Search Results (30)

Viral infections in stone fruit crops cause substantial economic losses across all sectors of production. Despite their significance, viruses affecting stone fruits remain under-investigated in Kazakhstan. Among these, plum pox virus (PPV, genus Potyvirus, family Potyviridae), commonly known as Sharka, is the most critical viral pathogen worldwide, severely threatening the sustainable cultivation of stone fruits and posing risks to food security. This study aimed to evaluate virus management strategies in stone fruit crops to facilitate the production of healthy planting material from valuable genotypes. Field surveys were conducted in plum and apricot orchards located in the Almaty region (Southeast Kazakhstan) and the Saryagash region (Southern Kazakhstan). Plant samples were tested for the presence of the following viruses: apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), PPV, prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), cherry green ring mottle virus (CGRMV), and myrobalan latent ringspot virus (MLRSV). Real-time RT-PCR diagnostics confirmed the presence of PPV in the ‘Stanley’ and ‘Ansar’ cultivars and Prunus armeniaca genotypes, while both PPV and ACLSV were detected in the ‘Ayana’ variety. Chemotherapy (Ribavirin), thermotherapy, cryotherapy, and shoot apical meristem (SAM) culture, both individually and in combination, were used to eliminate viruses and regenerate virus-free plants. Successful virus eradication was achieved for PPV and ACLSV. However, the ‘Stanley’ and ‘Ansar’ cultivars did not survive the treatment process, likely due to high thermo- or cryo-sensitivity. As a result of this research, an in vitro collection of virus-free plants was established, comprising eight rootstocks, six plum cultivars, and three apricot genotypes. Full article

Sharka disease, caused by the plum pox virus (PPV), negatively impacts stone fruit production, resulting in economic losses. It has been demonstrated that grafting the almond (Prunus dulcis (Miller) D.A. Webb) variety ‘Garrigues’ into susceptible peach (Prunus persica (L.) Batsch) rootstocks can result in PPV resistance. The molecular circuits related to grafting in Prunus species, however, have not been fully investigated. In this study, susceptible peach rootstocks ‘GF305’ were either heterografted with ‘Garrigues’ almond or homografted with the same cultivar. Peach samples were collected at two stages of scion development, with ungrafted plants utilized as controls. Profiles of transcripts, small RNAs (sRNAs), and DNA methylation were obtained and analyzed on a genome-wide scale. Homografting and heterografting significantly altered the transcriptome and methylome of peach rootstocks, with these modifications being more pronounced during the early stages of scion development. The profiles of sRNAs were significantly more impacted when almonds were used as a scion as opposed to peaches, likely due to the transmission of PPV-unrelated viral sequences. Gene expression differences resulting from DNA methylation alterations are more thoroughly documented at the promoter sequences of genes than within their bodies. This study suggests that the ‘Garrigues’ almond variety triggers a complex defense response in the peach rootstock, potentially involving the interplay of epigenetic modifications and small RNA-mediated priming of antiviral defenses, which ultimately may contribute to PPV resistance. Full article

Plum pox virus (PPV) is the etiological agent of sharka, the most important viral disease of stone fruit worldwide. In this study, a one-step reverse transcription real-time PCR test (RT-qPCR) was modified and translated as a one-step RT-droplet digital PCR (RT-ddPCR) for sensitive, direct, and accurate detection and quantification of PPV. The modified RT-qPCR and RT-ddPCR PPV detection tests were validated using both plant purified total RNA (TRNA) and crude extract as templates. The proposed tests were sensitive, specific, selective, repeatable, and reproducible in detecting PPV from fresh, lyophilized, and in vitro plant samples. RT-ddPCR was more sensitive than RT-qPCR in detecting PPV using purified TRNA while showing the same sensitivity using crude extract. This work highlights the robustness, time-saving, and cost-effective nature of the proposed one-step RT-ddPCR test, offering a potential reduction in resources for PPV detection and quantification even with raw extracts. Full article

