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In order to fill in a decade-long information gap regarding the biological, serological and molecular data for plum tree viruses in Bosnia and Herzegovina, a three-phase study combining symptom evaluation, and serological and molecular assays with high-throughput sequencing (HTS) technology was conducted. The most frequently observed symptoms were discolorations in the form of ring patterns, bands and irregular shapes, as well as vein banding. Sharka-associated symptoms in the form of ring patterns and semicircles were prevalent. A total of 468 plum tree samples were tested by ELISA for the presence of PPV, ApMV, PDV, PNRSV, PBNSPaV, ACLSV and MLRSV. An overall infection incidence of 51.9% was detected, with PPV being the most prevalent (48.7%), followed by PDV (2.99%), PNRSV (0.21%) and mixed infections of PPV+PDV (1.71%). RT-PCR-assisted strain typing in 45 samples revealed PPV-D as the most common strain (22.22%), followed by PPV-REC (6.66%). Mixed infections of PPV-D+PPV-REC were detected (6.66%). HTS enabled the recovery of a 9743 nts long sequence of PPV-D (PPV_O7/80, MW412433), which shared the highest nucleotide and amino acid identities with isolates S13 (LC375131) from Serbia, SVN1 (LC375132) from Slovenia and N9 (LC375129) from Bulgaria. The phylogenetic analysis of the whole genome placed the isolate of the D strain in a distinctive group with the Slovenian isolate SVN1 (LC375132). In addition, the (Cter)NIb/(Nter)CP fragment of a PPV-REC isolate (MW412434) obtained in this survey formed a separate group with previously known isolates from Bosnia and Herzegovina (BOS64Pl and BOS257Pl). European Cooperation in Science and Technology

The impact of plum pox virus (PPV) on sour cherry (Prunus cerasus L.) productivity has been studied by comparing the yield of PPV-infected and PPV-free fruit-bearing trees. A total of 152 16- to 17-year-old trees of nine cultivars and hybrids were surveyed in the production orchards (cultivar collection and hybrid testing plots) in the Republic of Tatarstan, Russia. Sixty trees tested positive for PPV using ELISA and RT-PCR. Among them, 58 PPV isolates belonged to the strain C and the other 2 isolates to the strain CV. For the cultivars Sevastyanovskaya, Shakirovskaya, hybrids 88-2 and 80-8, the average (2012 to 2019) productivity of infected trees was 38% to 45% lower than for PPV-free trees of the same cultivar or hybrid. No ilarviruses (prunus necrotic ringspot virus, prune dwarf virus, apple mosaic virus, American plum line pattern virus) were detected in PPV-infected trees, suggesting that reduced cherry productivity was attributed to the PPV infection. Thus, it was shown for the first time that PPV can reduce the productivity of at least some sour cherry cultivars and hybrids, and strain C isolates are responsible for crop losses.

To investigate the virus infections of sour and sweet cherries, various locations of Niğde province were examined during 2017. Ninety sweet and sour cherry leaf samples showing suspicious virus symptoms were collected and screened with virus-specific primers: Little cherry virus 1 (LChV1), Cherry necrotic rusty mottle virus (CNRMV), Prune dwarf virus (PDV), Prune necrotic ring spot virus (PNRSV), Apple mosaic virus (ApMV), Cherry green ring mottle virus (CGRMV), Cherry leaf roll virus (CLRV), Cherry mottle leaf virus (CMLV), Plum bark necrotic stem pitting associated virus (PBNSPaV), Cherry twisted leaf virus (CTLV), Apple stem grooving virus (ASGV), Little cherry virus 2 (LChV2), Cherry rusty leaf virus (CRLV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV). Based on RT-PCR analysis, no amplification was observed except LChV1 amplifications, dsRNA analysis resulted in one suspicious profile. To validate those results, more sensitive TaqMan Real-Time PCR analysis and sequence analysis were conducted and LChV1 was detected on 7 samples. It can be concluded that only a low quantity of LChV1 infections was observed on some of the screened cherry samples.

The aim of the present study was to determine whether chemotherapy with ribavirin can be used to eliminate Prune dwarf virus (PDV) from in vitro grown plants of infected sweet cherry cultivars ‘Tamara’ and ‘Amid’. Initial in vitro cultures were grown in Erlenmeyer flasks with 25 mL of MS medium (Murashige and Skoog, 1962), to which 1.5 mg L(‑1) BAP (6-benzylaminopurine) had been added. All cultures were cultivated in controlled environment chambers at 22±1°C. As soon as enough shoots were developed, individual shoots (5-10 mm in length) were excised from stock collections and transferred to treatment media with ribavirin. Ribavirin was added in concentration of 20 mg L(‑1) after autoclaving to the same MS medium as for multiplication. After a subculture period of 4 weeks, the apical part of the axis (about 3 mm in length comprising the apical meristem plus two-three primordial leaves) was dissected under a laminar flow hood and transferred to a fresh multiplication MS medium with 1.5 mg L(‑1) BAP for regeneration. The chemotherapy technique was able to eliminate PDV in high percentage according to the cultivar, ranging between 100.0 and 96.4% for cultivars ‘Tamara’ and ‘Amid’, respectively. In the course of chemotherapy cycles, ribavirin concentrations of 20 mg L(‑1), ‘Amid’ plants did not display symptoms of phytotoxicity and appeared vigorous and healthy. In contrary, 35% of ‘Tamara’ in vitro plants did not survive the ribavirin treatment. The obtained results demonstrate the effectiveness of the method to eliminate PDV by a combination of in vitro cultures, chemotherapy with ribavirin and subsequent removing of apical meristematic region.

The following agents were reported in Uruguay in stone fruit trees: PNRSV (Prunus necrotic ringspot virus), PDV (Prune dwarf virus), ACLSV (Apple chlorotic leafspot virus), ApMV (Apple mosaic virus) and PLMVd (Peach latent mosaic viroid). PNRSV and PDV are widespread, their plant to plant transmission in the orchard and by seed was confirmed and causes important losses in nurseries and commercial plantations. ACLSV, ApMV y PLMVd are not widely disseminated, and PPV (causal agent of sharka, Plum pox virus) was not found. Certification of fruit tree plants supervised by the INASE is mandatory. Healthy propagative materials of cultivars and rootstocks are available, but growers prefer the cheapest plants with less coverage of certification. A limited, uneven, and decreasing demand for healthy plants threatens the sustainability of the system. Phytosanitary requirements to prevent the entry of new pests (quarantine) are established by Ministry of Livestock, Agriculture and Fisheries. Official phytosanitary documents required are often difficult to obtain by the suppliers of new cultivars or rootstocks and the risk of pest introduction is high. For this reason, many countries have post entry quarantine stations where introduced plant materials are kept under surveillance until release to the industry. Some fruit producing regions that have important diseases perform plant eradication programs, control of vectors and replacement with certified plants; but, according to the current sanitary status of stone fruit trees in Uruguay, that would not be necessary yet. Conversely, it would be advisable to: 1) prevent the arrival of new pests, considering to set post quarantine plant stations, 2) keep an active surveillance to early detect sanitary problems, 3) encourage sanitary quality on certified plants and 4) develop new orchards using virus-free certified plants.

