato leaf roll virus was detected by double-antibody sandwich enzyme-linked immunosorbent assay and disease incidence was calculated using standard formula. The obtained result showed that the highest prevalence of potato leaf roll virus was detected from seed potato samples originated from the Raski region during 2018 (20.7%), while in the Moravicki region, only 2.3–11.1% of the potato leaf roll virus was detected every year. The average annual potato leaf roll virus infection was the highest in 2012 (8.4%) and 2018 (8.0%). For further confirmation of potato leaf roll virus infection, reverse transcription (RT)-PCR was performed using specific primers PLRVCPvEcoRI /PLRVCPcNcoI, designed to amplify a 650 bp fragment of the full-length coat protein gene. The PCR products derived from 26 isolates were directly sequenced using the same primer pair as in RT-PCR. The coat protein sequence analysis revealed that the Serbian potato leaf roll virus isolates showed very low nucleotide diversity (95.9–100%). They shared the highest nt identities of 98.08–99.36% with the sequences of potato leaf roll virus isolates deposited in the GenBank from other parts of the world. Phylogenetic analysis and the haplotype network of the coat protein gene sequences showed that the Serbian potato leaf roll virus isolates could be classified in two different groups indicating two possible introductions of the virus to Serbia. The results of this study confirmed the importance of potato leaf roll virus in seed potato production in Serbia. Additionally, this research highlights the need for a continuous monitoring of the potato seeds produced in Serbia as well as imported seeds for the presence of potato leaf roll virus.

Argentinian potato leafroll virus P0 protein: Novel activities for a previously known suppressor

AbstractThe potato leafroll virus (PLRV) P0 protein (P0PL) is a suppressor of RNA silencing. In this study, we showed that P0 protein from an Argentinian isolate of PLRV (P0PL‐Ar) has an additional activity not described for other PLRV or P0 proteins from poleroviruses. Besides reporting that P0PL‐Ar displays both local and systemic silencing suppressor activity, we demonstrated, for the first time, that P0PL‐Ar impedes accumulation of dsRNA‐derived siRNAs. We also showed that P0PL‐Ar interacts with Solanum tuberosum SKP1 orthologue (StSKP1) and triggers destabilization of ARGONAUTE 1 (AGO1) and that these actions are mediated by the F‐box‐like domain. A mutant in the GW/WG motif within the P0PL‐Ar F‐box‐like motif lost the suppression activity, the interaction with StSKP1 and abolished AGO1 decay. Interestingly, a mutant in the L76/P77 residues within the P0PL‐Ar F‐box‐like motif, which lost the suppression activity and the interaction with StSKP1, retained the capacity to enable AGO1 decay. Thus, unlike other P0 proteins of previously characterized poleroviruses, P0PL‐Ar seems to have a dual activity, according to the findings of this study. This protein would act at both an upstream and a downstream step of the RNA silencing pathway: upstream of Dicer‐like enzyme (DCL)‐mediated primary siRNA production and downstream at the RNA‐induced silencing complex (RISC) complex level. Our results contribute to the understanding of the different ways PLRV P0 proteins function as silencing suppressors.

RT-PCR Based Cloning and Sequencing of Potato Leaf Roll Virus-Coat Protein (PLRV-CP) Gene for Characterization as a Bangladeshi PLRV Isolate and Its Phylogenetic Analysis

An experiment was conducted in Molecular Biology and Plant Virology Laboratory under the Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh. Total RNA was extracted from Potato leaf roll virus (PLRV) positive leaves and complementary DNA (cDNA) were synthesized from total RNA. Reverse transcriptase polymerase chain reaction (RT-PCR) based detection conditions were optimized by using coat protein (CP) gene specific primers. In PCR amplification cDNA and in nucleotide sequencing PCR product was used as a template. A 346 bp amplicon of PLRV- CP gene was amplified and amplified gene region was sequenced. The expected nucleotide sequence of amplified PLRV-CP gene showed 95 to 98% homology when compared with the isolates sequences reported in Gene Bank database. This explored novel PLRV-CP gene was characterized as a PLRV Bangladeshi isolate (Accession number, Bankit 2274496, MN605963). PLRV-CP gene protein modeling was carried out using Expert Protein Analysis System (ExPaSy), DNASTAR’s protein tools server used for 3D protein modeling. Phylogenetic analysis was also carried out, the tree was made by using MEGA 4.0 software and maximum parsimony method was selected to construct phylogenetic tree. The RT-PCR based molecular technique optimized in this study, would be a useful for early detection, epidemiological studies of PLRV as well as in seed tubers certification program and the novel hyper variable sequenced region of PLRV-CP gene will be useful in pathogen derived resistance breeding program against the PLRV local strain.

