GF305 Peach Research Articles

Molecular and Biological Characteristics of a Peach Latent Mosaic Viroid PC Isolate in Peach from China: Base Mutations in Hairpin Stems and Implications for Symptomatology.

Peach latent mosaic viroid (PLMVd) infects peach trees in China and induces a conspicuous albino phenotype (peach calico, PC) that is closely associated with variants containing a 12-to-14 nucleotide hairpin insertion capped by a U-rich loop. Initially, PC disease distribution was limited to parts of Italy, and it was first detected in the field in China in 2019. To explore the molecular and biological characteristics of PLMVd PC isolates in peach in China, we conducted a comprehensive analysis of disease phenotype development and investigated the data-associated pathogenicity and in vivo dynamics of the Chinese isolate PC-A2 using slash-inoculation into GF-305 peach seedlings. Inoculated seedlings displayed PC symptoms much earlier following topping treatment, and PLMVd infectivity was further assessed using bioassay and semiquantitative RT-PCR experiments. Evolutionary analysis showed that the PC isolate and its progeny variants clustered into a single phylogroup distinct from reference PC-C40 isolates from Italy and PC-K1 and PC-K2 from South Korea. Some PC-A2 progeny variants from green leaves of PC-expressing seedlings showed unbalanced point mutations in hairpin stems compared with the PC-C40 reference sequence and constituted a new stem insertion type. The results reveal associations between the recessive phenotypes of peach albino symptoms and base variation in hairpin stem insertions relative to the PC-C40/chloroplastic heat shock protein 90 reference sequence.

The Expanding Menagerie of Prunus-Infecting Luteoviruses.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

Deep sequencing reveals the first fabavirus infecting peach

A disease causing smaller and cracked fruit affects peach [Prunus persica (L.) Batsch], resulting in significant decreases in yield and quality. In this study, peach tree leaves showing typical symptoms were subjected to deep sequencing of small RNAs for a complete survey of presumed causal viral pathogens. The results revealed two known viroids (Hop stunt viroid and Peach latent mosaic viroid), two known viruses (Apple chlorotic leaf spot trichovirus and Plum bark necrosis stem pitting-associated virus) and a novel virus provisionally named Peach leaf pitting-associated virus (PLPaV). Phylogenetic analysis based on RNA-dependent RNA polymerase placed PLPaV into a separate cluster under the genus Fabavirus in the family Secoviridae. The genome consists of two positive-sense single-stranded RNAs, i.e., RNA1 [6,357 nt, with a 48-nt poly(A) tail] and RNA2 [3,862 nt, with a 25-nt poly(A) containing two cytosines]. Biological tests of GF305 peach indicator seedlings indicated a leaf-pitting symptom rather than the smaller and cracked fruit symptoms related to virus and viroid infection. To our knowledge, this is the first report of a fabavirus infecting peach. PLPaV presents several new molecular and biological features that are absent in other fabaviruses, contributing to an overall better understanding of fabaviruses.

First Report of the Presence of Plum pox virus Rec Strain in France.

