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Article

Application of In Vitro Techniques for Elimination of Plum Pox Virus (PPV) and Apple Chlorotic Leaf Spot Virus (ACLSV) in Stone Fruits

by
Balnur Kabylbekova
1,*,
Toigul Nurseitova
1,2,*,
Zarina Yussupova
1,
Timur Turdiyev
1,3,
Irina Kovalchuk
1,
Svetlana Dolgikh
1,
Sagi Soltanbekov
1,
Aigerim Seisenova
1 and
Aigul Madenova
1,3
1
Kazakh Fruit and Vegetable Research Institute, 050060 Almaty, Kazakhstan
2
Faculty of Natural Sciences and Geography, Abai Kazakh National Pedagogical University, 050010 Almaty, Kazakhstan
3
Laboratory of Microclonal Reproduction of Plants, Kazakh National Agrarian Research University, 050010 Almaty, Kazakhstan
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(6), 633; https://doi.org/10.3390/horticulturae11060633
Submission received: 19 March 2025 / Revised: 26 May 2025 / Accepted: 1 June 2025 / Published: 5 June 2025
(This article belongs to the Section Propagation and Seeds)
Browse Figure
Versions Notes

Abstract

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.

1. Introduction

Viral diseases in fruits are of significant concern due to their economic impact in production areas, their frequency, and prevalence. Effective and mandatory measures for controlling viral diseases include timely and accurate virus diagnosis, removal of infected trees, use of virus-free planting materials, implementation of inspection and certification systems, and strict quarantine protocols [1,2,3,4].
Two strains of the plum pox virus (PPV), namely, PPV-D and PPV-W, have been identified in Kazakhstan [5,6]. Apple chlorotic leaf spot virus (ACLSV) is a common virus in apple-growing countries, affecting more than 60% of all cultivated varieties [7,8]. In Kazakhstan, it is also frequently found in combination with other viruses affecting pome crops [9]; however, there are no reports of its spread to stone fruits.
The apricot (Prunus armeniaca L. (syn. Armeniaca vulgaris Lam.)) is an endemic species listed as rare and endangered in the Red Book of Kazakhstan. The genetic diversity of apricots originates from Central Asia, with Kazakhstan representing the northern limit of its population [10,11]. The mountains of southeastern Kazakhstan are uniquely rich in wild fruit plants, hosting about 130 species belonging to 30 genera and 13 families, including relict plants, endemic species, and remnants of ancient landscapes, such as walnut, apple, and apricot forests [12]. Current apricot cultivars are believed to have originated from the wild Tien-Shan apricot. Some scientists posit that the original cultivated apricot should be considered the Tien-Shan wild apricot, which, along with apple trees, forms forests in the mountains and foothills of Kazakhstan.
Kazakhstan’s climatic conditions are favorable for cultivating wild fruits and their cultivars. In southeastern Kazakhstan, two common species of wild plum—Prunus sogdiana (cherry plum) and Prunus spinosa (blackthorn)—as well as relatives of the wild apricot, Prunus armeniaca, are of great interest to breeders and nurserymen as sources of hardiness, relative drought tolerance, and disease resistance.
In Kazakhstan, production orchards of all major stone fruits occupy 9377.1 hectares, of which only 1811.1 hectares are dedicated to plum and 4345.1 hectares to apricot cultivation (https://stat.gov.kz/ru/ (accessed on 1 December 2024)). Cultivation areas are decreasing and not expanding due to reduced yield and fruit quality, as well as the death of trees affected by viral diseases. Over the past decade, the Kazakh Fruit and Vegetable Research Institute (KazFaVRI) has lost about 30% of its introduced genotypes due to various biotic factors, including the spread of diseases. The institute’s collection currently includes 159 plum and apricot varieties. This situation necessitates the planting of virus-free planting material. The biodiversity of wild apricot represents a valuable gene pool for improving breeding programs aimed at resistance to abiotic and biotic factors, including viral diseases.
Stone fruits serve as hosts to numerous economically significant and pervasive plant RNA viruses, which fall into different classes. These viruses include Ilarvirus species, comprising apple mosaic virus (ApMV), prunus necrotic ring spot virus (PNRSV), prune dwarf virus (PDV), and American plum line pattern virus (APLPV); Potyvirus species, responsible for the disease “Sharka” caused by the PPV; and Trichovirus, such as ACLSV, which is considered to cause “Viruela” [13]. In total, 35 distinct viruses have been identified in apricots and plums, representing 15 genera and 9 families [14].
PPV is the most serious viral pathogen affecting stone fruits, including apricots, cherries, nectarines, peaches, and plums. Sharka negatively impacts the sustainable production of stone fruits worldwide and threatens food security. Symptoms of PPV include abnormalities in fruit morphology, loss of taste, and premature fruit drop, potentially resulting in 80–100% yield losses. Consequently, this disease holds economic significance in every country where stone fruits are cultivated [15,16,17]. To achieve successful eradication or effective management of the associated disease, it is necessary to ascertain the sensitivity of the virus in infected plants. In 2014, the first reports of Sharka virus detection in stone fruit crops in Kazakhstan were published [5], with PPV identified in various apricot and plum production locations within the country. However, PPV strains were not detected in wild apricot populations. Molecular genetic research conducted in the Trans-Ili Alatau revealed the existence of wild apricot forms exhibiting resistance to PPV [6,10]. Nonetheless, wild apricot trees showing PPV symptoms have been found in the Big Aksu Gorge of the Uygur Region. It is assumed that the virus spread to them from cultivated plantings located in that area. Since PPV is spread through infected plant material and sucking pests, the best approach to combat it involves eliminating infected trees or improving genotypes through biotechnological methods and establishing new plantations with healthy material.
In vitro cultivation is the most successful method for producing virus-free plants. Pathogen elimination can be achieved through various methods such as thermotherapy, meristem culture, and in vitro thermotherapy-based techniques combined with chemotherapy or cryotherapy [18,19]. Moreover, cryotherapy has been successfully used as a standalone method for virus elimination in apricot, as well as in combination with thermotherapy and chemotherapy [20,21,22]. This research aimed to develop and improve methods for virus-infected plant sanitation to facilitate the subsequent production of healthy planting material of valuable genotypes.

