Localisation and movement of Plum pox virus in apricot stem tissues

Localisation and movement of Plum pox virus (PPV), sharka disease, in stem tissues of susceptible and resistant apricot (Prunus armeniaca L.) cultivars was studied. Two different assays were performed. In the first assay, apricot cultivars were grafted on to a non‐inoculated GF305 peach rootstock and, after two months, the sprouted apricot was inoculated by chip‐grafting. In the second assay, apricot cultivars were grafted on to a previously chip‐inoculated GF305 showing strong PPV symptoms. Localisation of virus was studied in apricot stem by immuno‐tissue printing and sharka symptoms in GF305 and apricot leaves were also observed. Virus was mainly localised in the xylem, and sometimes in the cortex and pith. Results revealed that, while all the cultivars allowed limited virus movement from the inoculation point, only the susceptible cultivars (Screara, Bebeco and Colomer) allowed long distance movement and even showed symptoms in leaves.     

Analogues of virus resistance genes map to QTLs for resistance to sharka disease in <Emphasis Type="Italic">Prunus davidiana</Emphasis>

Plum pox virus (PPV), the causative agent of sharka disease in Prunoideae, is one of the most serious problems affecting stone fruit production in Europe and America. Resistance to PPV was previously described in a Prunus davidiana clone, P1908, and introduced into peach (Prunus persica) genotypes. Genetic resistance to PPV displays a complex pattern of quantitative inheritance. An analysis of quantitative trait loci (QTLs) for resistance was performed on an F1 interspecific peach population obtained from a cross between the susceptible nectarine cultivar Summergrand and P. davidiana. The hybrids were graft-inoculated with PPV in duplicate following a classical procedure. The incidence of infection was evaluated four times, over two vegetative cycles, by symptom observation and enzyme-linked immunoadsorbent assays (ELISA). Restriction of systemic downward movement of the PPV virus was also evaluated by testing the susceptible rootstocks. Using both analysis of variance and non-parametric tests, six genomic regions involved in PPV resistance were detected. Depending on the scoring data considered, between 22 and 51% of the phenotypic variance could be explained by the quantitative model. One QTL, located in the distal region of linkage group 1, maps in a genomic region that is syntenic to the location of a resistance gene previously identified in the apricot cv. Goldrich. Some QTLs appeared to be temporally specific, reflecting the environmental dependence of PPV-resistance scoring. Candidate gene fragments were amplified by PCR, isolated and mapped on the peach interspecific linkage map. We report here the co-localization of three analogues of virus resistance genes with two distinct genomic regions linked to PPV resistance in P. davidiana.Electronic Supplementary Material Supplementary material is available for this article at     

Transmission of PPV-M to Prunus persica by Brachycaudus schwartzi and Phorodon humuli (Hem., Aphididae)

Abstract:  Aphids are reported to be vectors of the most serious viral pathogen of the drupaceous species plum pox virus (PPV), but there is little direct experimental evidence of this. PPV (serotype M) is widespread in peach orchards even where there are severe control measures. Laboratory bioassays were conducted to study, under controlled conditions, the ability of Brachycaudus schwartzi (Börner) and Phorodon humuli (Schrank) to transmit PPV (serotype M). The results have shown that all the peach trees tested had evident symptoms of sharka and were positive to the RT-PCR analysis, confirming the ability of these two aphid species to transmit the virus.     

Genome‐wide characterization and expression analysis of genetic variants in sweet orange

Heterozyosity is an important feature of many plant genomes, and is related to heterosis. Sweet orange, a highly heterozygous species, is thought to have originated from an inter‐species hybrid between pummelo and mandarin. To investigate the heterozygosity of the sweet orange genome and examine how this heterozygosity affects gene expression, we characterized the genome of Valencia orange for single nucleotide variations (SNVs), small insertions and deletions (InDels) and structural variations (SVs), and determined their functional effects on protein‐coding genes and non‐coding sequences. Almost half of the genes containing large‐effect SNVs and InDels were expressed in a tissue‐specific manner. We identified 3542 large SVs (>50 bp), including deletions, insertions and inversions. Most of the 296 genes located in large‐deletion regions showed low expression levels. RNA‐Seq reads and DNA sequencing reads revealed that the alleles of 1062 genes were differentially expressed. In addition, we detected approximately 42 Mb of contigs that were not found in the reference genome of a haploid sweet orange by de novo assembly of unmapped reads, and annotated 134 protein‐coding genes within these contigs. We discuss how this heterozygosity affects the quality of genome assembly. This study advances our understanding of the genome architecture of sweet orange, and provides a global view of gene expression at heterozygous loci.     