Stone fruits are a valuable crop grown worldwide, however pathogens such as viruses threaten fruit production by reducing tree health and fruit yield. In an orchard within the Niagara region of Ontario, symptoms typical of viral infection such as chlorosis and leaf deformation were seen on sweet cherry (Prunus avium L.) trees. Next generation sequencing was performed on symptomatic and asymptomatic leaves and four viruses were identified. On the tree displaying the most severe symptoms, Prune dwarf virus (PDV), was the only virus detected. A survey conducted during this work showed 42% of cherry trees on a single orchard plot are infected by PDV. The first infectious clone of PDV was developed for molecular characterization of this virus. Introduction of the infectious clone into cherry revealed PDV caused dwarfing symptoms but did not induce the foliar symptoms found on orchard grown trees. A mass spectrometry (MS)-based label-free quantitative proteomic analysis was performed to identify host proteins affected by PDV infection. The results show in PDV infected cherry many defense related proteins are upregulated, and many photosynthesis-related proteins are downregulated. In the model plant cucumber (Cucumis sativus L.) infected by PDV, significant accumulation changes of proteins related to translation and photosynthesis were identified using proteomics, suggesting a possible role of these proteins in the viral infection cycle of PDV. Two proteins identified through proteomic analysis of cucumber were further studied. These proteins are predicted to be important in the infection cycle of PDV as both co-localized with the viral coat protein (CP) of PDV when visualized using confocal microscopy. Finally, to further understand the intra-host spread of PDV, the movement protein (MP) of PDV was characterized. In plant cells, MP expressed alone formed tubules, a typical structure for virus movement. Additionally, domains of MP crucial for tubule formation and subcellular localization were identified. Taken together, this work advances knowledge in the molecular biology of PDV and host impact caused by PDV infection. In the long run, these findings will assist the development of novel strategies against PDV for the sustainable production of cherry and related Prunus fruits.

Sweet cherry is an important fruit crop in Bulgaria. One of the most common viruses of sweet cherry and other stone fruits in the world is Prune dwarf virus (PDV). Field surveys were conducted in 43 commercial and 3 collection sweet and sour cherry orchards at 10 locations in six regions of Bulgaria. Leaf samples from 2090 trees showing and not showing virus-like symptoms were tested by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for PDV infection. The average PDV infection level in sweet and sour cherry was 14.4%. In 92.4% of the trees, PDV caused single infection, whereas in 7.6%, it caused mixed infection with Prunus necrotic ringspot virus (PNRSV). Visual observations were made to connect the symptoms with PDV infection. There were PDV symptoms in 56.2% of infected trees whereas, 43.8% of the trees were asymptomatic. The coat protein (CP) sequences of 14 isolates from four regions were determined. The obtained sequences were 654 nucleotides (nts) in length, translated to 218 amino acids. Sequence comparisons showed 87%–100% to 93%–100% identity at nucleotide and amino acid level, respectively. Phylogenetic analyses using nucleotide and amino acid sequences of the CP genes of PDV isolates from Bulgaria and 38 cherry isolates retrieved from the GenBank database indicated the existence of three and two phylogenetic groups, respectively. No association between the phylogenetic grouping of the studied isolates and the symptoms of infected trees and/or the sampling-region was observed.

Traditional almond cultivars are an interesting source of different traits for breeders. These old cultivars have been multiplied by grafting for a long time and usually have been accumulating different viruses. In this work a molecular characterization of the ilarviruses Prunus necrotic ring spot virus (PNRSV) and Prune dwarf virus (PDV) was performed in Spanish autochthonous almond cultivars. Results showed the generalized presence of PNRSV and PDV in the analysed collections with a presence of 90 and 68%, respectively, and a high percentage of coinfection (62%). Despite these viruses do not show noticeable symptoms in the tree nor fruits, this poor phytosanitary state urges to the adoption of new strategies for elimination of virus and cleaning these materials. The nucleotide sequence of the RNA 3 of the almond Spanish isolates of PRNSV (37 isolates) and PDV (23 isolates) revealed a nucleotide sequence identity percentage that ranged between 93.7-99.9% for PNRSV and 87.3-100% for PDV. The amino acid similarity percentage observed for the movement protein and coat protein ranged between 94.7-100% and 93.8-100% for PNRSV and 92.2-100% and 88.1-100% for PDV. Phylogenetic analysis based on the nucleotide sequence of the RNA 3 and the coat protein, grouped all PNRSV isolates into the PV32 (22 isolates) and PV96 (15 isolates) clusters; meanwhile all Spanish PDV isolates were grouped in the main cluster previously described by Pallas et al. (2012).