Pest categorisation of potato leafroll virus (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non‐EU isolates of potato leafroll virus (PLRV). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact and availability of control measures of non‐EU isolates of PLRV has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non‐EU isolates of PLRV are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non‐quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. This categorisation was performed considering two groups of PLRV isolates: those associated with the tomato yellow top disease (PLRV‐TYTV), not reported from the EU, and all other isolates (hereafter referred to as PLRV), with a worldwide distribution. Isolates of PLRV‐TYTV could potentially have an additional impact over the current situation in the EU and therefore meet all the criteria to qualify as a potential Union quarantine pest. All other non‐EU PLRV isolates, should they be introduced, are not expected to have additional impact and therefore do not meet this criterion to qualify as a potential Union quarantine pest.

Molecular, serological and biological characterizations of Potato Leaf Roll Virus in infected potato plants in Egypt, and its effects on plant cell organelles

Potato Leaf Roll Virus (PLRV) is one of the most serious viruses infecting potato plant (Solanum tuberosum L.) in Egypt. Indirect Enzyme Linked Immunosorbent Assay (Indirect- ELISA) results revealed that 70 % of the collected samples were infected with PLRV. A multiplex Polymerase Chain Reaction (PCR) was carried out using three different sets of primers, specific for both PLRV and Potato virus Y (PVY) isolates. For confirmation; the movement coat protein (MP) gene was isolated from the infected plant tissues, and a band with molecular size 336 bp was obtained using Reverse transcription-Polymerase chain reaction (RT-PCR). The DNA sequence of the Egyptian PLRV-Banha -MP gene was deposited in GenBank under an accession number of KR002119. Moreover, sequence analysis revealed that the Egyptian PLRV isolate was closely related to a New Zealand isolate of PLRV (GU002341), with identity of 100%. Transmission electron microscope (TEM) examination of PLRV showed isometric particles, with approximate size of 24-30 nm. The cytopathological examination of the potato plant infected with PLRV revealed many cellular effects such as; partially degraded and deformed chloroplast, starch with an increased size and color change, in addition to nucleus and cytoplasmic bridge. It could be concluded that PLRV is present in Egypt, infecting most of the potato cultivars. Moreover, four different strains of PLRV were detected based on the Single strand conformation polymorphism (SSCP) of the MP gene. The aims of the current study were to identify the PLRV infection of potato plant in Egypt using; molecular, serological and biological methods, in addition to studying the effect of this virus on potato cell organelles. This is the first record of the presence of four different strains of PLRV infecting potato in Egypt, using SSCP assay.