Plum pox virus (PPV) is the most detrimental virus in stone fruit crops (Prunus sp.). At least nine monophyletic PPV strains are recognized, three of which, PPV-D, PPV-M, and PPV-Rec, have broad distributions (2). PPV-Rec is characterized by a unique founding recombination event and has been reported mostly from Central and South-Central Europe (2). It is generally considered poorly adapted to peach, and the weak and transient symptoms it causes in the GF305 peach seedling indicator may complicate its biological detection (2). During surveys in the Alsace region of France in spring 2013, a plum orchard with trees (Prunus domestica cv. Quetsche d'Alsace 3066) showing dubious leaf symptoms possibly reminiscent of PPV infection was identified. Testing of material from this plant by ELISA (Bioreba AG, Switzerland) gave clear positive reactions, putting the overall infection rate of the orchard at 6.25%, while a second nearby orchard was found infected at a rate of 0.8%. The presence of PPV was confirmed by polymerase chain reaction (PCR) amplification using either the P1-P2 polyvalent primer pair or the P3M-P4b primer pair, which allows the specific amplification of isolates of the Rec and M strains (1). Sequencing of the 467-nt-long P3M-P4b PCR product (Genbank Accession No. KM035763), which spans the end of the NIb gene and the N-terminal hypervariable end of the coat protein gene, provided clear identification of the PPV isolate as belonging to the Rec strain, since it contained all the PPV-Rec specific mutations in the amplified region and showed 98.7 to 97.7% identity with a range of PPV Rec isolates mostly originating from the Balkans. Identification as a PPV-Rec isolate was also confirmed using a strain-specific reverse-transcription-PCR assay (3). This is, to our knowledge, the first report of the presence of PPV-Rec in France. This finding is worrisome given that PPV-Rec is considered well adapted to plum (2), the most important Prunus crop in Alsace. Further surveillance in Alsace during 2014 failed to provide evidence for the presence of PPV-Rec in other areas of the region away from the initial infection focus, which is currently undergoing eradication efforts.

Analysis of gene expression changes in peach leaves in response to Plum pox virus infection using RNA-Seq.

Differences in gene expression were studied after Plum pox virus (PPV, sharka disease) infection in peach GF305 leaves with and without sharka symptoms using RNA-Seq. For each sample, more than 80% of 100-nucleotide paired-end (PE) Illumina reads were aligned on the peach reference genome. In the symptomatic sample, a significant proportion of reads were mapped to PPV reference genomes (1.04% compared with 0.00002% in non-symptomatic leaves), allowing for the ultra-deep assembly of the complete genome of the PPV isolate used (9775 nucleotides, missing only 11 nucleotides at the 5' genome end). In addition, significant alternative splicing events were detected in 359 genes and 12 990 single nucleotide polymorphisms (SNPs) were identified, 425 of which could be annotated. Gene ontology annotation revealed that the high-ranking mRNA target genes associated with the expression of sharka symptoms are mainly related to the response to biotic stimuli, to lipid and carbohydrate metabolism and to the negative regulation of catalytic activity. A greater number of differentially expressed genes were observed in the early asymptomatic phase of PPV infection in comparison with the symptomatic phase. These early infection events were associated with the induction of genes related to pathogen resistance, such as jasmonic acid, chitinases, cytokinin glucosyl transferases and Lys-M proteins. Once the virus had accumulated, the overexpression of Dicer protein 2a genes suggested a gene silencing plant response that was suppressed by the virus HCPro and P1 proteins. These results illustrate the dynamic nature of the peach-PPV interaction at the transcriptome level and confirm that sharka symptom expression is a complex process that can be understood on the basis of changes in plant gene expression.

Susceptibility of Prunus rootstocks against Marcus and Dideron isolates of Plum pox virus by graft‐inoculation

AbstractSharka (Plum pox virus, PPV) is one of the most important diseases affecting stone fruits. While there is much information on the reaction of cultivars to PPV infection, details about rootstocks are scarce. In this study, we evaluated 28 stone fruit rootstocks belonging to different Prunus species against the Marcus and Dideron strains of PPV. Rootstocks were evaluated under controlled conditions during two growing seasons using two inoculation methods: direct inoculation of own‐rooted rootstocks and grafting onto PPV‐infected GF305 peach seedlings. Our results showed a generalised susceptibility of the rootstocks evaluated. Evaluating own‐rooted rootstocks was more efficient than the traditional method of grafting onto infected GF305 seedlings. As expected, PPV‐M was found to be more aggressive than PPV‐D, producing symptoms and occurring in a higher number of plants, as shown by ELISA. The most susceptible rootstocks were Myran, Viking, Myro pg Pecher, Julior, Myrobolan 29C, Rubira, Myrobolan B, MrS 2/5, Jaspi and MP8. The least susceptible were GF677, Myrotop, Citation and ZH6. These results highlight the necessity to breed PPV‐resistant rootstocks for the different stone fruit species.

