Apple Mosaic Virus Research Articles

Assessing the de novo assemblers: a metaviromic study of apple and first report of citrus concave gum-associated virus, apple rubbery wood virus 1 and 2 infecting apple in India

BackgroundThe choice of de novo assembler for high-throughput sequencing (HTS) data remains a pivotal factor in the HTS-based discovery of viral pathogens. This study assessed de novo assemblers, namely Trinity, SPAdes, and MEGAHIT for HTS datasets generated on the Illumina platform from 23 apple samples, representing 15 exotic and indigenous apple varieties and a rootstock. The assemblers were compared based on assembly quality metrics, including the largest contig, total assembly length, genome coverage, and N50.ResultsMEGAHIT was most efficient assembler according to the metrics evaluated in this study. By using multiple assemblers, near-complete genome sequences of citrus concave gum-associated virus (CCGaV), apple rubbery wood virus 1 (ARWV-1), ARWV-2, apple necrotic mosaic virus (ApNMV), apple mosaic virus, apple stem pitting virus, apple stem grooving virus, apple chlorotic leaf spot virus, apple hammerhead viroid and apple scar skin viroid were reconstructed. These viruses were further confirmed through Sanger sequencing in different apple cultivars. Among them, CCGaV, ARWV-1 and ARWV-2 were recorded from apples in India for the first time. The analysis of virus richness revealed that ApNMV was dominant, followed by ARWV-1 and CCGaV. Moreover, MEGAHIT identified novel single-nucleotide variants.ConclusionsOur analyses highlight the crucial role of assembly methods in reconstructing near-complete apple virus genomes from the Illumina reads. This study emphasizes the significance of employing multiple assemblers for de novo virus genome assembly in vegetatively propagated perennial fruit crops.

Apple chlorotic leaf spot virus infection affects the physiology and biochemistry of leaves and the quality of fruits of Tuscany (Italy) autochthonous apple varieties

AbstractThe present study focused on three autochthonous varieties of apple trees belonging to the old Tuscany germplasm (central Italy), Chitignano, Mora and Ruggine, and aimed to (a) evaluate the presence of the four main latent viruses of apple trees (apple chlorotic leaf spot virus [ACLSV], apple mosaic virus [ApMV], apple stem grooving virus [ASGV] and apple stem pitting virus [ASPV]) and (b) elucidate the biochemical and physiological responses of apple leaves and fruits challenged by ACLSV (the only virus detected). Indeed, despite no visible symptoms being reported, more than half of the assayed trees tested positive for ACLSV, although it occurred only in Mora and Ruggine. ACLSV impaired the photosynthetic performance of apple trees, especially in Ruggine leaves, but variety‐specific responses in terms of reorganization of photosynthetic pigment and primary metabolites were reported. Mora showed a higher ability to trigger the de‐epoxidation state of xanthophylls and to accumulate α‐tocopherol, while d‐glucose and sucrose increased only in infected Ruggine leaves, probably as a signalling response and/or an osmotic adjustment of this variety under ACLSV infection. Unexpectedly, ACLSV infection slightly increased pome size, but it dramatically reduced their quality (i.e., soluble solids content), again especially in Ruggine, the only variety where reductions of d‐fructose, d‐glucose and malic acid contents were reported. Overall, the evidence here reported could offer valuable insights to effectively cope with the detrimental viral diseases affecting this species and to protect the production of one of the most consumed and appreciated fruits in the world.

First report of apple hammerhead viroid infecting apple trees in Montenegro.

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.

First report of a new Carlavirus provisionally named rose virus B and apple mosaic virus in mixed infections of Rosa sp. in Mexico.