Plasma technology, relatively new in the fields of biomedicine, agriculture, and ecology, is the subject of intensive research as a prospective means of decontamination of various microorganisms (bacteria, viruses, and fungi). The objectives of the present study were to follow the effect of cold atmospheric plasma (CAP) treatment on in vitro grown plum plants (Prunus domestica L. ‘Kyustendilska sinya’ cv.) and the possibility of eradicating or inactivating plum pox virus (PPV) causing Sharka disease by CAP. The source tree is naturally co-infected by PPV (both M and D strains). In the experiments, two different plasma sources were used. First, a surface-wave-sustained Argon plasma torch and second, an underwater diaphragm discharge. For the treatments, nodal segments (10 mm in length) from in vitro cultured plum plants with or without one leaf were prepared. Apical shoots from treated plants (PPV-positive and negative clones as well non-treated controls) were cultivated in vitro for four passages. Then they were rooted and acclimatized to ex vitro conditions, and their virus status was observed periodically for more than 3 years after treatment for the appearance of Sharka symptoms. All plants, acclimatized to ex vitro conditions, were tested for PPV by immune capture–reverse transcription–polymerase chain reaction (IC-RT-PCR). As a first step in understanding the plasma treatment of living plants, a plasma treatment variant causing no damage must be established; this has been done in our previous works. Treatment of plants by plasma with parameters that have been carefully selected leads to better development than the non-treated plants. In the treated in vitro plants, no significant differences were found in the number and length of shoots compared to the control plantlets. In ex vitro acclimated plants, greater stem length was reported, but no differences in leaf number were observed. No significant differences in growth were recorded between the control and plants that were treated twice or three times. At this stage, 3 years after ex vitro cultivation in a greenhouse, Sharka symptoms were not registered on treated in vitro negative PPV plants, and the virus was not detected by IC-RT-PCR. Very mild symptoms were showing in CAP-treated PPV-positive plants. Development of typical Sharka symptoms on non-treated controls were observed. Full article

The recognition of pathogen effectors through the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. In addition to typical domains such as TIR, CC, NBS, and LRR, NLR proteins also contain some atypical integrated domains (IDs), the roles of which are rarely investigated. Here, we carefully screened the soybean (Glycine max) genome and identified the IDs that appeared in the soybean TNL-like proteins. Our results show that multiple IDs (36) are widely present in soybean TNL-like proteins. A total of 27 Gm-TNL-ID genes (soybean TNL-like gene encoding ID) were cloned and their antiviral activity towards the soybean mosaic virus (SMV)/tobacco mosaic virus (TMV) was verified. Two resistance (R) genes, SRA2 (SMV resistance gene contains AAA_22 domain) and SRZ4 (SMV resistance gene contains zf-RVT domain), were identified to possess broad-spectrum resistance characteristics towards six viruses including SMV, TMV, plum pox virus (PPV), cabbage leaf curl virus (CaLCuV), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV). The effects of Gm-TNL-ID^X (the domain of the Gm-TNL-ID gene after the TN domain) on the antiviral activity of a R protein SRC7^TN (we previously reported the TN domain of the soybean broad-spectrum resistance gene SRC7) were validated, and most of Gm-TNL-ID^X inhibits antiviral activity mediated by SRC7^TN, possibly through intramolecular interactions. Yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that seven Gm-TNL-ID^X interacted with SMV-component proteins. Truncation analysis on a broad-spectrum antiviral protein SRZ4 indicated that SRZ4^TIR is sufficient to mediate antiviral activity against SMV. Soybean cDNA library screening on SRZ4 identified 48 interacting proteins. In summary, our results indicate that the integration of IDs in soybean is widespread and frequent. The NLR-ID toolkit we provide is expected to be valuable for elucidating the functions of atypical NLR proteins in the plant immune system and lay the foundation for the development of engineering NLR for plant-disease control in the future. Full article

Sharka is a disease affecting stone fruit trees. It is caused by the Plum pox virus (PPV), with Myzus persicae being one of the most efficient aphid species in transmitting it within and among Prunus orchards. Other agricultural management strategies are also responsible for the spread of disease among trees, such as grafting and pruning. We present a mathematical model of impulsive differential equations to represent the dynamics of Sharka disease in the tree and vector population. We consider three transmission routes: grafting, pruning, and through aphid vectors. Grafting, pruning, and vector control occur as pulses at specific instants. Within the model, human risk perception towards disease influences these agricultural management strategies. Model results show that grafting with infected biological material has a significant impact on the spread of the disease. In addition, detecting infectious symptomatic and asymptomatic trees in the short term is critical to reduce disease spread. Furthermore, vector control to prevent aphid movement between trees is crucial for disease mitigation, as well as implementing awareness campaigns for Sharka disease in agricultural communities that provide a long-term impact on responsible pruning, grafting, and vector control. Full article