Plant growth stimulation in Prunus species plantlets by BTH or OTC treatments under in vitro conditions

The effects of benzothiadiazole (BTH) and l-2-oxothiazolidine-4-carboxylic acid (OTC) on the growth and viral content of micropropagated, Plum pox virus (PPV)-infected peach [(Prunus persica (L.) Batsch] ‘GF305’ plantlets were analyzed. Low BTH and OTC concentrations resulted in a significant increase in the growth of GF305 peach and plum plants, with greater effects in PPV-infected than in healthy GF305 peach plantlets. Neither BTH nor OTC reduced the virus content. In fact, the highest growth and viral contents coincided, especially with the 10μM BTH treatment. Differing effects on the antioxidative metabolism of PPV-infected GF305 peach plantlets were observed, depending on the compound and the concentration used: BTH decreased GSH, whereas OTC increased it. In PPV-infected plants, the 50μM OTC treatment produced a decrease in ascorbate peroxidase, catalase, and glutathione peroxidase, but an increase in superoxide dismutase. However, BTH produced a rise in peroxidase activity. Both 10μM BTH and 50μM OTC produced H2O2 accumulation that was correlated with the histochemical detection of H2O2 by 3,3′-diaminobenzidine staining. PPV infection induced NPR1 expression and a synergistic effect occurred in the presence of 50μM OTC, since this compound produced an up-regulation of NPR1 in both healthy and PPV-infected GF305 peach plantlets. The results showed that GSH, as previously suggested, and/or H2O2 could be involved in the regulation of NPR1 expression. Globally, the results show that both OTC and BTH improved the vigor of Prunus species, including peach and plum, under in vitro conditions, producing positive effects on growth, antioxidative metabolism and NPR1 expression. All of these improvements could be critical for more successful ex vitro acclimatization as well as for improved responses to different stresses.

First Report of Plum bark necrosis stem pitting-associated virus in Stone Fruit Trees in China.

Plum bark necrosis and stem pitting disease was first observed on a 'Black Beaut' plum (Prunus salicina Lindl.) in the United States in 1986 and later is several other countries. Plum bark necrosis stem pitting-associated virus (PBNSPaV; genus Ampelovirus, family Closteroviridae), the putative causal agent of the disease, infects many stone fruit species and causes decline, gummosis, flattening of scaffold branches, and stem necrotic pits in some diseased trees (1,3). An investigation of the incidence of PBNSPaV on stone fruit trees in China was conducted during 2009 and 2010. Leaf samples were collected from 47 trees, including peach (P. persica L. Batsch), nectarine (P. persica L. var. nucipersica Schneider), plum (P. domestica L.), ornamental plum (P. cerasifera Ehrb), sweet cherry (P. avium L.), and flowering cherry (P. serrulata L.), grown in Hubei, Henan, and Shandong provinces in central and northern China. Most of sampled trees showed trunk gummosis or stem pitting. The presence of PBNSPaV was tested by reverse transcription (RT)-PCR using primer set PBN195F/PBN195R (5'-CTGGTCTTCCTGCTACTCCTT-3'/5'-AAGCCCACAATCTCAGAGCG-3') designed for the detection of the coat protein (CP) gene of the virus. Total RNA was extracted from leaves using a CTAB protocol reported by Li et al. (2). Products of the expected size of 190 bp were amplified from 20 samples, including seven cultivated peach, four ornamental peach, one nectarine, two plum, one ornamental plum, three sweet cherry, and two flowering cherry samples. All trees positive for PBNSPaV showed stem pitting symptoms on the base of the trunk. To further confirm these results, a 590-base region of the heat shock protein 70 homolog (HSP70h) gene was amplified by RT-PCR using primers HSP-P1/HSP-P2 (5'-GGAATTGACTTCGGTACAAC-3'/5'-TCGAAAGTACCACCACCGAA-3'). Amplicons of the expected size were cloned into the vector pMD18-T (TaKaRa, Dalian, China) and sequenced by Genscript Corp. (Nanjing, China). Sequences of 18 PBNSPaV isolates were deposited in GenBank with Accession Nos. JF810177-JF810194. Sequence comparisons showed that the partial HSP70h gene from the Chinese PBNSPaV isolates shared 82.2 to 100% nucleotide (nt) and 94.0 to 100% amino acid (aa) similarities between them and 83.6 to 99.1% nt and 94 to 100% aa similarities with the corresponding region of the other PBNSPaV isolates deposited in GenBank. In July 2010, peach GF305 seedlings were inoculated by side grafting with budwoods from two PBNSPaV-positive ornamental peach plants. In June 2011, grooving symptom was observed on the stems of the seedlings and the virus was detected by RT-PCR. The results further confirmed PBNSPaV infection in China. These results show that PBNSPaV and the associated disease occur in main cultivated and ornamental Prunus species in China. Given the importance and the devastating symptoms of the disease, it is important to prevent virus spread by using virus-tested propagation materials.