The Niagara fruit belt is one of the richest fruit-producing areas in Canada, contributing to 90% of Ontario's tender fruits such as peach, plum and sweet cherry. Little cherry virus 1 (LCV1) of the genus Velarivirus is a causal agent of little cherry disease which has devastated cherry crops in many regions (Eastwell and Bernardy 1998, Jelkmann and Eastwell, 2011). From 2013 to 2018, foliar symptoms indicative of viral infection such as leaf deformation, ringspot, mottling, vein clearing, and reddening were found on sweet cherry trees grown in the Niagara region. To determine if these trees were infected by a virus, small RNAs (sRNAs) were isolated from separately pooled asymptomatic and symptomatic leaves using the mirPremier microRNA isolation kit (Sigma Aldrich Canada, Oakville, ON). The sRNAs were used to create two libraries (four leaves per library) with the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA). The sRNA libraries were separately sequenced with the MiSeq Desktop Sequencer (Illumina, San Diego, CA). In total, 5,380,196 reads were obtained and Trimmomatic (Bolger et al. 2014) was used to remove adaptors. The remaining 4,733,804 clean reads were assembled into contigs using Velvet 0.7.31 (Zerbino and Birney, 2008) and Oases 0.2.09 (Schulz et al. 2012) with minimum length of 75 nt (Supplementary Table 1). A BLASTn search (Altschul et al. 1997) of the contigs identified the presence of Cherry virus A (genus: Capillovirus), two members of the Ilarvirus genus (Prunus necrotic ringspot virus and Prune dwarf virus) in both libraries. LCV1 was only found in contigs derived from the symptomatic library. Of the clean reads, 22,016 were assembled into six contigs (with lengths ranging from 86 to 116 nt, Supplementary Table 1) mapping to LCV1, covering 7.07% of the viral genome. To confirm LCV1 infection, primers were designed from the assembled contigs and used for reverse transcription polymerase chain reaction (RT-PCR). Amplicons were sequenced and the terminal sequences were determined using 5' and 3' RACE Systems (Invitrogen, Burlington, ON). Degenerate primers were designed from multiple sequence alignments of published LCV1 genomes for amplification and primer walking to obtain the sequence of LCV1 (Table S2). The complete genome sequence of LCV1 has a length of 16,934 nt and was deposited in GenBank (accession no. MN508820). A BLASTn search showed that this isolate is nearly identical (99.6% sequence identity) to an isolate from California (accession no. MN131067). To determine the incidence of infection, a field survey was performed at the same location during spring months of 2014 to 2018 using RT-PCR with primers specific to the viral coat protein gene (Supplementary Tale 2). Among 46 cherry trees sampled, two (4.3%) trees were infected with LCV1 and showed negative results with CVA, PNRSV and PDV. Both trees displayed mild suturing of primary and secondary veins (Supplementary Figure 1). LCV1 has been identified in Western stone fruit producing regions (British Columbia in Canada, and Washington, California, and Oregon in the United States of America). To the best of our knowledge, this is the first report of LCV1 in any eastern region of Canada. The low incidence of LCV1 suggests that this virus is not widespread in this region. Routine monitoring and detection of LCV1 is required to prevent this devastating cherry disease from spreading in this region.

Prune dwarf virus (PDV) is one of the most dangerous pathogens of fruit trees worldwide. One of the most important proteins required for PDV infection is replicase. (P1 protein) which anchored viral RNA and builds replication complex along with RNA depended polymerase. Despite the importance of PDV as a pathogen, our knowledge regarding tissue/cellular localization and structure of PDV P1 protein is still incomplete. The aim of this work was to localize replicase distribution in leaf tissues and cells by immunofluorescent and immunogold labeling of Nicotiana tobaccum cv Samsun and development of a 3D model of PDV replicase. In this paper we demonstrate that PDV replication, is similar to that of Alfalfa mosaic virus and is strongly connected with tonoplasts. In addition, PDV replicase and coat protein (CP) were also found to be strongly associated with membranes of endoplasmic reticulum and, indicating the potential involvement of these membrane structures in the processes related to viral infection. Bioinformatic analyzes based on 3D modeling and structure prediction revealed that P1 protein has a potential transmembrane domain which enables protein anchoring to tonoplast during replication complex assembly.

Sharka caused by Plum pox virus (PPV) is the most harmful disease of stone fruits in Europe and elsewhere in the world. There is no highly PPV resistant cultivar of plum. Biotech approach has led to the development of resistance through genetic engineering. In this study, we evaluated a transgenic plum Prunus domestica L., clone C5 ( cv. HoneySweet), where the PPV resistance is based on RNA interference (RNAi). Resistance in C5 plums has been evaluated for PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV) in a regulated field trial in the Czech Republic for 12 years (2002–2013). Even under high and permanent infection pressure introduced through graft inoculation of the viruses, PPV has been detected in C5 trees only in several leaves situated close to the point of inoculum grafting in the first nine years. Mild symptoms of PPV disappeared year by year. No PPV symptoms were observed in the last three years and results of ELISA detection were negative. Similar results were obtained, when RT-PCR was used for PPV detection. No natural infection of PPV by aphids was recorded in C5 plums, even if PPV infected plants were growing in the same experimental field in close vicinity. Co-infections of PPV with PDV and/or ACLSV had practically no influence on stability of resistance in C5 trees. Twelve years of field testing in the Czech Republic clearly demonstrated the high level of resistance of C5 either to PPV infection by graft inoculation or natural infection through aphid vectors.

Plants in the genus Prunus of the family Rosaceae are important fruit and ornamental trees in China. In June of 2007, sweet cherry (Prunus avium) trees with mottling and mosaic symptoms were observed in a private garden near Kunming, Yunnan Province. Twenty-four samples, six each from sweet cherry, sour cherry (P. cerasus), flowering cherry (P. serrulata), and peach (P. persica) were collected from trees in private and community gardens in the area. The peach and sour and flowering cherry trees did not show any symptoms. Total nucleic acids were extracted using a cetyltrimethylammoniumbromide (CTAB) extraction method, and the extracts were tested for the following eight viruses by reverse transcription (RT)-PCR: American plum line pattern virus, Apple chlorotic leaf spot virus, Cherry green ring mottle virus, Cherry necrotic rusty mottle virus, Cherry virus A (CVA), Little cherry virus 1, Prune dwarf virus, and Prunus necrotic ringspot virus. Only CVA was detected in two symptomatic sweet cherry trees by RT-PCR with forward (5′-GTGGCATTCAACTAGCACCTAT-3′) and reverse (5′-TCAGCTGCCTCAGCTTGGC-3′) primers specific to an 873-bp fragment of the CVA replicase gene (2). The CVA infection of the two trees was confirmed by RT-PCR using primers CVA-7097U and CVA-7383L that amplified a 287-bp fragment from the 3′-untranslated region (UTR) of the virus (1). Amplicons from both amplifications were cloned and sequenced. Analysis of the predicted amino acid sequences of the 873-bp fragments (GenBank Accession Nos. EU862278 and EU862279) showed that they were 98% identical with each other and 97 to 98% with the type isolate of CVA from Germany (GenBank Accession No. NC_003689). The 286-bp sequences of the 3′-UTR (GenBank Accession Nos. FJ608982 and FJ608983) were 93% identical with each other and 93 to 98% with the type isolate. The sequence indicated that the three isolates were very similar and should be considered to be the same strain. CVA is a member of the genus Capillovirus in the family Flexiviridae and has been previously reported in Europe, North America, and Japan. The contribution of CVA to the symptoms observed and its distribution in China remain to be evaluated. To our knowledge, this is the first report of CVA in sweet cherry in China. References: (1) M. Isogai et al. J. Gen. Plant Pathol. 70:288. (2) W. Jelkmann. J. Gen. Virol. 76:2015, 1995.

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. References: (1) N. Capote et al. Int. Microbiol. 12:1, 2009. (2) M. Glasa et al. Ann. Appl. Biol. 140:279, 2002. (3) D. R. Parakh et al. Acta Hortic. 386:421, 1996. (4) S. Radičević et al. Genetika 44:285, 2012.

One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus.

