Sharka Disease Research Articles

Determination and partial molecular characterization of Plum pox virus in Bolu province

Plum pox virus (PPV), the causal agent of Sharka disease, causes yield, quality, and economic losses in stone fruits. PPV has been reported worldwide, especially in Europe. In studies to date, the presence of the virus has been identified as being restricted in different regions of Turkey. However, there is no record of PPV in Bolu province so far. Hence, surveys were carried out in Bolu province between 2016-2019, and a total of 306 samples were collected. To determine the presence of PPV, the samples were first tested by DAS-ELISA, and only three peach samples were found to be infected. DAS-ELISA results of infected samples were confirmed by RT-PCR using universal primers (P1/P2), then infected samples were identified at the strain level using strain-specific primers. The samples were found to be infected with the PPV-M (Marcus) strain and 243-bp long nucleotide sequences containing the partial coat protein gene of three isolates were deposited to NCBI. Phylogenetic analysis (Neighbor-Joining) generated by 38 representative PPV sequences indicated that Bolu isolates were clustered with PPV-M isolates and separated from other strains, as in BLAST analysis. To our knowledge, this is the first report of PPV in Bolu. This study reveals the necessity to carry out more extensive surveys to prevent the PPV dissemination in Bolu and to identify the complete genomes of the obtained isolates to determine their genetic variation. All the PPV-infected trees were destroyed as a consequence.

First Report of Plum Pox Virus on Plum in Uzbekistan

Apricot, sweet cherry, plum, and peach are economically important stone fruit crops in Uzbekistan. Plum pox virus from the genus Potyvirus in the family Potyviridae is the causal agent of sharka disease. Plum pox virus (PPV) is the most destructive viral pathogen of stone fruit trees. No information is available on PPV occurrence in Uzbekistan. In 2019, plum (Prunus domestica) trees exhibiting foliar pale green rings and spots were observed in a 21-year-old orchard of the local cultivar ‘Ispanskiy’ in the Tashkent region of Uzbekistan. One symptomatic leaf was taken from each of four trees growing at the corners of this orchard. The samples were tested for PPV by double antibody sandwich ELISA using reagent set SRA 31505/0500 (Agdia, U.S.A.) and immunocapture RT-PCR using the coating antibody CAB 31505/0500 (Agdia) and PPV-specific P1/P2 primers (Wetzel et al. 1992). PPV was readily detected in each sample by both methods. PCR products of the expected size (198 bp) were generated by RT-PCR using P1/PD strain-specific primers (Olmos et al. 1997), indicating that the isolates belonged to the strain D. No positive reaction was observed with primers specific to the PPV strains M, W, and C, indicating no mixed infection. All four isolates were recognized by triple antibody sandwich ELISA using PPV-D-specific antibody 4DG5 (K-12B kit, Agritest, Italy), thus confirming the results of the strain-specific RT-PCR. The 3′-terminal genomic sequences encompassing the coat protein (CP) gene and flanking sequences of the NIb gene and 3′-noncoding region were amplified using NIbF/4CPR1 primers (Chirkov et al. 2016), and the PCR products of about 1.3 kb were sequenced using the Sanger method. The sequence of isolates Uz2 and Uz4 was identical, and isolates Uz1, Uz2, and Uz3 shared 99.1 to 99.6% and 99.0 to 99.3% identity at the nucleotide and amino acid levels, respectively. The low genetic diversity of Uzbek isolates suggests their common origin. The ¹¹DAG¹³ motif responsible for aphid transmission, the PPV-D-specific ⁵⁵QPATKP⁶⁰ epitope, and the universal ⁹⁴DRDV(I)DAG¹⁰⁰ epitope (Candresse et al. 2011) were found in the deduced N-terminal sequence of the CP of all the isolates. The 3′-terminal genomic sequences of the Uz1, Uz2, and Uz3 isolates were deposited in GenBank under accession numbers MT038048 to MT038050. Phylogenetic analysis of the CP gene assigned Uz1, Uz2, and Uz3 to the cluster of PPV strain D isolates (LT600779 to LT600782) detected in a Prunus germplasm collection from neighboring Kazakhstan (James et al. 2017) and the isolate BIII/2 (GU461890) from Slovakia. According to BLASTn, the Uzbek isolates were most closely related to the Kazakh isolate P7R1 from plum (LT600780) (99.2 to 99.5% nucleotide identity). To the best of our knowledge, this is the first report of PPV from Uzbekistan. This finding expands the geographical distribution of the virus and encourages further studies on PPV prevalence, host range, and genetic variability to reduce its impact on the fruit-growing industry.