First Report of Cherry green ring mottle virus on Cherry and Peach Grown in China.

Cherry green ring mottle virus (CGRMV; a member of the genus Foveavirus in the family Flexiviridae) has a single-stranded, positive-sense RNA genome of approximately 8.4 kb (4). The viral infection on several Prunus spp. has been mainly reported in Japan, New Zealand, and some countries in Africa, Europe, and North America (3). The virus can cause leaf yellowing on sour and tart cherry. Sweet cherry plants are symptomless hosts of the virus. During the growing season of 2010, leaf samples were collected randomly from one ornamental cherry (Prunus serrulata L.) and 26 sweet cherry (P. avium (L.) L.) plants grown in Shangdong and Henan provinces in northern China and 64 peach (P. persica L. Batsch) plants grown in Hubei Province in central China and tested for the presence of CGRMV by reverse transcription (RT)-PCR. Total RNA was extracted from leaves using the CTAB protocol reported by Li et al (2). Primer set, CGRMV1/CGRMV2 (1), was used for the amplification of a 949-bp fragment, which contains the complete CP gene of 807 bp. PCR products with the expected size were identified in one ornamental cherry, seven sweet cherry, and eight peach plants. Although some of sampled plants showed leaf chlorosis, we did not find the specific association between the symptom and CGRMV infection. The obtained PCR products were cloned into the vector pMD18-T (TaKaRa, Dalian, China). Three independent clones from each isolate were sequenced by Genscript Corp., Nanjing, China. Results showed that CP sequences from the Chinese CGRMV isolates shared 87.7 to 99.8% nucleotide and 93.3 to 100% deduced amino acid similarities, and clones intra each isolate shared more than 99% nt similarities. The CP gene sequences of two representative isolates from cherry (YT-Ch-1) and peach (Pe-HB-18) were submitted to GenBank with Accession Nos. HQ539656 and JF810672, respectively. The neighbor-joining phylogenetic trees generated with nucleotide and amino acid sequences of CP genes by Clustal X v1.8 revealed that all Chinese CGRMV isolates fell into two well-resolved clades. Most of the Chinese CGRMV isolates (12 of 16 isolates, including the isolate YT-Ch-1) were grouped in a large clade represented by isolate ITA5 (GenBank Accession No. AF533159). Four isolates from peach (including the isolate Pe-HB-18) clustered into another clade represented by isolate ITA6 (GenBank Accession No. AF533160). In July 2010, peach GF305 seedlings were inoculated by side grafting with budwoods from two CGRMV positive cherry plants. In May 2011, some newly developed leaves from all inoculated plants showed vein yellowing. The CGRMV infection in these inoculated peach GF305 plants was detected by RT-PCR and protein A sandwich-ELISA using antiserum raised against the recombinant CP of CGRMV isolate YT-Ch-1 (unpublished data). These results further confirmed the CGRMV infection on field cherry plants as detected by RT-PCR. To our knowledge, this is the first record of the presence of CGRMV in ornamental and sweet cherry and peach plants in China, which provides valuable information for further evaluating the sanitary status of the virus in sweet cherry and peach orchards in China.