Rose (Rosa sp.) is an important ornamental plant in the cut flower industry around the world. This species is prone to hosting several viruses since it is propagated vegetatively, mainly by grafting (Mollov et al., 2013). In 2021, rose plants of unidentified variety with mosaic, vein yellowing, chlorotic line patterns, and interveinal chlorosis were observed in a rose plantation established in open field in Temixco, Morelos (Supplementary Figure 1). To determine the cause of symptoms was due to viral infection, nucleic acids were extracted from leaves by in-house CTAB procedure and DNase treated. A pooled RNA sample extracted from 4 symptomatic plants was sent to BGI Genomics (China) for high-throughput sequencing (HTS). A stranded mRNA library was prepared and sequenced on the DNBSEQ platform (BGI). A total number of 13,646,715 paired 150-bp clean reads were generated. The reads were assembled de novo into 79,309 contigs ranging from 78 to 15,817 nucleotides (nt) using SPAdes (Prjibelskiet et al., 2020). The contigs were subjected to BLASTx and BLASTn for annotation. A contig with a length of 8,842 nt (208x average coverage per nt) showed 90.6% identity to rose virus B (RVB) (MT473961), and was deposited in GenBank under accession number ON165234. Additionally, three contigs (ON165235-ON165237) corresponding to RNA1 (3,443 nt; 154x coverage), RNA2 (2,938 nt; 231x coverage), and RNA3 (1,897 nt; 232x coverage) of apple mosaic virus (ApMV) were identified. These contigs showed up to 98.4%, 89.7%, and 98.6% identity, respectively, to each corresponding RNA sequences of ApMV. No other viral sequence was identified from the constructed contigs. Subsequently, the presence of RVB was confirmed by RT-PCR performed with an aliquot of the pooled RNAspan style="font-family:'Times New Roman'; font-size:11pt"> with specific primers targeting the replicase and CP (Diaz-Lara et al., 2021). For ApMV, a new set of primers were designed: ApMV_RNA1F (5'-AAATCTCCCGAAAGGGCCTG-3')/ApMV_RNA1R (5'-TCACTCGTCGCATGGATGGATAGC-3'), ApMV_RNA2F (5'-TTGGTACGAGTCGTGGTTGGTTGG-3')/ApMV_RNA2R (5'-GGAAAACTGACCGCAAACCC-3'), and ApMV_RNA3F (5'-GGAGGTTAGAGGCCCGAATG-3')/ApMV_RNA3R (5'-CGCACAGGTGGTAACTCACT-3') which amplify segments of 444 bp, 546 bp, and 434 bp, respectively. The amplicons obtained for both viruses were subjected to Sanger sequencing, confirming the identity of RVB and ApMV. The sequences from the RVB replicase (ON165241) and CP (ON165240) showed 93.9% and 97.0% nt identity with an RVB isolate reported in the USA (MT473961). On the other hand, sequences from RNA1 (ON165238), RNA2, (OP413436), and RNA3 (ON165239) of ApMV had 99.2%, 89.2%, and 99% nt identity, respectively. Finally, the four symptomatic plants were individually tested by RT-PCR to identify RVB and ApMV. Interestingly, both viruses were detected in all the plants analyzed. ApMV (genus Ilarvirus) is associated with mosaic and mottling symptoms in rose (Thomas, 1984). It has been accepted that ApMV is present in rose plants in Mexico (Cardenas-Alonso, 1994), with no evidence to confirm it. RVB was identified in rose in USA, and this virus was classified as a new species of the genus Carlavirus (Diaz-Lara et al., 2021). In addition to RVB, rose virus A and rose virus C have also been reported in rose; however, the symptomatology linked to these viruses is unknown (Xing et al. 2021; Diaz-Lara et al., 2020). Recently, RVB and ApMV were reported in rose plants in Taiwan (Chen et al., 2022). To our knowledge, this is the first report of RVB and ApMV in a mixed infection in rose in Mexico.

First Report of Three Bunya-Like Viruses, Apple Luteovirus 1, and Apple Hammerhead Viroid in Apples from Hakkari, Türkiye

