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Article

Viral and Viroid Communities in Peach Cultivars Grown in Bulgaria

by
Mariyana Gozmanova
1,*,
Vesselin Baev
1,
Rumyana Valkova
1,
Elena Apostolova-Kuzova
1,
Stoyanka Jurac
1,
Galina Yahubyan
1,
Lilyana Nacheva
2 and
Snezhana Milusheva
2
1
Department of Molecular Biology, Faculty of Biology, University of Plovdiv, 24 Tsar Assen Str., 4000 Plovdiv, Bulgaria
2
Agricultural Academy, Fruit Growing Institute, 12 Ostromila Str., 4004 Plovdiv, Bulgaria
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(5), 503; https://doi.org/10.3390/horticulturae11050503
Submission received: 12 February 2025 / Revised: 30 April 2025 / Accepted: 6 May 2025 / Published: 7 May 2025
(This article belongs to the Section Biotic and Abiotic Stress)
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Versions Notes

Abstract

:
Peaches (Prunus persica L. Batsch) and nectarines (Prunus persica L. Batsch var. nectarina [Ait.] Maxim) are economically important stone fruits consumed worldwide, both fresh and processed. Viruses and viroids significantly constrain the cultivation and productivity of peach orchards. Climate change may alter vector populations and lead to shifts in agricultural practices, influencing the spread of these viruses and viroids. Additionally, market globalization further intensifies the pressure on peach crops by facilitating the movement of pathogens, increasing the incidence of virus-induced diseases. In this study, we identified the viral and viroid communities in five peach cultivars from Bulgaria and assessed their impact on symptom development. RNA sequencing of symptomatic leaf samples revealed the presence of common peach viruses, such as plum pox virus and prunus necrotic ringspot virus. Notably, we identified peach latent mosaic viroid and cherry green ring mottle virus in Bulgarian peach orchards for the first time. Furthermore, bioassays of indicator plants, ELISA, and Sanger sequencing were performed for each peach tree to complement the RNA sequencing data. These findings provide valuable insights into the composition of viral and viroid pathogens affecting peaches in Bulgaria and will support the development of targeted strategies for monitoring and managing these pathogens, contributing to the sustainable production of peaches in the region.

1. Introduction

Peaches (Prunus persica L. Batsch) and nectarines (Prunus persica L. Batsch var. nectarina [Ait] Maxim) are economically important stone fruits consumed globally, utilized both fresh and processed. Peach trees are the focus of intensive breeding programs aimed at selecting highly productive varieties that possess desired characteristics and exhibit resistance to both abiotic and biotic stressors [1,2].
The growth and production of peach and nectarine trees are severely impacted by numerous fungi, bacteria, nematodes, phytoplasmas, viruses, and viroids worldwide [3,4,5]. This includes several viral diseases, such as those caused by plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), apple chlorotic leafspot virus (ACLV), apple mosaic virus (ApMV), tomato black ring virus (TBRV), tomato ringspot virus (ToRSV), and peach latent mosaic viroid (PLMVd). Some of these pathogens can latently infect peaches, and the infected trees remain asymptomatic for an extended period until plant stressors trigger symptom development. Climate-change-induced shifts in plant virus vector populations and modifications in agricultural practices, combined with market globalization, could influence the spread of pathogens and the incidence of disease.
PPV, genus Potyvirus, is the causative agent of Sharka disease, one of the most damaging viral diseases affecting Prunus species [6]. The severity of symptoms can differ depending on the specific PPV strain, the cultivar, and environmental factors. This disease was first reported in Bulgaria in plums [7]. PNRSV is another important virus affecting peaches. Classified as an Ilarivirus, PNRSV spreads rapidly in orchards through vegetative propagation, seeds, and pollen [8,9]. While PNRSV infection can cause symptoms such as stunting, chlorosis, and tree decline, some strains may also be symptomless. Other viral pathogens, such as cherry green ring mottle virus (CGRMV) and certain strains of PLMVd, are widely distributed in Prunus species due to their latent nature, making their overall economic impact difficult to assess accurately. CGRMV is a Robigovirus in the Betaflexiviridae family that infects several Prunus species and causes symptoms in sour cherry [9]. PLMVd, belonging to the Pelamoviroid genus and Avsunviroidae family, has numerous variants, some of which remain latent, while others cause symptoms such as leaf mosaic, deformation, fruit cracking, and the premature aging of trees [10,11]. The severe peach calico disease, characterized by extreme albinism, is linked to a specific PLMVd variant [12,13,14].
Given the importance of peaches and nectarines to the region’s agriculture, a survey of virus infections was conducted on five peach cultivars in Bulgarian orchards. By integrating high-throughput RNA sequencing with diagnostic techniques such as ELISA (enzyme-linked immunosorbent assay), RT-PCR, and bioassays, this RNA virome study aimed to provide a comprehensive overview of the complexity of virus/viroid communities. As a result, we identified three virus species and one viroid species, all occurring in mixed infections. This discovery is particularly significant because it has implications for disease management strategies.

