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

Seven 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.     

Prunus necrotic ringspot virus: incidence on stone and pome fruits and diversity analysis

Stone fruits and pome fruits are cultivated commercially worldwide. In India, they are grown in temperate regions, which mainly includes Jammu and Kashmir, Uttarakhand, Himachal Pradesh and some North-Eastern states. In this study, an attempt has been made to identify the Prunus necrotic ringspot virus (PNRSV) infecting stone and pome fruits in India and to characterise them on the molecular level. Surveys were conducted in the temperate fruit-growing areas and incidence of PNRSV was detected by serological and molecular means in almond, apple, cherry, nectarine, peach, plum and wild cherry. Further diversity analysis of PNRSV was performed using bioinformatics tools such as clustalW, DNA Data Bank of Japan, MultAlin and Recombination Detection Programme. PNRSV was detected in plum, peach, cherry, almond, nectarine, wild cherry and apple. In the diversity analysis study on the basis of coat protein gene, it was found that the isolates showed identity levels from 82 to 100%. In a plum isolate, a stretch of amino acids from 207 to 221 was found variable from Indian and other isolates. In one of the Indian apple isolates, “NR” repeats at 41–44 position (characteristic of PV-32 group, Group I) were identified. Phylogenetic analysis revealed that Indian isolates are falling in Group-I. Movement protein was also amplified from peach and multiple alignment studies showed that N-terminus was mostly conserved, whereas the C-terminal was highly variable.     

Studies on rose mosaic disease in field-grown roses produced in the United Kingdom

Arabis mosaic virus (AMV) and prunus necrotic ringspot virus (PNRSV), separately or together, caused in field-grown roses the range of symptoms recognised as rose mosaic disease. PNRSV infection alone generally induced chlorotic line patterns, ring-spots or mottles in the leaves at some time during the growing season; AMV plus PNRSV normally caused chlorotic vein-banding. However, during prolonged periods of high temperatures (c. 21 °C or more) vein banding occurred in some roses infected only with PNRSV. Isolates of PNRSV from rose had particles which were similar in shape, protein mol. wt, density and sedimentation coefficients to previously described isolates of PNRSV from cherry, plum and rose; all were cherry serotypes. In graft-inoculated roses, apple serotypes of PNRSV induced stunting and chlorosis, puckering and distortion of leaves, which closely resembled symptoms associated with rose mosaic in the USA and chlorotic mottle rose mosaic in New Zealand. To avoid possible confusion in using the name rose mosaic it is suggested that the virus(es) present in roses should be named.     

Biological and molecular characterization of <Emphasis Type="Italic">Prunus necrotic ringspot virus</Emphasis> isolates from three rose cultivars

Prunus necrotic ringspot virus (PNRSV) is a rose and stone fruit tree pathogen. Three different PNRSV isolates, originating from three rose cultivars were studied. These PNRSV isolates were characterized using molecular techniques. Nearly the complete nucleotide sequence (1,630 nucleotides) of RNA3 of the isolate PNRSV-R1 has been determined (GenBank Acc. No. DQ003584). The sequence of the MP gene of the PNRSV-R1 isolate was determined, the first such results for a rose-derived PNRSV isolate. The reaction of PNRSV infection on test plants was also investigated. Cucumis sativus cv. Wisconsin, Cucurbita maxima cv. Buttercup and Cucurbita pepo cv. Melonowa Żółta appeared to be the most useful test plants for the differentiation of isolate-specific pathogenicity.     

Molecular diagnosis of apple virus and viroid pathogens from India

Apple is known to be susceptible to various virus and viroid pathogens. Symptomatic apple cultivars and rootstocks were collected and analyzed by ELISA and then through RT-PCR. The study reports the presence of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), the major apple viruses and Prunus necrotic ringspot virus (PNRSV), a minor apple virus, at the molecular level in India. Apple scar skin viroid (ASSVd) infection was also confirmed at the molecular level. Sporadic incidences of Tomato ringspot virus and Arabis mosaic virus infections were also detected by ELISA in nursery plants.     

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

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.     

Detection of Prunus necrotic ring spot virus in plum,cherry and almond by serological and molecular techniques from India

Stone fruits are cultivated in the temperate and sub-temperate regions of India. During surveys in stone fruit growing areas, viral symptoms were observed in almond, cherry and plum. These samples were brought to the laboratory for further detection at serological and molecular levels to check the presence of virus. In the present study, incidence of PNRSV is reported on plum (Prunus domestica), almond (Prunus dulcis) and cherry (Prunus avium) using serological and molecular techniques. Coat protein gene of PNRSV was amplified from almond, cherry and plum. This is the first molecular evidence of PNRSV on these stone fruits reported from India.     

Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

A cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 10⁷ fold. The McAb belonged to IgG_2a isotype and was diluted by 1.28 × 10⁵ folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.     