2. Materials and Methods

2.1. Field Survey and Plant Materials

The research objects were stone fruits—specifically, plum (Prunus domestica L.) and apricot (Prunus armeniaca L.) genotypes (Table 1). The plant materials were collected from the original Pomological collection of the KazFaVRI, and Big Aksu Gorge of the Uygur Region, specifically from the stone fruit locations in Almaty and Turkestan regions. The sampling was carried out during the plant vegetative period (from March to September), 2022–2024, and PCR research detection during autumn and winter.
Field monitoring with visual assessment of symptoms was carried out in all surveyed orchards. In this study, leaves and fruits of plants showing symptoms of viral infections were systematically selected for analysis. The main criterion for leaf selection was the detection of symptoms caused by PPV and ACLSV, which led to changes in the concentration of specific pigments, in particular chlorophyll. PPV-infected leaves usually had conspicuous necrotic spots, diffuse or well-defined ring patterns, yellowing of veins, and elongated linear lesions. Infected fruits develop severe pox symptoms with dark-colored rings or patches on the skin, brown or reddish discoloration in the flesh, and brown spots on the stones [23]. ACLSV is characterized by dark green sunken mottling on leaves. Symptoms of ACLSV are usually restricted to sunken spots, bands, or rings on the skin of fruit. These diagnostic symptoms served as reliable indicators for the presence of PPV and ACLSV, and the selected plant material was subsequently used for further investigation [24,25,26].
The samples were collected according to EPPO Standards [27]. Each genotype (sample) used in the research represented a single tree. From each tree, 15–20 fully developed leaves were sampled from east–west-north–south directions. The samples were stored in a refrigerator at +4 °C until the RNA samples were extracted.
Plant samples were tested in triplicate for ACLSV, ApMV, PPV, PDV, PNRSV, CGRMV, and MLRSV, as these viruses are listed in the EPPO regulation scheme for Prunus plant material [28].