2. Materials and Methods

2.1. Peach Survey and Sample Collection

A survey of viral infections was conducted in peach orchards across the Plovdiv province of Bulgaria during June 2022 and July 2023, encompassing four orchards in the region aged 6–7 years. Across the inspected orchards, the percentage of the trees manifesting virus-like symptoms varied between 5% and 35%. Leaves and fruits were systematically inspected for virus-associated symptoms, including chlorosis, leaf distortion, stunted growth, and fruit deformities. Observations of leaf symptoms were carried out in June and July, when the leaves were fully developed. Fruit symptom assessments were conducted on mature fruits, depending on the ripening period of the cultivars included in the study, between July and early September. Five symptomatic trees, each representing a distinct cultivar, were randomly selected for further in-depth virological analysis. Two trees were sampled from the orchard showing the highest incidence of virus-like symptoms, while one symptomatic tree was sampled from each of the remaining three orchards. From each tree, 10–12 symptomatic leaves were collected, pooled into a composite sample, and flash-frozen in liquid nitrogen to preserve RNA integrity prior to processing.

2.2. RNA Extraction, RT-PCR, and Sanger Sequencing

For RNA extraction, frozen leaf tissue was homogenized in liquid nitrogen, and 100 mg of pooled material per sample was processed using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer’s protocol. RNA concentration and quality were assessed using the Qubit-4 fluorometer. The first-strand cDNA was synthesized from 500 ng of individual RNA, utilizing a SCRIPT cDNA synthesis kit (Jena Bioscience, Jena, Germany) to perform sample denaturation, along with random hexamers and/or virus/viroid-specific primers. PCR reactions were conducted using Pfu X polymerase (Jena Bioscience) according to the manufacturer’s instructions. PCR amplicons were purified from agarose gel utilizing the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced in both directions by Eurofins Genomics (Ebersberg, Germany). RT-PCR was performed to individually test each of the peach trees included in the study with the primer pair CGRMV1/CGRMV2 for CGRMV [15], P1/P2 primers for PPV [16], primers I/II for PNRSV [17], and PLMVd-H/PLMVd-C primers for PLMVd [18]. Strain identification of the PPV isolates was carried out with primer pairs designed by Šubr et al. [19].

2.3. Long Non-Coding RNA Sequencing and Bioinformatics Analysis

RNA samples from each cultivar were pooled in approximately equal amounts and then sent to VirSeq Services, Plant Virus Department, Leibniz-Institut, Germany, for long non-coding RNA sequencing (lncRNA sequencing). Library construction was carried out in accordance with the Illumina DNA Prep Kit protocol, which included ribosomal RNA depletion treatment. Sequencing on a NextSeq2000 instrument generated paired-end reads (2 × 150 bp). The raw sequencing data were deposited into the SRA database, accompanied by the accession number PRJNA1219823. The raw reads were trimmed using TrimGalore tool (v0.6.7) with default settings, and taxonomic classification was executed using the Kraken 2 tool (v 2.1.3) against the core nt-Database (28 December 2024). The output from Kraken 2 was further analyzed and visualized employing the Pavian software (v1.0), elucidating the flow of reads across taxonomic levels.

2.4. Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS ELISA)

DAS ELISA [20] was performed using commercial antisera to detect PPV and PNRSV in each of the studied trees and indicator plants, following the protocol of the manufacturer, Bioreba AG (Reinach, Switzerland). The microplates were coated with polyclonal antisera diluted 1:1000 (v/v) in a carbonate buffer, pH 9.6. The antigen extracts were prepared by triturating the leaf samples in a ratio of 1:10 in a grinding buffer consisting of phosphate buffered saline (PBS), pH 7.4; 0.5 mL/L Tween 20; 20 g/L polyvynilpirrolidone (PVP); and 2 g/L sodium diethyldithiocarbamate. Alkaline-phosphatase-labeled polyclonal antibodies were diluted in a conjugate buffer (PBS pH 7.4, 0.5 mL/L Tween 20, 20 g/L PVP, and 2 g/L egg albumin) with a dilution of 1:1000 (v/v). The substrate p-nitrophenolphospate was dissolved in a diethanolamine buffer, pH 9.8, at a concentration of 1 mg/mL. The absorbance was recorded at a wavelength of 405 nm using the Multiscan EX (Thermo Fisher Scientific, Vantaa, Finland). Each sample was tested in two replicates. Samples that showed absorbance values greater than two times the absorbance of the negative controls were regarded as positive.

