Prunus Necrotic Ringspot Virus Isolates Research Articles

국내 복숭아에서 분리한 Prunus necrotic ringspot virus의 특성

In this paper, we report a characterization of Prunus necrotic ringspot virus (PNRSV) isolate. The virus was identified from `Yumyeong` peach showing mild mosaic on leaves in commercial orchard of `Umsung`, Chungbuk province in Korea. The virus isolate produced ringspot symptom on the inoculated cotyledons and systemic mosaic and malformation on the upper leaves of Cucumis sativus. Systemic mottles were appeared in Chenopodium quinoa. When the buds of the virus infected stem were grafted on the healthy young Prunus persica GF305 seedlings, line pattern with mosaic appeared within 3 months. Isometric virus-like particles were found in parenchyma cells and plasmodesmata of C. sativus leaves inoculated mechanically with the virus. The cDNA fragments of PNRSV coat protein (CP) region, approximately 675bp, were synthesized from genomic RNA extracted from virus-infected leaves by RT-PCR using specific primer pairs. Partial nucleotide sequences of the CP regions were determined and analyzed with the known PNRSV. The CP gene of PNRSVKorea isolates showed 93.9~94.7% similarity to the 4 known PNRSV isolates.

Genetic Diversity of Prunus necrotic ringspot virus Isolates Within a Cherry Orchard in New York.

A survey for Prunus necrotic ringspot virus (PNRSV) in an orchard of Prunus cerasus cv. Montmorency and Prunus avium cv. Hedelfingen in New York by enzyme-linked immunosorbent assay indicated an eightfold higher infection rate in sour cherry (33%, 32 of 96) than in sweet cherry (4%, 6 of 136) trees. The presence of PNRSV was confirmed by reverse transcription-polymerase chain reaction and amplification of the coat protein (CP) gene in total RNA from infected leaf tissue. Latent infection was prevalent in the majority of trees infected (87%, 33 of 38), while a few of them exhibited shock symptoms or had severely reduced growth (13%, 5 of 38). Asymptomatic PNRSV-infected trees clustered in spatial proximity to symptomatic trees. Sequence analysis of the CP gene (675 bp) indicated a population structure consisting of one predominant molecular variant for 10 isolates and six minor molecular variants for seven isolates. A high sequence identity was found between the CP gene of PNRSV isolates from cherry trees and other isolates from diverse hosts and various geographic origins at the nucleotide and amino acid levels (88 to 100%). Phylogenetic analyses showed a clustering of PNRSV isolates from cherry trees in New York in the predominant group PV-96.

Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus

Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.

First Report of Prunus necrotic ringspot virus in Peach in Mexico

Peach (Prunus persica (L.) Batsch) is one of the most important fruit crops in the temperate regions of Mexico. In 2006, during a survey conducted in commercial peach orchards in Puebla, Mexico for viral diseases, many trees were observed with foliar symptoms that included yellow mottle, ringspot, line patterns, and mosaic. Samples (flowers, young shoot tips, and leaves) were collected from 120 symptomatic trees in three locations (San Martin Texmelucan, Domingo Arenas, and Tepetzala). All samples were tested using double-antibody sandwich (DAS)-ELISA kits (Agdia, Inc., Elkhart, IN) for the presence of the following viruses: Apple mosaic virus, Plum pox virus, Prune dwarf virus, and Prunus necrotic ringspot virus (PNRSV). Sap extracts from young symptomatic leaves and shoots were used to mechanically inoculate Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Nicotiana tabacum cv. Xanthi, N. glutinosa, N. clevelandii, N. benthamiana, Datura stramonium, Capsicum annuum, and Solanum lycopersicum. Plants were kept in a greenhouse with approximate temperatures of 25 to 35°C, humidity of 70%, and 12 h of light. Sap extracts were also used for dsRNA extraction and analyses (2) and RNA extraction for use in reverse transcription (RT)-PCR with the Access RT-PCR system (Promega, Madison, WI) and primers that annealed to a conserved region in the PNRSV coat protein gene (1). The expected size amplicons of approximately 450 bp were generated from all field-collected samples. The PCR products from three geographically distinct PNRSV isolates (Domingo Arenas [Accession No. DQ979004], Tepetzala [Accession No. DQ979005], and San Martin Texmelucan [Accession No. EF456771]) were directly sequenced with a Genetic Analyzer 3100 (Applied Biosystems, Foster City, CA) and their nucleotide and deduced amino acids sequences were more than 93% identical to corresponding sequences of PNRSV available in the NCBI/GenBank database. PNRSV was the only virus detected by DAS-ELISA in flowers and young shoots from 60 of the symptomatic field samples tested from the three locations. DsRNA banding patterns were obtained from 40 field-collected symptomatic samples; all showed three bands of approximately 3.6, 2.5, and 1.8 kb, the expected sizes for RNAs 1, 2, and 3 of PNRSV, respectively. DsRNAs were not detected in asymptomatic plants. PNRSV transmission by mechanical inoculation induced mosaic symptoms in N. tabacum cv. Xanthi and necrotic local lesions in G. globosa. Although G. globosa is reported to be a systemic host of PNRSV and N. tabacum is not reported to be a host, symptomatic plants were positive for PNRSV in DAS-ELISA tests. It is possible that there was an additional virus not detected in our assays that was responsible for the unexpected reactions in the host range studies. To our knowledge, this is the first report of PNRSV in peach in Mexico.