Implication of peroxidase activity in development of healthy and PPV-infected micropropagated GF305 peach plants

In this work, we describe the micropropagation of PPV-infected GF305 peach plants for their use as a continuous source of PPV inoculums. We observed that PPV was maintained in each sub-culture as well as in the complete micropropagation cycle, including shoot proliferation, rooting and acclimatization to ex vitro conditions. We assayed the addition of fructose and ferulic acid to the multiplication media, to improve the quality and vigour of shoots. This increased the growth of explants and this effect was correlated with a reduction in POX activity, suggesting the possible participation of POX in the growth regulation of micropropagated GF305 shoots. In general, PPV-infected explants did not show evident Sharka symptoms under in vitro conditions, but some explants showed the typical interveinal chlorosis produced by PPV. However, all the PPV-acclimated plants developed normal Sharka symptoms. The data shows that shoot cultures can be used as a reservoir of PPV inoculums as well as to study different metabolic response of healthy and PPV-infected plantlets. In addition, this strategy could serve as a secure and constant source of both healthy and virus-infected plants for other molecular and biological studies.

Detection, characterization, epidemiology and eradication of Plum pox virus Marcus type in Spain.

Sharka disease caused by Plum pox virus (PPV) is widespread in a number of Spanish stone fruit-growing areas; only the common Dideron type (PPV-D) is prevalent. Systematic surveys of field and packing houses were conducted in 2002 for detection of the aggressive Marcus type (PPV-M). The focus of PPV-M was an infected cv. Royal Gem peach (Prunus persica L.) orchard in Zaragoza province (Ebro Valley, Aragon). Over three years, the virus has spread from this orchard to distant plots by means of grafting of infected plant material and to neighbouring plots by aphid transmission. Serological and molecular analyses of symptomatic trees and fruits confirmed the presence of PPV-M in these plots and segregation of the initial viral population into three serogroups and seven different haplotypes as a consequence of host/individual change or vector transmission. Not only did peach cv. Royal Gem become infected, but also cvs Calante and Gladys, in 4 orchards with a total area of 19.5 ha. Spatial analysis of the spread of infection showed a compound contagion process with long-range (up to 150 meters) and short-range movements to adjacent trees in the same row. The main aphid vectors present in the area were Aphis spiraecola Pagenstecher, A. gossypii Glover and Myzus persicae Sulz.. Experimental aphid transmission assays showed that the introduced PPV-M isolate was more efficiently transmitted to other peach cultivars from GF305 peach seedlings, rather than to P. persica cv. Gladys young trees. All tree blocks infected with PPV-M were uprooted. Eight years later (2010) no more PPV-M has been detected in commercial Prunus orchards in the area suggesting successful eradication.

Changes in the antioxidative metabolism induced by Apple chlorotic leaf spot virus infection in peach [ Prunus persica (L.) Batsch

Apple chlorotic leaf spot virus (ACLSV) is the causal agent of “viruela” disease, one of the limiting factors to apricot production in affected areas in the Southeast of Spain. In this work, the response of antioxidant enzymes to ACLSV infection of an indicator peach genotype, ‘GF305’, which is characterised by a great susceptibility to this virus, was studied before (short-period incubation) and after (long-period incubation) a cold treatment. Short-period ACLSV incubation caused significant changes in ascorbate peroxidase (APX), peroxidase (POX) and catalase (CAT) activities. In addition, long-period ACLSV incubation caused significant changes of activities in most of the antioxidant enzymes examined. The results show increases in the APX, dehydroascorbate reductase (DHAR), superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities, whereas POX suffered a decrease of about 34%. No changes in lipid peroxidation, measured as TBARS, were observed in peach leaves as a consequence of the long-period ACLSV incubation. Overall, the data show that long-period ACLSV incubation did not produce any symptoms in peach GF305 leaves or damage to membranes (no changes in lipid peroxidation), and this response was correlated with an increase in the antioxidant defences in leaves, such as the ASC-GSH cycle enzymes and the SOD and GST activities.