Cultivation of potato leafroll virus (PLRV) in mammalian continuous cell lines

Aim. To use the ability of potato leafroll virus (PLRV) to infect and multiply in mammalian continuous cell lines to purify PLRV isolates from the vegetative plant material, and to study the pathogenicity of those isolates for plants (after culturing in mammalian continuous cell line), to investigate morphological, physical-chemical, biological and antigen properties of PLRV isolates from mammalian cells and to study an alternative diagnostic method – the neutralization test in the mammalian continuous cell lines. Methods. The methods of cultivating animal viruses in the mammalian continuous cell line, microscopical biochemical, and serological methods, the method of artifi cial nutrition of aphids are detailed under Material and Methods. Results. It was demonstrated that successful cultivation of PLRV in mammalian continuous cell line allowed obtaining pure virus isolates from potato plants and aphids and preserving them for a long time (over a period of 7 years). The cultivation of PLRV in the mammalian continuous cell line did not impact its pathogenic properties and allowed transmitting the virus to plants. Continuous cells lines of pig embryonic kidney (PEKV), of kidney Syrian hamster (BHK- 21), of testicles of piglets (PTP), of kidneys of the bull (MDBC), and of carcinoma rabbit kidney (RK-13) were found to be sensitive to PLRV, Con tinuous cell lines of human (HeLa, Hep-2 and of African green monkey kidney (Vero) were not infected by the virus. The infectious activity of PLRV in the sensitive continuous cell lines was 20–8.5 lg TCD 50 /ml depending on the cell line. The isolates of PLRV were resistant to lipid- dissolving solvents, multiplied in a pH range from 4.0 till 10.0 and were thermoresistant at 50 oС in the absence of bivalent ions of magnesium, ТIP was in the range of 60–65 oС under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 °С. The use of neutralization test in the mammalian continuous cell line allowed isolation in pure culture and identifi cation of PLRV reliably in a time span of c. 14 days. Conclusions. It was proven that PLRV can be cultivated in the mammalian continuous cell lines of PEKV, ВНК-21, PTV, MDВК and RK-13. It was established that the cultivation of PLRV in these continuous cell lines did not impact its biological, pathogenic, antigenic and physical-chemical properties. The identifi cation of pure cultures of PLRV obtained in mammalian cells can be reliably performed by the use of neutralization reaction.

Biodiversity and full genome sequence of potato viruses Alfalfa mosaic virus and potato leaf roll virus in Egypt.

Solanum tuberosum (potato) is the second most important vegetable crop in Egypt. It is locally consumed, manufactured or supplied for export to Europe and other Arab countries. Potato is subject to infection by a number of plant viruses, which affect its yield and quality. Potato virus Y (PVY), potato leaf roll virus (PLRV), and Alfalfa mosaic virus (AMV) were detected in major potato-growing areas surveyed. Multiplex-RT-PCR assay was used for the detection of these three viruses in one reaction using three specific primer pairs designed to amplify genomic parts of each virus (1594 bp for PLRV, 795 bp for AMV, 801 bp for PVY). All three viruses were detected in a single reaction mixture in naturally infected field-grown potatoes. Multiplex RT-PCR improved sensitivity necessary for the early detection of infection. Incidence of single, double, or triple infection has been recorded in some locations. Full-length sequencing has been performed for an Egyptian FER isolate of PLRV. Through phylogenetic analysis, it was shown to occupy the same clade with isolate JokerMV10 from Germany. Complete nucleotide sequence of an Egyptian FER isolate of AMV and phylogenetic analysis was also performed; we propose that it is a new distinct strain of AMV belonging to a new subgroup IIC. This is the first complete nucleotide sequence of an Egyptian isolate of AMV. Genetic biodiversity of devastating potato viruses necessitates continuous monitoring of new genetic variants of such viruses.

First Complete Genome Sequence of Potato leafroll virus from Argentina.

ABSTRACTIn this study, we determined for the first time the complete genomic sequence of an Argentinian isolate of Potato leafroll virus (PLRV), the type species of the genus Polerovirus. The isolate sequenced came from a Solanum tuberosum plant that had been naturally infected with the virus. Isolate PLRV-AR had a nucleotide sequence identity between 94.4 and 97.3% with several known PLRV isolates worldwide.