Incidence of Prunus necrotic ring spot virus on Malus domestica in India

AbstractIn surveys of apple (Malus domestica) orchards in various parts of Himachal Pradesh, samples from trees showing necrotic symptoms on the leaves were collected and tested for detection of Prunus necrotic ringspot virus (PNRSV) initially by ELISA followed by RT‐PCR using coat protein gene primers. Positive results were obtained in samples from Kullu and Kalpa regions. The virus gene sequences showed 88–97% similarity to corresponding sequences of other PNRSV isolates deposited in the GenBank database using ncbi.nih.nlm.gov. Although the similarity was high, there were some distinct differences with the Spanish isolate. This is the first report of PNRSV in apple from India.

Studies on Prunus Necrotic Ringspot Virus (PNRSV) Occurring on Lily

This study was to molecular identify Prunus necrotic ringspot virus (PNRSV) occurring on lily. Lily corms were randomly selected and grown in the greenhouse. The total RNAs were extracted from younger leaves, and partial amplification of the CP gene was performed by RT-PCR with primer pairs specific for PNRSV. An expected cDNA fragment of about 450 bp was amplified from lily, cloned into pGEM-T-easy vector, sequenced, and shared 84.5-99.1% homology with the 27 PNRSV isolates reported previously at the nucleotide level, indicating that lily is a new natural host of PNRSV.

Evidence for the occurrence of Prunus necrotic ringspot virus on peach in India by serological and molecular methods

Prunus necrotic ringspot virus (PNRSV) is a widespread virus that causes tremendous losses on stone fruits. Necrotic symptoms typical of the virus were observed on the leaves of peach trees, Prunus persica ‘Elberta’, during a survey of the Kullu area of Himachal Pradesh, a northern state of India. PNRSV-specific antibodies were used for serological detection, and positive results were obtained. Molecular detection of the coat-protein (CP) gene of the PNRSV by means of reverse transcription – polymerase chain, with a primer pair that amplifies the complete CP region, amplified the ~700-bp product, which showed a maximum of 98% identity with an isolate from the United States. Multiple alignment of the CP sequence with that of other PNRSV isolates showed 87%–100% identity at the nucleotide and amino acid level.

Studies on Parameters Influencing the Performance of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in Detecting Prunus Necrotic Ringpot Virus (PNRSV)

In order to have a more detailed understanding of the various factors influencing a reverse transcriptase polymerase chain reaction (RT-PCR), a number of important parameters such as Mg+2, primer, enzyme concentration and others were optimized for the detection of Prunus necrotic ringspot virus (PNRSV). Using a PNRSV isolate with a pair of primers, complementary DNA of viral genome as template, and an appropriate enzyme together with magnesium chloride, the following optimal conditions were identified: primer concentration between 0.2 and 0.0002 pmol µl-1 and 0.06–2 units µl-1 for Taq DNA polymerase enzyme for a 50 µl reaction volume when other parameters were optimum; magnesium chloride concentration less than 2.5 mM; dNTP concentration between 1 and 10 mM. The optimum cDNA amount should be ~360 ng for a 50 µl reaction mixture. When these optimized concentrations and/or values of the main PCR parameters were brought together for a new RT-PCR, a clear and a reliable PNRSV detection having no background was performed from both growth-chamber and field-grown PNRSV-infected plants.