Deep Sequencing of the Small RNAs Derived from Two Symptomatic Variants of a Chloroplastic Viroid: Implications for Their Genesis and for Pathogenesis

Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21–24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (−) RNAs presenting a biased distribution of their 5′ nucleotide, and adopting a hotspot profile along the genomic (+) and (−) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12–14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery.

CONTROVERSIAL CONSIDERATIONS BETWEEN NUCLEOTIDE DIVERSITY OF THE 3' TERMINAL REGION OF PLUM POX VIRUS AND DIFFERENCES IN VIRAL SEROTYPES

Plum pox virus (PPV) is a potyvirus causing economic losses to stone fruit trees. In the present study we have analyzed more than 40 PPV isolates collected from different western, eastern, central European and north African countries To preserve the originality of these isolates, the majority was maintained in GF305 peach (Prunus persica), some in apricot (P. armeniaca), plum (P. domestica, P.insititia), or ornamental (P. tomentosa) trees. Serological tests conducted in 2004, 2005 and 2006 with polyclonal antisera by Agdia (USA) validated the high rate of response with all PPV tested. Two primer pairs were designed to amplify 1200 nucleotides (nt) of the 3‘ region of the PPV genome spanning from the carboxy-terminal part of the Nib cistron to the carboxy- terminus of CP, and 500 nt starting from the conserved EcoRI site of the CP cistron to the polyA tail, respectively. Nucleotide sequences of these isolates were determined either from the amplified fragment or the recombinant plasmid cloned into E. coli. Two regions with variable nucleotides were found, the first 110 nt upstream the cleavage site, and the second in nucleotides encoding the cleavage site of NIa-Pro between the Nib and CP cistrons. Sequence homology among the viral isolates was very high. However, phylogenetic analysis of the sequences coding for CP indicated that diversity resided in the amino-terminal section. This finding was corroborated by the use of monoclonal antibodies to PPV D (Durviz, Spain) and PPV M (Agritest, Italy). We discuss the results of these analyses and, particularly, the significance of these nucleotide diversities.

Identification of Plum pox virus Pathogenicity Determinants in Herbaceous and Woody Hosts

Plum pox virus (PPV) is a member of the genus Potyvirus that is able to infect a large variety of plant species, including trees of the genus Prunus, its natural host. When some PPV isolates are propagated for an extended time in herbaceous plants, their ability to infect trees is reduced. The molecular basis of this change in host infectivity is poorly understood. We report the construction of hybrid viruses from cDNA clones of two D-strain isolates of PPV, PPV-D and PPV-R, which differ in their host range. PPV-D can infect GF305 peach seedlings efficiently, however, it is unable to infect Nicotiana clevelandii plants. Conversely, PPV-R infects N. clevelandii, but not GF305 peach seedlings. The analyses of the hybrid viruses showed that, although determinants of PPV pathogenicity are extensively spread throughout the PPV genome, the 3' terminal region of the PPV-R genome, including the 3' noncoding region and the coding regions for the coat protein (CP), NIb, and part of NIa protein, is sufficient to confer infectivity of N. clevelandii in a PPV-D background. Our data demonstrate a high concentration of amino acid substitutions in the CP and a host-specific effect of a deletion at the N terminus of this protein in PPV pathogenicity in peach and N. clevelandii infectivity experiments. These results suggest that relevant host specificity determinants are located in the N-terminal region of the CP. The analyses of the PPV-R and PPV-D chimeras also showed that key host-specific pathogenicity determinants lie in the 5' terminal third of the PPV genome, a region that spans proteins P1, HCPro, and P3. The selection of mutations in only a few specific residues in proteins P1, P3, and 6K1 after partial adaptation of a chimeric virus (BD-GFP) to N. clevelandii further suggests a relevant role for these proteins in host adaptation.