The present study focused on trees of Tuscany autochthonous plum cv. Coscia di Monaca in order to evaluate the presence of viruses and elucidate the physiological and biochemical responses to virus infections under real field conditions. Among the several investigated viruses, plums tested positive only to plum pox virus (PPV) and plum bark necrosis steam pitting associated virus (PBNSPaV), occurring as both singular and co-infections. This is the first report of PBNSPaV in a Tuscany orchard. Furthermore, the present study not only confirmed the detrimental effects of PPV on the carbon dioxide assimilation rate due to both stomatal limitations and mesophyll impairments, but also showed that although PBNSPaV did not induce such photosynthetic impairments when occurring as singular infection, it enhanced this damaging effect when present as a co-infection with PPV, as confirmed by a severe decrease in the chlorophyll content. Infection-specific responses in terms of accessory pigments (i.e., carotenoids and xanthophylls), as well as sugars and organic acids, were also reported, these being likely related to photoprotective mechanisms and osmotic regulations under virus-induced oxidative stress. Overall, the results here presented represent an important step to fill knowledge gaps about the interaction of plant viruses and autochthonous Prunus cultivars. Full article

Plum pox virus (PPV) infects Prunus trees across the globe, causing the serious Sharka disease. Breeding programs in the past 20 years have been successful, generating plum varieties hypersensitive to PPV that show resistance in the field. Recently, a single tree displaying typical PPV symptoms was detected in an orchard of resistant plums. The tree was eradicated, and infected material was propagated under controlled conditions to study the new PPV isolate. Performing overlapping PCR analysis, the viral sequence was reconstructed, cloned and tested for infectivity in different ‘Jojo’-based resistant plums. The results confirmed that the isolate, named PPV-D ‘Herrenberg’ (PPVD-H), was able to infect all these varieties. Analyses of chimeras between PPVD-H and a PPV-D standard isolate (PPVD) revealed that the NIa region of PPD-H, carrying three amino acid changes, was enough to break the resistance of these plums. Experiments with single and double mutants showed that all changes were essential to preserve the escaping phenotype. Additionally, one of the changes at the VPg-NIapro junction suggested the involvement of controlled endopeptidase cleavage in the viral response. Transient expression experiments in Nicotiana benthamiana confirmed that NIa cleavage in PPVD-H was reduced, compared to PPVD, linking the observed behavior to an NIa cleavage modulation. Full article

Plum pox virus (PPV) is a causal agent of the stone fruit tree sharka disease that often causes enormous economic losses. Due to its worldwide distribution and economic importance, rapid and reliable diagnostic technologies are becoming increasingly important for successful management of sharka disease. In this study, we have produced two super-sensitive and specific anti-PPV monoclonal antibodies (i.e., MAbs 13H4 and 4A11). Using these two MAbs, we have now developed a dot enzyme-linked immunosorbent assay (dot-ELISA) and a colloidal gold immunochromatographic strip (CGICS) assay. These two technologies can be used to quickly and reliably detect PPV. The results of these sensitivity assays confirmed that the dot-ELISA and CGICS assays could detect PPV infection in apricot tree leaf crude extracts diluted up to 1:5120 and 1:6400 (w/v), respectively. Further analyses using field-collected apricot tree leaf samples showed that the detection endpoint of the dot-ELISA was ~26 times above that obtained through RT-PCR, and the CGICS was as sensitive as RT-PCR. This present study is to broaden the knowledge about detection limits of dot-ELISA and CGICS for PPV monitoring. We consider that these newly developed dot-ELISA and CGICS are particularly useful for large scale PPV surveys in fields. Full article

