The aim of the study was to investigate the early response of apple fruit infected with Penicillium expansum (P. expansum) to blue light-emitting diode (LED) light (BLL) irradiation. To focus our study on the interaction between apple fruit, the pathogen, and BLL, the effect of BLL was also studied on apples without P. expansum and P. expansum grown on malt extract agar (MEA). Transcriptome analysis revealed that the most pronounced responses among biological processes were observed in inoculated apples under BLL. The upregulated processes included water transport, response to heat, and response to high light intensity. The defence response of apples was enhanced by the upregulation of thaumatin-like proteins and caffeic acid 3-O-methyltransferase, while the cellular response to phosphate deficiency and the regulation of multicellular organism development were downregulated. In P. expansum grown on apples under BLL, transcriptome analysis revealed downregulation of genes related to signalling, response to organic compounds, and regulation of metabolic and biosynthetic processes, while genes involved in the biosynthesis of secondary metabolites were upregulated. In addition, the expression of patulin cluster genes was predominantly downregulated in P. expansum. The significant upregulation of genes related to cryptochrome inhibition, defence response, and caffeic acid metabolism in apples under BLL, together with the reduced virulence of P. expansum, contributes to the inhibition of fungal growth.

The grapevine (Vitis vinifera L.) is one of the most important horticultural crops, with thousands of varieties cultivated worldwide. In this study, we analyzed chloroplast SNV markers using a whole-genome shotgun sequencing approach to investigate the genetic diversity and phylogeny of 409 cultivated V. vinifera accessions originating from nine countries across Southeast and Central Europe, as well as a heterogeneous set of additional accessions maintained by INRAE. Shotgun sequencing allowed high coverage, enabling the detection of 93 SNVs across 24 chloroplast genes, including 11 non-synonymous variants. The ycf1 gene showed the highest variability, consistent with its role in species differentiation. Haplotype analysis revealed 102 distinct haplotypes, with clear geographic structuring: ATT predominated in the eastern Mediterranean, ATA in western Europe, and GTA mainly in a heterogeneous group of varieties from a French collection. To validate the shotgun approach, seven SNV markers were analyzed using target capture sequencing, confirming the accuracy of detected variants with only minimal discrepancies, which is mostly attributable to homopolymeric regions and low-frequency alleles. Phylogenetic analyses using both trees and networks delineated three major haplotype clusters, reflecting human-mediated dispersal of grapevine cultivars through historical viticultural practices. This study represents the largest chloroplast genome analysis of cultivated V. vinifera to date, providing a large cpDNA resource for assessing chloroplast diversity and maternal haplotype structure in cultivated grapevine. The results highlight the power of combining high-throughput sequencing and chloroplast genomics for population-level studies in perennial crops.

In this study, we describe a novel virus, tentatively named Verticillium nonalfalfae virus M (VnaVM), identified in the phytopathogenic fungus Verticillium nonalfalfae isolated from hop (Humulus lupulus L.). The VnaVM genome consists of six double-stranded RNA (dsRNA) segments ranging from 1051 to 2401 bp. DsRNAs 1–3 encode an RNA-dependent RNA polymerase (RdRp), a hypothetical protein and a methyltransferase (MTR), respectively, while dsRNA6 encodes a proline-alanine-serine-rich protein (PASrp). These four proteins share less than 58

Hemp (Cannabis sativa L.) is one of the oldest cultivated plants in the world. It is a wind-pollinated and heterozygous species, and diverse phenotypes can occur within population varieties. In our study, three different hemp varieties—(‘Carmagnola Selected’ (CS), ‘Tiborszallasi’ (TS) and ‘Finola selection’ (FS))—were grown. Based on visual characteristics, two, five and four phenotypes were identified within CS, TS and FS, respectively. According to Cannabis sativa L. transcriptome data from the Sequence Read Archive (SRA), 4631 single-nucleotide polymorphism (SNP) positions were identified to develop capture probes. DNA was isolated from 171 plants representing selected phenotypes of three cultivars. Next-generation sequencing (NGS) libraries were constructed and hybridized with capture probes for target enrichment. The population structure of the samples was analyzed using SNP data for each genotype. Based on genotype profiles, CS formed a single cluster, while TS and FS were each grouped into two clusters, with phenotypes randomly distributed among them. The GWAS results were visualized using Manhattan plots. Fourteen significant SNPs surpassing the false discovery rate (FDR) of 0.01 were identified for delta-9-tetrahydrocannabinol (delta-9-THC). For cannabigerol (CBG), 12 significant SNPs were detected, and for myrcene, one SNP exceeded the 0.01 FDR threshold. However, plausible genes located 1000 bp to the left and right of the SNP position were identified for all significant SNPs.

