Snezhana Milusheva

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.

Cherry virus A (CVA) is widespread in sweet cherry ( Prunus avium L.) across Europe, North and South America, Asia, and Oceania. However, until now, the presence of CVA in Bulgaria has not been reported. This study focused on two critical aspects: the occurrence of CVA in sweet cherry in Bulgaria's two main fruit production areas—the South Central and Southeast regions—and the probability of its vertical (seed) transmission. CVA was identified through virus-specific amplification of the coat protein (CP) gene, and in vitro embryo culture was used to assess vertical transmission. Our results indicate that CVA is present in sweet cherry in both surveyed regions, detected in 23.5 and 21.4% of the tested samples from the South Central and Southeast regions, respectively. Phylogenetic analysis of the CP nucleotide sequence revealed that Bulgarian CVA isolates were clustered into two groups based on their geographical origin. Moreover, CVA was detected in seeds collected from naturally infected trees and their seed progenies. To the best of our knowledge, this is the first report of CVA in Bulgaria and the first evidence of its vertical transmission in sweet cherry.

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.

Recently, cold atmospheric plasma (CAP) is under investigation for possibility to be applied for inactivation of pathogens in medicine, food technologies, water cleaning technologies and agriculture. The aim of the current study is to investigate the effect of CAP on microplants, propagated in vitro from plum tree (Prunus domestica L., cv. ‘Kyustendilska sinya’) naturally co-infected by M and D strains of Plum pox virus (PPV) and in that respect the possibility for CAP application for virus inactivation. In the present work, we have used two types of plasma sources for biological systems treatments: a surface-wave-sustained Argon plasma torch and an underwater diaphragm discharge. These enabled several variants of plasma treatment to be performed. Based on the data of IC-RT-PCR tests of the microplants on the third subculture after treatment, it was found the most effective variant was the reiterated plasma torch tip treatment to nodal segments without leaves in gas medium. The strain specific RT-PCR analysis results of PPV positive CAP-treated microplants showed that only PPV-M strain was identified after treatment, although the starting material was co-infected by both strains. The results obtained from IC-RT-PCR and strain specific RT-PCR of the acclimatized ex vitro plants have been in agreement with the data from molecular analyses of the microplants tested. These are the first experiments on CAP ability for inactivation of PPV from tissue of living woody plants even if in in vitro conditions. The completed estimation of this approach for obtaining of PPV-free plum plants will be made after more prolonged observation and testing of the ex vitro plants.

ISHS I Balkan Symposium on Fruit Growing SCREENING OF PLUM ELITES FOR RESISTANCE TO PLUM POX VIRUS UNDER FIELD CONDITIONS