Development and validation of an SNP-based new set of markers useful for early selection for Sharka disease in apricot (P. armeniaca)

Development and validation of an SNP-based new set of markers useful for early selection for Sharka disease in apricot (<i>P. armeniaca</i>)

Statistical model for Plum pox virus prediction in Prunus nursery blocks using vector and virus incidence data in four different European ecological areas

AbstractPlum pox virus (PPV) is the causal agent of sharka, the most devastating virus disease of Prunus species. PPV is transmitted by vegetative propagation, particularly by grafting, as well as by aphid species in a nonpersistent manner. The objective of this paper was to evaluate the prevalence and diversity of PPV‐vector aphid species in representative European areas of prunus cultivation. Four experimental nursery plots were established in Skierniewice (Poland), Liria (Spain), Plovdiv (Bulgaria) and Bistrita (Romania). Aphid population was weekly monitored using the sticky shoot method in spring and/or autumn in each nursery plot. Furthermore, we estimated the relative efficiency factor for the main PPV‐vector aphid species using bibliographic and reference data. Aphid diversity in each nursery plot was assessed using the Shannon index and the number of estimated aphid species was calculated by the rarefaction method. The highest diversity of aphid species population was reported in Plovdiv in spring while the lowest diversity was found in Liria in spring. A cluster analysis based on Morisita–Horn similarity index was performed to study the differences in species composition between the four nursery plots and seasons. Aphid population was clearly grouped by location and season. Results showed Aphis spiraecola and Hyalopterus pruni complex were typically spring aphid species, while Anoecia corni and Rhopalosiphum padi dominated autumn catch. Regarding PPV‐vector aphid species present in nursery plots, A. spiraecola was the most prevalent PPV‐vector aphid species in Liria and in Bistrita, showing a high relative efficiency factor of PPV transmission (0.91). Consequently, A. spiraecola should be considered a key actor in the spread of PPV in these regions. Hyalopterus pruni complex was the most prevalent PPV‐vector aphid species in Skierniewice and Plovdiv, showing a very low relative efficiency factor of PPV transmission (0.09). Therefore, the role of H. pruni complex in spread of PPV in these regions can be considered negligible. Furthermore, we statistically demonstrated that the presence of specific PPV‐vector aphid species is associated with the spread of the disease, whereas the biodiversity of aphid species population does not affect the spread of PPV. Finally, the advantages of the use of vector pressure index in the management of sharka disease are discussed.