First Report of the Presence of Plum pox virus Rec Strain in Turkey.

Plum pox virus (PPV) is a detrimental virus in stone fruit crops. Six strains of PPV are recognized, one of which, PPV-Rec, represents a group of isolates sharing a unique founding recombination event (2). This strain has been reported only from central and south-central Europe. Its distribution is of interest because PPV-Rec is reported to induce only weak and transient symptoms in GF305 peach seedlings, which may complicate its detection using this widely used indicator (2). During a field trip in May 2006, a Japanese plum (Prunus salicina) tree showing leaf symptoms reminiscent of PPV infection was identified in Isparta, Turkey. A leaf sample tested by a serological lateral flow PPV Pocket Diagnostic (Central Science Laboratory, Sand Hutton, UK) gave a weak positive reaction. The presence of PPV was confirmed by grafting onto GF305 peach and by PCR amplification and sequencing of a short P3M-P4b PCR product (1; positions 8446 to 8912 on PPV-BOR3; GenBank Accession No. AY028309) spanning the end of the NIb gene and the N-terminal hypervariable end of the coat protein gene. Comparison of the sequence obtained (GenBank Accession No. EF051630) with databases unambiguously identified the isolate as belonging to the Rec strain because it contained all the PPV-Rec specific mutations in the amplified region. In keeping with this identification, the symptoms observed in GF305 were very weak, consisting only of slight vein clearing on a few leaves. This is, to our knowledge, the first report of the presence of PPV-Rec in Turkey.

Micropropagation of ornamental Prunus spp. and GF305 peach, a Prunus viral indicator

A micropropagation approach was developed for nine ornamental Prunus species, P. americana, P. cistena, P. glandulosa, P. serrulata 'Kwanzan', P. laurocerasus, P. sargentii, P. tomentosa, P. triloba, P. virginiana 'Schubert', commercially important in North America, and GF305 peach, commonly used for Prunus virus indexing. The micropropagation cycle based on proliferation of vegetative tissues includes establishment of tissue culture through introduction of shoot meristems in vitro, shoot proliferation, root induction and plant acclimatization steps and can be completed in 5 months. A meristem sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation in ornamental Prunus was obtained through the use of 1 mg l(-1) 6-benzyladenine. For GF305 peach, alteration in the sugar composition, fructose instead of sucrose, and addition of 1 mg l(-1 )ferulic acid had a significant impact on the shoot proliferation rate and maintenance of long-term in vitro culture. Rooting and plant acclimatization conditions were improved using a two-step protocol with a 4-day root induction in indole-3-butiric acid (IBA)-containing media with consequent 3-week root elongation in IBA-free media. One-month incubation of rooted shoots in a vermiculite-based medium resulted in additional shoot and root growth and provided better acclimatization and plant recovery. The micropropagation approach can be used for maintenance of the clonal properties for Prunus spp. as well as a protocol to support meristem therapy against viral infection.

Variants of Peach latent mosaic viroid inducing peach calico: uneven distribution in infected plants and requirements of the insertion containing the pathogenicity determinant

