Experimental Transmission of Plum Pox Virus (PPV) to Prunus Mahaleb and Prunus avium

Sharka caused by plum pox virus (PPV) is a disease spread in France since 1970, and causing severe damages essentially on apricot but also on plums and peach. Cherry is generally considered as not infected by PPV. Experimental transmissions by chip budding or aphids allowed to show that 3 isolates of PPV can multiply inside three cherry rootstocks (P. Mahaleb cv.‘SL 64′, P. avium cv.‘F 12-1′, and P. avium*P. pseudocerasus cv. ‘Colt') (Tables 1 and 2). But generally, the virus remained localized to the infection site and disappeared quickly (Table 3). Typical symptoms of chlorotic ringspot or vein clearing are also limited to the leaves probed by the aphids. The fact that no translocation was detected is discussed.     

Serological detection of Sharka quarantine disease (Plum pox virus) on stone fruit trees in Golestan province

Sharka disease is one of the most damaging diseases of fruit trees in the world, which is caused by Plum pox virus (PPV) that belongs to the genus Potyvirus in the family Potyviridae. Each year, this virus decreases the yield and causes substantial economic damages to its host plants worldwide. This virus is quarantined in Iran but in recent years, suspicious symptoms of the disease were observed in different grown areas, such as Golestan province. During 2010, 420 samples with mosaic, chlorosis, necrosis, ring pattern, blotches, etc. symptoms were collected from the gardens in Golestan province that included 100 samples from plum, 100 from peach and 240 from nectarine. These samples were evaluated using a double-antibody sandwich-ELISA (DAS-ELISA) method and a polyclonal antibody. The results of this survey indicated that among the total of 420 samples, none of them showed positive reaction in DAS-ELISA test.     

Development of a high-resolution melting approach for reliable and cost-effective genotyping of PPVres locus in apricot (<Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">armeniaca</Emphasis>)

Sharka, caused by plum pox virus, is the most important viral disease of stone fruits. Important progresses have been recently achieved in apricot (Prunus armeniaca), identifying a major locus on chromosome 1 which explains most of the variability for plum pox virus (PPV) resistance trait. A set of molecular markers associated with the resistance has been developed and validated in different genetic backgrounds, endorsing their application for breeding purposes. Particularly for complex traits as the PPV resistance, requiring long and expensive phenotyping procedures, marker-assisted selection (MAS) bears a great potential to improve the efficiency of conventional breeding. In this work, novel HRM (high-resolution melting) assays were designed for the genotyping of resistant/susceptible alleles at PPV resistance (PPVres) locus. The assays were tested on 51 apricot cultivars and breeding selections already phenotyped for PPV resistance and cross-validated with standard short simple repeat marker data. We demonstrated that three HRM assays, PGS1.21_SNP, PGS1.24_SNP, and ZP002_DEL, represent a reliable, quick, and cost-effective genotyping approach, particularly suitable as high-throughput screening method for large-scale breeding programs.     

Aphid (Hemiptera: Aphididae) species composition and potential aphid vectors of plum pox virus in Pennsylvania peach orchards

Plum pox, an invasive disease recently identified in Pennsylvania stone fruit orchards, is caused by the aphid-transmitted Plum pox virus (genus Potyvirus, family Potyviridae, PPV). To identify potential vectors, we described the aphid species communities and the seasonal dynamics of the dominant aphid species within Pennsylvania peach orchards. Aphids were trapped weekly in 2002 and 2003 from mid-April through mid-November within two central Pennsylvania orchards by using yellow and green water pan traps. In total, 42 aphid species were identified from both orchards over 2 yr. Within orchards, actual species richness ranged from 24 to 30 species. The Abundance Based Coverage Estimator predicted species richness to range from 30 to 36 species, indicating that trap catches were identifying most aphid species expected to occur in the orchard. Three species, Rhopalosiphum maidis (Fitch), Aphis spiraecola Patch, and Myzus persicae (Sulzer), were consistently dominant across locations and years. Orchard-trapped populations of these three species peaked in a similar chronological sequence each year. As expected, trap color influenced the total number and distribution of the predominate species collected. However, the same dominant species occurred in both yellow and green traps. Based on the seasonal population dynamics reported here and on published vector efficacy studies, the most probable significant PPV vector was identified as A. spiraecola. If the PPV pathogen escapes current quarantine or if subsequent reintroductions of PPV occur, these data will be useful for developing plum pox management strategies.     