2.2. Extraction of Total RNA and RT-PCR

RNA was extracted from the leaves of all the above plants using a commercial extraction kit, “PhytoSorb®” (SINTOL® (http://www.syntol.ru (accessed on 13 January 2023)), modified according to Mekuria et al. [29]. A total of 200 mg of fresh or frozen leaf tissue was ground to a fine powder in liquid nitrogen, mixed with 2.0 mL of extraction buffer containing 4.4% (w/v) PVP-40 (SINTOL® (http://www.syntol.ru ((accessed on 16 January 2023)) and 1% (w/v) sodium metabisulfite, and briefly vortexed. The quality and quantity of RNA were measured using a NanoDrop 1000 UV/VIS spectrophotometer (ThermoFisher, Waltham, MA, USA). RT-PCR was performed using the One-Step RT-PCR Kit (Qiagen) in a 10 µL reaction volume (Table 2). The reverse transcription reaction and RT-PCR were conducted using a Rotor-Gene instrument (Qiagen, Germantown, MD, USA). The RNA samples were stored at −20 °C. The RNA of the studied plants was assessed for purity using spectrophotometry, with extinction indicators of 280/260 nm and 260/230 nm, ranging from 1.8 to 2.3.
Reagents from LETGEN® (https://letgenbio.com/en/ (accessed on 18 March 2023)) were employed for the detection of PPV and other viruses. The kit from LETGEN® contains primers specific to PPV, and its probe is based on TaqMan® technology.

2.3. In Vitro Cultures and Clonal Micropropagation

The plant apical shoot tips (1–3 cm) from infected trees were placed in aseptic culture. For sterilization, 0.1% HgCl₂ was employed, with an exposure period of 4.0–4.5 min for apricot and 3.0–3.5 min for plum materials [30]. Subsequently, the shoots were washed three times with sterile distilled water. The microplants were cultured in vitro in Magenta boxes, with 25 mL of medium allotted to each. The pre-cultivation phase (lasting one month) and subsequent adaptation were conducted in MS medium [31], comprising 75% N, 0.5 mg/L BAP, 1.0 mg/L GA, and 0.1 mg/L IBA. The temperature was maintained at 24 ± 0.5 °C with a variable light regime, comprising 16 h of illumination (25 μmol m−2s−1) and 8 h of darkness (n = 15). For obtaining the needed number of infected in vitro plants, three passages of 30 days each were conducted.

2.4. Management of Viral Infections

For treatment, we used a treatment regimen successfully applied to other crops, in particular, to berry crops [1], pome fruits [7,32,33,34,35], and stone fruits [20,21,22]. We combined several techniques and carried out the following therapy. For this set of experiments, ‘Stanley’, ‘Ansar’, and ‘Ayana’ plum cultivars and the wild form of P. armeniaca apricot were used in three replicates.

2.5. Thermotherapy + Shoot Apical Meristem (SAM)

This treatment was carried out for 12 days in a climate cabinet (Daihan climate chamber, HHP400, Osan, Republic of Korea) with the following settings: 36 ± 0.5 °C, 70% humidity, 16 h of illumination (25 μmol m−2s−1), and 8 h of darkness [36].
For virus testing, after treatment, the shoot apical meristems of microplants (1.5–3 mm) were transferred to a standard medium containing 75% N and 0.2 mg/L BAP for regrowth. After 30 days of growth under standard conditions (growing environment with a 16 h light period at 25 μmol m−2s−1, 8 h darkness, +24 °C), leaves were collected from in vitro propagated plants for the PCR assay.

2.6. Chemotherapy (Treatment with Ribavirin) + SAM

The plants were kept in MS medium with 75% N and 25 mg/L ribavirin (Virozole, Sigma-Aldrich, St. Louis, MO, USA) for 60 days [37]. Cultures were grown at 24 °C under a 16 h photoperiod with an average of 40 μmol m−2s−1 radiation provided by cool-white fluorescent lamps (Philips, Poland). After this treatment, the procedure for virus testing described in the Thermotherapy + SAM method was applied.