Sweet cherry is an economically important stone fruit grown in temperate regions of Turkey and the world with increasing production. With an annual production of 480.748 tons, Turkey is the leading sweet cherry producers in the world providing about 20% of world production. Isparta province, especially Uluborlu district, is one of the most important sweet cherry production and exportation centers of Turkey (Anonymous, 2014). Although sweet cherry production has been increasing recently, pathogens, especially viruses, cause diseases reducing the yield and quality of fruits leading to significant economical losses. A number of viruses reported to cause diseases in sweet cherry (Myrta and Savino, 2008; Nemeth, 1986). Many studies in different regions showed that Prune dwarf virus (PDV) is the most widespread virus in sweet cherry production areas. It is generally found as mixed infections with Prunus necroctic ring spot virus (PNRSV) and Apple necroctic leaf spot virus (ACLSV) (Cevik et al., 2011; Mandic et al., 2007; Myrta and Savino, 2008). Previous studies reported that PDV infection rate was 67.2–80% in Mediterranean countries (Myrta et al., 2003; Myrta and Savino, 2008), 37.6% in Serbia (Mandic et al., 2007) and 5–55% in Turkey (Caglayan-Yildizgordu and Hurigil, 1996; Cevik et al., 2011; Gumus et al., 2007). Occurrence of PDV has previously been reported in different growing regions of Turkey including Aegean (Dunez 1986; Gumus et al., 2007), Eastern Mediterranean (Caglayan-Yildizgordu and Hurugil 1996; Sertkaya et al., 2004), Western Mediterranean (Cevik et al., 2011), Eastern Anatolia (Sipahioglu et al., 1999), Central Anatolia (Elibuyuk, 2003). PDV is able to infect sweet cherry, sour cherry, apricot, plum and peach as well as other Prunus species by mechanical inoculation, grafting, pollens and seeds transmission (Nemeth, 1986). While PDV is commonly found in stone fruit production areas, severity of symptoms may vary based on host, virus strain and/or temperature (Diekmann and Putter, 1996). Trees infected with PDV generally have small and narrow or curled leaves, short internodes, and PDV also cause cholorotic ring spots especially on sweet cherry and sour cherry leaves (Sutic et al., 1999). Generally, PDV does not affect organs other than leaves, but mixed infection with PNRSV cause reduction in number of fruit buds and formation of leafless shoots (Kunze, 1988). PDV belongs to Ilarvirus genus in the Bromoviridae family and has a tripartite single-stranded positive-sense RNA genome. The segments of the genome are encapsidated by a single coat protein (CP) into three isometric particles with a diameter of 19–26 nm. The virus particles contain 14% nucleic acid and 86% protein and the CP is about 24 kDa (Brunt et al., 1996; Halk and Fulton, 1978). The CP gene has been used as target for serological and molecular detections as well as molecular characterization and genetic diversity studies of PDV isolates from different geographical origins and host plants (Bachmanet et al., 1994; Boulila, 2010; Ulubas Serce et al., 2009; Vaskova et al., 2000; Youssef et al., 2002). The CP gene sequences of 11 PDV isolates from plum, sweet cherry and peach trees in Central Europe were determined. Comparison of the CP gene sequences of these isolates and two previously sequenced isolates showed that the CP gene are highly conserved with more than 88% identity in both nucleotide (nt) and amino acid (aa) sequences. The results also showed that the C-terminal half of the CP was more conserved than the N-terminal half. No association with sequence variation within the CP gene and the geographic origins and/or hosts of PDV isolates was observed (Vaskova et al., 2000). Comparison of the CP gene sequences of peach and plum isolates from Egypt and a sweet cherry isolate from the USA with previously sequenced peach isolate from the USA (Bachman et al., 1994) showed 97% and 98% nt sequence identity, respectively (Youssef et al., 2002). The CP gene sequence of an almond isolate from Tunisia was determined and compared with previously sequenced PDV isolates in the GenBank databases. It showed 86–96% sequence similarity with the CP gene of PDV isolates from different host and geographical origins. Phylogenetic analysis of 32 PDV isolates revealed that they were divided into four distinct groups mostly correlated with geographical origin or recombination within the CP gene (Boulila, 2010). Sequence analyses of the CP gene of 10 PDV isolates, one from apricot and 9 from sweet cherry trees grown in different regions of Turkey, showed 87–99% and 84–99% sequence identity at nt and aa level, respectively. Phylogenetic analysis of these isolates and other PDV isolates from different countries and various Prunus hosts revealed four distinct groups of isolates clustered largely based on their hosts. Group I and IV contained only sweet cherry and almond isolates, respectively. However, group II composed of apricot and sweet cherry isolates and the group III was most divers containing isolates from plum, peach, and sweet cherry (Ulubas Serce et al., 2009). In this study, sweet cherry isolates of PDV from Isparta Province of Turkey, which is one of the most important sweet cherry production and export centers, was identified by double antibody sandwich (DAS)-ELISA and RT-PCR methods. The CP gene of isolates representing different districts were sequenced and compared with previously determined CP gene sequences of PDV isolates from different geographical origins and various Prunus hosts.

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection of four viruses: Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Little cherry virus-2 (LChV-2) and Cherry green ring mottle virus (CGRMV). Random hexamer primer was used for cDNA synthesis. Detection primers were designed against the genomic sequences of four viruses. The reaction conditions were firstly optimized by selecting primers combinations and standardizing the individual primer proportion and PCR protocols. Then, sensitivity of multiplex RT-PCR was evaluated. The assay could detect all four viruses in diluted cDNA (10 −6 , about 8.64 × 10 −4 ng μL −1 ) and RNA (10 −1 , about 0.2 μg). The reliability of the assay was evaluated by cloning and sequence alignments. The developed multiplex RT-PCR method was then used to test virus infections from field samples of sweet cherry in this paper. It will be quite helpful for plant quarantine and certification programs. To our knowledge, it is the first report of the multiplex RT-PCR assay for simultaneous detection of PNRSV, PDV, LChV-2 and CGRMV.

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.

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.

Although Prune dwarf virus (PDV) is one of the most common viruses infecting stone fruits (Prunus spp.), most of the molecular diversity studies have so far focused on the capsid protein (CP) gene. An alternative diagnostic primer pair targeting the movement protein (MP) gene was used in the present work, increasing then polyvalence of PDV detection in cherry samples from Slovakia as compared to the previously published primers amplifying 5′MP-intergenic region-CP-3′UTR fragment. The nearly complete sequence of the RNA3 from 24 Slovak PDV isolates was determined, together with 19 partial sequences of the MP gene for other isolates. Even though the isolates originated from a geographically limited region, analyses showed a high degree of nucleotide sequence diversity between isolates. No clear-cut phylogenetic grouping based on host or geographical origin could be observed. The topology of the MP and CP trees were incongruent for some isolates. Highly supported recombination signals were detected in five PDV isolates, with breakpoints located at different positions in the central part of RNA3, within or close to the intergenic region. Taken together these results extend our understanding of the variability of PDV and for the first time highlight the contribution of recombination to the evolutionary history of this virus.