2.5. Biological Assay

A biological assay was performed on the indicator plant GF 305 (Prunus persica). Bud sticks, used as a source of inocula, were taken from three infected peach trees: cv. Summerset (S1), cv. Glohaven (S2), and cv. Royal Gem (21/29). Prior to inoculation, all experimental rootstocks of GF 305 were tested for the presence of the target viruses and viroid using RT-PCR or ELISA. A minimum of three GF 305 plants were inoculated with the selected isolates by chip budding with two or more buds. Non-inoculated trees were kept as negative controls. The biological reactions of the inoculated GF 305 plants were monitored for at least 18 months after inoculation. All inoculated plants and non-inoculated controls were analyzed using ELISA for PPV and PNRSV and RT-PCR for all of the studied pathogens.

3. Results

In the diagnostic survey performed in 2022–2023 in Bulgarian peach and nectarine orchards, four peach trees (cv. Royal Gem, Laskino, Summerset, and Glohaven) and one nectarine tree (cv. Fantasia) showing virus-like symptoms were selected for virome analysis. The symptoms on the leaves included chlorotic blotches, vein clearing, rings and bands surrounding the veins, mottling, mosaic patterns, necrosis, and distortion. Light rings and patterns, discoloration, and malformations were recorded on the fruit of these plants (Figure 1, Table 1).
lncRNA sequencing was chosen for our study due to its unbiased detection of poly-A- and non-poly-A-tailed RNA viruses and the manageable computational demands of the associated data analysis. The lncRNA sequencing of the pooled samples generated 9,363,024 raw reads, with viral sequences accounting for 0.3%. A wide variety of viral taxa were identified, representing well-established plant viruses and viruses that infect non-plant organisms.
The plant virus family Potyviridae (67% of total viral reads) was the most abundant, indicating its dominant representation within the viral community. A high percentage of Potyviruses was expected, as they have a broad range of plant hosts, including many stone fruits. The families Betaflexiviridae (15.2%), Bromoviridae (7.3%), Avsunviroidae (6.9%), Tombusviridae (2.5%), and Virgaviridae (0.4%) were also represented within the viral community. At the family level, non-plant viruses in Nodaviridae were detected at 0.4% (Supplementary Table S1, Figure 2). Although these non-plant viruses are present at low levels in plant tissues, typically infecting animals or invertebrates, they highlight the wide range of viral diversity in the sample.
At the species level, PPV (66.7%), CGRMV (14.7%), PNRSV (7.3%), PLMVd (6.9%), peach-associated luteovirus (PaLV, 1.2%), and nectarine stem pitting-associated virus (NSPaV, 1.1%) were highlighted as key representatives within their genera (Figure 2). Since sequencing was performed on a bulk sample, we subsequently employed multiple diagnostic techniques to confirm the presence of PLMVd, PPV, PNRSV, and CGRMV in the individual trees (Table 2). Among the targeted pathogens, PPV and PLMVd were identified in all of the studied trees.
PLMVd was the most consistently detected pathogen across all of the analyzed peach and nectarine tree samples, with positive results obtained using both RT-PCR and Sanger sequencing. Its widespread presence suggests a high infection incidence within the tested population. Amplicons from two samples were successfully sequenced and deposited in the GenBank Nucleotide Sequence Database under accession numbers PV037676 and PV037677. The remaining sequences exhibited ambiguous nucleotide positions, likely reflecting mixed infections involving multiple PLMVd variants.
ELISA is a robust and reliable method for detecting plant viruses, particularly when targeting well-characterized pathogens such as PPV and PNRSV. However, commercial ELISA kits for CGRMV are currently not available, limiting serological detection options for this pathogen. To complement the obtained ELISA results for PPV and PNRSV, RT-PCR assays were performed. The PPV-M strain was specifically identified using primer pairs targeting the genomic region spanning the C-terminus of the viral replicase and the N-terminus of the coat protein [19] in the five studied trees. ELISA and RT-PCR analysis confirmed the presence of PNRSV only in cv. Summerset (sample S1). Using primers specific to the coat protein gene [15], CGRMV was identified only in cv. Summerset (S1) and cv. Glohaven (S2). Amplicon sequencing showed the same sequence in the two cultivars, which was deposited in GenBank under accession number PV102403.
Molecular and serological analyses revealed mixed infections within individual trees. These assays indicated the presence of PLMVd and PPV in cvs. Royal Gem, Fantasia, and Laskino. Additionally, PLMVd, PPV, and CGRMV were detected in cv. Glohaven, while PLMVd, PPV, CGRMV, and PNRSV were identified in cv. Summerset. These findings underscore the complexity of viral infections in individual trees, where multiple pathogens coexist.
The bioassay is a classical method in virus research, as it provides valuable insight into the pathogenicity, symptom development, transmission, and host specificity of viral and viroid infections. The first virus-like symptoms on inoculated GF 305 plants were observed approximately 2 to 3 months after inoculation. The symptoms recorded on GF 305 grafted with inocula from the studied trees was likely associated with a mixed virus/viroid infection. The most frequent virus-like symptoms observed on GF 305 inoculated with buds from cv. Summerset (S1, co-infected with PLMVd, PPV, PNRSV, and CGRMV) were leaf distortion, vein clearing, chlorotic rings, and irregular yellowish areas, differing in size, which later pervaded the leaf blades (Figure 3A). The indicator plants grafted with buds from cv. Glohaven (S2, mixed infected by PLMVd, PPV, and CGRMV) exhibited vein clearing, chlorotic blotches, and rings. GF 305 seedlings grafted with inocula from cv. Royal Gem (21/29, co-infected by PPV and PLMVd) reacted with chlorotic patterns and rings, vein clearing, and leaf deformations (Figure 3B). To confirm the presence of PLMVd, PPV, PNRSV, and CGRMV in the grafted indicator seedlings, we performed RT-PCR and ELISA. These diagnostic methods validated the ability of the analyzed viruses and viroid to infect the indicator plants.