2.7. Cryotherapy + SAM

Microplants for cryopreservation were prepared using in vitro culture under standard culture room conditions. A cold acclimation process, serving as preliminary preparation for cryopreservation, was conducted at +4 °C with a 16 h light (25 μmol m⁻2 s⁻1) and 8 h dark photoperiod for three weeks in a Daihan climate chamber (HHP400, Gyeonggi-do, Republic of Korea). The binocular microscope “BioLar” (Przeźmierowo, Poland) was used to isolate the meristem. Cryopreservation in liquid nitrogen was carried out by the vitrification method with 0.3 M sucrose and cryoprotectant PVS2 [38]. Meristems were frozen in liquid nitrogen (−196 °C) in 1.0 mL CryoTube™ cryovials (Thermo Scientific™, Waltham, MA USA). The protocol for meristem tissue regeneration following cryotherapy involved transferring the cryovials from liquid nitrogen to water baths: initially at +45 °C for one minute, followed by +25 °C for one minute [39]. After thawing, the meristem was transferred to a nutrient medium of MS N 75%, BAP 0.5 mg/L, GA 1.0 mg/L, IBA 0.1 mg/L to restore growth and development. Leaves from regenerated plants after cryotherapy were used for virus testing.

2.8. Chemotherapy + Thermotherapy + SAM + Cryotherapy + SAM

Plants were subjected to in vitro chemotherapy for 14 days on a medium supplemented with ribavirin at a concentration of 25 mg/L in standard conditions (24 °C under a 16-h photoperiod with an average of 40 μmol m−2s−1 radiation). Thereafter, in vitro plants without passages were transferred to the climate cabinet (36 ± 0.5 °C, 70% humidity, 16 h of illumination (25 μmol m−2s−1), and 8 h of darkness) for thermotherapy for 12 days. After thermotherapy, the isolated apical meristems were prepared for cryotherapy as described above in the section titled Cryotherapy + SAM.
Each therapy was evaluated by assessing plant survival rates and virus elimination confirmed by PCR, as well as the multiplication capacity of regenerated plants.

2.9. Data Collection

Observations and counts were carried out monthly, and the condition and number of shoots formed were noted. From the number of shoots formed, the multiplication coefficient was calculated on average for 1 passage for each genotype, using the following formula [30]:
P = a b × c
P = Multiplication coefficient;
a = Number of newly formed shoots;
b = Number of shoots planted for reproduction;
c = Number of passages.