To investigate the occurrence of viruses in peach, leaf samples were collected from peach trees in commercial orchard of six areas in Korea. Reverse transcription polymerase chain reaction (RT-PCR) was used to identify the presence of the following stone fruit viruses: Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV) and Plum pox virus (PPV). About 65.0% of the 515 samples were infected with ACLSV and PNRSV. Virus-like symptoms showing mosaic on leaves was observed in ACLSV infected peach trees. However, PNRSV infected peach trees showed no symptoms. These viral DNAs by sequence analysis were confirmed 4 ACLSV isolates and 3 PNRSV isolates. The Korean peach isolates of ACLSV and PNRSV showed 70-99% and 88-99% amino acid sequence identities, respectively, with those reported previously and their amino acid sequence identities with each other were approximately 95% and 88%, respectively. Phylogenetic analysis indicated that the Korean ACLSV isolates belong to the A group of ACLSV. The Korean PNRSV isolates reported in this study were grouped into I (PV32), II (PV96) and III (PE5) groups.

Autochthonous genotypes of fruit species are very important source of genetic variability and valuable material for breeding work. Fruit Research Institute-Cacak has a long tradition of studying autochthonous genotypes of temperate fruits sporadically spread and preserved in some localities in Serbia. Over 2005-2006, the following properties of nine autochthonous sour cherry genotypes grown in Feketic region were investigated: flowering and ripening time, pomological properties, biochemical composition of fruits and field resistance to causal agents of cherry diseases - cherry leaf spot (Blumeriella jaapii (Rehm.) v. Arx.), shot-hole (Clasterosporium carpophilum (Lev.) Aderh.) and brown rot (Monilinia laxa /Ader et Ruhl./ Honey ex Whetz.). The genotypes were tested for the presence of Prune dwarf virus and Prunus necrotic ring spot virus. In majority of genotypes fruits were large, with exceptional organoleptical properties, whereas ripening time was in the first ten or twenty days of June. The highest fruit weight was observed in F-1 genotype (8.1 g). The highest soluble solids and total sugars content were found in F- 4 genotype (17.60% and 14.25%, respectively). As for field resistance to causal agents of diseases and good pomo-technological properties, F-1, F-2, F-3, F-7 and F-8 genotypes were singled out. [Projekat Ministarstva nauke Republike Srbije, br. TR31064]

The use of riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the simultaneous detection of up to six different pathogens using a non-radioactive molecular hybridization procedure. In the present report, we describe the development of a unique polyprobe (poly10) with the capacity to detect viruses and viroids commonly found infecting fruit trees. The poly10 covers eight viruses: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV), Plum bark necrosis and stem pitting-associated virus (PBNSPaV) and two viroids: Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). Poly10 is comparable to the individual riboprobes in terms of end-point dilution limit and specificity, allowing the detection of up to 2.5 picograms of viral or viroidal RNA. However, the polyprobe requires a hybridization temperature of 60 degrees C instead of the standard 68 degrees C. The validation of the new simultaneous detection strategy was confirmed by the analysis of 60 field samples, which came from seven different hosts. The use of the polyprobe as an alternative to other routinely used detection methods is discussed., A. P. M. is the recipient of a JAE-Pre contract from the Consejo Superior de Investigaciones Cientificas (CSIC). We thank L. Corachan for her excellent technical assistance. This work was supported by grant BIO2008-03528 from the Spanish granting agency DGICYT and by grant ACOMP/2010/214 from the Generalitat Valenciana.

A survey was carried out on 38 commercial and experimental stone fruit orchards located in major growing areas of stone fruit trees in Poland to determine the incidence of lesser known viruses and phytoplasmas. Leaf samples from 145 sweet cherry and 102 sour cherry trees were tested for Little cherry virus 1 (LChV-1), Little cherry virus 2 (LChV-2), Cherry green ring mottle virus (CGRMV), Cherry mottle leaf virus (CMLV), and Cherry necrotic rusty mottle virus (CNRMV) using RT-PCR. Sixty samples collected from peach and 20 apricot trees were also tested for CGRMV. Eleven out of 145 sweet cherry and three out of 102 sour cherry trees were infected by LChV-1. CGRMV was detected in 10 sweet cherry, four sour cherry, 14 peach and two apricot trees. No LChV-2, CMLV and CNRMV were detected in any of the tested trees. Phloem tissue from samples of shoots collected from 145 sweet cherry, 102 sour cherry, 128 peach, 37 apricot, five nectarine and 20 European as well as Japanese plum trees were tested for phytoplasmas. The nested PCR of the extracted DNA with universal and specific primer pairs showed the presence of phytoplasmas in six sweet cherry, three sour cherry, nine peach, four apricot, one nectarine and three Japanese plum trees. The RFLP patterns of 16S rDNA fragments after digestion with RsaI, MseI, AluI, and SspI endonucleases indicated that selected stone fruit trees were infected by two distinct phytoplasmas belonging to the apple proliferation group. The stone fruit trees infected by LChV-1, CGRMV and phytoplasmas were grown in orchards localised in all seven regions

A protocol of multiplex RT-PCR in a one-tube system for the detection of the most common stone fruit trees viruses [e.g., plum pox virus (PPV), prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV)], including the internal control of NADH dehydrogenase subunit 5 (nad5) gene are described here. The method specificity was tested on more than 80 different samples with various isolates and strains of the viruses. It showed that the targeted viruses produced the expected amplicons, whereas all other related viruses produced only the nad5 internal control amplicon. The method sensitivity was evaluated by comparing it with Simplex RT-PCR with the same primers; no significant differences in detection limits were recorded. Furthermore, the competitiveness of the primers in the assay was tested by serial RNA dilutions of samples with mixed and single infections. The least competitive was the internal control nad5 gene primer pair; therefore, there is a reduced risk of false negatives as all the other primers tend to be more efficient in the given primer cocktail than in the primers for internal control.