The efficiency of RNA interference for conferring stable resistance to plum pox virus

Plum transformed with an intron hairpin RNA CP (ihpRNA-CP) was resistant to plum pox virus (PPV) infection through the specific process of RNA silencing involving both small interfering-RNA (siRNA) and a methylated virus transgene. Silencing specifically targeted the PPV genome and led to the degradation of viral RNA in the model plant species Nicotiana benthamiana and the natural Prunus domestica host. Plums inoculated with the five major PPV strains, three widespread PPV strains (D, M, and Rec), and the atypical EA strain did not allow systemic spread of PPV in greenhouse-grown transgenic ihRNA-CP plum over multiple cycles of vegetative growth and cold-induced dormancy. PPV ihRNA-CP N. benthamiana displayed an immunity reaction and also allowed for the testing of PPV-C, a strain that was unable to infect P. domestica. This stable resistance demonstrated in plum based on the accumulation of siRNA can prevent PPV infection and can also act as a “curative” when PPV is inoculated through graft inoculation, through a recovery reaction. Regardless PPV strain variability based on geography, host species, epidemiology and serotypes of the CP protein and substitutions of nucleotides at the NH₂-terminus of CP of the major five PPV strains tested, we show that the use of a PPV-CP intron hairpin (ihp) RNA is an effective strategy to specifically target the PPV genome. We provide methods and tools that demonstrate a reliable path towards developing PPV resistance suitable for protecting stone fruit orchards.     

Interaction of Plum Pox Virus with Specific Colloidal Gold-Labeled Antibodies and Development of Immunochromatographic Assay of the Virus

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in sam- ples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by “sandwich”-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.     

Plum Pox Virus in Japanese Plum from Argentina: Serological Detection and Molecular Characterization of an Isolate from cv. Red Beauty

A Plum pox virus (PPV) isolate detected in a Japanese plum orchard in Pocito (San Juan, Argentina) was transmitted mechanically to Prunus tomentosa and Nicotiana benthamiana. DAS‐ELISA and DASI‐ELISA indicated the virus presence and serological relationship with D‐strain isolates; IC‐RT‐PCR amplified a 1.2‐kb fragment of the virus genome encoding the CP‐3′ nc region. The analysis of the sequence showed the presence of the DAG motif at the 5′ end of the capsid protein and the Rsa I and Alu I sites at the 3′ end. The phylogenetic relationships and multiple alignment with PPV isolates from NCBI database indicated greatest (+98%) homology with the D strain and close identity with MNAT1 ( AF360579 ) USA peach isolate. The sequence analysed showed two amino acid mutations towards the 5′ N‐terminus of CP (the most variable region) with respect to a consensus of PPV D‐strain isolates. This is the first molecular characterization of 3′terminal genome region of PPV isolate to confirm D strain in a Japanese plum from Argentina.     

Molecular Cloning of DNA Complementary to the RNA-Genome of Plum Pox Virus (PPV)

Genomic RNA of plum pox virus (PPV) was used as a template for the synthesis of complementary DNA (cDNA). The generated cDNA molecules were subsequently cloned into pBR 322. A physical map covering 9700 bases of the PPV genome was constructed from 8, clones by hybridization and restriction endonuclease digestion. Clone pPPV-NAT 309, starting at the 3′-end, with an 866 bp insert was used in Northern- and Dot-hybridizations for the detection of single-stranded viral RNA in total nucleic acid as well as in sap preparations of PPV infected Nicotiana clevelandii. The nucleotide sequence of this clone was determined, the amino acid sequence of the coat protein C-terminal part was deduced and compared with four other coat proteins of potyviruses.     

Spread and competitiveness of Plum 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.     