3. Results

This study involved both field and laboratory surveys of stone fruit plantations located in the Almaty and Turkestan regions of Kazakhstan. A total of six plum, nine apricot, and eight rootstock accessions were examined from the Almaty region, along with two plum and two apricot accessions from the Saryagash region (Turkestan region), resulting in 29 accessions overall. ACLSV was detected in only two plum cultivars, whereas PPV demonstrated a broader distribution, particularly among plum accessions in the Almaty region. In total, seven plum and one apricot accession tested positive for either PPV or ACLSV, exhibiting both symptomatic and latent infections (Table 2, Figure 1). No infections were detected for ApMV, PDV, PNRSV, CGRMV, or MLRSV. The results of PPV and ACLSV diagnostics are summarized in Table 2.
Table 2 presents the results of RT-PCR testing of plant leaves collected from field collections and wild populations. The analysis revealed that plum cultivars ‘Ayana’, ‘Stanley’, and ‘Ansar’, apricot cultivar ‘Manitoba’, as well as P. armeniaca Wild Apricot form-3, were infected with PPV. Notably, the plum cultivar ‘Ayana’ was found to be mixed-infected with both PPV and ACLSV, while the cultivar ‘Ispanskaya’ tested positive for ACLSV. Visual symptoms were inconsistent with PCR results—several asymptomatic plants tested positive, and some symptomatic ones were PCR-negative—indicating that visual diagnosis alone is unreliable. Rootstocks tested PCR-negative despite some showing symptoms, suggesting tolerance or latent infection. Cultivars such as ‘Stanley’ and ‘Manitoba’ emerged as potential virus carriers.
To develop material free of PPV and ACLSV, three in vitro treatments and their combination were evaluated. The ‘Stanley’, ‘Ansar’, and ‘Ayana’ plum cultivars and the wild form of apricot P. armeniaca were used in the experiment (Table 3).
Table 3 shows the efficacy of four different in vitro treatments in eliminating viruses and promoting regeneration in stone fruit varieties previously infected with PPV and/or ACLSV. The sanitation methods were assessed based on the percentage of viable plant regeneration after therapy, and phytosanitary status as determined by PCR, alongside the plant multiplication coefficient post-treatment. Thermotherapy + SAM demonstrated moderate efficacy: although P. armeniaca failed to survive, other cultivars, especially ‘Stanley’ and ‘Ansar’, achieved 100% virus elimination with multiplication coefficients of 5.6 and 3.0, respectively. However, ‘Ayana’ remained infected with both viruses despite a 40% survival rate. Chemotherapy + SAM showed improved viability in ‘Ayana’ (60%), but it eliminated only PPV, with ACLSV persisting. Cryotherapy + SAM provided a more balanced outcome: ‘Stanley’ showed the highest survival (38.5%) with virus-free status and moderate propagation (1.3). P. armeniaca survived with PPV still present, but ‘Ansar’ again failed to regenerate. ‘Ayana’ retained only ACLSV, with a higher multiplication rate (1.9) than in Chemotherapy + SAM.
Chemotherapy + Thermotherapy + Cryotherapy + SAM showed mixed results. Neither ‘Stanley’ nor ‘Ansar’ survived. However, P. armeniaca and ‘Ayana’ demonstrated a notable absence of virus replication, suggesting a form of virus suppression rather than full elimination. Their multiplication coefficients were relatively high at 2.4 and 3.4, respectively.