The Region of Murcia is one of the main apricot growing areas in Spain. Murcia apricot culture is affected by important virus diseases. In the 1960s, "viruela" disease symptoms were described for the first time in this Region. Apricot trees infected with this disease produce fruits with irregular and ring spots and fruit malformations that make them non-commercial. However, the causal agent of this disease persists without a clear identification to date. In this study, 40 apricot samples (leaf and fruit) with and without "viruela" symptoms were collected in three different locations in the Murcia Region during the harvest time. All samples were analyzed by both ELISA and RT-PCR tests to detect Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Plum pox virus (PPV) and Apple chlorotic leaf spot virus (ACLSV). The results showed the higher sensibility of the RT-PCR approach in comparison with the ELISA test. All the leaves and fruits showing strong "viruela" symptoms were ACLSV positive by RT-PCR. In addition, we detected other viruses as PNRSV (21 % of leaves and 50% of fruits) and ApMV (4% of leaves and 7% of fruits). These results indicate that ACLSV is strictly related to the "viruela" disease. The synergism of ACLSV with other viruses in the expression of "viruela" symptoms is also discussed.

Virus infection seriously limits stone fruit production. The most important viral pathogen is Plum pox virus (PPV). However, other viruses, such as Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) also represent major threats. In this paper, results of in vitro treatments are compared with those of in vivo thermotherapy (i.e., treatment of plants in a peat substrate) to eliminate PPV, PDV, and PNRSV from three apricot varieties, including 'Bergeron', 'Leskora', and 'Marlen'. Therapy takes 45 days under temperature 37°C (in light) and 35°C (in dark) and photoperiod 16h (light) and 8h (dark) in both cases. Following treatment, shoot tips (3 mm long) were collected from plants and cultured in vitro. Plants originating from these shoot tips were tested by RT-PCR and ELISA following transfer to non-sterile conditions for either 6 or 12 months, respectively. No differences were detected between the two methods of treatment. However, differences in numbers of dead plants during treatment and during culture of apical segments.

The operational capacity of a duplex RT-PCR method for simultaneous detection of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) has been established by nine European laboratories. A total of 576 samples from Prunus trees with known sanitary status, corresponding to 32 samples in two repetitions for each laboratory, were analysed. The level of sensitivity achieved by the method was 98.3% for PDV and 90.4% for PNRSV. The specificity was 87.4% for PDV and 94.3% for PNRSV. The unilateral 95% confidence intervals were calculated for all these values. Cohen's Kappa coefficient of repeatability and reproducibility of the technique indicated a strong agreement between data. Likelihood ratios were 7.50 (positive) and 0.02 (negative) for PDV. For PNRSV, the positive likelihood ratio was 15.00 while the negative likelihood ratio was 0.11. In addition, post-test probabilities of infection were calculated to manage the risk associated with the routine use of this method. This allows an accurate test result interpretation to facilitate the integration of this new technique into a certification scheme.

Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

Polak J. (2007): Viruses of blackthorn and road-bordering trees of plum, myrobalan, sweet and sour cherries in the Czech Republic. Plant Protect. Sci., 43: 1–4. The distribution of Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic ringspot virus (ACLSV) and Apple mosaic virus (ApMV) in naturally growing shrubs of blackthorn and road-bordering trees of plum and myrobalan, and of PPV, PDV, PNRSV and Cherry leafroll virus (CLRV) in sweet and sour cherry trees were investigated. The most widely distributed viruses were PPV in plums (74% of the investigated trees were infected); PPV, PDV, and PNRSV in myrobalans (26%, 11% and 18%, respectively), PDV in blackthorns (27%), and PDV and PNRSV in cherries (25% and 22%). PPV was not detected in sweet and sour cherries. The incidence of ACLSV and ApMV was negligible in individually growing trees of the genus Prunus in the Czech Republic.

Virus infection seriously limits stone fruit production in Europe and in the Mediterranean region. The most important viral pathogen is Plum pox virus (PPV), however, other viruses, e.g. Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) also represent a major threat. Experiments on in vitro thermotherapy and subsequent meristem culture led to virus-free plant material, which could be multiplied rapidly by micropropagation techniques. In vitro cultures of peach, plum and apricot cultivars were established. Cultures initiated from different buds of the same cultivar were treated as different clones. The cultures were inoculated onto modified MS medium containing BAP, IBA and GA 3 and kept at 22°C under a 16/8-hr photoperiod.[...]

An apple chlorotic leaf spot virus (ACLSV) isolate was detected by TAS-ELISA and RT-PCR in an ornamental dwarf fl owering almond (Prunus glandulosa Thunb.). This plant, maintained at the Centre for Plant Health, Sidney, B.C., Canada, has been showing transient leaf symptoms during the spring seasons. A 390-bp fragment and a 1,350-bp product, in the RNA polymerase and the coat protein viral coding regions, respectively, were amplifi ed by RT-PCR from the infected plant. A sequence comparison of the 390- bp fragment of this ACLSV isolate (designated as AL1292) with other published isolates, revealed a similarity of 81% to 84% at the nucleotide level and 88% to 100% at the amino acid level. In contrast to other ACLSV isolates, AL1292 has an exceptionally narrow range of experimental herbaceous and woody hosts, as determined by mechanical and graft inoculation assays. These standard bioassays may not be effective for the detection of the AL1292 isolate because of its limited host range. The results we report in this paper confi rm P. glandulosa as a natural host of this virus. Currently it is not known how ACLSV is spread, other than by bud-grafting and possibly by root grafts. The use of virus-tested source plants for the preparation of planting material will minimize its spread. Prunus glandulosa Thunb. is a deciduous, multistemmed shrub, known by the common name dwarf fl owering almond (U.S. Depart- ment of Agriculture, Agricultural Research Service, The Germplasm Resources Informa- tion Network). Native to China and Japan, it has been grown mainly for its fl owers coming into full bloom in early spring before new leaves develop. Plants of P. glandulosa 'Alba' bear single white fl owers while 'Sinensis' (also known as 'Rosea Plena') has double pink fl ow- ers. Dwarf fl owering almond was introduced to North America as an ornamental fl owering bush in 1683 (Adams, 2004). In western Europe, it is known in France since 1785 and later was cultivated in England mainly for cut fl owers (Flower Council of Holland, 2002). Propagation of planting material from seeds is possible but requires stratifi cation before sowing and may take up to 18 months to germinate. Vegetative propagation by rooting cuttings is less time-consuming and, therefore, has been applied routinely for production of planting material. As is the case for other vegetatively propagated plants, transmission of viruses in nursery stock of P. glandulosa is a potential problem. Prunus glandulosa, as well as other plants in the genus Prunus, grown for fruits and as ornamentals, are natural hosts of viruses like prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV) and plum pox virus (PPV) (Nemeth, 1986). Virus-infected plants often express disease symptoms but may also be symptomless. Apple chlorotic leaf spot virus (ACLSV), the type member of the genus Trichovirus, was reported from the rosaceous species Prunus, Malus, and Pyrus (Martelli et al., 1994) and is also known to occur in one- seed hawthorn (Crataegus monogyna) and medlar (Mespilus germanica) (Nemeth, 1986). ACLSV isolates vary greatly in their biologi- cal and molecular properties. Some strains are latent and others may cause symptoms like bark split, dark green sunken mottle of leaves and even graft incompatibility (Nemeth, 1986). Symptoms in Prunus species are generally more severe than in Malus and Pyrus spp. (Dunez and Delbos, 1988). Based on bioassays results, Colin and Verhoyen (1975) suggested that leaf symptoms observed in P. glandulosa 'Sinensis' were caused by ACLSV. In this study, we applied biological, serological and molecular methods for the detection and characterization of an ACLSV isolate in the same host. The results we report herein confi rm P. glandulosa as a natural host of this virus.