Verticillium wilt of hop (Humulus lupulus L.), caused by the soil-borne pathogen Verticillium nonalfalfae, is a devastating disease with no effective chemical control. In European hop-growing regions, breeding resistant cultivars is the most effective strategy. The lack of response differences in earlier studies suggests constitutive resistance. We therefore conducted a genome-wide association study (GWAS) using a phased hop genome assembly to improve detection of Verticillium resistance loci. A bi-parental population of 142 genotypes, derived from a cross between resistant Wye Target and susceptible BL2/1, was phenotyped for Verticillium wilt resistance and genotyped by sequencing. Association analyses with five statistical models (MLM in TASSEL 5, MLM, MLMM, FarmCPU and BLINK in GAPIT) did not identify any significant SNPs; however, several candidate loci were identified using exploratory threshold, particularly in the phase 2 genome assembly, including a wall-associated kinase (WAK) consistently detected across both genome phases and all models. GWAS results were further assessed with a Random Forest model, which identified SNPs of high feature importance and showed adequate predictive power (accuracy ≈ 0.4, correlation ≈ 0.8) for preliminary breeding screening. These findings provide an initial set of candidate markers and exploratory prediction models for Verticillium wilt resistance in hop, representing a valuable genomic resource for future marker-assisted selection and breeding strategies.

Background/objectives: Grapevine (Vitis vinifera L.) is one of the most economically and culturally important fruit crops worldwide and hosts more than 100 viruses. Viral infections can cause severe yield losses, but plants can adapt to infection through changes in miRNA-mediated regulatory pathways. MicroRNAs are key regulators of plant development and stress responses. Several prediction tools are available for miRNA detection from small RNA sequencing data, each relying on different algorithms. The aim of this study was to compare miRNA predictions generated by three widely used tools (miRador, ShortStack, and miRDeep2) and to evaluate how viral coinfections influence miRNA expression in grapevine. Methods: Two grapevine cultivars, Refošk (“Terrano”) and Zeleni Sauvignon (“Sauvignon Vert”), were analyzed. Small RNA sequencing was performed on virus-free plants and plants coinfected with grapevine Pinot gris virus (GPGV), grapevine rupestris stem pitting-associated virus (GRSPaV), and grapevine rupestris vein feathering virus (GRVFV). Three miRNA prediction tools were used to identify miRNAs annotated in public databases. Differential expression analysis was performed separately for each tool and by using an integrated approach that combined all three datasets. The expression of selected miRNAs was further evaluated using stem-loop RT-qPCR. Results: The three prediction tools detected markedly different numbers of miRNAs, resulting in largely distinct sets of differentially expressed miRNAs and limited overlap between individual analyses. The integrated approach yielded a separate set of differentially expressed miRNAs, most of which overlapped with at least one individual dataset. Stem-loop RT-qPCR analysis supported the differential expression of several selected miRNAs. Conclusions: This study provides new insight into miRNA expression in grapevine under mixed-virus infection and demonstrates that miRNA profiling outcomes are strongly influenced by the choice of bioinformatic prediction tool. Our results highlight the importance of integrated analytical strategies combined with experimental validation to obtain robust and biologically meaningful interpretations of miRNA expression in plants.

RNA silencing is one of the major defence activities against viral pathogens in plants. Silencing signals are initiated by Dicer-like proteins (DCLs) to generate viral-derived small RNAs (sRNAs). Viral sRNAs are then loaded into Argonaute proteins to form an RNA-induced silencing complex to guide cleavage of target RNAs based on sequence homology. While the model regarding RNA silencing-mediated defence against viral pathogens is largely established based on extensive studies using the model plant Arabidopsis thaliana, there are diverse sets of silencing components in other plants, especially in domesticated crops. Here, we tracked the expansion of solanaceous-specific DCL2 genes during the course of evolution. We found that the DCL2a gene in tomato chromosome 6 is likely an evolutionarily new gene copy. We also found that DCL2b is more prone to be induced by viral pathogens in tomato plants, which is dependent on the combinations of cultivar and viral pathogen. Both DCL2a and DCL2b are critical to suppress the accumulation titre of a subviral agent, potato spindle tuber viroid (PSTVd). We noticed an unusually high accumulation of viral sRNAs shorter than 20 nt (16- to 19-nt in length) in viroid-infected tomato cv. Heinz 1706. Using synthetic small interfering RNAs, we demonstrated that shorter size sRNAs may also play a role in suppressing target RNAs, which can be interfered with by a viral suppressor of silencing, P19. Altogether, we provided further insights into the expansion of functional DCL2 family members in the Solanaceae family and their roles in combating viral and subviral agents.