Türkiye is a major apple fruit producer in the crossroads of Europe and the Middle East. Several reports have described the presence of multiple viruses affecting apple production in Türkiye, including apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leafspot virus (ACLSV), and apple mosaic virus (ApMV) (Kurçman 1977; Fidan 1994; Çağlayan et al. 2003). However, there are no reports of the presence of the recently discovered bunya-like viruses citrus concave gum-associated virus (CCGaV), and apple rubbery wood viruses 1 and 2 (ARWV1 and 2), as well as apple luteovirus 1 (ALV-1), and apple hammerhead viroid (AHVd) in Türkiye, all of which have been previously reported in other apple-producing countries (Wright et al. 2018; Liu et al. 2018; Zhang et al. 2014). Leaves from one Gala, two Granny Smith, and one Golden Delicious apple trees showing mild symptoms of curling, chlorosis, and yellowing were collected from four different orchards in the province of Hakkari, southeast Türkiye during June 2022 and sent to USDA APHIS Plant Germplasm Quarantine Program (under permit) for virus and viroid HTS-based diagnostics. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s guidelines to prepare RNAseq libraries using the TruSeq Stranded Total RNA Library Plant Kit (Illumina, Inc) as described in Malapi-Wight et al. (2021). Libraries were sequenced on the NextSeq500 sequencer (PE 2x75), and approximately 45 million reads were obtained per each sample on average. Bioinformatic analysis was performed as described in Costa et al. (2022) using Phytopipe, where unclassified pathogen-derived reads were de novo assembled and contigs were compared to the NCBI viral nucleotide and protein databases by BlastN and BlastX respectively using a 10-4 e-value cutoff. Nearly complete genome contigs were obtained for ACLSV (OR640150) and ASPV (OR640151) in all four samples and for ASGV (OR640152) in 3 of the 4 samples. The average BlastN identity to sequences in GenBank was 92.3% for ACLSV, ranging from 89-94 %. BlastN identity for ASPV was 86%, ranging from 81-92 % while the ASGV average BlastN identity was 98.2%. Nearly complete genomes with average genome coverage of 92.4% and 95.6% for RNA1 and RNA2 of CCGaV (OR640153 and OR640154), were found in two of the four samples with BlastN identity of 94.7% and 94.8% to GenBank sequences. Additionally, nearly complete genome of the large (L), medium (M), and small (S) segments for ARWV1 were found in two samples with average genome coverage of 99.9%, 99.4%, and 100% respectively and BlastN identity of 98.8%, 95.2%, and 98.4% (OR640155, OR640156, OR640157). ARWV2 contigs were also found in 1 sample where M and S segments had a coverage of 99.8% and BlastN identity of 95.4% (OR640158 and OR640159). The nearly complete genome of ALV-1 was also found in two of four samples with genome coverage of 94.1% and an average BlastN identity of 93.4% (OR640160). AHVd was found in one of the Granny Smith trees with 19,260 mapped reads to the reference GenBank MH049335.1 and identity of 98.3% (OR640149). The HTS findings of CCGaV, ARWV1, ARWV2, and ALV-1, from Türkiye were later confirmed by Sanger sequencing using custom-designed primers targeting the coat protein, the RNA-dependent RNA polymerase, or ~390bp for the AHVd genome (Supplementary Table 1). To further learn about the incidence of these agents, we tested 12 other apple samples from six different neighboring orchards and found them at 18.8% rate for CCGaV, 12.5% for both ARWV1 and ARWV2, 25% for ALV-1, and 37.5% for AHVd respectively. To our knowledge, this is the first report of the apple viruses CCGaV, ARWV1, ARWV2, and ALV-1, and the AHVd viroid in Türkiye. Further studies of the impact of these agents on orchard’s health are necessary, including their prevalence in high apple production regions of Türkiye.

Comparison of three different techniques for eradication of Apple mosaic virus (ApMV) from hazelnut (Corylus avellana L.)

Comparison of three different techniques for eradication of Apple mosaic virus (ApMV) from hazelnut (Corylus avellana L.)

Sanitation of Apple Cultivars from AP Phytoplasma and ApMV and ACLSV Viruses Using In Vitro Culture and Cryo-Knife Therapy in Liquid Nitrogen

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.

The Virome of Babaco (Vasconcellea × heilbornii) Expands to Include New Members of the Rhabdoviridae and Bromoviridae.

Babaco (Vasconcellea × heilbornii) is a subtropical species in the Caricaceae family. The plant is native to Ecuador and represents an important crop for hundreds of families. The objective of this study was to characterize, at the genomic level, two new babaco viruses identified by high-throughput sequencing. The viruses, an ilarvirus and a nucleorhabdovirus, were found in a symptomatic babaco plant from a commercial nursery in the Azuay province of Ecuador. The tripartite genome of the new ilarvirus, provisionally named babaco ilarvirus 1 (BabIV-1), is related to subgroup 3 ilarviruses, including apple mosaic virus, apple necrotic mosaic virus, and prunus necrotic ringspot virus as the closest relatives. The genome of the nucleorhabdovirus, provisionally named babaco nucleorhabdovirus 1 (BabRV-1), showed the closest relation with joa yellow blotch-associated virus and potato yellow dwarf nucleorhabdovirus. Molecular-based detection methods found BabIV-1 and BabRV-1 in 21% and 36%, respectively, of plants surveyed in a commercial babaco nursery, highlighting the importance of enforcing virus testing and nursery certification programs for babaco.