4. Discussion

Southern Bulgarian regions are the leading areas for peach production in the country. Due to unfavorable climatic conditions, the country’s average yield decreased from 9519.7 kg/ha in 2022 to 7297.3 kg/ha in 2023 [21]. Another reason for yield losses in peaches that must be taken into account is the pressure of various pathogens.
Traditional biological, serological, and molecular diagnostic methods have limitations in detecting all viral agents, especially those present in low quantities or emerging viral genotypes. These approaches often fail to capture the full diversity of viral populations. To address these challenges and expand our understanding, researchers worldwide have employed various NGS technologies [22,23].
Using the NGS technique, a comprehensive survey of six Korean peach cultivars identified five viruses in the family Betaflexyviridae, a novel Tymovirus, and two viroids [24]. The technique facilitated the identification of SNPs, allowing for mutation frequency analysis in each detected virus or viroid. NGS of small RNAs was used to characterize the viral profiles, including PPV, PLMVd, NSPaV, and PaLV, in Hungarian peaches [25]. In our study, bulk RNA sequencing of five Bulgarian peach cultivars identified the same pathogens. Previously, a significant increase only in PPV infection was reported, with approximately 60% of peach orchards in Southern Bulgaria being affected by PPV [26].
PLMVd is widely distributed, and its presence has been documented in neighboring countries of Bulgaria [27,28,29]. Recently, a relatively high incidence of PLMVd was reported among Mediterranean countries [30]. Here, we report the presence of PLMVd in peach orchards in Bulgaria for the first time, thereby expanding its known range in Europe. PLMVd can be transmitted by the green peach aphid (Myzus persicae) and pollen [11], which may facilitate its spread to a new territory. PLMVd was detected in co-infections with CGRMV and PNRSV in symptomatic nectarine trees in China [31]. Similarly, studies in Chile identified PLMVd and PNRSV as the most frequent mixed infections in stone fruit trees [32]. Notably, PNRSV co-infection with PDV leads to peach stunt disease and progressive tree decline, resulting in yield losses of up to 55% [33]. CGRMV was detected in Tennessee peach orchards through transcriptome analysis [34]. In our study, we observed PLMVd, CGRMV, PPV, and PNRSV in mixed infections in cv. Summerset, and PLMVd, CGRMV, and PPV co-infected cv. Glohaven, further supporting the occurrence of complex viral interactions in Bulgarian peaches. PNRSV was previously found with an infection incidence of 18.7% in peach orchards in the Plovdiv region [35], emphasizing the need for early detection to control disease spread and prevent new infections.
Our NGS data revealed sequence reads corresponding to PaLV and NSPaV of the family Luteoviridae in Bulgarian peach cultivars, representing their first potential detection in the country; these preliminary findings will require further validation and characterization in future studies. In previous research, NSPaV was identified using metagenomic analysis in stunted nectarine trees grafted onto peach rootstock [36]. Recently, four new Luteovirus species were identified by high-throughput RNA sequencing in wild, cultivated, and ornamental Prunus gardens [37]. Given the potential economic and ecological impact of Luteoviruses, further investigation is essential to determine their prevalence, diversity, and effects in Bulgarian orchards.
Overall, our study provides an initial analysis of viral communities in peach trees from Bulgarian orchards, revealing the presence of viruses and viroids from various taxonomic groups. These findings emphasize the complex interactions between viruses and viroids within horticultural crops, underscoring the challenges they pose for accurate diagnosis and effective management.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11050503/s1. Table S1: Viral community structure at the family level in symptomatic peaches and nectarines from Bulgaria.