Apricot breeding for tolerance to Plum pox potyvirus in Nikita Botanical Gardens

For the fi rst time, the results of studying of varieties and breeding forms of apricot in the gene pool collection of the Nikita Botanical Garden – the National Scientifi c Center of the Russian Academy of Sciences (NBS – NSC) (Yalta, Crimea) on the tolerance to the plum Sharka disease – Plum pox potyvirus (PPV), are presented in the article. The collection includes varieties from diff erent ecological and geographical groups that give us the possibility to present an objective comparative assessment of their susceptibility to phytopathogens. Based on long-term monitoring of the apricot orchards on PPV susceptibility in the collection of NBG-NSC, single aff ected plants were discovered and exterminated. Molecular genetic approaches to apricot breeding for tolerance were demonstrated and PPV tolerant genotypes were selected. According to the results of the research, hybridization was carried out. During the entire period of the research, in order to create genotypes tolerant to PPV, 28 738 fl owers were pollinated and 2 908 hybrid seeds were obtained from which plants were grown. Under in situ and in vitro conditions, new breeding forms tolerant to PPV were obtained. The prospect of biotechnological methods usage for inducing the development of hybrid apricot embryos was shown. The conditions for their culture (trophic, hormonal, and physical) were determined, viable seedlings and plants were obtained in vitro and planted for further adaptation ex vitro and to the open ground. New created plants were evaluated and breeding forms were selected, characterized by tolerance to PPV, good fruit quality and high stable yield. Based on the comprehensive studies, two breeding forms of apricot (‘97 -11 ’and ’97-17’) tolerant to PPV and promising for breeding and commercial cultivation were identifi ed.

Common and Strain-Specific Post-Translational Modifications of the Potyvirus Plum pox virus Coat Protein in Different Hosts.

Phosphorylation and O-GlcNAcylation are widespread post-translational modifications (PTMs), often sharing protein targets. Numerous studies have reported the phosphorylation of plant viral proteins. In plants, research on O-GlcNAcylation lags behind that of other eukaryotes, and information about O-GlcNAcylated plant viral proteins is extremely scarce. The potyvirus Plum pox virus (PPV) causes sharka disease in Prunus trees and also infects a wide range of experimental hosts. Capsid protein (CP) from virions of PPV-R isolate purified from herbaceous plants can be extensively modified by O-GlcNAcylation and phosphorylation. In this study, a combination of proteomics and biochemical approaches was employed to broaden knowledge of PPV CP PTMs. CP proved to be modified regardless of whether or not it was assembled into mature particles. PTMs of CP occurred in the natural host Prunus persica, similarly to what happens in herbaceous plants. Additionally, we observed that O-GlcNAcylation and phosphorylation were general features of different PPV strains, suggesting that these modifications contribute to general strategies deployed during plant-virus interactions. Interestingly, phosphorylation at a casein kinase II motif conserved among potyviral CPs exhibited strain specificity in PPV; however, it did not display the critical role attributed to the same modification in the CP of another potyvirus, Potato virus A.

Molecular Plant-Plum Pox Virus Interactions.

Plum pox virus, the agent that causes sharka disease, is among the most important plant viral pathogens, affecting Prunus trees across the globe. The fabric of interactions that the virus is able to establish with the plant regulates its life cycle, including RNA uncoating, translation, replication, virion assembly, and movement. In addition, plant-virus interactions are strongly conditioned by host specificities, which determine infection outcomes, including resistance. This review attempts to summarize the latest knowledge regarding Plum pox virus-host interactions, giving a comprehensive overview of their relevance for viral infection and plant survival, including the latest advances in genetic engineering of resistant species.

Agrobacterium-Mediated Transformation of Russian Commercial Plum cv. "Startovaya" (Prunus domestica L.) With Virus-Derived Hairpin RNA Construct Confers Durable Resistance to PPV Infection in Mature Plants.

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum “Startovaya” (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for β-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, ImmunoStrip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene construct encoding intron-spliced hairpin RNAs provided a highly effective protection of plum trees against permanent viral infection. At the same time, this observation indicates the lack of the systemic spread of resistance from GM tissues to an infected plum graft even after years of joint growth.

Impact of Sharka disease on tree growth, productivity and fruit quality of apricot (Prunus armeniaca L.)