Global Population Structure of Apple Mosaic Virus (ApMV, Genus Ilarvirus).

The gene sequence data for apple mosaic virus (ApMV) in NCBI GenBank were analyzed to determine the phylogeny and population structure of the virus at a global level. The phylogenies of the movement protein (MP) and coat protein (CP) genes, encoded by RNA3, were shown to be identical and consisted of three lineages but did not closely correlate with those of P1 and P2, suggesting the presence of recombinant isolates. Recombination Detection Program (RDP v.4.56) detected significant recombination signal in the P1 region of K75R1 (KY883318) and Apple (HE574162) and the P2 region of Apple (HE574163) and CITH GD (MN822138). Observation on several diversity parameters suggested that the isolates in group 3 had higher divergence among them, compared to isolates in groups 1 and 2. The neutrality tests assigned positive values to P1, indicating that only this region experiencing balanced or contracting selection. Comparisons of the three phylogroups demonstrated high Fixation index (FST) values and confirmed genetic separation and the lack of gene flow among them. Additionally, ±500 bp of partial MP + 'intergenic region' + partial CP coding regions of two Turkish isolates from apple and seven from hazelnut were sequenced and determined that their phylogenetic positions fell within group 1 and 3, respectively.

Quantitative Assessment of Apple Mosaic Disease Severity Based on Hyperspectral Images and Chlorophyll Content

The infection of Apple mosaic virus (ApMV) can severely damage the cellular structure of apple leaves, leading to a decrease in leaf chlorophyll content (LCC) and reduced fruit yield. In this study, we propose a novel method that utilizes hyperspectral imaging (HSI) technology to non-destructively monitor ApMV-infected apple leaves and predict LCC as a quantitative indicator of disease severity. LCC data were collected from 360 ApMV-infected leaves, and optimal wavelengths were selected using competitive adaptive reweighted sampling algorithms. A high-precision LCC inversion model was constructed based on Boosting and Stacking strategies, with a validation set Rv2 of 0.9644, outperforming traditional ensemble learning models. The model was used to invert the LCC distribution image and calculate the average and coefficient of variation (CV) of LCC for each leaf. Our findings indicate that the average and CV of LCC were highly correlated with disease severity, and their combination with sensitive wavelengths enabled the accurate identification of disease severity (validation set overall accuracy = 98.89%). Our approach considers the role of plant chemical composition and provides a comprehensive evaluation of disease severity at the leaf scale. Overall, our study presents an effective way to monitor and evaluate the health status of apple leaves, offering a quantifiable index of disease severity that can aid in disease prevention and control.

Development of one step colorimetric RT-LAMP assays for rapid detection of Apple mosaic virus and Prunus necrotic ringspot virus

Apple mosaic virus (ApMV) and Prunus necrotic ringspot virus (PNRSV), belonging to genus Ilarvirus, cause significant losses to rose and other plants of the family Rosaceae. They are easily transmitted through mechanical or vegetative means. In our previous study, the occurrence of ApMV and PNRSV in rose plants was reported. In this study, as a first step towards the development of a colorimetric Reverse Transcriptase – Loop Mediated Isothermal Amplification (RT-LAMP) assay, two primer sets were designed, each containing six primers (F3, B3, FIP, BIP, LF and LB) targeting the coat protein genes of ApMV and PNRSV. After incubation of RT-LAMP reaction mix at an isothermal temperature (65 °C/30 min), the amplified products were visually confirmed with the nucleic acid intercalation dye SYBR Green I and the indicator dye Hydroxy-Naphthol Blue. The developed assays were virus specific and showed no cross amplification. Their sensitivity was 103 times higher than that of the corresponding RT-PCRs. The LAMP assays developed in this study are inexpensive, rapid and reliable for the early detection of ApMV and PNRSV, and could therefore be used in plant quarantine to control the risk of their spread.