Author Contributions

Conceptualization, S.M. and M.G.; methodology, S.M., R.V., E.A.-K., and S.J.; software, V.B.; validation, M.G., R.V., E.A.-K., and S.J.; formal analysis, V.B.; investigation, M.G., V.B., R.V., E.A.-K., S.J., G.Y., L.N., and S.M.; resources, S.M., M.G., V.B., and L.N.; data curation, V.B.; writing—original draft preparation, M.G. and S.M.; writing—G.Y., M.G., R.V., and S.M.; visualization, V.B.; supervision, S.M.; project administration, S.M.; funding acquisition, S.M. and M.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the European Union’s NextGenerationEU, the National Recovery and Resilience Plan of the Republic of Bulgaria, project BG-RRP-2.004-0001-C01, and the Bulgarian National Science Fund under Grant No. KP-06-N 36/10 (KП-06-H 36/10) from 2019. The APC was funded by the Bulgarian National Science Fund, Grant No. KP-06-N 36/10. NGS was funded by the European Virus Archive Global (EVAg) project, through the European Union’s Horizon 2020 research and innovation program under grant agreement no. 871029.

Data Availability Statement

The raw NGS data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1219823. The data will be publicly available following the scheduled release date of 1 February 2026.

Acknowledgments

The authors are thankful to W. Menzel and P. Margaria (Leibniz Institute DSMZ) for their support in the framework of the EVAg Trans-National Access program.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Li, Y.; Cao, K.; Zhu, G.; Fang, W.; Chen, C.; Wang, X.; Zhao, P.; Guo, J.; Ding, T.; Guan, L.; et al. Genomic Analyses of an Extensive Collection of Wild and Cultivated Accessions Provide New Insights into Peach Breeding History. Genome Biol. 2019, 20, 36. [Google Scholar] [CrossRef]
  2. Petruccelli, R.; Bonetti, A.; Ciaccheri, L.; Ieri, F.; Ganino, T.; Faraloni, C. Evaluation of the Fruit Quality and Phytochemical Compounds in Peach and Nectarine Cultivars. Plants 2023, 12, 1618. [Google Scholar] [CrossRef]
  3. Umer, M.; Liu, J.; You, H.; Xu, C.; Dong, K.; Luo, N.; Kong, L.; Li, X.; Hong, N.; Wang, G.; et al. Genomic, Morphological and Biological Traits of the Viruses Infecting Major Fruit Trees. Viruses 2019, 11, 515. [Google Scholar] [CrossRef]
  4. Hou, W.; Li, S.; Massart, S. Is There a “Biological Desert” With the Discovery of New Plant Viruses? A Retrospective Analysis for New Fruit Tree Viruses. Front. Microbiol. 2020, 11, 592816. [Google Scholar] [CrossRef]
  5. Martelli, G.P.; Flores, R.; Schneider, B. CHAPTER 3: Classification of Pome and Stone Fruit Viruses, Viroids, and Phytoplasmas. In Virus and Virus-Like Diseases of Pome and Stone Fruits; The American Phytopathological Society: Minnesota, MN, USA, 2011. [Google Scholar]
  6. Rubio, M.; Martínez-Gómez, P.; Marais, A.; Sánchez-Navarro, J.A.; Pallás, V.; Candresse, T. Recent Advances and Prospects in Prunus Virology. Ann. Appl. Biol. 2017, 171, 125–138. [Google Scholar] [CrossRef]
  7. Atanasoff, D. Plum pox. A New Viral Disease. In Annuaire de L’ Universite de Sofia, Faculte d’Agronomie et de Silviculture, Volume 1932/33; Grafika: Sofia, Bulgaria, 1933; pp. 49–70, (In Bulgarian, English Summury). [Google Scholar]
  8. Pallas, V.; Aparicio, F.; Herranz, M.C.; Amari, K.; Sanchez-Pina, M.A.; Myrta, A.; Sanchez-Navarro, J.A. Ilarviruses of Prunus Spp.: A Continued Concern for Fruit Trees. Phytopathology 2012, 102, 1108–1120. [Google Scholar] [CrossRef]
  9. Jelkmann, W.; Rott, M.; Uyemoto, J.K. Chapter 23: Cherry Green Ring Mottle Virus. In Virus and Virus-like Diseases of Pome and Stone Fruits; The American Phytopathological Society: Minnesota, MN, USA, 2011. [Google Scholar]