In this study, the influence of Sharka disease, caused by Plum pox virus (PPV), on tree growth, yield attributes, financial losses, fruit physical properties, nutritive compounds, phenolic and flavonoid contents and antioxidant power was evaluated in a susceptible apricot (Prunus armeniaca L.) genotype. The results showed that Sharka significantly reduced tree growth, yield and economic results in PPV infected trees in comparison with PPV uninfected trees. The Sharka did not change fruit and stone weight and fruit shape, but significantly decreased flesh rate and flesh firmness. Soluble solids content (SSC) and titratable acidity (TA) were higher in symptomatic fruits, whereas ripening and sweetness index, sucrose and total sugars contents were smaller in fruits from PPV infected trees. Differences in content of invert sugars were not significant. Finally, PPV significantly reduced total phenolic (TPC) and total flavonoid contents (TFC) and total antioxidant capacity (TAC) in comparison with their contents in fruits from PPV uninfected trees.

Phenotypic, molecular and phytohormonal evidence of Plum pox virus silencing in susceptible apricot genotypes

Plum pox virus (PPV; sharka disease) is the most important limiting factor for apricot production in affected areas. The generation of new resistant cultivars is the definitive solution to this problem. For this reason, resistance to PPV is one of the main objectives of breeding programmes. During the evaluation of resistance in these programmes, a reduction of sharka symptoms has been observed after the infection of virus in some susceptible cultivars. In this study, we present the results of phenotypic, gene expression and phytohormonal profile changes induced by PPV infection in susceptible apricot cultivars. At the phenotypic level, a reduction in the severity of symptoms during the second infection cycle of the study was observed. This reduction of symptoms could be explained by a silencing effect on the PPV. In line with this idea, the transcriptomic analyses revealed that the early response to PPV is associated with an induction of genes involved in pathogen resistance such as those encoding jasmonic acid, resistance proteins, chitinases, etc. However, the overexpression of the Dicer protein 2a gene in leaves with strong symptoms may indicate the suppression of a gene silencing mechanism in the plant by PPV HCPro and P1 PPV proteins. Salicylic acid appeared to be involved in susceptible infected genotypes. This could also be related to the gene silencing response elicited by the virus. The successful silencing of the virus could be the origin of the reduction in sharka symptoms observed after the infection of some susceptible cultivars.

Multi-resistance of apricot cultivars to the most important diseases in Spain

Spain is one of the most important producers of fresh apricots. Traditional production was based on several local cultivars of good quality and well adapted to industry demands. Nevertheless, most of these cultivars are susceptible to important diseases that affect production and fruit quality. These diseases are sharka (Plum pox virus; PPV), viruela (Apple chlorotic leaf spot virus; ACLSV), and fruit degeneration disease (Hop stunt viroid; HSVd). In the Department of Plant Breeding of CEBAS-CSIC, we study these diseases, looking for sources of resistance to be used in breeding programmes. Using foreign sources of resistance to PPV, our department has released new cultivars with excellent agronomic behaviour that are widely cultivated in our country and abroad today. More recently, we have identified two new sources of resistance to ACLSV. We are combining traditional breeding methods with molecular markers to study the interactions among the resistance to these different pathogens with the objective of accumulating both types of resistance in good-quality cultivars. ACLSV-resistant sources were crossed with PPV-resistant selections homozygous for PGS1.21 and PGS1.24 markers, linked to PPV resistance. Descendants obtained are being evaluated against PPV and ACLSV separately as well as against both diseases simultaneously, in order to detect possible interactions. Despite much work, we have not yet identified sources of resistance to HSVd within apricot species to date, although this deficiency will be addressed by looking for such sources in wild relative apricot species.

