Necrotic Ringspot Virus Research Articles

Detection and partial molecular characterization of Plum pox virus on almond trees in Turkey

Almond (Prunus dulcis) is one of the well known stone fruit species grown for its unripe fruits and delicious seeds in Turkey. In the Trakya region, however, some prevailing virus infections have reduced almond yields and quality. In ten districts of Trakya, 260 leaf samples were collected from affected almond trees in June 2010. DAS–ELISA assays and RT-PCR tests were employed for the identification of viruses. As a result of these detection studies, five of the 260 leaf samples gathered from symptomatic almond trees had Plum pox virus (PPV), 81 of them had Prunus necrotic ringspot virus (PNRSV), and 11 samples contained Prune dwarf virus (PDV). Only four out of 260 samples had a mixture of these viruses. Partial nucleotide sequences of five almond isolates of PPV were determined and compared with 17 other PPV isolates in databases. Computer analysis of obtained and published nucleotide sequences showed identity ranged from 75.72% to 96.87%. Of the five PPV almond isolates obtained, however, there was a close nucleotide identity of 95.82–96.61% to Turkish isolates. Phylogenetic analysis of nucleotides and amino acids showed that five PPV isolates of almond from the Trakya Region of Turkey were clustered in the same subgroup with PPV-T Turkish isolates in GenBank. Therefore we can consider almond isolates of PPV as PPV-T strain, like the two other isolates from apricot trees in Turkey.

Identification and characterization of Lisianthus necrotic ringspot virus, a novel distinct tospovirus species causing necrotic disease of lisianthus (Eustoma grandiflorum)

In 2007, lisianthus (Eustoma grandiflorum) plants with necrotic ringspots on the leaves were found in Kochi Prefecture, Japan. Tospovirus-like spherical enveloped particles with ca. 160 nm in diameter were observed with electron microscopy. The complete nucleotide sequence of the S RNA segment of the virus was determined, and phylogenetic analysis using deduced amino acid sequences of the nucleocapsid protein and the nonstructural S protein indicated that the virus is phylogenetically distinct from any known tospovirus species. The results suggest that the virus is a new member of the genus Tospovirus, in the family Bunyaviridae. The virus is the fifth distinct tospovirus occurring naturally in lisianthus in Japan. The necrotic symptoms were reproduced on lisianthus seedlings after mechanical inoculation. The host range of the virus isolate on several test plants was also examined.

First Report of Cherry necrotic rusty mottle virus Infecting Sweet Cherry Trees in Korea.

Cherry necrotic rusty mottle virus (CNRMV), an unassigned member in the family Betaflexiviridae, has been reported in sweet cherry in North America, Europe, New Zealand, Japan, China, and Chile. The virus causes brown, angular necrotic spots, shot holes on the leaves, gum blisters, and necrosis of the bark in several cultivars (1). During the 2012 growing season, 154 sweet cherry trees were tested for the presence of CNRMV by RT-PCR. Samples were randomly collected from 11 orchards located in Gyeonggi and Gyeongsang provinces in Korea. RNA was extracted from leaves using the NucliSENS easyMAG system (bioMérieux, Boxtel, The Netherlands). The primer pair CGRMV1/2 (2) was used to amplify the coat protein region of CNRMV. Although none of the collected samples showed any notable symptoms, CNRMV PCR products of the expected size (949 bp) were obtained from three sweet cherry samples from one orchard in Gyeonggi province. The PCR products were cloned into a pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of the three Korean sequences obtained (GenBank Accession Nos. AB822635, AB822636, and AB822637) showed 97% nucleotide sequence identity with a flowering cherry isolate from Japan (EU188439), and shared 98.8 to 99.6% nucleotide and 99.6 to 100% amino acid similarities to each other. The CNRMV positive samples were also tested for Apple chlorotic leaf spot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV-1), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. One of the three CNRMV-positive samples was also infected with CVA. To confirm CNRMV infection by wood indexing, Prunus serrulata cv. Kwanzan plants were graft-inoculated with chip buds from the CNRMV-positive sweet cherry trees. At 3 to 4 weeks post-inoculation, the Kwanzan plants showed quick decline with leaves wilting and dying; CNRMV infection of the indicators was confirmed by RT-PCR. To our knowledge, this is the first report of CNRMV infection of sweet cherry trees in Korea. Screening for CNRMV in propagation nurseries should minimize spread of this virus within Korea.