Examination of an isolate of Potato leaf roll virus that does not induce visible symptoms in the greenhouse

Over the last 30 years the importance of Potato leaf roll virus (PLRV) in commercial potato and seed potato production has decreased considerably. Since PLRV is transmitted by aphids in a persistent manner it can be controlled by applying a systemic insecticide. However, the development of insecticide resistance in the main vectors of PLRV Myzus persicae, Aulacorthum solani, Rhopalosiphoninus latysiphon, Aphis fabae, A. nasturtii, A. frangulae and Macrosiphum euphorbiae, and the development of isolates of PLRV that do not induce visible symptoms in some potato cultivars may lead to a resurgence in the significance of PLRV. Isolates of this type were found repeatedly during growing-on tests in Lower Saxony, Germany. In this study we examined such a symptomless isolate. The visible symptoms induced by this isolate in different potato cultivars were compared with those induced by isolates causing typical symptoms of a PLRV infection. By using quantitative real-time PCR the quantifiable amount of viral RNA was determined. Under climate chamber conditions all the isolates tested induced similar symptoms and did not differ in viral RNA content. Complete sequences for the tested isolates were obtained and used in a phylogenetic analysis. All the PLRV isolates compared were very similar at the molecular level. Several motifs that could play a role in symptom expression were analyzed, but none of them were correlated with the absence of symptoms in potato plants during growing-on tests. The discrepancy between the observations recorded in the growing-on tests and our experiments are discussed.

Possible correlations between the characteristics of Potato leafroll virus isolates occurring in different geographical regions in Tunisia

Biological and molecular characterization supported by transmission efficiency, symptom expression and Open Reading Frame 0 (ORF0) nucleotide sequence analysis were carried out to assess nine isolates of Potato leafroll virus (PLRV) collected from three Tunisian geographic and bioclimatic zones. Plant-to-plant transmission by Tunisian Myzus persicae aphid clones showed high transmission efficiency for all isolates tested. Symptom expression analysis on a Physalis floridana plant test distinguished viral isolates as very severe, severe and mild. The ORF0 sequences of the Tunisian PLRV isolates showed an assignment to two aggregates when compared with GenBank PLRV sequences. A significant correlation between symptom severity and ORF0 nucleotide sequence or between symptom severity and geographic origins of the PLRV isolates was established. However, the transmission efficiency and the ORF0 sequence were not affected by the bioclimatic origin. No significant correlation between transmission and symptom or between transmission and the ORF0 sequence was detected.

Complete Genome Sequence of Potato leafroll virus Isolates Infecting Potato in the Different Geographical Areas of India Shows Low Level Genetic Diversity

Five Potato leafroll virus (PLRV) isolates were collected from five states representing different potato growing parts of India. The ssRNA genome sequences of these isolates were determined. The genome comprised of 5,883 nucleotides and deduced genome organization resembled other PLRV isolates. About 97.6-98.7% similarities was observed within the Indian isolates and were more close to European, Canadian, African, American and Czech isolates (95.8-98.6%) than to an Australian isolate (92.9-93.4%). These isolates were 43.7-53.1% similar to other poleroviruses and 29.1-29.3% to Barley yellow dwarf virus, a luteovirus. Out of five isolates, the isolate PBI-6 was recombinant one as detected by RDP3 software. Multiple sequence alignment of nucleotide and amino acid sequences of different ORFs indicated that the ORF 3 and ORF 4, corresponding to coat protein and movement proteins are more conserved than other ORFs. Amino acid changes specific to Indian isolates were observed and it was more in ORF 2 than in ORF 0, ORF 3 and ORF 4. This is the first report of complete genome sequence of PLRV isolates from India, which reveals low level genetic diversity.

Genetic analysis of Iranian population of Potato leafroll virus based on ORF0

Potato leafroll virus (PLRV) is a destructive virus of potatoes and responsible for high yield losses wherever potatoes are grown. In this study, DNA fragments containing ORF0 from each of nine PLRV isolates was sequenced. Sequence analysis data using 36 isolates from 12 different countries including 14 Iranian isolates showed that the identities of ORF0 at both nucleotide and amino acid levels between the Iranian isolates were 96-100% and these isolates were more similar to the European PLRV isolates than to the other isolates. Furthermore, phylogenetic and population genetic analysis were carried out on the basis of full-length ORF0 and overlapping and non-overlapping regions of ORF0 and ORF1 (ORF0/1) which revealed that PLRV isolates were not geographically resolved. Also, we identified negative selection with different ratios for each of the mentioned genomic regions suggesting effects of F-box motif and -1 frameshift on ORF0 non-overlapping region and ORF0/1 in the selection pressure, respectively. Five recombination events were detected in the Iranian, Australian, and European isolates suggesting an important role for this phenomenon in influencing genetic diversity within this virus population.