Bursa ilinde şarka hastalığının (Plum pox virus) yaygınlığının belirlenmesi

Plum pox virus (PPV)’nin neden olduğu şarka hastalığı sert çekirdekli meyve ağaçlarında ciddi ekonomik kayıplara neden olan en önemli viral hastalıktır. Bursa ilinde sert çekirdekli meyve bahçelerinde şarka hastalığının yaygınlığını belirlemek amacıyla sürveyler gerçekleştirilmiştir. Örnekler Yenişehir, Gürsu, Osmangazi, Mudanya, Orhangazi ve İnegöl ilçelerinden nisan-haziran 2015 tarihleri arasında toplanmıştır. Toplam 166 tane sert çekirdekli meyve ağacından (erik, şeftali, nektarin ve kiraz) ve badem ağacından toplanan yaprak örnekleri test edilmiştir. Toplanan örneklerde virüs varlığını tespit etmek için DAS-ELISA (double antibody sandwich enzyme linked immunosorbent) testi kullanılmıştır. DAS-ELISA sonuçlarına göre, Bursa'da PPV’nin yaygınlığı ortalama %24.1 olarak bulunmuştur. En yüksek PPV enfeksiyonu, %57.1 oranı ile İnegöl'de belirlenmiştir. Orhangazi, Gürsu, Yenişehir, Osmangazi ve Mudanya ilçelerinin sırası ile %50.0, %27.3, %25.0, %10.0 ve %7.1 oranında PPV ile enfekteli olduğu bulunmuştur. En yüksek PPV enfeksiyonu erikte (%41.7) saptanmış, bunu nektarin (%40) ve şeftali (%16.5) izlemiştir. PPV, kiraz ve badem ağaçlarında tespit edilmemiştir.

Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes

BackgroundPlum pox virus (PPV), causing Sharka disease, is one of the main limiting factors for Prunus production worldwide. In apricot (Prunus armeniaca L.) the major PPV resistance locus (PPVres), comprising ~ 196 kb, has been mapped to the upper part of linkage group 1. Within the PPVres, 68 genomic variants linked in coupling to PPV resistance were identified within 23 predicted transcripts according to peach genome annotation. Taking into account the predicted functions inferred from sequence homology, some members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing genes were pointed as PPV resistance candidate genes.ResultsHere, we have characterized the global apricot transcriptome response to PPV-D infection identifying six PPVres locus genes (ParP-1 to ParP-6) differentially expressed in resistant/susceptible cultivars. Two of them (ParP-3 and ParP-4), that encode MATHd proteins, appear clearly down-regulated in resistant cultivars, as confirmed by qRT-PCR. Concurrently, variant calling was performed using whole-genome sequencing data of 24 apricot cultivars (10 PPV-resistant and 14 PPV-susceptible) and 2 wild relatives (PPV-susceptible). ParP-3 and ParP-4, named as PrunusarmeniacaPPVresMATHd-containing genes (ParPMC), are the only 2 genes having allelic variants linked in coupling to PPV resistance. ParPMC1 has 1 nsSNP, while ParPMC2 has 15 variants, including a 5-bp deletion within the second exon that produces a frameshift mutation. ParPMC1 and ParPMC2 are adjacent and highly homologous (87.5% identity) suggesting they are paralogs originated from a tandem duplication. Cultivars carrying the ParPMC2 resistant (mutated) allele show lack of expression in both ParPMC2 and especially ParPMC1.ConclusionsAccordingly, we hypothesize that ParPMC2 is a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. As a whole, results strongly support ParPMC1 and/or ParPMC2 as host susceptibility genes required for PPV infection which silencing may confer PPV resistance trait. This finding may facilitate resistance breeding by marker-assisted selection and pave the way for gene edition approaches in Prunus.

Generation of Transgenic Rootstock Plum ((Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)) Using Hairpin-RNA Construct for Resistance to the Plum pox virus

