Search Results (12)

Apples (Malus domestica), one of Iran’s oldest cultivated fruit crops, hold considerable economic importance. In this study, 170 apple leaf samples representing various commercial cultivars were collected across the country. RT-PCR screening targeted five common apple-infecting viruses and two viroids: apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple green crinkle-associated virus (AGCaV), apple mosaic virus (ApMV), apple scar skin viroid (ASSVd), and hop stunt viroid (HSVd). To identify additional or novel agents, 40 RT-PCR-negative samples were pooled into two composite groups and analyzed using next-generation sequencing (NGS). NGS was also performed on individual samples with mixed infections to retrieve full genomes. RT-PCR confirmed the presence of ACLSV, ASPV, ASGV, AGCaV, ApMV, and HSVd. NGS further revealed three additional pathogens: citrus concave gum-associated virus (CCGaV), apple hammerhead viroid (AHVd), and apricot vein clearing-associated virus (AVCaV), which were subsequently detected across the collection by RT-PCR. AGCaV was most prevalent (47.6%), followed by ACLSV (45.8%), HSVd (27.6%), AVCaV (20.5%), ASGV (17%), AHVd (15.2%), ASPV (14.1%), CCGaV (4.7%), and ApMV (3.5%). Mixed infections occurred in 67% of samples. Phylogenetic analysis based on CP genes (ACLSV, ASGV, AGCaV) and full genomes (AVCaV, AHVd) clustered Iranian isolates together, suggesting a common origin. This is the first report in Iran of AGCaV, CCGaV, ApMV, and AVCaV in apple, and notably, the first global report of AVCaV in a non-Prunus host. The findings provide the first comprehensive assessment of the sanitary status of apple trees in Iran. Full article

Kazakhstan is recognized as one of the primary centers of origin of the wild apple Malus sieversii, concentrated mainly in the mountains like Trans-Ile and Zhongar Alatau, as well as parts of the Tarbagatay, Talas Alatau, and Karatau ranges. As the wild progenitor of Malus domestica, M. sieversii harbors a critical genetic diversity essential for apple breeding and conservation efforts. However, its natural populations are increasingly threatened by latent viral infection, which weakens trees, reduces reproduction, and hinders regeneration. In this study, the spread of apple chlorotic leaf spot virus (ACLSV) and apple stem pitting virus (ASPV) was documented in four wild apple populations, with detection rates of 50.2% and 42.2%, respectively. Mixed infections were observed in 28.8% of sampled trees. Apple stem grooving virus (ASGV) was detected exclusively in cultivated orchards, whereas apple mosaic virus (ApMV) and apple necrotic mosaic virus (ApNMV) were not found in either wild forests or cultivated orchards. Using Geographic Information System (GIS) technology, we developed the first spatial distribution maps of these viruses in wild apple forests in the Tian Shan region, revealing site-specific variation and infection rates. These results underscore the importance of monitoring viral infections in wild M. sieversii populations to preserve genetically valuable, virus-free germplasm critical for apple breeding, crop improvement, and sustainable orchard management. Full article

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

A sudden, unexplained decline and collapse of young apple trees on dwarfing and semi-dwarfing rootstocks has been reported across North America over the past decade. Although viruses have been detected in declining trees, no information is available on their potential causal role in the decline phenomenon. To this end, virus-inoculated apple trees were established in a high-density experimental orchard and monitored over five years. Tree decline was observed in year 4 (2022), resulting in 17% mortality, with declining trees exhibiting marked vascular tissue necrosis. However, none of the eight viruses and one viroid detected in the experimental orchard was significantly more prevalent in declining trees. Extreme temperature fluctuations in January 2022, followed by a severe water deficit in summer 2022, were recorded at the experimental orchard. Similar but distinct observations were made in a nearby commercial orchard with foliar nutrient imbalances documented in trees exhibiting symptoms of rapid decline. Together, our findings suggest that viruses are not primarily responsible for the rapid decline phenomenon and highlight the need for future work to investigate the roles of tree physiology and water stress in tree decline, as well as the potential efficacy of horticultural mitigation practices. Full article