Since the discovery of Citrus bark cracking viroid (CBCVd) in hops in 2007, affected hop-growing countries such as Slovenia and Germany have been actively pursuing efficient and easily accessible diagnostic tools that could contribute to the early detection of CBCVd in the field. In the early stages of CBCVd infection, typical symptoms or subtle signs of spread may not be evident. Detection becomes feasible only when the plant begins to display symptoms. Unfortunately, there is currently no treatment available, which requires the removal of infected plants as the only viable solution. However, this approach leads to significant economic losses on a large scale. This work demonstrates the development and study of a sensitive, selective, and label-free impedimetric genosensor for the detection of CBCVd in total RNA hop samples. The genosensor is based on a supporting glassy carbon electrode modified with streptavidin-agarose beads, which serve as an effective immobilization layer for a biotinylated single-stranded DNA capture probe. The integration of a 2D-layered Ti3C2Tx MXene into the sensing architecture resulted in a significantly improved electroanalytical performance of the genosensor. Several fabrication and operational parameters were optimized, such as the streptavidin-agarose beads deposition time, capture probe immobilization time and concentration, and sample incubation time. The optimized genosensor exhibited a limit of detection of only 0.5 fg μL⁻¹ (5.5 fmol L−1) in combination with a one-hour incubation with the denatured total RNA hop extract, thus eliminating the need for an additional and laborious amplification step.

This study evaluates the susceptibility of various hop varieties to three viroids: Hop Latent Viroid (HLVd), Hop Stunt Viroid (HSVd), and Citrus Bark Cracking Viroid (CBCVd), all of which pose significant threats to Slovenian hop production. The experiment was conducted under in vitro conditions, involving the cultivation of 13 different hop varieties in tissue cultures, alongside the in vitro synthesis of viroid transcripts and subsequent inoculation of the plants. The plants were then monitored for growth, development, and their responses to viroid infection. The findings reveal the complex interactions between hops and viroids, with successful inoculation observed in all varieties. However, infection rates varied, with HSVd showing the highest rates of infection, followed by CBCVd and HLVd. This research enhances the understanding of viroid dynamics in hop plants, which is crucial for developing effective disease management strategies. Despite the insights gained, no resistance or tolerance to the viroids was identified in any of the tested hop varieties.

RNA silencing is one of the major defence activities against viral pathogens in plants. Silencing signals are initiated by Dicer‐like proteins (DCLs) to generate viral‐derived small RNAs (sRNAs). Viral sRNAs are then loaded into Argonaute proteins to form an RNA‐induced silencing complex to guide cleavage of target RNAs based on sequence homology. While the model regarding RNA silencing‐mediated defence against viral pathogens is largely established based on extensive studies using the model plant Arabidopsis thaliana , there are diverse sets of silencing components in other plants, especially in domesticated crops. Here, we tracked the expansion of solanaceous‐specific DCL2 genes during the course of evolution. We found that the DCL2a gene in tomato chromosome 6 is likely an evolutionarily new gene copy. We also found that DCL2b is more prone to be induced by viral pathogens in tomato plants, which is dependent on the combinations of cultivar and viral pathogen. Both DCL2a and DCL2b are critical to suppress the accumulation titre of a subviral agent, potato spindle tuber viroid (PSTVd). We noticed an unusually high accumulation of viral sRNAs shorter than 20 nt (16‐ to 19‐nt in length) in viroid‐infected tomato cv. Heinz 1706. Using synthetic small interfering RNAs, we demonstrated that shorter size sRNAs may also play a role in suppressing target RNAs, which can be interfered with by a viral suppressor of silencing, P19. Altogether, we provided further insights into the expansion of functional DCL2 family members in the Solanaceae family and their roles in combating viral and subviral agents.