The use of Prunus rootstocks that are resistant to plum pox virus (PPV) is an important agronomic strategy to combat the spread of the Sharka disease in nurseries and orchards. Despite remarkable progress in developing stone fruit rootstocks to adapt to various stresses, breeding that ensures durable virus resistance has not yet been achieved. For this reason, the engineering of PPV resistant plants through genetic transformation is a very promising approach to control sharka disease. The aim of the present study is to produce transgenic plants of the clonal rootstock ‘Elita’, which is resistant to PPV using ribonucleic acid interference (RNAi) technology. The genetic construct containing the self-complementary fragments of the plum pox virus coat protein (PPV-CP) gene sequence were used to induce the mechanism of post-transcriptional gene silencing to ensure virus resistance. Transgenic plants have been produced after agrobacterium-mediated transformation of in vitro explanted leaves. The results of polymerase chain reaction (PCR) and Southern blotting analyses confirmed the stable genomic integration of the PPV-CP sense and antisense intron-hairpin-RNA sequence. The functionality of the introduced expression cassette was confirmed by the activity of including the uidA gene into the transferring T-DNA. To our knowledge, this is the first interspecific plum rootstock produced by genetic engineering to achieve PPV resistance.

Sharka (Plum pox virus): a forgotten disease in Iran.

Plum pox or sharka, a viral disease caused by Plum pox virus (PPV), severely affects the production of Prunus species in Europe. The first evidence of sharka was reported in Iran in 2000 . Due to the economic impact of this disease on crop production, recent advances in the term of biology, epidemiology, and disease management are provided in this paper to assure awareness among growers and professionals involved in Prunus production. This study will provide fundamental knowledge about this virus to guaranty the successful detection and controlling of sharka disease in Iran.

Genetic dissection of Sharka disease tolerance in peach (P. persica L. Batsch)

BackgroundPlum pox virus (PPV), agent of Sharka disease, is the most important quarantine pathogen of peach (P. persica L. Batsch). Extensive evaluation of peach germplasm has highlighted the lack of resistant sources, while suggesting the presence of a quantitative disease resistance, expressed as reduction in the intensity of symptoms. Unravelling the genetic architecture of peach response to PPV infection is essential for pyramiding resistant genes and for developing more tolerant varieties. For this purpose, a genome-wide association (GWA) approach was applied in a panel of accessions phenotyped for virus susceptibility and genotyped with the IPSC peach 9 K SNP Array, and coupled with an high-coverage resequencing of the tolerant accession ‘Kamarat’.ResultsGenome-wide association identified three highly significant associated loci on chromosome 2 and 3, accounting for most of the reduction in PPV-M susceptibility within the analysed peach population. The exploration of associated intervals through whole-genome comparison of the tolerant accession ‘Kamarat’ and other susceptible accessions, including the PPV-resistant wild-related species P. davidiana, allow the identification of allelic variants in promising candidate genes, including an RTM2-like gene already characterized in A. thaliana.ConclusionsThe present study is the first effort to identify genetic factors involved in Sharka disease in peach germplasm through a GWA approach. We provide evidence of the presence of quantitative resistant loci in a collection of peach accessions, identifying major loci and highly informative SNPs that could be useful for marker assisted selection. These results could serve as reference bases for future research aimed at the comprehension of genetic mechanism regulating the complex peach-PPV interaction.

A fast and efficient protocol for small RNA extraction in Japanese plum and other Prunus species

Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum ( Prunus domestica ) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV- Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV–P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion: We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species. Normal 0 false false false ES-CL X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:ES-CL;} Background: Results: Conclusions:

Spread and competitiveness ofPlum Pox Virus:Rec and -D strains in experimental plum orchard

Sharka disease caused by Plum pox virus (PPV) is endemic in Bulgaria. The speed and mode of spread of PPV-Rec and PPV-D isolates, molecularly characterised in advance and introduced in an experimental plum orchard to serve as permanent sources of infection was studied from 2008 to 2015. The trees were visually inspected for symptoms development and tested by DASI-ELISA twice each year. The positive samples were serologically (-M and -D specific MAbs) and molecularly (IC-RT-PCR) strain-typed, and sequenced in (Cter)NIb-(Nter)CP coding region. Artificially inoculated trees were found infected in the spring of 2009. After 7 years 43 trees (11.2%) became naturally infected. The majority of the trees were infected by PPV-Rec (65%), while only one tree (2.5%) by PPV-D. Nevertheless PPV-M was not introduced in the orchard, this strain was found in single and mixed infections with PPV-Rec strain. None of the isolates in naturally infected trees was identical to PPV-Rec and PPV-D inoculums, while several of them were a 100% identical to the respective PPV-Rec, PPV-D or PPV-M isolates from neighbourhood-infected trees. After the initial random pattern of PPV spread, aggregation of the new infections around the trees infected in previous years was observed. The presence of only Phorodon humuli and Hyalopterus pruni complex, known as PPV vectors and their poor abundance (5.0% from all caught aphids), together with some specific features of the landscape influenced the slow natural development of PPV in the experimental orchard.