국내 양앵두나무에서 발생한 Cherry green ring mottle virus 동정

2012년 국내 양앵두나무에서 발생하는 CGRMV를 조사하기 위해 화성, 평택, 경주, 김천, 대구, 영주 음성의 양앵두 재배과원 7개 지역에서 잎 시료 154점을 채집하였다. 채집한 시료에 대해 CGRMV 유전자 검정을 수행한 결과 6점의 시료에서 807 bp 크기의 PCR 증폭산물이 검출되었다. 이들 PCR 증폭산물은 클로닝과 유전자 염기서열 분석 결과 GeneBank에 등록된 외국의 CGRMV 분리주들과 88% 이상의 외피단백질 유전자 염기서열 상동성을 보였다. 국내 양앵두나무에서 분리된 분리주들, CGR-KO 1-6 간에는 98.8-99.8%의 염기서열 및 99.6-100%의 아미노산 서열 상동성을 나타내었다. 국내 CGRMV 분리주들은 외피단백질 유전자 계통도 분석에서 기존에 분류된 I, II, III 그룹 중 II 그룹에 속하였다. 또한 CGRMV가 감염된 국내 양앵두나무 이병주들은 ACLSV 등 10종 바이러스에 대해서도 RT-PCR을 수행한 결과 모든 시료에서 ACLSV가 검출되어 CGRMV와 ACLSV가 복합 감염된 것을 확인하였다. During the 2012 growing season, 154 leaf samples were collected from sweet cherry trees in Hwaseong, Pyeongtaek, Gyeongju, Kimcheon, Daegu, Yeongju and Eumseong and tested for the presence of Cherry green ring mottle virus (CGRMV). PCR products of the expected size (807 bp) were obtained from 6 samples. The PCR products were cloned and sequenced. The nucleotide sequences of the clones showed over 88% identities to published coat protein sequences of CGRMV isolates in the GenBank database. The sequences of CGRMV isolates, CGR-KO 1-6 shared 98.8 to 99.8% nucleotide and 99.6 to 100% amino acid similarities. Phylogenetic analysis indicated that the Korean CGRMV isolates belong to the group II of CGRMV coat protein genes. The CGRMV infected sweet cherry trees were also tested for Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Cherry necrotic rusty mottle virus (CNRMV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV1), Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. All of the tested trees were also infected with ACLSV.

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.

First report of Prunus necrotic ringspot virus infecting bindweed in Iran.

Prunus necrotic ringspot virus (PNRSV) affects rosaceous plants worldwide. In 2011 and 2012, 50 symptomatic bindweeds (Convolvulus arvensis) were collected from rose gardens in Alburz, Mazandaran and Tehran provinces of Iran. Samples were tested for the presence of PNRSV using DAS-ELISA and dot-immunobinding assay (DIBA) with a polyclonal antiserum (Agdia, USA). PNRSV was detected in 38% of the samples with symptoms such as line pattern, mottle and marginal necrosis in all visited regions. Five isolates from bindweed plants were used for bioassays on cucumber (Cucumis sativus) after single lesion transfer on cowpea (Vigna unguiculata). Inoculated cowpea plants developed chlorotic spotting on the young leaves and cucumber plants reacted with systemic mosaic, vein banding, and malformations. To confirm the presence of PNRSV in diseased plants, total RNA was extracted from symptomatic bindweed and herbaceous plants with the RNeasy Plant Mini Kit (Vivantis, Malaysia) and analyzed by RT-PCR using the PNRSV-specific primers Ilar 1 and Ilar 2 (Moury et al., 2000). The expected 210 bp fragment was amplified by RT-PCR from samples of symptomatic tissues while no amplification products were obtained when water or total RNA from symptomless plants were used as template. In Iran, PNRSV was first detected in rose samples from Tehran province (Rakhshandehroo et al., 2006). To the best of our knowledge, this is the first report of PNRSV in Convolvulus arvensis in Iran.