Detection and identification ofPrunus necrotic ringspot virusinPelargonium

Rose geraniums (Pelargonium spp.) are important essential oil bearing plants of great demand in national and international markets. Geraniums are susceptible to several viruses; however, the incidence of Prunus necrotic ring spot virus (PNRSV) was not reported until recently. During the survey of geranium plantations in experimental fields of the Institute of Himalayan Bioresource Technology, Palampur, India, Ilarvirus-like symptoms were observed. To identify the causal virus, the plants were tested by enzyme-linked immunosorbent assay using antibodies of different Ilarviruses. Initial results showed the presence of PNRSV in geranium. This diagnosis was confirmed by Northern hybridisation, reverse transcription polymerase chain reaction, restriction enzyme digestion and sequencing. Coat protein gene sequence showed 97–99% amino acid homology with other PNRSV isolates. It was concluded that rose geraniums are natural hosts of this virus.

RT-PCR Detection and Molecular Characterization of Prunus necrotic ringspot virus Isolates Occurring in Turkey

Sour cherry (Prunus cerasus L.), sweet cherry (P. avium L.), peach [P. persica (L.) Batsch], nectarine [P. persica (L.) Batsch var. nucipersica (Suckow) C.K. Schneid], apricot (P. armeniaca L.) and plum (P. domestica L.) trees either known to be infected, or shown to be so during a survey of different stone fruit growing regions of Turkey, were tested for the occurrence of Prunus necrotic ringspot virus (PNRSV) using double antibody sandwich (DAS)–enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). DAS–ELISA detected virus in only 31 of 486 samples; an additional 20 samples (total infection 51) were found to be infected by PNRSV when RT-PCR was used. The highest virus incidence occurred in nectarine followed by that in peach. In order to identify specific virus strains, the PCR products amplified from the capsid protein (CP) gene of the 20 isolates selected from different provinces were subjected to restriction fragment length polymorphic (RFLP) analysis. The PCR products were digested by eight different restriction endonucleases. Most of the PNRSV isolates were identified as members of group PV96, but a single isolate was a member of group PV32; none of the isolates belonged to group PE5. Phylogenetic analysis divided the virus isolates into three additional major and three minor groups. No relation was found between the geographic distribution and the grouping of the PNRSV isolates based on RFLP.

Prunus necrotic ringspot virus isolates in stone fruit germplasm accessions and cultivars in Israel

Summary Prunus necrotic ringspot virus (PNRSV) was detected in almonds, plum and apricot germplasm accessions and local almond cultivars in Israel. PNRSV was widespread both in wild and cultivated almond trees and uncommon in wild apricots and plums. The possible variation among the PNRSV isolates was initially evaluated by restriction analysis of PCR products representing the CP gene with the endonuclease RsaI and followed by nucleotide sequence analysis of selected isolates. It was concluded that all 13 isolates belong to group PV96, the largest cluster of PNRSV isolates, described previously. Two PNRSV isolates, one from a plum accession and one from an almond cultivar, were found to be distinct members of group PV96 with unique nucleotide modifications not found in other documented isolates of this virus. However, no PNRSV isolate typical to a specific host and/or to the Middle East region could be identified. This study expands the body of data on variability of PNRSV isolates and highlights the importance of assessing the virus status of germplasm collections by applying reliable diagnostic and differentiating methods.

RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport. In this study, putative RNA-binding properties of the PNRSV MP were studied. The PNRSV MP was produced in Escherichia coli using an expression vector. Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity. Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio. The different responses of the complexes to urea treatment strongly suggested that they have different structural properties. Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88. This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated. This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates. Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships. The evolutionary implications of this observation are discussed.

Biological and molecular characterisation of Prunus necrotic ringspot virus isolates and possible approaches to their phylogenetic typing

SummarySeven isolates of Prunus necrotic ringspot virus (PNRSV) originating from Slovakia were subjected to biological tests under glasshouse conditions. Mainly mild symptoms were observed on chip‐budded test cherry rootstocks. The complete sequence for the capsid protein (CP) gene of four isolates was determined. All sequences were 675 nucleotides long and clustered in the largest of four groups delineated by phylogenetic analyses of all so far known PNRSV CP sequences. A set of restriction endonucleases was suggested to differentiate four isolate clusters by restriction enzyme digestion of CP sequences.

The molecular variability analysis of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus sheds light on the minimal requirements for the synthesis of its subgenomic RNA.

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen PNRSV isolates characterized in this study revealed that two of them showed a 12-nt deletion that led to the disappearance of the most proximal hairpin to the initiation site. Interestingly, the only hairpin found in these two isolates is very similar in primary and secondary structure to the one previously shown in Brome mosaic virus to be required for the synthesis of the subgenomic RNA. In this hairpin, the molecular diversity was concentrated mostly at the loop whereas compensatory mutations were observed at the base of the stem strongly suggesting its functional relevance. The evolutionary implications of these observations are discussed.