Sharka disease (Plum pox virus) in Turkey: the past, present and future

Sharka disease (caused by Plum pox virus, PPV) was first reported on plum trees in Edirne province of Turkey in 1968. It was later found in Ankara (1972), then in the Marmara region (Bilecik, Bursa, Izmit, Istanbul and Tekirdag provinces, 1984) and the Aegean region (Izmir, 1986). Further studies in the Aegean and Marmara regions in 1994 showed that PPV was more widespread, and it was detected on plums, apricots and peach trees in more provinces such as Aydin, Balikesir, Canakkale, Izmir and Manisa. After that, new survey studies were conducted in different provinces and the virus was also detected in the Mediterranean region (Kahramanmaras, Adana, Mersin, Hatay, Isparta), Aegean region (Afyon, Kutahya), Central Anatolia (Aksaray, Kayseri, Konya), Marmara region (Kirklareli) and Black Sea region (Samsun). Recently, extensive surveys in Prunus orchards and nurseries were initiated by the Turkish Agriculture, Food and Livestock Ministry within the framework of a National Sharka Project. As a result of this project, important stone-fruit-growing regions were surveyed, new PPV outbreaks were identified and eradication programmes were strictly applied. Serological and molecular characterization studies of Turkish PPV isolates showed that two common strains were PPV-M and PPV-T, of which M appears in recently infected regions, whereas T appears in regions where PPV infection has been known for years. PPV-D has mostly been found as mixed infections with PPV-M, but it has recently also been found as a single infection in Central Anatolia. The most abundant aphid species in PPV-infected regions of Turkey were found to be Myzus persicae, Hyalopterus pruni, Aphis gossypii and Aphis spiraecola, and these species had the highest percentage of viruliferous aphids. Experimental transmission trials confirmed that Myzus persicae was the most efficient vector for PPV-T. When some of the local apricot cultivars such as ‘Hacihaliloglu’, ‘Cologlu’ and ‘Sekerpare’ were crossed with PPV-resistant apricot cultivars like ‘Stark Early Orange’, ‘Harcot’, ‘Harleyne’ and ‘Stella’, some progeny were found to be tolerant to PPV. Molecular screening and phenotyping studies are still under way.

Development and improvement of detection technologies to control sharka disease

First reported in Bulgaria in 1933, Plum pox virus (PPV), the causal agent of sharka disease, has spread throughout Europe, despite its classification as a quarantine pest. In recent years, PPV has reached North Africa, America, and Japan. Sharka disease causes severe yield losses to the stone-fruit industry. As is typical for plant virus diseases, there is neither cure nor effective treatment of infected trees. Detection and quarantine measures are relied upon to delay or halt virus spread. PPV detection has led to the harmonization of preventive measures such as spring season surveys of nurseries and orchards, the use of certified virus-free plants, removal of infected trees, and creation of quarantine zones. In summer, while a majority of susceptible cultivars exhibit PPV symptoms, some tolerant cultivars are symptomless, and infected fruits become marketable. This situation led us to analyze fruits through RT-PCR to evaluate levels of PPV in these symptomless marketed fruits. Because evidence of PPV infection was previously found in some fruit samples, this study may have implications for the export and marketing of fruits from PPV-tolerant plum cultivars.