Chloroplast protection in plum pox virus‐infected peach plants by L‐2‐oxo‐4‐thiazolidine‐carboxylic acid treatments: effect in the proteome

Sharka, a disease caused by plum pox virus (PPV), has a significant economic impact on fruit tree production. In this work, we analysed the effect of (2,1,3)‐benzothiadiazole (BTH) and L‐2‐oxo‐4‐thiazolidine‐carboxylic acid (OTC) on plant growth and virus content. OTC reduced sharka symptom, stimulated plant growth and alleviated PPV‐induced oxidative stress, indicated by a lack of changes in some oxidative stress parameters. PPV infection reduced chloroplast electron transport efficiency. However, in the presence of BTH or OTC, no changes in the chlorophyll fluorescence parameters were observed. PPV produced an alteration in chloroplast ultrastructure, giving rise to a decrease in starch contents that was less dramatic in OTC‐treated plants. Furthermore, PPV reduced the abundance of proteins associated with photosynthesis, carbohydrate and amino acid metabolism and photorespiration. These changes did not take place in OTC‐treated plants, and increases in the expression of proteins related with the aforementioned processes, including ADP‐glucose pyrophosphorylase, were produced, which correlated with the lower decrease in starch contents observed in PPV‐infected plants treated with OTC. The results suggested that OTC treatment provides protection to the photosynthetic machinery and/or the chloroplast metabolism in PPV‐infected peaches. Thus, OTC could have practical implications in agriculture in improving the vigour of different plant species as well as in immunizing plants against pathogens.     

Oxidative stress induced by long-term plum pox virus infection in peach (Prunus persica)

In this study, the effect of long-term plum pox virus (PPV) infection on the response of certain antioxidant enzymes at the subcellular level was studied in peach plants ( Prunus persica (L.) Batch) (cv. GF305), which are characterized by great susceptibility to the virus. In infected plants, a decrease in the efficiency of excitation energy capture by PSII ( F _v'/ F _m') was observed, which was accompanied by a decrease in non-photochemical quenching (NPQ). p -Hydroxy-mercury benzoic acid (pHMB)-insensitive ascorbate peroxidase (APX) activity (class III peroxidase) was detected in both chloroplast and soluble fractions. In soluble fractions from inoculated peaches, a significant increase in pHMB-sensitive APX activity and a significant decrease in superoxide dismutase (SOD) activity were observed. These changes were correlated with the observations in isolated chloroplasts, where an increase in both pHMB-sensitive and pHMB-insensitive APX activities was observed, whereas significant decreases in SOD, monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activities were produced. According to these results, as a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and protein oxidation, was produced in peach leaves, which was monitored by the diaminobenzidine (DAB) peroxidase-coupled H₂O₂ probe. PPV infection produced an alteration in chloroplast ultrastructure, giving rise to dilated thylakoid membranes. PPV-infected peach leaves showed a decreased amount of starch in chloroplasts from palisade parenchyma, as well as an increase in the number and size of plastoglobuli, in relation to control plants. The results suggest that long-term PPV infection produces an oxidative stress, and that an antioxidative metabolism imbalance may be related to the progress of PPV infection and symptoms in peach plants.     

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.     

Comparative genomics analysis in Prunoideae to identify biologically relevant polymorphisms