Detection and characterization of stone fruit virus diseases in Tunisia

A preliminary survey was conducted in Tunisia to identify stone fruit (almond, plum, peach) virus diseases occurring in orchards and mother block stands. Two ilarviruses, Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV) infecting almond and plum and a trichovirus, Apple chlorotic leaf spot virus (ACLSV) found in a peach orchard in northern Tunisia, were detected. These viruses were detected by means of herbaceous and woody indicators (cucumber, peach GF 305), serology (DAS-ELISA), electron microscopy (DIP, ISEM, Decoration) and reverse transcription-polymerase chain reaction-based assay (RT-PCR). Furthermore, typing PNRSV isolates was done by bioassays using various species and cultivars of Cucurbitaceae, serotyping with specific monoclonal antibodies (Mabs) in ELISA-DASI and RFLP analysis. Characterization work showed that serotyping with Mabs was the best mean to distinguish between PNRSV isolates.

SEROLOGICAL CHARACTERISATION OF MEDITERRANEAN PRUNUS NECROTIC RINGSPOT VIRUS ISOLATES

SUMMARY Prunus necrotic ringspot virus (PNRSV), a worldwide pathogen of stone fruits, has many isolates with different biological, serological, and molecular properties. Monoclonal antibodies (MAbs) to a peach isolate of PNRSV were used to investigate the serological variability of PNRSV isolates by DASI-ELISA. Isolates from different stone fruit species and different Mediterranean countries were first identified with polyclonal antisera (PAbs), then 81 isolates were selected from Albania (3), Cyprus (10), Greece (5), Italy (31), Lebanon (6), Malta (5), Tunisia (7), Turkey (7) and Spain (1) and characterised against 10 single MAbs. Six U.S. isolates were included as controls. The virus showed high serological variability as shown by the identification of 34 serogroups, many of which were host-specific (64%) and country-specific (67%).

Survey of Prunus necrotic ringspot virus in Rose and Its Variability in Rose and Prunus spp.

ABSTRACT A survey for viruses in rose propagated in Europe resulted in detection of only Prunus necrotic ringspot virus (PNRSV) among seven viruses screened. Four percent of cut-flower roses from different sources were infected with PNRSV. Progression of the disease under greenhouse conditions was very slow, which should make this virus easy to eradicate through sanitary selection. Comparison of the partial coat protein gene sequences for three representative rose isolates indicated that they do not form a distinct phylogenetic group and show close relations to Prunus spp. isolates. However, a comparison of the reactivity of monoclonal antibodies raised against these isolates showed that the most prevalent PNRSV serotype in rose was different from the most prevalent serotype in Prunus spp. All of the 27 rose isolates tested infected P. persica seedlings, whereas three of the four PNRSV isolates tested from Prunus spp. were poorly infectious in Rosa indica plants. These data suggest adaptation of PNRSV isolates from Prunus spp., but not from rose, to their host plants. The test methodologies developed here to evaluate PNRSV pathogenicity in Prunus spp. and rose could also help to screen for resistant genotypes.

Simultaneous Detection of the Three Ilarviruses Affecting Stone Fruit Trees by Nonisotopic Molecular Hybridization and Multiplex Reverse-Transcription Polymerase Chain Reaction

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Molecular Variability Among Isolates of Prunus Necrotic Ringspot Virus from Different Prunus spp.

ABSTRACT Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5' untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of the CP, the amino acid residues in this region, which were previously thought to be functionally important in the replication cycle of the virus, were strictly conserved. No clear correlation with the type of symptom or host specificity could be observed. The validity of this grouping was confirmed when other isolates recently characterized by other authors were included in these analyses.

Conformation polymorphism of RNA transcripts derived from various PNRSV isolates is affected by the temperature of electrophoresis

Polymorphism of heterologous duplexes of RNA transcripts of prunus necrotic ringspot virus isolates was studied at different temperatures of electrophoresis. Duplexes made from complementary RNA transcripts originating from four virus isolates differed in their electrophoretic mobilities. The differential mobilities of double-stranded RNA transcripts increased with increasing temperature of electrophoresis. The mobility of single-stranded RNA, on the other hand, though affected by temperature during electrophoresis, was not simply correlated with sequence divergence. This finding further supports the hypothesis that transcript conformation is the basis for the differential mobility phenomenon.