4. Discussion

The current study was designed to identify viral pathogens in stone fruit collections and to evaluate virus management strategies using in vitro techniques. Previously, field collections of stone fruit had not been tested for viruses, and studies on virus sanitation had not been conducted. The conducted research is of great importance for Kazakhstan.
Constant monitoring of field plants with visual evaluation of symptoms, timely and sensitive diagnostics [40,41,42,43], and identification of resistant genotypes [44] are key to controlling plant diseases. Viral infections cannot be cured with fungicides, and the best way to prevent viral diseases is to start with certified virus-free nursery stock [2,3]. The production of virus-free plant material cannot be successfully implemented without biotechnological methods of sanitation and propagation with optimization of in vitro genotype-specific conditions [45,46,47,48].
In vitro treatment of rootstocks and seedlings used for fruit crop multiplication is crucial for the development of virus-free plants. Thermotherapy, or heat treatment, is a widely recognized method for sanitizing plant material from viral infections [28,32,35]. Tissue culture techniques are extensively used in viral eradication efforts because they provide sterile conditions and utilize meristematic tissues, where viral concentrations are generally low or absent, since many viruses cannot penetrate the meristem [49].
Our study’s survival rates varied significantly between genotypes and treatments, reflecting genotype-specific responses. Thermotherapy + SAM treatment proved effective for ‘Stanley’ and ‘Ansar’ (Table 3). These improvements are similar to those observed with in vitro thermotherapy for apricot [50], peach [51] other woody plants [52,53,54]. ‘Stanley’ was the most responsive to treatment, achieving complete virus elimination with the highest multiplication coefficient. Conversely, ‘Ansar’ consistently failed under all therapies except the first (Table 3).
Chemotherapy + SAM using ribavirin was less effective overall in terms of virus elimination, plant survival, and multiplication, particularly for ‘Stanley’ (1.9) and ‘Ayana’ (0.6) (Table 3). Paunovic et al. demonstrated that low concentrations of ribavirin (10 and 20 mg L⁻1) were entirely ineffective for PPV elimination [37], whereas PPV was successfully eliminated using MS medium with 25 mg L⁻1 rimantadine for three weeks without heat treatment. Despite these findings, ribavirin remains widely regarded as the most effective antiviral agent [55,56]. Some studies recommend combining chemotherapy with thermotherapy and other methods for more efficient sanitation. This approach has been validated for the elimination of ACLSV, ASGV, and ASPV in apple [57], as well as CVA in cherry [58].
Cryotherapy was first applied to plum rootstocks against PPV by Brison et al. (1997) [59] and has since shown promise in virus elimination for apple [7,60], grapevine [61], and plum [62]. In addition to preserving genetic resources, cryopreservation in liquid nitrogen serves as an effective method for virus elimination, especially when combined with other treatments. Several researchers, investigated virus elimination from PPV-infected apricot shoot tips using in vitro thermotherapy, chemotherapy, and cryotherapy. The highest survival rate (100%) was observed with quercetin application, while ribavirin yielded 80% survival. A two-stage freezing technique involving pre-adaptation in MS medium at +4 °C, PVS2 exposure, and rapid immersion in liquid nitrogen for 60 min achieved virus elimination in apricot, although only 10% of apical tips survived [20,21]. Similar techniques were effective for quince, eliminating ACLSV and ASGV (apple stem grooving virus) [63], and these findings align with the current study. Genotypic differences in plant response to temperature stress are particularly relevant in cryotherapy, where stress is often the primary response mechanism [64]. This may explain the death of the ‘Stanley’ and ‘Ansar’ genotypes during Chemotherapy + Thermotherapy + Cryotherapy + SAM and Cryotherapy + SAM (Table 3). In general, the combination of cryotherapy, thermotherapy, and chemotherapy has proven effective in eliminating viruses such as ACLSV, ASGV, AHVd (apple hammerhead viroid), and ApMV in both pome and stone fruit crops [35,65,66,67].
This study concludes that the most effective treatments for viral eradication and virus-free plant production were Thermotherapy + SAM and Chemotherapy + Thermotherapy + Cryotherapy + SAM. Complete virus elimination was achieved in the ‘Stanley’, ‘Ayana’, and P. armeniaca genotypes, resulting in the successful production of PPV- and ACLSV-free plants. These outcomes emphasize the influence of genotype-specific stress responses in cryotherapy protocols [39]. The death of ‘Ansar’ plants was probably due to their sensitivity to high temperatures and chemical components of the medium, such as ribavirin and the cryoprotectant PVS2, which may have toxic effects on sensitive genotypes. Remarkably, ‘Ayana’, despite being co-infected with both viruses, showed the highest survival in Chemotherapy + SAM and virus suppression in the full combination treatment. While the complete treatment combination proved optimal for Prunus armeniaca and ‘Ayana’, it was too aggressive for other genotypes.
These contrasting responses among genotypes highlight the importance of understanding how specific treatments affect both virus suppression and plant survival. In this regard, temperature plays a key role. At elevated temperatures, plant growth accelerates due to increased cell division and rapid development of the apical meristem. At the same time, accelerated cell division can restrict virus movement, leading to a phenomenon referred to as “escape from infection” [1,35,58]. When exposed to ultra-low temperatures with liquid nitrogen, only cells located in the apical meristem typically survive [39] and are able to self-renew, divide, differentiate, and regenerate into new virus-free plants [68]. Therefore, optimizing temperature conditions and selecting non-toxic or minimally toxic cryoprotectants is essential.
This further supports the idea that sanitation treatments must be tailored to specific genotypes. The results highlight the importance of customizing virus elimination strategies according to both the viral profile and the genetic background of the plant material. Ultimately, this study supports the integration of in vitro therapies into phytosanitary certification programs for virus-free propagation of Prunus spp.