Identification, characterisation and detection of a new tospovirus on sweet pepper

A putative virus-induced disorder showing mottling and deformation on leaves and fruits of sweet pepper was observed in Taiwan in 2009. Crude sap of symptomatic sweet pepper leaves reacted positively in enzyme-linked immunosorbent assay (ELISA) with antisera to tospoviruses in the Watermelon silver mottle virus (WSMoV) serogroup. A virus culture, TwPep3, isolated from the symptomatic sweet pepper revealed isometric virions measuring about 80–100 nm in diameter via electron microscopy. Seedlings of sweet pepper inoculated with TwPep3 showed similar symptoms as those observed in the field. A 0.9-kb viral genome of isolate TwPep3 could be amplified using degenerate primers for L RNA conserved region of tospoviruses but not using those of tobamoviruses, potyviruses and Cucumber mosaic virus. The partial L RNA sequence of isolate TwPep3 (KF383955) shared 75.1% and 82.4% nucleotide identities with those of WSMoV and Tomato necrotic ringspot virus (TNRV), respectively. To determine the taxonomic relationship of isolate TwPep3, the full length of S RNA (KF383956) was sequenced. Sequence analyses showed that the nucleocapsid (N) gene of TwPep3 shared 82–83% and 87.9–89% nucleotide and amino acid sequence identities, respectively, with those of five isolates of TNRV, the most closely related tospovirus. Moreover, the NSs proteins and S RNAs of TwPep3 and TNRV shared only 78.7% and 77.6% amino acid and nucleotide sequence identities, respectively. Based on our results, isolate TwPep3 should be considered as a new tospovirus and we propose the name Pepper chlorotic spot virus (PCSV). A specific primer pair designed in the N gene was successfully used in reverse transcription-polymerase chain reaction for PCSV diagnostic work.

First Report of Prune dwarf virus and Prunus necrotic ringspot virus on Peach in Montenegro.