Previous characterization of Peach latent mosaic viroid (PLMVd) variants from a single peach calico (PC) isolate showed that PC symptoms are induced by variants with a 12-13 nt insertion at a specific position and folding into a hairpin with a U-rich loop. Here, this study was extended to two other PC isolates. PLMVd variants with insertions similar to those reported previously (type 1), predominated in one isolate (PC-P2). The second (PC-P1), in addition to these variants, contained others with insertions in the same position and of the same size, but with the hairpin capped by a GA-rich loop (type 2). When symptomatic and non-symptomatic tissues from both isolates were used to inoculate GF-305 peach seedlings, they reproduced the phenotype of the inoculum source, indicating that variants differing in pathogenicity are unevenly distributed within single plants. Moreover, characterization of the progeny from inoculations with the PC-P1 source showed that variants with insertions of type 1 and 2 were predominant in the symptomatic and non-symptomatic seedlings, respectively, confirming the association between PC and variants with type 1 but not type 2 insertions. Inoculations with dimeric in vitro transcripts from PLMVd variants with type 1, type 2 and with a chimeric insertion showed that the variant with type 2 insertion was latent and established that the U-rich capping loop has a major role in PC, although the adjacent stem may also have some influence. Insertions can be acquired and lost during infection, suggesting that latent variants can evolve into pathogenic variants and vice versa.

Analysis of recombinant Plum pox virus (PPV) isolates from Serbia confirms genetic homogeneity and supports a regional origin for the PPV-Rec subgroup

The recent observation of the frequent occurrence of natural recombinant Plum pox virus (PPV) isolates has led to the identification of a distinct PPV subgroup, named PPV-Rec. The diversity, origin and geographical spread of the recombinant PPV isolates belonging to this subgroup remain, however, relatively poorly known. In an effort to further our understanding of these isolates, eight PPV isolates from Serbia, the country from which the first such recombinant (PPV-o6) originated, were characterized. Depending on the genomic region targeted by different typing assays, seven of the eight isolates tested presented discrepancies in their typing behavior. Sequence analysis of the (Cter)NIb-(Nter)CP region confirmed the recombinant nature of these seven isolates which all presented an identical recombination breakpoint identical to previously characterized PPV-Rec isolates. Biological indexing and immunoblot analysis provided indications that asymptomatic infection of the GF305 peach indicator and migration of the coat protein as a double-band in immunoblots may represent conserved and discriminating properties of PPV-Rec isolates. The genetic diversity of PPV-Rec isolates from former Yugoslavia (Serbia, Bosnia and Herzegovina) was estimated to be twice as large as that of the PPV-Rec isolates obtained from all other countries to date (Albania, Bulgaria, Czech republic, Germany, Hungary and Slovakia). These last results are consistent with the hypothesis that former Yugoslavia is the center of dispersion of PPV-Rec. Taken together, the results presented here provide further evidence for the wide distribution and temporal genetic stability of these natural PPV recombinant isolates and provide for the first time a possible scenario for their dispersion throughout central and eastern Europe.

Resistance to Plum Pox Virus (Dideron Isolate RB3.30) in a Group of California Almonds and Transfer of Resistance to Peach

Sharka [(plum pox virus (PPV)] mainly affects Prunus species, including apricot (Prunus armeniaca L.), peach (Prunus persica L.), plum (Prunus salicina Lindl., Prunus domestica L.), and, to a lesser degree, sweet (Prunus avium L.) and sour cherry (Prunus cerasus L.). Level of resistance to a Dideron isolate of PPV in seven California almond [P. dulcis (Miller) D.A. Webb], five processing peach cultivars, and two peach rootstocks was evaluated. In addition, almond and peach selections resulting from interspecific almond × peach hybridization and subsequent gene introgression were tested. Evaluations were conducted in controlled facilities after grafting the test genotypes onto inoculated GF305 peach rootstocks. Leaves were evaluated for PPV symptoms during three consecutive cycles of growth. ELISA-DASI and RT-PCR analysis were also employed to verify the presence or absence of PPV. Peach cultivars and rootstocks showed sharka symptoms and were ELISA-DASI or RT-PCR positive for some growth cycles, indicating their susceptibility to PPV. Almond cultivars and almond × peach hybrids did not show symptoms and were ELISA-DASI and RT-PCR negative, demonstrating resistance to PPV. Two (almond × peach) F2 selections as well as two of three backcrossed peach selections also showed a resistant behavior against the PPV-D isolate. Results demonstrate a high level of resistance in almond and indicate potential for PPV resistance transfer to commercial peach cultivars.