Reliable identification of hop (Humulus lupulus L.) cultivars is important for quality control and authentication in the brewing industry. DNA-based methods provide a powerful tool for this purpose, but isolation of highquality DNA from processed hop materials such as cones and pellets can be challenging due to the presence of PCR inhibitors, including polyphenols, polysaccharides, and bitter acids. In this study we compared four cetyltrimethylammonium bromide (CTAB)-based DNA extraction protocols for hop cones and pellets, with the aim of improving yield and purity of DNA used for microsatellite genotyping. The tested methods included the standard CTAB protocol, CTAB supplemented with polyvinylpyrrolidone (PVP40), CTAB with PVP40 and activated charcoal, and CTAB with PVP10 and liquid nitrogen grinding. Additionally, a hexane pretreatment step was evaluated with the aim to reduce the amount of PCR inhibitory compounds. DNA quality was assessed using NanoVue, Qubit, and agarose gel electrophoresis. Agarose gels showed intact highmolecular-weight DNA with minor RNA traces. Microsatellite genotyping confirmed consistent allele profiles across the first three extraction methods, thus confirming the suitability of CTAB-based methods for reliable hop genotyping.

Since the discovery of Citrus bark cracking viroid (CBCVd) in hops in 2007, affected hop-growing countries such as Slovenia and Germany have been actively pursuing efficient and easily accessible diagnostic tools that could contribute to the early detection of CBCVd in the field. In the early stages of CBCVd infection, typical symptoms or subtle signs of spread may not be evident. Detection becomes feasible only when the plant begins to display symptoms. Unfortunately, there is currently no treatment available, which requires the removal of infected plants as the only viable solution. However, this approach leads to significant economic losses on a large scale. This work demonstrates the development and study of a sensitive, selective, and label-free impedimetric genosensor for the detection of CBCVd in total RNA hop samples. The genosensor is based on a supporting glassy carbon electrode modified with streptavidin-agarose beads, which serve as an effective immobilization layer for a biotinylated single-stranded DNA capture probe. The integration of a 2D-layered Ti3C2Tx 3 C 2 T x MXene into the sensing architecture resulted in a significantly improved electroanalytical performance of the genosensor. Several fabrication and operational parameters were optimized, such as the streptavidin-agarose beads deposition time, capture probe immobilization time and concentration, and sample incubation time. The optimized genosensor exhibited a limit of detection of only 0.5 fg mu L-1- 1 (5.5 fmol L- 1 ) in combination with a one-hour incubation with the denatured total RNA hop extract, thus eliminating the need for an additional and laborious amplification step.

The red palm weevil, Rhynchophorus ferrugineus, is a destructive, invasive pest to a diverse range of palm plantations globally. Commonly used broad-range chemical insecticides for insect control pose high risks to non-target organisms, humans, and the environment. A bio-rational approach of screening natural small-molecule inhibitors that specifically target R. ferrugineus proteins critical to its life processes can pave the way for developing novel bioinsecticides. Digestive enzymes (DEs), which impair feeding on plants (herbivory), are promising targets. We generated de novo transcriptomes, annotated DE-related genes from the R. ferrugineus gut and abdomen, manually annotated the DE gene family from the recently available genome and our transcriptome data, and reported 34 glycosidases, 85 lipases, and 201 proteases. We identified several tandem duplicates and allelic variants from the lipase and protease families, notably, 10 RferLip and 21 RferPro alleles, which emerged primarily through indels and single-site substitution. These alleles may confer enhanced digestive lipolysis and proteolysis. Phylogenetic analyses identified and classified different subfamilies of DEs and revealed close evolutionary relationships with other coleopterans. We assessed select candidate DEs’ activity and the potential for inhibition in silico to better understand the herbivory arsenal. In silico analysis revealed that the selected enzymes exhibited similar ligand-binding affinity to their corresponding substrate, except for protease aminopeptidase N, RferPro40, which exhibited poorer affinity to the inhibitor bestatin. Overall, our study serves as a foundation for further functional analysis and offers a novel target for the development of a novel bio-rational insecticide for R. ferrugineus.