A sensitive and reliable one step RT-PCR reaction with an internal control has been developed to detect and differentiate eight important viruses that affect stone fruit tress: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV) and Plum bark necrosis stem pitting associated virus (PBNSPaV). In addition, we investigated the detection limit and the efficiency of three different nucleic acid extraction methods that avoid the use of organic solvents, for both multiplex RT-PCR and dot-blot hybridisation assays. The primer cocktail was used to analyse 38 stone fruits originating from nine different countries and six species. A large number of virus combinations was detected and up to three different viruses were observed in five samples. A decrease in sensitivity was observed when the primer cocktail contained more than five different pair primers. However, comparative analyses showed that the multiplex RT-PCR containing the eight virus pair primers was even more sensitive than the ELISA or molecular hybridisation assays. The use of the multiplex RT-PCR technology in routine diagnosis of stone fruit tree viruses is discussed.

Prunus necrotic ringspotvirus (PNRSV) and Prune dwarf virus (PDV) are among the most widespread and economical important viruses in Prunus spp. Enzyme-linked immune-sorbent assay (ELISA) was used to determine the incidence of PNRSV and PDV in lots of stone fruit species- plum, apricot, peach, sweet cherry, sour cherry, almond and many of their rootstocks between 2000 and 2004. These viruses were found in all areas tested in South Bulgaria. Presence of range of Ilarviruses was as follows: PNRSV from 0.58% in P. mahaleb to 27.7% in sour cherry, and PDV from 1.4% in sour cherry to 15.8% in sweet cherry. Use of ELISA to monitor virus incidence in Prunus spp. is a valuable tool for reducing the incidence of viruses, which are transmitted by pollen and seeds.

Field surveys were carried out in the main stone-fruit growing areas of Morocco to evaluate the sanitary status of commercial orchards, varietal collections and nurseries. The presence of virus and virus-like diseases was checked by ELISA, sap transmission to herbaceous hosts, testing on woody indicators and molecular hybridization (dot-blot and tissue-printing). 1211 samples (382 almond, 339 peach, 291 plum, 150 apricot and 49 cherry) were tested by ELISA for the presence of Prunus necrotic ring spot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Plum pox virus (PPV). The overall average of virus infection rate was 16.4%, whereas that of single species was: 22.6% for almond, 17.8% for plum, 15% for peach, 10.2% for cherry, and 2.7% for apricot. The following viruses were detected: PNRSV, PDV, ACLSV and ApMV. 565 samples were tested by dot-blot and tissue-printing hybridization for the presence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). 48 samples were infected, 41 by PLMVd and 7 by HSVd. In addition, nested-PCR tests identified Plum bark necrosis and stem-pitting associated virus (PBNSPaV) in a few almond trees affected by stem pitting.

Despite the fact that Apricot ‘Viruela’ disease (apricot pox) was described more than thirty years ago, no conclusive evidence has been reported for the identification of its causal agent. To shed light on the aetiology of the disease we first analysed ten mature fruits from trees showing no, mild and severe symptoms of 'viruela' (two trees for each) to look for the presence of Apple chlorotic leaf spot virus (ACLSV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Plum pox virus (PPV) and Hop stunt viroid (HSVd). From the fruits which showed mild symptoms of 'viruela', asymptomatic and symptomatic areas were separately extracted and analysed. The presence of ACLSV and HSVd was detected in all the symptomatic areas of the mature fruits. PNRSV was detected in two of the four affected trees, whereas PDV and PPV were not detected. A large scale indexing for ACLSV and HSVd was then made with 20 'healthy' mature fruits and 120 mature fruits showing symptoms of 'viruela'. In all 120 mature fruits showing symptoms of the disease the presence of ACLSV and HSVd was recorded by non-isotopic tissue-blot hybridisation, whereas the other 20 'healthy' fruits were ACLSV-free and 18 out of the 20 were positive for HSVd. The latter result can be explained by the high incidence of HSVd in apricot trees previously observed in the Murcia Region (Canizares et al., 1998). Our results, then, are consistent with those which suggest an association between ACLSV and apricot 'viruela' disease.

Sucha J., Svobodova L. , 2010. Incidence of Prune dwarf virus and Prunus necrotic ring spot virus in orchards of sweet and sour cherry in the Czech Republic – Short communication . Hort. Sci. (Prague), 37: 118–120. During 2006–2008 field surveys were carried out in the important cherry growing areas of the Czech Republic to assess the incidence of dwarf virus and Prunus necrotic ring spot virus in commercial orchards and nurseries. A total of 1,438 samples from 1,198 sweet cherry trees and from 240 sour cherry trees were tested by ELISA for the presence of Prune dwarf virus and Prunus necrotic ring spot virus. The overall average infection level was 17.7%. The most infected species were sour cherry trees (22.5%). The most frequently detected virus was Prune dwarf virus (10.9%). Prunus necrotic ring spot virus occurred in 6.3% of samples. Our study provided an indication of a sanitary status of sweet and sour cherry in commercial orchards and nurseries in the Czech Republic.

Sequences of the capsid protein gene and the preceding intergenic region of eleven isolates of prune dwarf virus from central Europe were determined. The isolates were obtained from plum, cherry and peach trees. Comparison of all sequenced isolates (including two sequences published previously) revealed high (88%) conservation of the capsid protein gene. The highest degree of identity was observed in the C-terminal half, where only 13 amino acid substitutions could be observed in contrast to the N-terminal half with 22 substitutions. No reasonable correlation between amino acid substitutions and host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other ilarviruses revealed apple mosaic virus, elm mottle virus, lilac ring mottle virus and prunus necrotic ringspot virus as the most related to prune dwarf virus. Unlike the isolates of related prunus necrotic ringspot virus all the isolates of prune dwarf virus shared extensive conservation of the intergenic region. Portions of RNA3 were selected for design of universal primers for PCR detection.