  10. Flores, R.; Delgado, S.; Rodio, M.E.; Ambrós, S.; Hernández, C.; Di Serio, F. Peach Latent Mosaic Viroid: Not so Latent. Mol. Plant Pathol. 2006, 7, 209–221. [Google Scholar] [CrossRef]
  11. Flores, R.; Di Serio, F.; Hernández, C.; Barba, M.; Hadidi, A. CHAPTER 40: Peach Latent Mosaic Viroid and Its Diseases in Peach Trees. In Virus and Virus-like Diseases of Pome and Stone Fruits; The American Phytopathological Society: Minnesota, MN, USA, 2011; pp. 219–224. [Google Scholar]
  12. Malfitano, M.; Di Serio, F.; Covelli, L.; Ragozzino, A.; Hernández, C.; Flores, R. Peach Latent Mosaic Viroid Variants Inducing Peach Calico (Extreme Chlorosis) Contain a Characteristic Insertion That Is Responsible for This Symptomatology. Virology 2003, 313, 492–501. [Google Scholar] [CrossRef]
  13. Rodio, M.E.; Delgado, S.; Flores, R.; Di Serio, F. Variants of Peach Latent Mosaic Viroid Inducing Peach Calico: Uneven Distribution in Infected Plants and Requirements of the Insertion Containing the Pathogenecity Determinant. J. Gen. Virol. 2006, 87, 231–240. [Google Scholar] [CrossRef]
  14. Navarro, B.; Gisel, A.; Rodio, M.E.; Delgado, S.; Flores, R.; Di Serio, F. Small RNAs Containing the Pathogenic Determinant of a Chloroplast-replicating Viroid Guide the Degradation of a Host MRNA as Predicted by RNA Silencing. Plant J. 2012, 70, 991–1003. [Google Scholar] [CrossRef]
  15. Li, R.; Mock, R. An Improved Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay for the Detection of Two Cherry Flexiviruses in Prunus spp. J. Virol. Methods 2005, 129, 162–169. [Google Scholar] [CrossRef] [PubMed]
  16. Wetzel, T.; Candresse, T.; Ravelonandro, M.; Dunez, J. A Polymerase Chain Reaction Assay Adapted to Plum Pox Potyvirus Detection. J. Virol. Methods 1991, 33, 355–365. [Google Scholar] [CrossRef] [PubMed]
  17. Rosner, A.; Maslenin, L.; Spiegel, S. The use of short and long PCR products for improved detection of prunus necrotic ringspot virus in woody plants. J. Virol. Methods 1997, 67, 135–141. [Google Scholar] [CrossRef] [PubMed]
  18. Loreti, S.; Faggioli, F.; Cardoni, M.; Mordenti, G.; Babini, A.R.; Pollini, C.P.; Barba, M. Comparison of Different Diagnostic Methods for Detection of Peach Latent Mosaic Viroid. EPPO Bull. 1999, 29, 433–438. [Google Scholar] [CrossRef]
  19. Šubr, Z.; Pittnerová, S.; Glasa, M. A Simplified RT-PCR-Based Detection of Recombinant Plum Pox Virus Isolates. Acta Virol. 2004, 48, 173–176. [Google Scholar]
  20. Clark, M.F.; Adams, A.N. Characteristics of the Microplate Method of Enzyme Linked Immunosorbent Assay for the Detection of Plant Viruses. J. Gen. Virol. 1977, 34, 475–483. [Google Scholar] [CrossRef]
  21. FAOSTAT FAOSTAT Online Database. Available online: https://www.fao.org/faostat/en/#data/QC (accessed on 10 January 2025).
  22. Visser, M.; Bester, R.; Burger, J.T.; Maree, H.J. Next-Generation Sequencing for Virus Detection: Covering All the Bases. Virol. J. 2016, 13, 85. [Google Scholar] [CrossRef]
  23. Pecman, A.; Kutnjak, D.; Gutiérrez-Aguirre, I.; Adams, I.; Fox, A.; Boonham, N.; Ravnikar, M. Next Generation Sequencing for Detection and Discovery of Plant Viruses and Viroids: Comparison of Two Approaches. Front. Microbiol. 2017, 8, 1998. [Google Scholar] [CrossRef]
  24. Jo, Y.; Lian, S.; Chu, H.; Cho, J.K.; Yoo, S.H.; Choi, H.; Yoon, J.Y.; Choi, S.K.; Lee, B.C.; Cho, W.K. Peach RNA Viromes in Six Different Peach Cultivars. Sci. Rep. 2018, 8, 1844. [Google Scholar] [CrossRef]