Viroids are small, naked, infectious single-stranded RNA molecules that exploit host factors to replicate. Some viroids have been linked to severe diseases in agricultural crops, including the recent outbreak of Cocadviroid rimocitri, previously known as Citrus bark cracking viroid (CBCVd), in hop plants (Humulus lupulus). Numerous studies have demonstrated the involvement of viroid-derived RNA in viroid pathogenesis through interactions with RNAi host factors, leading to alterations in gene expression, metabolism, and phenotype. Recent research efforts have also focused on elucidating viroid-induced changes in DNA methylation patterns via the RNA-directed DNA methylation pathway. In this study, we conducted an epigenome analysis of CBCVd-infected hop plants to provide novel evidence supporting the putative role of DNA methylation in CBCVd viroid pathogenesis. Our findings revealed that several genes involved in pathogen interaction pathways, such as MAPK signaling and LRR, exhibit hypomethylation, suggesting that their increased transcription enhances the host's ability to counteract the pathogen. Intriguingly, genes associated with RNA transcription and encoding key proteins, such as POL II, POL IV, and POL V, display hypermethylation, highlighting the significance of DNA methylation as a defense mechanism.IMPORTANCEViroids are emerging as a substantial threat to various crops; however, our understanding of the molecular mechanisms governing their pathogenesis and the host's defense remains incomplete. This knowledge gap leaves crop disease management reliant on unsustainable strategies. Our research seeks to address this issue by examining the complex world of infected hop plants. Specifically, we are investigating the DNA methylation processes, providing insights into the less-explored aspects of the host's response to viroid interaction. Our aim was to unravel the complexities of how viroids influence the molecular landscape within plants and the corresponding host defenses. By understanding these interactions, we hope to provide insights that lead to more sustainable ways to protect crops and keep agriculture resilient against viroid-related threats.

The quality of water for use in producing crops can be threatened by different environmental factors, including contamination by plant pathogens. Owing to the limited availability of water and affected by climate change, crop production systems using irrigation has expanded worldwide. Plant pathogens in irrigation water can spread rapidly and cause substantial economic losses. Different water sources can serve as pathways for water-borne plant pathogens, including certain viroids, which are the smallest plant pathogens causing severe diseases in susceptible hosts. In the last decade, a new viroid disease named severe hop stunt disease caused by Cocadviroid rimocitri (citrus bark cracking viroid, CBCVd), has threatened hop (Humulus lupulus L.) production in Slovenia. Like other viroids, CBCVd is a single-stranded pathogenic RNA that primarily spreads through mechanical means, such as contaminated plant sap residue on tools and machines, as well as from the remains of infected plants. Given that hop production relies heavily on irrigation, it is important to assess the risk of CBCVd transmission through contaminated water sources. In our study, we assessed water as a potential pathway for CBCVd transmission through four experiments. CBCVd was detectable in stored contaminated water for at least 16 weeks and remained infective for up to 3 of these weeks. In a dilution experiment, CBCVd was detectable in directly homogenised contaminated plant sap up to a 10−5 dilution, while mechanical inoculations demonstrated infectivity up to a 10−2 dilution. In an experiment in which hop plants were watered with CBCVd-contaminated water once a week for a month, viroid transmission to the plants could not be confirmed. However, in a hydroponic system experiment, examining the release of CBCVd and Cocadviroid latenshumuli (hop latent viroid, HLVd) from the roots of infected plants into the nutrient solution, we confirmed both their release and subsequent transmission to bait plants. Our results indicate that water can serve as a pathway for CBCVd transmission, but this is highly dependent on the plant cultivation system. While no transmission occurred when plants were watered with contaminated water, and infectivity declined rapidly with sap dilution, CBCVd remained detectable in the water solution for at least 16 weeks and infectious for up to 3 weeks. This highlights the importance of preventing CBCVd contamination in hop irrigation systems through infected plants or their remains.

Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

The Asian palm weevil, Rhynchophorus ferrugineus, is a tremendously important agricultural pest primarily adapted to palm trees and causes severe destruction, threatening sustainable palm cultivation worldwide. The host plant selection of this weevil is mainly attributed to the functional specialization of odorant receptors (ORs) that detect palm-derived volatiles. Yet, ligands are known for only two ORs of R. ferrugineus, and we still lack information on the mechanisms of palm tree detection. This study identified a highly expressed antennal R. ferrugineus OR, RferOR2, thanks to newly generated transcriptomic data. The phylogenetic analysis revealed that RferOR2 belongs to the major coleopteran OR group 2A and is closely related to a sister clade containing an R. ferrugineus OR (RferOR41) tuned to the non-host plant volatile and antagonist, α-pinene. Functional characterization of RferOR2 via heterologous expression in Drosophila olfactory neurons revealed that this receptor is tuned to several ecologically relevant palm-emitted odors, most notably ethyl and methyl ester compounds, but not to any of the pheromone compounds tested, including the R. ferrugineus aggregation pheromone. We did not evidence any differential expression of RferOR2 in the antennae of both sexes, suggesting males and females detect these compounds equally. Next, we used the newly identified RferOR2 ligands to demonstrate that including synthetic palm ester volatiles as single compounds and in combinations in pheromone-based mass trapping has a synergistic attractiveness effect to R. ferrugineus aggregation pheromone, resulting in significantly increased weevil catches. Our study identified a key OR from a palm weevil species tuned to several ecologically relevant palm volatiles and represents a significant step forward in understanding the chemosensory mechanisms of host detection in palm weevils. Our study also defines RferOR2 as an essential model for exploring the molecular basis of host detection in other palm weevil species. Finally, our work showed that insect OR deorphanization could aid in identifying novel behaviorally active volatiles that can interfere with weevil host-searching behavior in sustainable pest management applications.