In September and October 2011, samples were collected from mature peach trees (~17 years old) exhibiting symptoms of chlorotic rings and spots, vein clearing, mosaic, necrosis, leaf distortion, stunting, and rosette formation in a major commercial orchard (~80 ha) near Podgorica, Montenegro. Samples were collected from nine different peach varieties (cvs. Adriana, Caldesi, Gloria, Maria Marta, May Crest, Morsiani, Rita Star, Spring Belle, and Spring Crest). Samples (n = 58) were tested using DAS-ELISA for the presence of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV). Commercial positive and negative controls were included in each ELISA (antisera and controls supplied by BIOREBA AG, Reinach, Switzerland). Only one symptomatic sample from cv. Gloria tested positive for PDV (sample reference: 399/11), a further 11 samples (cvs. Rita Star [six], May Crest [four] and Spring Crest [one]) were positive for PNRSV. Samples were also tested for Plum pox virus (PPV) by real-time RT-PCR (1). The PDV positive sample (399/11) showing mosaic was in mixed infection with PPV, as were 6 of the 11 PNRSV samples, including sample 373/11 with yellow mottling and leaf distortion symptoms. On single-infected PNRSV, sample 368/11 chlorotic line patterns and leaf deformations were observed. To confirm the presence of PDV and PNRSV, positive samples were also tested by RT-PCR. Total RNA was extracted using RNeasy Plant Mini kit (Qiagen, Hilden, Germany). RT-PCR was performed with primer pairs PDV2F/PDV1R (3) and MG1/MG2 (2) specific for PDV and PNRSV, respectively. Amplicons of the expected size, 173 bp for PDV and 675 bp for PNRSV, were obtained from corresponding ELISA-positive samples. Amplified products from three samples (PDV 399/11 and PNRSV 368/11 and 373/11) were cloned into pGEM-T Easy Vector (Promega, Madison, WI) then sent for sequence analysis (MWG-Biotech AG, Edersberg, Germany). Sequence data was compared to sequences published in GenBank. Analysis of sequence obtained from isolate 399/11 (cv. Gloria) corresponded to partial CP gene of PDV, with a high degree of similarity to isolates reported from other parts of the world ranging from 94.2 to 95.9%, showing highest similarity with isolate Ch 137 (L28145). Sequence analyses of CP gene from PNRSV isolates 368/11 (JX569825) and 373/11 (JX569826) proved to be 89.3 to 99.7% identical with corresponding sequences of isolates previously described. In particular, the Montenegrin PNRSV isolates were most closely related to Chilean NctCl.augl isolate from nectarine (EF565253). To demonstrate that the virus was infectious, seedlings of peach cv. GF305 were side grafted with bud-woods from PDV (sample 399/11) and PNRSV-positive samples (samples 368/11 and 373/11) and a healthy control sample. Grafted seedlings were kept in a greenhouse with a under 16-h light regime at 22 to 24°C and observed for symptom development. No symptoms were observed in grafted plants with the healthy control. All plants inoculated with virus-positive samples exhibited stunted vegetation and mild mottle with no difference in symptoms between the two viruses. Indicator plants of peach cv. GF305 inoculated with PPV dual-infected samples (399/11 and 373/11) were subsequently shown to be positive for PPV by real-time RT-PCR. Subsequent DAS-ELISA test on samples from experimentally inoculated trees using specific antisera as described above confirmed PDV and PNRSV infections as expected. These viruses have recently been reported from sour cherry (Prunus cerasus L.) in Serbia (4), ~600 km to the northeast. However, to our knowledge, this is the first report on the occurrence of PDV and PNRSV in Montenegro.

First Report of Rose yellow vein virus in Rosa sp. in New Zealand.

Rose is the top selling cut flower in New Zealand and is the most popular garden plant in the world. Several virus-like diseases have been described in roses, but the causal agents for many remain unknown. Most of the described viruses infecting rose belong to the genera Ilarvirus and Nepovirus. Only recently, a number of new viruses have been or are in the process of being characterized (1,2,3,4). In January 2011, 10 rose samples showing virus-like symptoms were collected from the Wanganui region on the North Island of New Zealand. Total nucleic acid was extracted from these samples using an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). PCR and reverse transcription (RT)-PCR was conducted using specific primers for Arabis mosaic virus (ArMV), Cherry leaf roll virus, Prunus necrotic ringspot virus (PNRSV), Rosa rugosa leaf distortion virus, Rose spring dwarf associated virus, Rose yellow leaf virus, Rose yellow mosaic virus, Rose yellow vein virus (RYVV), and Strawberry latent ringspot virus. Samples were also tested using generic primers for carlavirus, potexvirus, potyvirus, tombusvirus, and phytoplasmas. Two samples (cvs. Pauls Himalayan Musk and Bloomfield) were positive for ArMV, four samples (cvs. Leda, Rosa Mundi, Charles de Mills, and Indica Major) were positive for PNRSV, and two samples (cvs. Leda and Zephirine Drouhin) were positive for RYVV. Samples were negative for all other tested viruses and phytoplasmas. RYVV was detected using two sets of primers (D. Mollov, personal communication) designed to amplify fragments of estimated sizes of 797 bp and 684 bp of the movement protein (MP) and coat protein (CP) genes of RYVV, respectively. RYVV amplicons were sequenced directly (GenBank Accession Nos. JX887423 to JX887426). A BLASTn search of the MP and CP fragments showed the highest nucleotide identity of 98% and 96 to 97%, respectively, with the type isolate of RYVV (JX028536). RYVV has been reported as the causal agent of a vein yellowing disease in rose (2). Symptoms observed in the 'Leda' sample infected with PNRSV and RYVV (vein yellowing and chlorotic mottle in the apex of leaves) were not typical of PNRSV, so they may be caused by RYVV. Symptoms in samples of cv. Zephirine Drouhin (curling of leaves and mottle), observed in both RYVV-positive and -negative samples, may not be associated with RYVV infection. This suggests that vein yellowing may be influenced by cultivar. RYVV has been reported in several rose cultivars, but only in the United States (2). To the best of our knowledge, this is the first report of RYVV infecting rose in New Zealand, where it is likely that the virus has been present for some time. The virus may have a much wider geographical distribution than that reported as the virus was only recently characterized (3).