  25. Barath, D.; Jaksa-Czotter, N.; Varga, T.; Varallyay, E. Viromes of Hungarian Peach Trees Identified by High-Throughput Sequencing of Small RNAs. Plants 2022, 11, 1591. [Google Scholar] [CrossRef]
  26. Milusheva, S.; Dimitrova, N.; Pandova, S. Plum pox virus strains identified in peach in South Central region of Bulgaria. Fruit Grow. Res. 2023, XXXIX, 140–144. [Google Scholar] [CrossRef]
  27. Kyriakopoulou, P.E.; Giunchedi, L.; Hadidi, A. Peach latent mosaic and pome fruit viroids in naturally infected cultivated pear (Pyrus communis) and wild pear (P. amygdaliformis): Implication on possible origin of these viroids in the Mediterranean Region. J. Plant Pathol. 2001, 83, 51–62. [Google Scholar]
  28. Gümüs, M.; Paylan, I.C.; Matic, S.; Myrta, A.; Sipahioglu, H.M.; Erkan, S. Occurrence and Distribution of Stone Fruit Viruses and Viroids in Commercial Plantings of Prunus Species in Western Anatolia, Turkey. J. Plant Pathol. 2007, 89, 265–268. [Google Scholar]
  29. Boubourakas, I.N.; Arambatzis, C.; Kyriakopoulou, P.E.; Dovas, C. Amelioration of a Reverse Transcription Polymerase Chain Reaction (RT-PCR) for the Detection of ASSVd, PBCVd and PLMVd Viroids, and Their Presence in Cultivated and Wild Pome and Stone Fruits in Greece. Acta Hortic. 2006, 781, 519–528. [Google Scholar] [CrossRef]
  30. Kaponi, M.; Kyriakopoulou, P.E.; Hadidi, A. Viroids of the Mediterranean Basin. Viruses 2024, 16, 612. [Google Scholar] [CrossRef]
  31. Xu, Y.; Li, S.; Na, C.; Yang, L.; Lu, M. Analyses of Virus/Viroid Communities in Nectarine Trees by next-Generation Sequencing and Insight into Viral Synergisms Implication in Host Disease Symptoms. Sci. Rep. 2019, 9, 12261. [Google Scholar] [CrossRef]
  32. Fiore, N.; Zamorano, A.; Pino, A.M.; González, F.; Rosales, I.M.; Sánchez-Navarro, J.A.; Pallás, V. Survey of Stone Fruit Viruses and Viroids in Chile. J. Plant Pathol. 2016, 98, 631–635. [Google Scholar] [CrossRef]
  33. Tayal, M.; Rodriguez Bonilla, F.; Powell, G.; Cieniewicz, E. Pollen-Borne Ilarviruses of Peach: Biology, Ecology, and Disease Management. J. Integr. Pest Manag. 2024, 15, 23. [Google Scholar] [CrossRef]
  34. Dias, N.P.; Hu, R.; Hensley, D.D.; Hansen, Z.R.; Domier, L.L.; Hajimorad, M.R. A Survey for Viruses and Viroids of Peach in Tennessee Orchards by RNA Sequencing. Plant Health Prog. 2022, 23, 265–268. [Google Scholar] [CrossRef]
  35. Milusheva, S.A.; Borisova, A.Z. The Incidence of Prunus Necrotic Ringspot and Prune Dwarf Viruses in Prunus Species in South Bulgaria. Biotechnol. Biotechnol. Equip. 2005, 19, 42–45. [Google Scholar] [CrossRef]
  36. Bag, S.; Al Rwahnih, M.; Li, A.; Gonzalez, A.; Rowhani, A.; Uyemoto, J.K.; Sudarshana, M.R. Detection of a New Luteovirus in Imported Nectarine Trees: A Case Study to Propose Adoption of Metagenomics in Post-Entry Quarantine. Phytopathology 2015, 105, 840–846. [Google Scholar] [CrossRef] [PubMed]
  37. Khalili, M.; Candresse, T.; Koloniuk, I.; Safarova, D.; Brans, Y.; Faure, C.; Delmas, M.; Massart, S.; Aranda, M.A.; Caglayan, K.; et al. The Expanding Menagerie of Prunus-Infecting Luteoviruses. Phytopathology 2023, 113, 345–354. [Google Scholar] [CrossRef] [PubMed]
Horticulturae 11 00503 g001
Figure 1. Virus-like symptoms observed on selected trees. (a) Fruit symptoms on nectarine cv. Fantasia (sample 2/4); (b) leaf symptoms on peach cv. Royal Gem (sample 21/29).
Figure 1. Virus-like symptoms observed on selected trees. (a) Fruit symptoms on nectarine cv. Fantasia (sample 2/4); (b) leaf symptoms on peach cv. Royal Gem (sample 21/29).