Simultaneous detection of five viruses and two viroids affecting apples through a DNA macroarray chip

Multiple infections of various viruses and viroids in apple trees are common and have caused a significant loss in the world apple industry. To provide an early detection of any of those possible pathogens at the molecular level, a multiplex DNA macroarray chip was designed and developed for a simultaneous identification of five common apple viruses and two viroids including apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple mosaic virus (ApMV), apple necrosis mosaic virus (ApNMV), apple scar skin viroid (ASSVd), and apple dimple fruit viroid (ADFVd). The macroarray with a 23 bp probe arranged with the coat protein (CP) gene or a target DNA segment of each viruses and viroids has demonstrated a high specificity and sensitivity without any competitions, inhibitions or cross-interferences when it was tested against more than a mixture of viral and viroid samples. To our best knowledge, this is the first report on the simultaneous detection of five different apple viruses and two viroids through using a DNA macroarray, therefore, we suggest that this detection protocol and procedure be used for any apple viral diagnosis before setting up a production nursery for virus-free apple seedlings.

Metagenomic analysis of plant viruses in tropical fresh and wastewater

AbstractDue to considerable losses in the agricultural sector, phytopathogenic viruses are of significant economic importance. Several phytopathogenic viruses have been detected in water bodies. These can be transmitted to their hosts through water contact. However, relatively few studies concentrate on the presence and impact of plant‐infecting viruses in water environments. In the present study, we used a metagenomic approach to determine viral phytopathogen diversity in wastewater, freshwater, and sludge from the southernmost tropical region in Mexico. We detected 15 different phytopathogenic viruses that belonged to the family Virgaviridae, genus Tobamovirus. Pepper mild mottle virus, tomato brown rugose fruit virus, and tropical soda apple mosaic virus had the highest number of reads. Phylogenetic analysis of the coat protein (CP) amino acid sequence revealed a close relationship between the solanaceous plant‐infecting viruses and the reference sequences found in GenBank. On the other hand, the CP amino acid sequence of Fabaceae and Cucurbitaceae‐infecting viruses did not show such a close relationship when compared with GenBank reference sequences. These results suggested that detected viral sequences could originate from new strains or species of local viruses likely from the consumption of locally cultivated produce, whereas those infecting solanaceous plants might come from consumption of processed or imported food to Mexico. The prevalence of a high diversity of plant viruses in wastewater and rivers and their potential infectivity raise concerns about the use of contaminated freshwater and wastewater in agriculture in Mexico.

Tissue and Time Optimization for Real-Time Detection of Apple Mosaic Virus and Apple Necrotic Mosaic Virus Associated with Mosaic Disease of Apple (Malus domestica).

Besides apple mosaic virus (ApMV), apple necrotic mosaic virus (ApNMV) has also been found to be associated with apple mosaic disease. Both viruses are unevenly distributed throughout the plant and their titer decreases variably with high temperatures, hence requiring proper tissue and time for early and real-time detection within plants. The present study was carried out to understand the distribution and titer of ApMV and ApNMV in apple trees from different plant parts (spatial) during different seasons (temporal) for the optimization of tissue and time for their timely detection. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) was carried out to detect and quantify both viruses in the various plant parts of apple trees during different seasons. Depending on the availability of tissue, both ApMV and ApNMV were detected in all the plant parts during the spring season using RT-PCR. During the summer, both viruses were detected only in seeds and fruits, whereas they were detected in leaves and pedicel during the autumn season. The RT-qPCR results showed that during the spring, the ApMV and ApNMV expression was higher in leaves, whereas in the summer and autumn, the titer was mostly detected in seeds and leaves, respectively. The leaves in the spring and autumn seasons and the seeds in the summer season can be used as detection tissues through RT-PCR for early and rapid detection of ApMV and ApNMV. This study was validated on 7 cultivars of apples infected with both viruses. This will help to accurately sample and index the planting material well ahead of time, which will aid in the production of virus-free, quality planting material.