Effect of viruses on agronomic and brewing characteristics of four hop (Humulus lupulus) cultivars in Australia

The effect of Hop latent virus (HpLV), Hop mosaic virus (HpMV), and Prunus necrotic ring spot virus [PNRSV (apple, A, and intermediate, I, serotypes)], on the survival of softwood cuttings, the vigour (height) of early season growth, cone yield, and the levels of brewing organic acids in mature plants, was assessed in four hop (Humulus lupulus) cultivars in Tasmania, Australia. Virus infections were associated with an increase in the mortality of softwood cuttings following propagation. In all cultivars, height of early growth was a poor indicator of the effect of viruses on cone yield and levels of brewing organic acids (alpha and beta acids). In cv. ‘Nugget’, infection by the virus combinations studied was not associated with reductions in cone yield, however plants infected by PNRSV‐I, in 2000, had 11% lower alpha acids and 7% lower beta acids. In ‘Opal’, infection by HpLV and HpMV were the most deleterious to cone yield, however the effect of HpMV was ameliorated when in combination with PNRSV‐I. Reductions in alpha and beta acid content were attributable only to mixed infections of HpLV + HpMV in combination with either serotype of PNRSV. In ‘Pride of Ringwood’, yield loss was mostly attributable to HpMV and to a lesser extent, HpLV. Some ameliorations in cone yield loss occurred in plants containing a mixed infection between HpMV and HpLV or either of the ilarvirus serotypes. Both of the ilarviruses and HpMV caused reductions in alpha acid content. In ‘Victoria’, cone yield loss was mostly attributable to combinations of viruses such as HpLV + PNRSV‐I and HpLV + HpMV. The deleterious effect of HpLV + HpMV was ameliorated by PNRSV‐A and to a lesser extent, PNRSV‐I. Infection by the virus combinations studied did not significantly affect alpha and beta acid levels in either year. Results suggested the effect of viruses and their combinations differed between cultivars and varied between seasons. This information, when combined with knowledge of the rates of virus re‐infection, can be used to recommend control strategies for the Australian hop industry.     

An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus‐induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense‐orientated target gene sequence of 100‐200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV‐based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E‐knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector‐mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees.     

Rootstock‐to‐scion transfer of transgene‐derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus‐resistant transgenic rootstocks developed through siRNA‐mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock‐to‐scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV‐hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV‐hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long‐distance (1.2 m) transfer of PNRSV‐hpRNA‐derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for ‘using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.     

Molecular Evidence for Recombination in <Emphasis Type="Italic">Prunus</Emphasis> Necrotic Ringspot Virus

Genetic RNA recombination plays an important role in viral evolution. The evolutionary history of Prunus necrotic ringspot virus (PNRSV) has been extensively studied, but knowledge of recombination in its genome is still lacking. To investigate the recombination events in this virus, 67 accessions composed by 62 isolates retrieved from the databanks and five Tunisian isolates described in this study were analyzed. The use of RECCO algorithm which is based on cost minimization allowed us to detect several breakpoints in the coat protein gene (CP) of three out of five isolates from Tunisia and one from Poland. Moreover, a recombination signal was also detected in the putative cell-to-cell movement protein-encoding gene of an isolate from the USA. Tajima Neutrality test implemented in MEGA4 program indicated the occurrence of a high level of deletion/insertion events in the sequences. The evolutionary historical relationships were determined by constructing a dendrogram using neighbor joining, minimum evolution, maximum parsimony, maximum likelihood, and unweighted pair group method with arithmetic mean (UPGMA). The first four analyses gave similar results. Three classical groups (PE 5, PV 32, and PV 96) were delineated. The recombinant isolates from Tunisia clustered in a distinct clade except for one nonrecombinant (Ghernghezel) which revealed to be a member of PV 32 group. In contrast, UPGMA algorithm divided the Tunisian isolates in three distinct subgroups. Apart from recombination, reassortment is still an open question among many others and may also represent another way to explore the genetic diversity of PNRSV.     

Apple mosaic virus isolated from hop plants in Japan

A strain of apple mosaic virus was isolated from hop plants in Japan. The virus was purified from young hop plants and back-inoculated to virus-free hop plants obtained by meristem tip culture. Inoculated plants developed chlorotic spots, ringspots and a band pattern accompanied by necrosis in the inoculated and systemically infected leaves. Shoot tips of infected plants sometimes became necrotic and these symptoms resembled those of a ring- and band-pattern mosaiclike disease prevalent in hop gardens in Japan. Since apple mosaic virus was recovered from infected plants, it is likely that the virus was the causal agent of this disease. Agar gel double diffusion tests and ELISA showed the hop virus to be serologically closely related to apple mosaic virus (ApMV), and distantly related to prunus necrotic ringspot virus (PNRSV). The virus had a narrow host range, and infected only cucumber of 18 species of Cucurbitaceae, Chenopodiaceae, Leguminosae or Solanaceae inoculated. It produced chlorotic spots on the inoculated cotyledons of cucumber, but no systemic infection. By contrast, ApMV from apple and PNRSV from peach had wide host ranges and infected cucumber plants systemically.     