Apple hammerhead viroid (AHVd, Pelamoviroid, Avsunviroidae) is one of the five viroids infecting apples. It has been identified on all continents except Australia since its viroid nature was confirmed (DiSerio et al. 2018; CABI and EPPO 2022). AHVd has been found in apple trees showing leaf mosaic, ringspot and dieback (Hamdi et al., 2021). Apple (Malus domestica Borkh.) and its wild relatives are traditionally grown in Montenegro. With an annual production of 7767 tons on 216 ha, it is the second most important fruit tree (after plum) in the country (Anonymous 2022). In a 2020-2022 survey, 29 apple trees exhibiting virus-like symptoms (e.g. mosaic, necrosis) were sampled throughout Montenegro, including 16 locations in eight municipalities (Podgorica, Danilovgrad, Niksic, Mojkovac, Bijelo Polje, Berane, Pljevlja and Savnik). Small RNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Life Technologies) and pooled into three bulk samples. Each bulk contained 9 to 10 samples. Libraries of sRNAs were constructed using the Ion Total RNA-Seq Kit v2 and barcoded using the Xpress RNA-Seq Barcode 1–16 Kit (Ion Torrent) according to the manufacturer's instructions. Small RNA library sequencing was performed on Illumina platform (Novogene Europe) yielding 9.9, 9.8 and 18.6 million reads in the three libraries. The CLC Genomics Workbench software was used to demultiplex the reads into pools using the 'Demultiplex Reads' tool. The online program VirusDetect (Zheng et al. 2017) was used for virus/viroid detection and identification. Besides viruses known to infect apple (apple stem grooving virus, apple stem pitting virus, apple mosaic virus), contigs mapping to AHVd were identified in all three bulks enabling full AHVd genomes reconstruction. To verify AHVd presence, all 29 apple samples were tested by reverse transcription-polymerase chain reaction (RT-PCR) using the AHVd PG13f/PG12r primers (Messmer et al. 2017). AHVd amplicons were obtained in three samples (30/21, 32/21 and 38/21) from bulk 1 and two samples (47/21 and 55/21) from bulk 2, while all samples from bulk 3 tested negative potentially due to the low titer of the pathogen or nucleotide mismatches at the 3’ end of the primers. The three amplicons from bulk 1 were Sanger sequenced and partial AHVd genomes over 200 nts were obtained from two of them (30/21 and 32/21) (GenBank acc. nos. OQ863319 and OR020603). Furthermore, three full consensus AHVd genomes were assembled in Geneious Prime by mapping Sanger sequences onto contigs from Virus Detect and named 30/21, 32/21 and 38/21 (acc. nos. PP133245, -46, and -47, respectively). All three genomes exhibited conserved hammerhead motifs (Messmer et al. 2017). In BLASTn analysis, the isolate 30/21 from Montenegro shared the highest nt identity (98.8%) with the isolate SA-36 (ON564299) from Czechia, while 32/21 and 38/21 showed the highest identities (95.4% and 92.3%) with isolates SD17_2-3 (MK188691) from Canada and JF2 (ON564298) from Czechia, respectively. To the best of our knowledge, this is the first report of AHVd infecting Malus domestica in Montenegro. The AHVd-positive samples 30/21 and 32/21 originated from at least two-decade-old apple trees from Niksic, whilst 38/21 came from a 40-year-old tree from Mojkovac district, suggesting that this viroid has long been present in different parts of the country. The AHVd discovery in Montenegro should be considered in any phytosanitary regulations and pome fruit certification program in the country.