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Figure 2. Sankey diagram displaying the plant virus community structure in five symptomatic peach and nectarine trees. The colored columns from left to right correspond proportionality to the viral taxa’s abundance from domain to genus levels.
Figure 2. Sankey diagram displaying the plant virus community structure in five symptomatic peach and nectarine trees. The colored columns from left to right correspond proportionality to the viral taxa’s abundance from domain to genus levels.
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Figure 3. Symptoms on GF 305 peach indicator seedlings inoculated using bud sticks of sample S1 from Summerset peach (A) and sample 21/29 from Royal Gem peach (B).
Figure 3. Symptoms on GF 305 peach indicator seedlings inoculated using bud sticks of sample S1 from Summerset peach (A) and sample 21/29 from Royal Gem peach (B).
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Table 1. List of peach and nectarine samples used for lncRNA sequencing.
Table 1. List of peach and nectarine samples used for lncRNA sequencing.
SampleSpeciesCultivarsCollection YearCollection Location Symptoms 1
21/29Prunus persicaRoyal Gem 2022PlovdivCB, VC, FRP
2/4Prunus persica var. nectarinaFantasia2022Assenovgrad
Plovdiv		CB, FDM
15/6Prunus persicaLaskino2023PlovdivVC, R
S1Prunus persicaSummerset2023PlovdivCB, R, FRP
S2Prunus persicaGlohaven2023PlovdivCB, VC, FRP
1 CB—chlorotic blotches and rings on the leaves; FDM—discoloration and malformations on the fruits; FRP—rings and patterns on the fruits; R—rings on the fruits; VC—vein clearing.
Table 2. Identification of plant viruses and viroids in individual peach and nectarine trees using bioassays, ELISA, RT-PCR, and Sanger sequencing.
Table 2. Identification of plant viruses and viroids in individual peach and nectarine trees using bioassays, ELISA, RT-PCR, and Sanger sequencing.
SampleMethod Pathogen
PLMVdPPVPNRSVCGRMV
21/29Biotest++−−
ELISAN/A+−N/A
RT-PCR++−−
Sequencing+ndN/AN/A
2/4Biotestndndndnd
ELISAN/A+−N/A
RT-PCR++−−
Sequencing+ndN/AN/A
15/6Biotestndndndnd
ELISAN/A+−N/A
RT-PCR++−−
Sequencing+ndN/AN/A
S1Biotest++++
ELISAN/A++N/A
RT-PCR++++
Sequencing+ndnd+
S2Biotest++−+
ELISA++−NA
RT-PCR++−+
Sequencing+ndN/A+
Abbreviations: N/A—not applicable; nd—no data; (+) positive reaction; (−) negative reaction; PLMVd—peach latent mosaic viroid; PPV—plum pox virus; PNRSV—prunus necrotic ringspot virus; CGRMV—cherry green ring mottle virus.
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Gozmanova, M.; Baev, V.; Valkova, R.; Apostolova-Kuzova, E.; Jurac, S.; Yahubyan, G.; Nacheva, L.; Milusheva, S. Viral and Viroid Communities in Peach Cultivars Grown in Bulgaria. Horticulturae 2025, 11, 503. https://doi.org/10.3390/horticulturae11050503

AMA Style

Gozmanova M, Baev V, Valkova R, Apostolova-Kuzova E, Jurac S, Yahubyan G, Nacheva L, Milusheva S. Viral and Viroid Communities in Peach Cultivars Grown in Bulgaria. Horticulturae. 2025; 11(5):503. https://doi.org/10.3390/horticulturae11050503

Chicago/Turabian Style

Gozmanova, Mariyana, Vesselin Baev, Rumyana Valkova, Elena Apostolova-Kuzova, Stoyanka Jurac, Galina Yahubyan, Lilyana Nacheva, and Snezhana Milusheva. 2025. "Viral and Viroid Communities in Peach Cultivars Grown in Bulgaria" Horticulturae 11, no. 5: 503. https://doi.org/10.3390/horticulturae11050503

APA Style

Gozmanova, M., Baev, V., Valkova, R., Apostolova-Kuzova, E., Jurac, S., Yahubyan, G., Nacheva, L., & Milusheva, S. (2025). Viral and Viroid Communities in Peach Cultivars Grown in Bulgaria. Horticulturae, 11(5), 503. https://doi.org/10.3390/horticulturae11050503

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