Flanking the major Plum pox virus resistance locus in apricot with co-dominant markers (SSRs) derived from candidate resistance genes

Plum pox virus (PPV) is a potyvirus that causes sharka disease in infested stone fruit trees (Prunus species, peach, apricot, plum). In apricots, the resistance is controlled by a major quantitative trait locus that explains up to 70% of the phenotypic variance; it is localised in the upper part of linkage group 1. In this report, we transformed candidate genes that mapped in the region of the apricot resistance locus into polymerase chain reaction markers (SSCP and SSR) and tested for their co-localisation with the major PPV resistance locus in related and unrelated populations. Three populations of F1 and F2 individuals issued from crosses between the PPV-resistant cultivar ‘Stark Early Orange’ or ‘Goldrich’ and three susceptible parents were used in this study. Molecular-marker data were collected to determine the linkage relationship between the PPV resistance locus in apricots and markers that target candidate disease-resistance genes. In addition, SSR markers linked to resistance-gene candidates were mapped to positions flanking the PPV resistance locus in different apricot populations. Therefore, we demonstrate that this strategy helps to saturate the major genomic region controlling resistance to PPV in apricot with valuable co-dominant markers. O. Sicard and G. Marandel have contributed equally to this work.     

Silencing of the Host Factor eIF(iso)4E Gene Confers Plum Pox Virus Resistance in Plum

Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.     

Interspecific transfer of resistance to Plum pox virus from almond to peach by grafting

Grafting almond variety ‘Garrigues’ onto ‘GF305’ peach seedlings heavily infected by Plum pox virus (PPV) progressively produces the disappearance of viral symptoms and drastically reduces virus accumulation in ‘GF305’ rootstock, in most cases to undetectable levels. This response appears to be specific between almond and peach, as it was not consistently observed by grafting ‘Garrigues’ onto other Prunus species such as plum (‘Adesoto’) or apricot (‘Real Fino’). The ability to induce resistance to PPV in ‘GF305’ was transmitted to the sexual descendants of Garrigues. Furthermore, grafting ‘Garrigues’ onto ‘GF305’ before PPV inoculation completely prevented virus infection, showing that the resistance is constitutive and not induced by the virus. This fact suggests that resistance may be due to the transfer of a defence factor from ‘Garrigues’ almond through the graft union and its interaction with specific factors of ‘GF305’ peach to produce the antiviral response. These results open new avenues to potential protection against PPV in peach, the most economically important species among stone fruits.     

Messung von Merkmalen der quantitativen Scharkaresistenz der Pflaume und Nachweis ihrer Wechselbeziehungen

Measurement of the characteristics of the quantitative resistance in plum to the plum pox virus and evidence for their interrelations Two plum clones, with different degrees of susceptibility towards the plum pox virus (PPV), were inoculated with either a weakly or strongly pathegonic strain of PPV. The symptoms which developed in the older leaves (i. e. the 3rd to 5th basal leaves) were measured by visual rating as well as with the aid of a UV-VIS-spectralphotometer SPECORD M 40. Additionally, the relative virus concentrations in these leaves were determined by ELISA. Significant differences were established between the infected leaves of the PPV-sensitive and PPV-resistant plum clones, irrespective of the method of measurement. The values recorded for the weakly and strongly pathogenic PPV strains were significantly different only with the PPV-sensitive clone. A significant correlation was established between the visual ratings and the colormetrical values or the ELISA-values, as well as between the individual colormetrical values a, L and b. In the system plum-PPV, the b-values are the most important in the evaluation of the severity of the symptoms and in the qualitative colour changes.     

A genetic linkage map for an apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) BC<Subscript>1</Subscript> population mapping <Emphasis Type="Italic">plum pox virus</Emphasis> resistance

Plum pox virus (sharka; PPV) can cause severe crop loss in economically important Prunus species such as peach, plum, apricot, and cherry. Of these species, certain apricot cultivars (‘Stark Early Orange’, ‘Goldrich’, ‘Harlayne’) display significant levels of resistance to the disease and are the genetic substrate for studies of several xlaboratories working cooperatively to genetically characterize and mark the resistance locus or loci for marker-assisted breeding. The goals of the work presented in this communication are the characterization of the genetics of PPV resistance in ‘Stark Early Orange’ and the development of co-dominant molecular markers for marker-assisted selection (MAS) in PPV resistance breeding. We present the first genetic linkage map for an apricot backcross population of ‘Stark Early Orange’ and the susceptible cultivar ‘Vestar’ that segregates for resistance to PPV. This map is comprised of 357 loci (330 amplified fragment length polymorphisms (AFLPs), 26 simple sequence repeats (SSRs), and 1 morphological marker for PPV resistance) assigned to eight linkage groups. Twenty-two of the mapped SSRs are shared in common with genetic reference map for Prunus (T × E; Joobeur et al. 1998) and anchor our apricot map to the general Prunus map. A PPV resistance locus was mapped in linkage group 1 and four AFLP markers segregating with the PPV resistance trait, identified through bulk segregant analysis, facilitated the development of SSRs in this region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lalli, D.A. and Salava, J. contributed equally to this work.     