Two Golden Queen peach blocks, one propagated from virus-free scions (Block A), the other from non-indexed sources (Block B), were ELISA-tested 5-7 years after planting for prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV). Trees in Block A were 18% infected by PNRSV at year 5 and 60% by year 7, at which time 1% also had PDV. Block B was 100% infected by PNRSV and 9% with PDV by year 5, increasing to 15% by year 7. Between years 5 and 7, salable yield reduced by 18% as a result of PNRSV-infection. Dual infection was likely to have reduced average salable yield by 32% (9.6 t/ha) and gross returns by $4,805/ha.

Prune dwarf virus (PDV) is one of the major positive RNA viruses which cause economical damages in stone fruit trees. The symptoms of PDV vary between different stone fruits namely sour and sweet cherry, almond, peach, apricot and plum including leaf narrowing, leaf chlorosis, vein clearing, mosaic, leaf whitening, leathery leaf, bushy branches and stunt trees. During the years 2011 and 2012, 251 leaf samples were collected for detection of PDV in stone fruit orchards of Charmahal-va-Bakhtiari province. DAS-ELISA test proved PDV presence serologically. Then, total RNA were extracted and tested by two-step RT-PCR which replicated partial and full coat protein sequence of PDV. One hundred and eighty one out of total samples (251 samples) showed PDV infection using serological and two-step RT-PCR assays, hence, incidence of PDV in Charmahal-va-Bakhtiari province was confirmed. This is the first report of PDV in stone fruit orchards of Charmahal-va-Bakhtiari province and in Iran.

Detection of viruses presence were carried out for the 225 a of pome and stone fruit trees from the collection of the Genetic Resources Institute of University of Banja Luka, located within the Botanical Garden of the University, tested by DAS-ELISA. The pome fruit trees were analyzed on presence of the following viruses: Apple Chlorotic Leaf Spot Virus (ACLSV), Apple Stem Grooving Virus (ASGV), Apple Stem Pitting Virus (ASPV) and Apple Mosaic Virus (ApMV). The stone fruits were analyzed on presence of Plum Pox Virus (PPV), Prune Dwarf Virus (PDV) and Prunus Necrotic Ring Spot Virus (PNRSV). All samples were tested serologically by DAS-ELISA. In addition to this, virus negative pear and apple accessions were tested for 'Candidatus Phytoplasma mali' and 'Candidatus Phytoplasma pyri' presence using nested-PCR/RFLP analyses.

Natural spread of Prune dwarf virus (PDV) and Prunus necrotic ring-spot virus (PNRSV) was monitored among healthy trees of six peach (Prunus persica (L.) Batsch) cultivars interplanted into a mature peach orchard between virus-infected trees of the cultivar Carson. Spread of PDV to a few trees of cvs. Andross, Carson, and Halford occurred during bloom just after the dormant test trees were planted in the orchard, as indicated by serological and biological assays during the next season. In contrast, virus infections in cvs. Dr. Davis, Loadel, and Ross were not detected until the fourth to sixth growing seasons. By the seventh season, cumulative transmissions for PDV and PNRSV were 41 and 9, respectively, which included double virus infections in five test trees. Significant differences in cumulative transmission of PDV but not of PNRSV occurred by the fourth season (P = 0.0020). Overall, virus infections among the six cultivars ranged from 70 to 100%.

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.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; POLAK J., KUMAR J., KRSKA B., RAVELONANDRO M. (2012): Biotech/GM crops in horticulture: Plum cv. HoneySweet resistant to Plum pox virus. Plant Protect. Sci., 48 (Special Issue): S43-S48. Commercialisation of Biotech/GM (Biotech) crops started in 1995. Not only field crops, but also horticultural transgenic crops are under development and are beginning to be commercialised. Genetic engineering has the potential to revolutionise fruit tree breeding. The development of transgenic fruit cultivars is in progress. Over the past 20 years an international public sector research team has collaborated in the development of HoneySweet plum which is highly resistant to Plum pox virus (PPV) the most devastating disease of plums and other stone fruits. HoneySweet was deregulated in the USA in 2010. HoneySweet (aka C5) has been evaluated for eleven years (2002-2012) in a regulated field trial in the Czech Republic for the resistance to PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV), all of them being serious diseases of plum. Even under the high and permanent infection pressure produced through grafting, PPV has only been detected in HoneySweet trees in several leaves and fruits situated close to the point of inoculum grafting. The lack of infection spread in HoneySweet demonstrates its high level of PPV resistance. Co-infections of PPV with PDV and/or ACLSV had practically no influence on the quantity and quality of HoneySweet fruit which are large, sweet, and of a high eating quality. In many respects, they are superior to the fruits of the well-known cultivar Stanley. Many fruit growers and fruit tree nurseries in the Czech Republic are supportive of the deregulation of HoneySweet plum to help improve the plum production and control the spread of PPV.

Six sap‐transmitted viruses were identified during a study of 434 flowering cherries in the North Island of New Zealand. These included Prunus necrotic ringspot ilarvirus (PNRSV), apple mosaic ilarvirus (ApMV), flowering cherry virus B (FCVB), strawberry latent ringspot virus (SLRV), prune dwarf ilarvirus (PDV), and flowering cherry virus I (FCVI). Of these, ApMV, FCVB, SLRV, and FCVI were new records for this host. FCVB and FCVI appear to be newly described viruses with characteristics of members of the bromovirus and ilarvirus groups respectively. The most common virus was PNRSV (30.6%); the other viruses ranged in incidence from 10.2% (FCVB) to 0.5% (PDV). A further 43 virus isolates were categorised into nine classes based on symptoms in Chenopodium quinoa and cucumber, and were not further characterised in this study. Repeated sampling of 30 flowering cherry trees during late winter and early spring showed that the enzyme‐linked immunosorbent assay (ELISA) was more sensitive for detecting PN...

Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural infection pressure. Transgenic clone C5, subsequently named "HoneySweet", showed high resistance to PPV. None of the C5 trees became naturally infected by aphids for more than ten years. The resistance of C5 was based on post-transcriptional gene silencing (PTGS). In the second experiment we assessed the effect of two heterologous viruses (Prunus necrotic ringspot virus and Prune dwarf virus) on the efficacy and stability of PTGS - mediated resistance to PPV displayed by the C5 plum. The engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses. Some PPV-CP transgenic plum clones that are susceptible to PPV including C2, C3, C4 and PT3 display a constitutive transcription of PPV-CP sequence. In the third experiment we used these plants to assess the environmental safety issues related to potential hazards concerning the emergence of PPV variants. The serological and molecular variability of PPV detected in transgenic and in conventional plums revealed that the transgenic plums do not affect the diversity of indigenous PPV populations. We suggest that the safety and efficacy data developed in Romania and other European countries and in the United States of America over the last 15 years justifies an expansion of the field tests in Romania and in other countries that are experiencing the damages to plum production caused by PPV.