Molecular adaptation within the coat protein-encoding gene of Tunisian almond isolates of Prunus necrotic ringspot virus

The sequence alignments of five Tunisian isolates of Prunus necrotic ringspot virus (PNRSV) were searched for evidence of recombination and diversifying selection. Since failing to account for recombination can elevate the false positive error rate in positive selection inference, a genetic algorithm (GARD) was used first and led to the detection of potential recombination events in the coat protein-encoding gene of that virus. The Recco algorithm confirmed these results by identifying, additionally, the potential recombinants. For neutrality testing and evaluation of nucleotide polymorphism in PNRSV CP gene, Tajima’s D, and Fu and Li’s D and F statistical tests were used. About selection inference, eight algorithms (SLAC, FEL, IFEL, REL, FUBAR, MEME, PARRIS, and GA branch) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of PNRSV capsid. Inferred phylogenies pointed out, in addition to the three classical groups (PE-5, PV-32, and PV-96), the delineation of a fourth cluster having the new proposed designation SW6, and a fifth clade comprising four Tunisian PNRSV isolates which underwent recombination and selective pressure and to which the name Tunisian outgroup was allocated.     

Detection and Molecular Variability of Apple Stem Grooving Virus in Shaanxi,China

Apple stem grooving virus (ASGV) is one of the economically important latent viruses that are distributed in apple production areas worldwide. The presence of ASGV in apple trees was studied by serological assay and molecular biology methods. A total of 550 apple leaf samples from 14 different areas in Shaanxi were tested by DAS‐ELISA, and the results revealed an ASGV infection level of 55%. Those samples were also examined by RT‐PCR, and an infection level of 67% was found. Fourteen complete coat protein gene sequences of ASGV were obtained; phylogenetic analysis revealed that these 14 sequences separated into two clusters regardless of the geographic origin or host plants. To our knowledge, this is the first report of molecular variability analysis of ASGV in apple trees in China.     

Responses of micropropagated peach cultivars to thermotherapy for the elimination of prunus necrotic ringspot virus

Peach (Prunus persica (L.) Batsch) shoot-cultures infected with prunus necrotic ringspot virus (PNRSV) were selected for evaluating the responses of in vitro grown shoots of cvs Hermosa and Summerset to thermotherapy. The survival of shoot-cultures during thermotherapy was improved by selection of the optimum concentration of 6-benzylamino purine in the medium and optimum age of shoots for treatment. Alternating high and low temperature thermotherapy regimes were more effective in decreasing virus titre than constant high temperatures. Of the regimes tested, the most effective inhibition of PNRSV combined with a high survival of shoots was obtained by applying 38°C for 16 h in light alternating with 28 ^CC for 8 h in darkness for 18 days for Hermosa and 22 days for Summerset. Following this treatment 90% of Hermosa and 40% of Summerset shoot-cultures were virus-free as determined by enzyme-linked immunosorbent assay. Relatively large (about 10 mm) apices excised from these shots regenerated into virus-free plants. The advantage of the in vitro system for thermotherapy is discussed.     

The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian apple R12740-7A map to the same linkage group of apple

Russian apple R12740-7A is the designation for an accession grown from seed collected in Russia, which was found to be highly resistant to apple scab. The resistance has historically been attributed to a naturally pyramided complex involving three major genes: one race-nonspecific gene, Vr, conditioning resistance to all known races, plus two race-specific genes. The race-nonspecific gene was identified as an independently segregating gene by Dayton and Williams (1968) and is referred to in this paper as Vr-DW. The first researchers to study the scab resistance gene complex in Russian apple never described the phenotype conditioned by the race-nonspecific gene. Later, Aldwinckle et al. (1976) associated the name Vr with a scab resistance gene conditioning distinctive stellate necrotic reactions, which we refer to as Vr-A in order to distinguish it from Vr-DW. We show that the segregation ratios in progenies from the scab differential hosts 2 and 4 that are derived from Russian apple, crossed with susceptible cultivars were consistent with a single gene conditioning resistance in each host. The genes have been named Vh2 and Vh4, respectively. Resistant segregants from host 2 showed stellate necrotic reactions, while those from host 4 showed hypersensitive reactions. Both the phenotypes and the genetic maps for the genes in the respective hosts were very similar to those of the genes previously named Vr-A and Vx, respectively, in an F1 family of Russian apple. We showed that race 2 of V. inaequalis isolated from host 2 was able to infect resistant descendants of the non-differential accession PRI 442-23 as well as host 2. The descendants of PRI 442-23 were expected to carry the race-nonspecific Vr-DW gene, but in fact carry Vr-A. We conclude that the Vh2 gene in host 2 and Vr-A are the same, and that the Vh4 gene in host 4 and Vx are the same. However, a major finding of this study is that the latter gene mapped to linkage group 2 of apple instead of linkage group 10 as suggested from previous research. With the two race-specific genes from Russian apple defined now, we discuss the nature of the race-nonspecific Vr-DW gene in this accession. We also report the identification of a new scab resistance gene, VT57, from either Golden Delicious or Red Dougherty, which conditions chlorotic resistance reactions and is linked to Vh2.