Incidence and Molecular Analysis ofPotato leafroll virusin Iran

Potato leafroll virus (PLRV) is one of the most prevalent potato viruses in Iran. This report describes the distribution of PLRV in four Provinces from south-eastern, southern, north-eastern and north-western Iran and phylogenic relationships of Iranian PLRV to other previously reported PLRV isolates. PLRV was detected in c.15% (126 of 836) of symptomatic potato samples (showing yellowing and leaf roll) by double antibody sandwich enzyme-linked immunosorbent assay. The coat protein (CP) gene of four isolates was amplified by reverse transcription-polymerase chain reaction using specific primers. The nucleotide sequence showed a high degree of sequence identities between all PLRV isolates. Three of the four Iranian isolates were 100°% identical at the amino acid level (for the domain sequenced), but the fourth isolate differed by an amino acid. For isolate PLRV-Ke, we amplified the open reading frame (ORFO) (which overlaps the 5' end of the ORF1) and the sequence analysis indicated that the amino acid sequences of the ORFO and the 5' end of the ORF1 showed identity of 92.7-100% and 90.2-99% with that of the GenBank PLRV isolates, respectively. In contrast to the amino acid sequence of the CP, a constructed phylogenetic tree using the amino acid sequence of the ORFO differentiated the Iranian isolate (PLRV-Ke) from some European and African isolates.

Short communication: Molecular analysis of Potato leafroll virus isolates from the Czech Republic

The complete genomes of three Czech isolates VIRUBRA 1/045, VIRUBRA 1/046, and VIRUBRA 1/047 of Potato leafroll virus (PLRV) were sequenced and compared with 13 complete sequences of PLRV isolates available in GenBank. Among the Czech isolates, VIRUBRA 1/046 and 1/047 showed the highest nucleotide (nt) identity (98.7%). PLRV was the most conserved virus in both open reading frames (ORFs) 3 and 4. The most variable regions were ORFs 0 and Rap1. Interestingly, isolate VIRUBRA 1/045 significantly differed from the other two Czech isolates in ORFs 0 and 1. Moreover, we identified mutations in the amino acid (aa) sequences, which were specific for the Czech isolates. Phylogenetic analysis based on ORF0 showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on sequence analysis of the genome sequences of PLRV isolates from the Czech Republic.

BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF TUNISIAN ISOLATES OF POTATO LEAFROLL VIRUS

SUMMARY Nine Tunisian isolates of Potato leafroll virus (PLRV) were collected from potato plants (cv Spunta) with similar secondary leafroll symptoms. These PLRV isolates were readily transmitted by Myzus persicae (Sulzer) from potato plants to Physalis floridana test plants inducing symptoms of differing severities. In order to investigate the genetic variability between the PLRV isolates, the ORF0 regions of the Tunisian PLRV isolates were sequenced and compared with that of a French isolate (Fr5) that induces mild symptoms and 24 ORF0 PLRV sequences available in Genbank. The Tunisian isolates could be classified in two of the three groupings of the phylogenetic tree obtained. A tentative correlation between symptom expression in P. floridana and specific changes in the ORF0 sequences is discussed.

Diagnosis and molecular analysis of Potato leafroll virus isolates in Tunisia

Potato leafroll virus (PLRV) is a major constraint to potato production in North Africa. Serological (sandwich and cocktail ELISA) and molecular (RT‐PCR) tests were used to detect PLRV in 131 potato samples collected in different areas of Tunisia. RT‐PCR proved to be usable as a routine diagnostic test for epidemiological purposes, being more sensitive and reliable, and less time‐consuming, than serological tests. One RT‐PCR‐amplified portion of ORF3 (336 nt) was cloned and sequenced, and used for molecular characterization of Tunisian PLRV isolates. These showed high sequence identity with PLRV retrieved from GenBank.