Sharka disease, caused by the Plum pox virus (PPV), is one of the most harmful, quarantine viral diseases that affect stone fruit crops. The absence of natural resistance to the virus in stone fruits has become a decisive factor for the use of genetic transformation methods to obtain stable forms. The eIF(iso)4G and eIF(iso)4E genes encode translation initiation factors used in the PPV life cycle. In the presented study, the effect of silencing these genes using the RNA interference method on the resistance of sour cherry rootstock 146-2 plants (Prunus pumila L. x Prunus tomentosa Thunb) to the sharka disease was studied. Two vectors have been created for the genetic transformation of plants, with self-complementary sequences of the eIF(iso)4G and eIF(iso)4E gene fragments. The hairpin expression cassette contains a strong promoter of the peach ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) gene, as well as an intron and terminator of the same gene. We used the pMF1 vector containing recombinase R and a codA-nptII gene which makes it possible to obtain intragenic marker-free plants. A successful genetic transformation was carried out by the AGL0 strain of A. tumefaciens. Whole leaves of shoots cultivated in vitro were used as a source of explants. Eight independent transgenic lines of rootstock 146-2 were obtained in experiments (sixlines with a hairpin to the eIF(iso)4G gene and two lines with a hairpin to the eIF(iso)4E gene). Their status was confirmed by the PCR and Southern blotting. The obtained plants were acclimatized in a greenhouse. The silencing of the eIF(iso)4G and eIF(iso)4E genes in transgenic plants was confirmed by the quantitative PCR. The presence of specific small interfering (si) RNAs was confirmed by the method of Northern blotting. Plants of all transgenic rootstock lines were infected with PPV by the method of grafting with infected buds. Resistance to the PPV infection of the obtained transgenic plants was carried out by using an enzyme immunoassay. The ELISA results showed that silencing the eIF(iso)4G gene did not lead to increased resistance while silencing the eIF(iso)4E factor gene led to increased resistance to the PPV, and the one line’s plants showed no signs of infection for two years after infecting. The work demonstrates a (promising) approach in which the creation of stone cultures resistant to the plum pox virus can be achieved by suppressing the genes of translation initiation factors in clonal rootstocks. Full article

Following pathogen infection, plants have developed diverse mechanisms that direct their immune systems towards more robust induction of defense responses against recurrent environmental stresses. The induced resistances could be inherited by the progenies, rendering them more tolerant to stressful events. Although within-generational induction of tolerance to abiotic stress is a well-documented phenomenon in virus-infected plants, the transgenerational inheritance of tolerance to abiotic stresses in their progenies has not been explored. Here, we show that infection of Nicotiana benthamiana plants by Potato virus X (PVX) and by a chimeric Plum pox virus (PPV) expressing the P25 pathogenicity protein of PVX (PPV-P25), but not by PPV, conferred tolerance to both salt and osmotic stresses to the progeny, which correlated with the level of virulence of the pathogen. This transgenerational tolerance to abiotic stresses in the progeny was partially sustained even if the plants experience a virus-free generation. Moreover, progenies from a Dicer-like3 mutant mimicked the enhanced tolerance to abiotic stress observed in progenies of PVX-infected wild-type plants. This phenotype was shown irrespective of whether Dicer-like3 parents were infected, suggesting the involvement of 24-nt small interfering RNAs in the transgenerational tolerance to abiotic stress induced by virus infection. RNAseq analysis supported the upregulation of genes related to protein folding and response to stress in the progeny of PVX-infected plants. From an environmental point of view, the significance of virus-induced transgenerational tolerance to abiotic stress could be questionable, as its induction was offset by major reproductive costs arising from a detrimental effect on seed production. Full article

Peach cultivars (’Elberta’, ‘Red Top’, and ’Dixie Red’) were studied for their in vitro adoptability and performance in producing virus-free plantlets. The thermotherapy method with increasing temperatures (25 °C to 37 °C) was applied for the elimination of famous peach infecting plum pox viruses (PPVs) and prunus necrotic ringspot virus (PNRSV), and the DS-ELISA test and RT-PCR technique were performed to confirm the production of virus-free microshoots. The application of 30 mg L⁻¹ of Fe-EDDHA treatment resulted in the best performance for culture establishment of all cultivars, where the best subsequent morphological performance in terms of branch and leaf numbers was recorded for the ’Dixie Red’ cultivar in MS medium, supplemented with 0.5 mgL⁻¹ of gibberellic acid (GA₃) and 0.5 mg L⁻¹ of 6-Benzylaminopurine (BAP). At the regeneration stage, the highest (26.96 mm) and lowest (18.43 mm) shoot lengths were obtained from the ’Dixie Red’ cultivar treated with GA₃ (2 mg L⁻¹) + thidiazuron (TDZ) (2.5 mg L⁻¹) and the ’Red Top’ cultivar treated with GA₃ (1 mg L⁻¹) + TDZ (1 mg L⁻¹), respectively. The leaf numbers were affected by the application of growth regulators, where the ’Elberta’ cultivar under GA₃ (2 mg L⁻¹) + TDZ (2.5 mg L⁻¹) treatment showed the highest numbers and the ’Red Top’ cultivar under GA₃ (1 mg L⁻¹) + TDZ (1 mg L⁻¹) showed the lowest mean values. The thermotherapy treatment and micropropagation of shoot tips resulted in 100% virus-free plantlets, as confirmed by both applied diagnostic methods. The result of the application of the rooting stage with growth regulators on ’Elberta’ plantlets showed the best performance (90%) in ½ MS medium supplemented with 0.5–1 mg L⁻¹ of IBA, which was significantly higher than the same treatment in MS medium. The obtained results should constitute the basis for further optimization of the multiplication and rooting of virus-free peach plantlets to be served for nurseries and planation orchards. Full article