Virusi i viroidi kruške

One of the most common fruit species in the world is the pears. Compared to stone and small fruit trees, pears and other pome fruits are infected by a lesser number of viruses and viroids. The majority of commercial pear cultivars do not exhibit symptoms on their leaves, fruits, or other plant parts due to viruses. Only foliage and fruits show symptoms in susceptible cultivars. Apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leafspot virus (ACLSV), and apple mosaic virus (ApMV) are widespread and economically significant pear viruses. Pear blister canker viroid (PBCVd) and apple scar skin viroid (ASSVd) are the most dangerous viroids infecting pears.

The effect of the Pharmaiod preparation in the virus elimination of pear plants using the thermotherapy method

Currently, the main methods of virus elimination of fruit crops are dry-air thermotherapy, meristem culture and chemotherapy. The use of a complex of physical and chemical methods makes it possible to increase the efficiency of virus elimination. Improving the methods of recovery must be carried out in relation to certain types of viruses, the biological characteristics of the crop and variety. The purpose of the research is to study the effect of the Pharmaiod preparation on biometric, physiological parameters and the release of harmful viruses-free pear plants in the process of dry-air thermotherapy. Improvement of pear plants of varieties Letnyaya Zabava, Zolotoy Vityaz, hybrids R-11-9, R-10-3 and R-2-4 from harmful latent viruses of Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) were carried out in 2020-2021 at the temperature of 38 °C for 3 months using a thermal chamber designed by the Federal Horticultural Research Centеr for Breeding, Agrotechnology and Nursery. Biometric parameters in pear plants depended on varietal characteristics, the duration of therapy and the concentration of the Pharmaiod preparation. In most of the varieties and forms studied, after 3 months of thermotherapy, the Pharmaiod preparation at a concentration of 1 ml/l contributed to an increase in the length of 1 shoot of pear plants by 1.4-3.1 times compared with the control. Under the conditions of a thermal chamber, according to a set of indicators, pear hybrids R-11-9 and R-10-3 were characterized by the highest heat resistance; medium heat resistance was shown by Letnyaya Zabava and Zolotoy Vityaz varieties; low – by R-2-4 hybrid. Higher heat-resistant varieties and forms were characterized by a more restrained growth of shoots in length compared to lower heat-resistant ones. An average negative significant correlation at the 5% significance level was established between the water content in the leaves and the length of 1 shoot (r = -0.52). The yield of virus-free pear plants depended on the type of virus. The yield of pear plants free from 4 major harmful viruses as a result of dry-air thermotherapy without the use of the Pharmaiod preparation was 50 %, with the use of the drug it was 60 %.

Rose Virome Analysis and Identification of a Novel Ilarvirus in Taiwan.

Rose (Rosa spp.), especially R. hybrida, is one of the most popular ornamental plants in the world and the third largest cut flower crop in Taiwan. Rose mosaic disease (RMD), showing mosaic, line patterns and ringspots on leaves, is a common rose disease caused by the complex infection of various viruses. Due to pests and diseases, the rose planting area in Taiwan has been decreasing since 2008; however, no rose virus disease has been reported in the past five decades. In the spring of 2020, rose samples showing RMD-like symptoms were observed at an organic farm in Chiayi, central Taiwan. The virome in the farm was analyzed by RNA-seq. Rose genomic sequences were filtered from the obtained reads. The remaining reads were de novo assembled to generate 294 contigs, 50 of which were annotated as viral sequences corresponding to 10 viruses. Through reverse transcription-polymerase chain reaction validation, a total of seven viruses were detected, including six known rose viruses, namely apple mosaic virus, prunus necrotic ringspot virus, rose partitivirus, apple stem grooving virus, rose spring dwarf-associated virus and rose cryptic virus 1, and a novel ilarvirus. After completing the whole genome sequencing and sequence analysis, the unknown ilarvirus was demonstrated as a putative new species, tentatively named rose ilarvirus 2. This is the first report of the rose virus disease in Taiwan.

Sequencing and Variant Detection of Eight Abundant Plant-Infecting Tobamoviruses across Southern California Wastewater.