Genetic engineering of Plum pox virus resistance: ‘HoneySweet’ plum—from concept to product

Sharka disease, caused by Plum pox virus (PPV) was first recorded in Bulgaria during the early twentieth century and since that first report, the disease has progressively spread throughout Europe and more recently to Asia, Africa, North and South America. Few PPV resistance genes have been found to naturally occur in Prunus and this has led to the investigation of biotech approaches to the development of resistance through genetic engineering (GE). A notable example of the utility of this approach is ‘HoneySweet’ plum. PPV protection in this case is based on RNA interference (RNAi) and resistance has been shown to be highly effective, stable, durable, and heritable as a dominant trait. Extensive testing and risk assessment of ‘HoneySweet’ in laboratory, greenhouse and in the field for over 20 years has demonstrated not only the effectiveness but also the safety of the technology. ‘HoneySweet’ has been cleared for cultivation in the USA. By the appropriate regulatory agencies. The development and regulatory approval of ‘HoneySweet’ demonstrate the ability of RNAi technology to contribute to the sustainability of stone fruit production in regions impacted by PPV. Although it has taken almost 100 years since the identification of sharka, we are now able to effectively protect stone fruit species against this disease through the application of GE.     

Quantitative trait analysis of resistance to plum pox virus in the apricot F1 progeny “Harlayne” × “Vestar”

Plum pox virus (PPV) is a devastating stone fruit disease of major importance, and better understanding of the genetic control of resistance to this trait would be useful for more efficient development of resistant cultivars. Previous studies have reported a locus of major effect from PPV resistance on linkage group 1. The current study confirms these results by mapping plum pox virus resistance in a F1 progeny issued from a cross between “Harlayne”, as a PPV-resistant parent, and “Vestar” as a susceptible parent. The hybrids were grafted simultaneously and subsequently inoculated with the PPV-M and D strains. The symptom scoring on leaves was performed nine times over two vegetative cycles. Marker–trait associations were analyzed using the Kruskal–Wallis (KW) non-parametric test, and the PPV resistance loci were mapped using composite interval mapping (CIM). We show that both analyses (KW and CIM) highlighted the upper part of linkage group 1 of the apricot “Harlayne” genitor.     

Identification of simple sequence repeat markers tightly linked to plum pox virus resistance in apricot

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit production in Europe and America. Attempts to stop the disease through the eradication of infected trees have been unsuccessful. Introgression of PPV resistance for crop improvement is therefore the most important goal in Prunus breeding programs. Due to time- and labour-consuming protocols, phenotyping for sharka is still the major bottleneck in the breeding pipeline. In this context, screening of seedlings at early stages of development and marker-assisted selection (MAS) provide the best solution for enhancing breeding efficiency. In this study, we generated 42 simple sequence repeat (SSR) markers from the peach genome assembly v1.0 and an apricot bacterial artificial chromosome clone identified in the physical map of the PPV resistance locus previously defined in apricot. Using a linkage mapping approach, we found SSR markers tightly linked to PPV resistance trait in all our progenies. Three SSR markers, PGS1.21 PGS1.23 and PGS1.24, showed allelic variants associated with PPV resistance with no recombinants in the crosses analysed. These markers unambiguously discriminated resistant from susceptible accessions in different genetic backgrounds. The results presented here are the first successful application of their use in MAS for breeding resistance in Prunus species.     

Detection of Double-Stranded RNA (DSRNA) in Crude Extracts of Virus-Infected Plants by Indirect ELISA

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by indirect ELISA (ELISA-I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)-infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA-I 50 days after inoculation. ELISA values then obtained with the In-Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.