A viral chimera in which the P1-HCPro bi-cistron of a plum pox virus construct (PPV-GFP) was replaced by that of potato virus Y (PVY) spread slowly systemically in Nicotiana benthamiana plants and accumulated to levels that were 5−10% those of parental PPV-GFP. We tested whether consecutive mechanical passages could increase its virulence, and found that after several passages, chimera titers rose and symptoms increased. We sequenced over half the genome of passaged chimera lineages infecting two plants. The regions sequenced were 5′NCR-P1-HCPro-P3; Vpg/NIa; GFP-CP, because of being potential sites for mutations/deletions leading to adaptation. We found few substitutions, all non-synonymous: two in one chimera (nt 2053 HCPro, and 5733 Vpg/NIa), and three in the other (2359 HCPro, 5729 Vpg/NIa, 9466 CP). HCPro substitutions 2053 AUU(Ile)→ACU(Thr), and 2359 CUG(Leu)→CGG(Arg) occurred at positions where single nucleotide polymorphisms were observed in NGS libraries of sRNA reads from agroinfiltrated plants (generation 1). Remarkably, position 2053 was the only one in the sequenced protein-encoding genome in which polymorphisms were common to the four libraries, suggesting that selective pressure existed to alter that specific nucleotide, previous to any passage. Mutations 5729 and 5733 in the Vpg by contrast did not correlate with polymorphisms in generation 1 libraries. Reverse genetics showed that substitution 2053 alone increased several-fold viral local accumulation, speed of systemic spread, and systemic titers. Full article

In recent years, an important renewal of apricot cultivars is taking place worldwide, with the introduction of many new releases. Self-incompatible genotypes tolerant to the sharka disease caused by the plum pox virus (PPV), which can severely reduce fruit production and quality, are being used as parents in most breeding programs. As a result, the self-incompatibility trait present in most of those accessions can be transmitted to the offspring, leading to the release of new self-incompatible cultivars. This situation can considerably affect apricot management, since pollination requirements were traditionally not considered in this crop and information is lacking for many cultivars. Thus, the objective of this work was to determine the pollination requirements of a group of new apricot cultivars by molecular identification of the S-alleles through PCR amplification of RNase and SFB regions with different primer combinations. The S-genotype of 66 apricot cultivars is reported, 41 for the first time. Forty-nine cultivars were considered self-compatible and 12 self-incompatible, which were allocated in their corresponding incompatibility groups. Additionally, the available information was reviewed and added to the new results obtained, resulting in a compilation of the pollination requirements of 235 apricot cultivars. This information will allow an efficient selection of parents in apricot breeding programs, the proper design of new orchards, and the identification and solution of production problems associated with a lack of fruit set in established orchards. The diversity at the S-locus observed in the cultivars developed in breeding programs indicates a possible genetic bottleneck due to the use of a reduced number of parents. Full article

Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a broad host range. In our work, the expression vectors pAD/pAD-agro based on the plum pox virus (PPV) genome were used for the heterologous expression of different foreign polypeptides: alfalfa mosaic virus capsid protein (AMV CP), zucchini yellow mosaic virus capsid protein (ZYMV CP), the small heat-shock protein of Cronobacter sakazakii fused with hexahistidine (sHSP-his), a fragment of influenza A virus hemagglutinin (HA2-2), influenza A virus protein PB1-F2, SARS-CoV-2 nucleocapsid protein (CoN2-his), and its N- and C-terminal fragments (CoN-1-his and CoN3-his, respectively), each fused with a hexahistidine anchor. Particular proteins differed in their accumulation, tissue localization, stability, and solubility. The accumulation rate of produced polypeptides varied from low (N, hemagglutinin fragment) to relatively high (plant viral CPs, N-terminal fragment of N, PB1-F2). Some proteins preferentially accumulated in roots (sHSP, hemagglutinin fragment, PB1-F2), showing signs of proteolytic degradation in leaf tissues. Thus, each expression requires an individual approach and optimization. Here, we summarize our several-year experiments and discuss the usefulness of the pAD/pADep vector system. Full article