Occurrence of Stone Fruit Viruses in Plum Orchards in Latvia

To evaluate the occurrence of nine viruses infecting Prunus a large-scale survey and sampling in Latvian plum orchards was carried out. Occurrence of Apple mosaic virus (ApMV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), and Plum pox virus (PPV) was investigated by RT-PCR and DAS ELISA detection methods. The detection rates of both methods were compared. Screening of occurrence of Strawberry latent ringspot virus (SLRSV), Arabis mosaic virus (ArMV), Tomato ringspot virus (ToRSV) and Petunia asteroid mosaic virus (PeAMV) was performed by DAS-ELISA. In total, 38% of the tested trees by RT-PCR were infected at least with one of the analysed viruses. Among those 30.7% were infected with PNRSV and 16.4% with PDV, while ApMV, ACLSV and PPV were detected in few samples. The most widespread mixed infection was the combination of PDV+PNRSV. Observed symptoms characteristic for PPV were confirmed with RT-PCR and D strain was detected. Comparative analyses showed that detection rates by RT-PCR and DAS ELISA in plums depended on the particular virus tested. The results obtained in this study revealed that commonly grown plum cultivars in Latvia are infected with economically important stone fruit viruses and highlight the need to implement a programme to produce and propagate virus-free planting material.

A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach.

Molecular characterization and intermolecular interaction of coat protein of Prunus necrotic ringspot virus: implications for virus assembly

Coat protein (CP) and RNA3 from Prunus necrotic ringspot virus (PNRSV-rose), the most prevalent virus infecting rose in India, were characterized and regions in the coat protein important for self-interaction, during dimer formation were identified. The sequence analysis of CP and partial RNA 3 revealed that the rose isolate of PNRSV in India belongs to PV-32 group of PNRSV isolates. Apart from the already established group specific features of PV-32 group member's additional group-specific and host specific features were also identified. Presence of methionine at position 90 in the amino acid sequence alignment of PNRSV CP gene (belonging to PV-32 group) was identified as the specific conserved feature for the rose isolates of PNRSV. As protein-protein interaction plays a vital role in the infection process, an attempt was made to identify the portions of PNRSV CP responsible for self-interaction using yeast two-hybrid system. It was found (after analysis of the deletion clones) that the C-terminal region of PNRSV CP (amino acids 153-226) plays a vital role in this interaction during dimer formation. N-terminal of PNRSV CP is previously known to be involved in CP-RNA interactions, but our results also suggested that N-terminal of PNRSV CP represented by amino acids 1-77 also interacts with C-terminal (amino acids 153-226) in yeast two-hybrid system, suggesting its probable involvement in the CP-CP interaction.

A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by prunus necrotic ringspot virus and peach latent mosaic viroid

BackgroundMicroarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees.ResultsUpon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection.ConclusionsOur results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10−4) and RNA (10−3, except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.