Tobamoviruses are agriculturally relevant viruses that cause crop losses and have infected plants in many regions of the world. These viruses are frequently found in municipal wastewater, likely coming from human diet and industrial waste across wastewater catchment areas. As part of a large wastewater-based epidemiology study across Southern California, we analyzed RNA sequence data from 275 influent wastewater samples obtained from eight wastewater treatment plants with a catchment area of approximately 16 million people from July 2020 to August 2021. We assembled 1,083 high-quality genomes, enumerated viral sequencing reads, and detected thousands of single nucleotide variants from eight common tobamoviruses: bell pepper mottle virus, cucumber green mottle mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, tomato brown rugose fruit virus, tomato mosaic virus, tomato mottle mosaic virus, and tropical soda apple mosaic virus. We show that single nucleotide variants had amino acid-altering consequences along with synonymous mutations, which represents potential evolution with functional consequences in genomes of these viruses. Our study shows the importance of wastewater sequencing to monitor the genomic diversity of these plant-infecting viruses, and we suggest that our data could be used to continue tracking the genomic variability of such pathogens. IMPORTANCE Diseases caused by viruses in the genus Tobamovirus cause crop losses around the world. As with other viruses, mutation occurring in the virus's genomes can have functional consequences and may alter viral infectivity. Many of these plant-infecting viruses have been found in wastewater, likely coming from human consumption of infected plants and produce. By sequencing RNA extracted from influent wastewater obtained from eight wastewater treatment plants in Southern California, we assembled high-quality viral genomes and detected thousands of single nucleotide variants from eight tobamoviruses. Our study shows that Tobamovirus genomes vary at many positions, which may have important consequences when designing assays for the detection of these viruses by agricultural or environmental scientists.

Evaluation of Apple Chlorotic Leaf Spot Virus Impact on Fruit Yield and Vegetative Growth in Apple Cultivars ‘Antonovka’ and ‘Gita’

Abstract The negative influence of pathogens on plant development is well known. However, the negative impact depends on various factors: cultivar and rootstock resistance, nutrition, the environment, the virulence, and concentration of pathogens in the plant tissue. The evaluation of apple chlorotic leaf spot virus (ACLSV) influence on apple tree growth and production was performed at Institute of Horticulture (LatHort) from 2012 to 2020. Virus-free and ACLSV-infected trees from the traditional cultivar ‘Antonovka’ and new scab resistant cultivar ‘Gita’ were evaluated on dwarfing rootstock B.396. The presence of four common apple viruses (apple mosaic virus (ApMV), ACLSV, apple stem grooving virus (ASGV), apple stem pitting virus (ASPV)) was assayed in the planting year and repeated at the end of the study period. The vigour of planting material, growth, yield, and yield efficiency were compared between virus negative and ACLSV-infected trees. During eight growing seasons (2013–2020), no statistically significant negative impact of ACLSV infection on tree growth or yield was observed for the cultivars. A larger canopy volume was observed for ACLSV-infected trees (p = 0.01), but the cumulative yield per canopy volume was equivalent between virus-negative trees and ACLSV-infected trees (p = 0.07).

Viral Infection Control in the Essential Oil-Bearing Rose Nursery: Collection Maintenance and Monitoring

Viral diseases affecting the essential oil rose, which is a valuable object of agricultural production, may have a significant negative impact on the economic value of this crop. Hence, the study and control of potentially dangerous viruses is essential to improving the quality of cultivars of this raw plant material, to enable production of valuable derivatives. The diversity of viruses affecting Rosa L. plants manifests itself in their conditional division into those that are specific to this crop, and those that are hosted by other plants. Representatives of both groups are found in different countries, however, a low number of viruses identified have been thoroughly studied through the use of experimental methods. In particular, with regard to many viruses, the issue of their spread remains open. The viruses infecting Rosa L. plants along with other crops are described in the literature in detail, as the range of hosts they affect is rather wide and well-studied. It is also possible to single out the three most significant viruses affecting this host—Prunus necrotic ringspot virus, Apple mosaic virus and Arabis mosaic virus which individually, or collectively, cause viral diseases that manifest themselves in mosaic symptoms. The most likely mechanisms for the spread of the Rosa L. species viruses are vegetative propagation procedures and transmission by various pests. These presumptions underlie viral infection control methods, including a well-thought-out planting scheme and provision of accurate plant care, which considers plant disinfection, disease monitoring associated with diagnostics and obtaining virus-free material through biotechnology techniques.