5. Conclusions

This study aimed to investigate virus management techniques in stone fruit crops to enable the production of healthy plant material from valuable genotypes. The results demonstrated that Thermotherapy + SAM is most effective for complete PPV elimination in specific genotypes, while the combination of several in vitro treatments is effective in eliminating viruses from plants mixed-infected with PPV and ACLSV. Also, these findings highlight the necessity of routine molecular diagnostics for effective virus detection and control, particularly in breeding and certification programs. These findings highlight the potential application of a combination of in vitro treatments to establish a virus-free nursery system, particularly for producing basic and pre-basic category planting material.
However, it is important to note that certain genotypes are sensitive to high and ultra-low temperatures and/or toxic drugs (ribavirin, cryoprotectants), which should be considered in further applications. Future research should focus on identifying a genotype with resistance to PPV, combinations of methods for diagnosing viral infections in orchards and nurseries, optimization of conditions, development of universal combinations for several varieties and viruses, and selection of optimal temperature conditions to enhance the reproducibility and applicability of the method in large-scale production systems.

Author Contributions

Conceptualization, B.K., I.K. and A.M.; methodology, B.K. and T.T.; validation, I.K. and A.M.; formal analysis, Z.Y. and A.S.; investigation, T.N., T.T. and S.S.; resources, S.S.; data curation, S.D.; writing—original draft preparation, B.K., T.N., Z.Y. and A.S.; writing—review and editing, B.K. with assistance from AI-based language tools, S.D., A.M. and I.K.; visualization, T.T. and A.M.; project administration, B.K.; funding acquisition, B.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financed by the Ministry of Science and Higher Education of the Republic of Kazakhstan project: AP14869380.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

This study was conducted as part of the PhD research of Toigul Nurseitova at Abai Kazakh National Pedagogical University, under the supervision of Kanat Galymbek and Balnur Kabylbekova.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 00633 g001
Figure 1. Specific symptoms of plum pox virus on fruits, chlorotic spots, and on stones, distinct discolored rings of the apricot cultivar “Manitoba”, Almaty location.
Figure 1. Specific symptoms of plum pox virus on fruits, chlorotic spots, and on stones, distinct discolored rings of the apricot cultivar “Manitoba”, Almaty location.
Horticulturae 11 00633 g001
Table 1. Locations and names of plant materials collected for virus analysis.
Table 1. Locations and names of plant materials collected for virus analysis.
NoCropsSelected Samples for Research
Location: Almaty Region (South East Kazakhstan)
1PlumAyana
2 Zhomart
3 Stanley
4 Renclod Talgarskiy
5 Agyl
6 Ansar
7ApricotNikitskiy krasnoshekiy
8 Burshtynoviy
9 Kolkhozniy
10 Alexander
11 Balkhiya
12 Manitoba
13 Baikalov 9-9
14 Persikoviy
17 P. armeniaca Wild Apricot form-3
18RootstocksVSV-1
19 VVA-1
20 St. Julien
21 Fortuna
22 Druzhba
23 Kuban 86
24 Pumiselect
25 Evrika-99
Location: Saryagash Region (South Kazakhstan)
26PlumStanley
27 Ispanskaya
28ApricotNikitskiy krasnoshekiy
29 Manitoba
Table 2. The presence of apple chlorotic leaf spot virus (ACLSV) and plum pox virus (PPV) by visual assessment and PCR methods in plum and apricot plantations in Almaty and Turkestan regions.
Table 2. The presence of apple chlorotic leaf spot virus (ACLSV) and plum pox virus (PPV) by visual assessment and PCR methods in plum and apricot plantations in Almaty and Turkestan regions.
NoName of Cultivar/RootstockVisual Manifestation of the VirusVirus Detection by PCR
ACLSVPPVACLSVPPV
Almaty Region (South-East Kazakhstan)
Plum
1Ayana--++
2Zhomart-on the leaves--
3Stanley -on the leaves and fruits-+
4Renclod Talgarskiy----
5Agyl----
6Ansar---+
Apricot
7Nikitskiy krasnoshekiy-on the leaves--
8Burshtynoviy----
9Kolkhozniy ----
10Alexander ----
11Balkhiya ----
12Manitoba-on the fruits and seeds-+
13Baikalov 9-9----
14Persikoviy ----
17P. armeniaca Wild Apricot form-3-on the leaves-+
Rootstocks
18VSV-1 ----
19VVA-1 ----
20St. Julien-on the leaves--
21Fortuna-on the leaves--
22Druzhba----
23Kuban 86----
24Pumiselect----
25Evrika-99----
Saryagash Region (South Kazakhstan)
Plum
26Stanley----
27Ispanskaya on the leaves-+-
Apricot
28Nikitskiy krasnoshekiy----
29Manitoba----
Table 3. Effect of combination of in vitro therapy on elimination of plum pox virus (PPV) and apple chlorotic leaf spot virus (ACLSV), and plant propagation.
Table 3. Effect of combination of in vitro therapy on elimination of plum pox virus (PPV) and apple chlorotic leaf spot virus (ACLSV), and plant propagation.
TreatmentName of Cultivar/RootstockPhytosanitary Status Before Therapy (PCR)*** Number of Viable Plants After Treatment/%Phytosanitary Status After Therapy (PCR)** Plant Multiplication Coefficient
Thermotherapy + Shoot apical meristem (SAM)StanleyPPV *3/20Virus-free5.6
AnsarPPV3/20Virus-free3.0
P. armeniacaPPV0/died unknown0
AyanaPPV, ACLSV6/40PPV, ACLSV3.5
Chemotherapy + SAM
Ribavirin,
25 mg/L 		StanleyPPV5/33.3Virus-free1.9
AnsarPPV0/diedunknown0
P. armeniacaPPV0/diedunknown0
AyanaPPV, ACLSV9/60ACLSV0.6
Cryotherapy + SAMStanleyPPV7/38.8Virus-free1.3
AnsarPPV0/diedunknown0
P. armeniacaPPV6/28.5PPV2.2
AyanaPPV, ACLSV4/21.0ACLSV1.9
Chemotherapy + Thermotherapy + Cryotherapy + SAMStanleyPPV0/diedunknown0
AnsarPPV0/diedunknown0
P. armeniacaPPV8/38Virus-free2.4
AyanaPPV, ACLSV4/19.0Virus-free3.4
** Plant multiplication coefficient after 4 weeks of cultivation—the ratio of the number of plants obtained to the number of original plants. *** Percentage of viable plants after treatment—the ratio of living plants to the original number of plants studied.
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Kabylbekova, B.; Nurseitova, T.; Yussupova, Z.; Turdiyev, T.; Kovalchuk, I.; Dolgikh, S.; Soltanbekov, S.; Seisenova, A.; Madenova, A. Application of In Vitro Techniques for Elimination of Plum Pox Virus (PPV) and Apple Chlorotic Leaf Spot Virus (ACLSV) in Stone Fruits. Horticulturae 2025, 11, 633. https://doi.org/10.3390/horticulturae11060633