Genomic Segments RNA1 and RNA2 of Prunus necrotic ringspot virus Codetermine Viral Pathogenicity to Adapt to Alternating Natural Prunus Hosts

Prunus necrotic ringspot virus (PNRSV) affects Prunus fruit production worldwide. To date, numerous PNRSV isolates with diverse pathological properties have been documented. To study the pathogenicity of PNRSV, which directly or indirectly determines the economic losses of infected fruit trees, we have recently sequenced the complete genome of peach isolate Pch12 and cherry isolate Chr3, belonging to the pathogenically aggressive PV32 group and mild PV96 group, respectively. Here, we constructed the Chr3- and Pch12-derived full-length cDNA clones that were infectious in the experimental host cucumber and their respective natural Prunus hosts. Pch12-derived clones induced much more severe symptoms than Chr3 in cucumber, and the pathogenicity discrepancy between Chr3 and Pch12 was associated with virus accumulation. By reassortment of genomic segments, swapping of partial genomic segments, and site-directed mutagenesis, we identified the 3' terminal nucleotide sequence (1C region) in RNA1 and amino acid K at residue 279 in RNA2-encoded P2 as the severe virulence determinants in Pch12. Gain-of-function experiments demonstrated that both the 1C region and K279 of Pch12 were required for severe virulence and high levels of viral accumulation. Our results suggest that PNRSV RNA1 and RNA2 codetermine viral pathogenicity to adapt to alternating natural Prunus hosts, likely through mediating viral accumulation.

Relationship of viruses and viroids with apricot “viruela” disease

In this study, 34 apricot orchards in south-eastern Spain planted with the Spanish cultivar ‘Bulida’ and showing “viruela” symptoms were studied for 2 years. Leaf and fruit samples from four trees in each orchard, either with or without “viruela” symptoms, were collected and analysed by multiplex RT-PCR for the detection of American plum line pattern virus (APLPV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Apricot latent virus (ApLV), Plum bark necrosis and stem pitting-associated virus (PBNSPaV), Plum pox virus (PPV), Prune dwarf virus (PDV), and Prunus necrotic ring spot virus (PNRSV). In addition, molecular hybridization assays were performed for the detection of Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). All fruits showing “viruela” symptoms were infected with ACLSV and HSVd, suggesting that these pathogens may be responsible for “viruela” disease. Other viruses including PNRSV, PBNSPaV, ApLV, PDV, ApMV and PPV, were detected to a lesser degree. Detection of ACLSV and HSVd in samples without symptoms could be explained by the influence of environmental conditions and/or the physiological stage of fruits on the expression of symptoms.

Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi‐mediated silencing

Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, 'Gisela 6' (GI 148-1) and 'Gisela 7'(GI 148-8)', which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic 'Gisela 6' exhibited no symptoms but a significant PNRSV titre, while the transgenic 'Gisela 6' had no symptoms and minimal PNRSV titre. The nontransgenic 'Gisela 7' trees died, while the transgenic 'Gisela 7' trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes.

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.

A survey of viruses infecting Rosa spp. in New Zealand

A survey was carried out to confirm which viruses infecting Rosa spp. are present in New Zealand and to determine the extent of their distribution. Eighty nine rose samples were collected from different regions across New Zealand and from various sources as follows, private and public gardens, a commercial glasshouse and native bush. The samples were tested by RT-PCR for 17 viruses known to infect roses. Of the 89 samples tested, 48 % were infected with Rose cryptic virus-1, 22 % with Prunus necrotic ringspot virus, 20 % with Rose spring dwarf-associated virus, 10 % with Rose yellow vein virus, 2 % with Arabis mosaic virus and 35 % of the samples tested negative. The viruses were detected in some samples as single infections and sometimes as mixed infections (2 or 3 viruses together). Viruses were detected in all regions sampled in both the North and South Islands of New Zealand. Rose cryptic virus-1 and Rose spring dwarf associated virus were detected for the first time in New Zealand and Rose yellow vein virus, which had only been recently detected in 2011, was found to be widespread. Viral symptoms included leaf chlorosis and chlorotic line patterns; leaf mottling and puckering; vein clearing and banding; misshapen and balled (rosette) leaves and flower break.

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

AbstractA 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 IgG2a 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.