Many viruses occur in apple (Malus domestica (Borkh.)), but no information is available on their seed transmissibility. Here, we report that six viruses infecting apple trees, namely, apple chlorotic leaf spot virus (ACLSV), apple green crinkle-associated virus (AGCaV), apple rubbery wood virus 2 (ARWV2), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and citrus concave gum-associated virus (CCGaV) occur in seeds extracted from apple fruits produced by infected maternal trees. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) assays revealed the presence of these six viruses in untreated apple seeds with incidence rates ranging from 20% to 96%. Furthermore, ASPV was detected by RT-PCR in the flesh and peel of fruits produced by infected maternal trees, as well as from seeds extracted from apple fruits sold for fresh consumption. Finally, a large-scale seedling grow-out experiment failed to detect ACLSV, ASGV, or ASPV in over 1000 progeny derived from sodium hypochlorite surface sterilized seeds extracted from fruits produced by infected maternal trees, suggesting no detectable transmission via embryonic tissue. This is the first report on the seedborne nature of apple-infecting viruses. Full article

Apple chlorotic leaf spot virus (ACLSV) (genus, Trichovirus; family, Betaflexiviridae) is a widespread, deleterious, and the most damaging pathogen of pome and fruit trees including domesticated apple (Malus × domestica Borkh.), to which it is transmitted by grafting and pruning. The positive-sense, single-stranded RNA virus is 600–700 nm long and has a genome of 74.7–7.56 kbp in size, minus the poly-A tail and 3′- and 5′-untranslated regions. The genome has three overlapping open reading frames (ORFs) that encode a replication-associated protein (Rep), movement protein (MP), and coat protein (CP). RNA interference (RNAi)-mediated antiviral defense in eukaryotes has evolved to control infections in plant viruses. The objective of this study was to analyze locus-derived microRNAs (mdm-miRNAs) in the apple genome with potential for targeting ACLSV +ssRNA-encoded mRNAs, using a predictive approach that involves four algorithms. The goal is to mobilize the in silico-predicted endogenous mdm-miRNAs and trigger the RNAi pathway experimentally in apple trees to evaluate antiviral resistance to ACLSV. Experimentally validated apple (2n = 2X = 34) mdm-miRNAs (n = 322) were obtained from the miRBase database and aligned to the ACLSV genome (KU870525). Of the 322 targeting mature locus-derived mdm-miRNAs analyzed, nine apple mdm-miRNA homologs (mdm-miR395k, mdm-miR5225c, and mdm-miR7121 (a, b, c, d, e, f, g, h) were predicted by all “four algorithms”, whereas fifty-eight mdm-miRNAs were identified as consensus binding sites by the combined results of two algorithms. The miRanda, RNA22, and TAPIR algorithms predicted binding of mdm-miR395k at nucleotide position 4691 and identified it as the most effective interacting mdm-miRNA targeting the virus ORF1 sequence. An integrated Circos plot was generated to validate the accuracy of target prediction and determine if apple mdm-miRNAs could bind to the predicted ACLSV mRNA target(s). A genome-wide in silico-predicted miRNA-mediated target gene regulatory network was implicated to validate interactions necessary to warrant in vivo analysis. The availability of validated locus-derived microRNAs (mdm-miRNAs) with predicted potential to target ACLSV in infected apple trees represents the first step toward development of ACLSV-resistant apple trees. Full article

Systemic infections with phytoplasmas and viruses threaten the production of healthy plant material under the fruit species certification system. We tested the possibility of sanitation using in vitro culture and cryotherapy. The starting material of the cultivars Golden Delicious (clones A and B), Virginia Crab, and Panenské zlepšené was taken from in vivo plants that tested positive for apple proliferation phytoplasma. The Táborita cultivar was obtained from already established in vitro cultures that had tested positive for apple proliferation phytoplasma, apple mosaic virus, and apple chlorotic leaf spot virus. Cultivars Golden Delicious A, Virginia Crab, and Panenské zlepšené were sanitated from the phytoplasma in the first step, i.e., by sterilization and a subsequent transfer to in vitro conditions. Golden Delicious B remained infected with the phytoplasma, and both viruses, after the in vitro culture phase and together with Táborita, were subjected to cryotherapy by vitrification. In Golden Delicious B, three out of thirteen initial shoot tips regenerated after a liquid nitrogen treatment. Four mericlones were regenerated from 10 initial cryopreserved shoot tips of Táborita. None of the three pathogens were detected by PCR in the regenerated Golden Delicious B mericlones. On the contrary, in the case of Táborita, infection with all the pathogens was detected after regeneration. The results obtained indicate the potential applicability of in vitro cultivation techniques or, if necessary, subsequent cryopreservation as a method for sanitizing against systemic microbial contamination. However, further research on the relationship between pathogens and specific genotypes is needed. Full article