AMA Style

Kabylbekova B, Nurseitova T, Yussupova Z, Turdiyev T, Kovalchuk I, Dolgikh S, Soltanbekov S, Seisenova A, Madenova A. Application of In Vitro Techniques for Elimination of Plum Pox Virus (PPV) and Apple Chlorotic Leaf Spot Virus (ACLSV) in Stone Fruits. Horticulturae. 2025; 11(6):633. https://doi.org/10.3390/horticulturae11060633

Chicago/Turabian Style

Kabylbekova, Balnur, Toigul Nurseitova, Zarina Yussupova, Timur Turdiyev, Irina Kovalchuk, Svetlana Dolgikh, Sagi Soltanbekov, Aigerim Seisenova, and Aigul Madenova. 2025. "Application of In Vitro Techniques for Elimination of Plum Pox Virus (PPV) and Apple Chlorotic Leaf Spot Virus (ACLSV) in Stone Fruits" Horticulturae 11, no. 6: 633. https://doi.org/10.3390/horticulturae11060633

APA Style

Kabylbekova, B., Nurseitova, T., Yussupova, Z., Turdiyev, T., Kovalchuk, I., Dolgikh, S., Soltanbekov, S., Seisenova, A., & Madenova, A. (2025). Application of In Vitro Techniques for Elimination of Plum Pox Virus (PPV) and Apple Chlorotic Leaf Spot Virus (ACLSV) in Stone Fruits. Horticulturae, 11(6), 633. https://doi.org/10.3390/horticulturae11060633

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