Prunus is an economically important genus with a wide range of physiological and biological variability. Using the peach genome as a reference, sequencing reads from four almond accessions and one sweet cherry cultivar were used for comparative analysis of these three Prunus species. Reference mapping enabled the identification of many biological relevant polymorphisms within the individuals. Examining the depth of the polymorphisms and the overall scaffold coverage, we identified many potentially interesting regions including hundreds of small scaffolds with no coverage from any individual. Non‐sense mutations account for about 70 000 of the 13 million identified single nucleotide polymorphisms (SNPs). Blast2GO analyses on these non‐sense SNPs revealed several interesting results. First, non‐sense SNPs were not evenly distributed across all gene ontology terms. Specifically, in comparison with peach, sweet cherry is found to have non‐sense SNPs in two 1‐aminocyclopropane‐1‐carboxylate synthase (ACS) genes and two 1‐aminocyclopropane‐1‐carboxylate oxidase (ACO) genes. These polymorphisms may be at the root of the nonclimacteric ripening of sweet cherry. A set of candidate genes associated with bitterness in almond were identified by comparing sweet and bitter almond sequences. To the best of our knowledge, this is the first report in plants of non‐sense SNP abundance in a genus being linked to specific GO terms.     

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

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

Horticultural mineral oil influences Plum pox virus transmission by Myzus persicae

The residual activity of horticultural mineral oil (HMO) on the ability of green peach aphids, Myzus persicae (Sulzer), (GPA) to transmit Plum pox virus (PPV) to peach was measured by infection rates of detached leaves from plants sprayed with either HMO or water as a control that were inoculated using transfer of 25 viruliferous aphids per leaf at 0, 2, 4, 7, 9, 11 and 14 days after treatment (DAT). Persistent effects of HMO residue on the probing and feeding behaviours of GPA were also monitored with the electrical penetration graph (EPG) system. For glasshouse‐grown peach seedlings, the residual activity of HMO reduced PPV infection rates by more than 58% for up to 4 DAT following an initial reduction of approximately 81%. EPG recordings of GPA feeding behaviour showed that HMO significantly delayed first feeding probes and first intracellular punctures by more than 50 min without changing the ensuing stylet penetration behaviour. Applying HMO reduced virus infection rates for up to a week depending on the environmental conditions. EPG monitoring of aphid probing showed that HMO reduced the mean duration and mean number of potential drop (PD) phase feeding occurrences, compared with the water control. A reduction in the PD that has been shown to be related to the transmission of non‐persistently transmitted viruses may partly explain the reduction in PPV infection rates.     

Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses

Plant viruses cause a wide array of disease symptoms and cytopathic effects. Although some of these changes are virus specific, many appear to be common even among diverse viruses. Currently, little is known about the underlying molecular determinants. To identify gene expression changes that are concomitant with virus symptoms, we performed comparative expression profiling experiments on Nicotiana benthamiana leaves infected with one of three different fruit tree viruses that produce distinct symptoms: Plum pox potyvirus (PPV; leaf distortion and mosaic), Tomato ringspot nepovirus (ToRSV; tissue necrosis and general chlorosis), and Prunus necrotic ringspot ilarvirus (PNRSV; subtle chlorotic mottling). The numbers of statistically significant genes identified were consistent with the severity of the observed symptoms: 1,082 (ToRSV), 744 (PPV), and 89 (PNRSV). In all, 56% of the gene expression changes found in PPV-infected leaves also were altered by ToRSV, 87% of which changed in the same direction. Both PPV- and ToRSV-infected leaves showed widespread repression of genes associated with plastid functions. PPV uniquely induced the expression of large numbers of cytosolic ribosomal genes whereas ToRSV repressed the expression of plastidic ribosomal genes. How these and other observed expression changes might be associated with symptom development are discussed.     

Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation

Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus identification, strain differentiation, and de novo assembly of virus genomes. In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for sRNA library construction and deep sequencing. Each library generated between 5-7 million sRNA reads, of which more than 90% were from the tomato genome. Upon in-silico subtraction of the tomato sRNAs, the remaining highly enriched, virus-like siRNA pools were assembled with or without reference virus or viroid genomes. A complete genome was assembled for Potato spindle tuber viroid (PSTVd) using siRNA alone. In addition, a near complete virus genome (98%) also was assembled for Pepino mosaic virus (PepMV). A common mixed infection of two strains of PepMV (EU and US1), which shared 82% of genome nucleotide sequence identity, also could be differentially assembled into their respective genomes. Using de novo assembly, a novel potyvirus with less than 60% overall genome nucleotide sequence identity to other known viruses was discovered and its full genome sequence obtained. Taken together, these data suggest that the sRNA deep sequencing technology will likely become an efficient and powerful generic tool for virus identification in plants and animals.     

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.     

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.