Viral seed transmission causes the spread of many plant viral diseases. Pyrusbetulifolia and P. calleryana are important rootstock germplasms for pear production in China. This study revealed the widespread infection of apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and apple stem pitting virus (ASPV) in maternal trees of P. betulifolia and P. calleryana by nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assays. Seeds from eight P. betulifolia and two P. calleryana trees had positive rates of 15.9–73.9%, 0–21.2%, and 40.4% for ASGV, ASPV, and ACLSV, respectively. At the cotyledon and 6–8 true leaf stages, seedlings grown from seeds of infected trees gave positive rates of 5.4% and 9.3% for ASGV, 6.7% and 15.6% for ACLSV, and 0% and 2.7% for ASPV, respectively. Incidence in nursery P. betulifolia seedlings of 10.1%, 5.3%, and 3.5% were determined for ASGV, ACLSV, and ASPV, respectively. The nucleotide sequences of coat protein (CP) and movement protein coding genes of both ASGV and ASPV, and CP gene of ACLSV from maternal trees, seeds, and seedlings were analyzed. Sequence identities and phylogenetic comparison with corresponding sequences from GenBank demonstrated that molecular variation occurred within ASGV, ACLSV, and ASPV isolates, with most sequences determined here had close relationships with reported isolates infecting pear or formed independent clades. This is the first report on the seed transmission and the molecular characteristics of these viruses infecting two rootstock species. These findings provided important evidence in management effort for pear viral diseases. Full article

Many viruses have been found associated with apple mosaic disease in different parts of the world. In order to reveal and characterize the viruses and viroids in symptomatic apple plants, next-generation sequencing (RNA seq.) of rRNA-depleted total RNA using Illumina Hiseq2500 was applied to two cultivars, Oregon Spur and Golden Delicious, with symptoms of mosaic and necrosis and one cultivar, Red Fuji, which was asymptomatic. The RNA sequencing detected five viruses, viz., apple necrotic mosaic virus (ApNMV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and one viroid i.e., apple hammerhead viroid (AHVd). RT-PCR amplification and sequencing also confirmed the presence of all these five viruses and viroids detected in HTS of total RNA. The complete genomes of five viruses and AHVd were reconstructed. The phylogenetic analysis of these viruses and AHVd revealed genetic diversity by forming subclusters with isolates from other countries. Recombination events were observed in all five viruses while single-nucleotide variants were detected only in ApMV and ApNMV. The absence of ApMV and ApNMV in asymptomatic samples from the same cultivars in an RT-PCR assay indicated that these two viruses are associated with mosaic disease of apples in India. This is the first viral genome analysis of symptomatic and asymptomatic apple plants and the first report of genome characterization of viruses associated with apple mosaic disease from India. High-throughput RNA sequencing is a powerful tool to characterize the genome of viruses and viroids in plants previously undetected by conventional methods. This would also help in the indexing and certification of large-scale germplasm. Full article

Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or a combination of both could effectively eradicate ACLSV, ASGV, and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36 °C (day) and 32 °C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for one day followed by exposure to PVS2 for 60 or 75 min at 22 °C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Following treatments, shoot tips were warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting great potential for producing virus- and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm. Full article

Loquat (Eriobotrya japonica) is an important crop in Spain. To date, only one viral species, apple stem pitting virus (ASPV), has been detected in Spanish loquat orchards. In this study, the presence of additional viruses infecting this crop in Spain was investigated. RT-PCR and high-throughput sequencing (HTS) of symptomatic loquat plants led to first-time detection and characterization of apple stem grooving virus (ASGV), also known as citrus tatter leaf virus (CTLV), and apple chlorotic leaf spot virus (ACLSV) from Spain with description of nearly complete genomic sequences. The frequency of ACLSV infection was the highest, with over 30% of the samples testing positive and were also detected as coinfections with ASGV and ASPV, although most of the samples infected were symptomless. Studies on all the full-length sequences available in the databases were performed in order to establish the phylogenetic relationships of the Spanish isolates of these two viral species. Moreover, apple hammerhead viroid (AHVd) was also detected to infect loquat, the first host different from apple reported for this viroid to date. Full article

One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus. Full article