Viral Metatranscriptomic Analysis to Reveal the Diversity of Viruses Infecting Satsuma Mandarin (Citrus unshiu) in Korea.

Citrus cultivation plays a pivotal role, making a significant contribution to global fruit production and dietary consumption. Accurate identification of viral pathogens is imperative for the effective management of plant viral disease in citrus crops. High-throughput sequencing serves as an alternative approach, enabling comprehensive pathogen identification on a large scale without requiring pre-existing information. In this study, we employed HTS to investigate viral pathogens infecting citrus in three different regions of South Korea: Jejudo (Jeju), Wando-gun (Wando), and Dangjin-si (Dangjin). The results unveiled diverse viruses and viroids that exhibited regional variations. Notably, alongside the identification of well-known citrus viruses such as satsuma dwarf virus, citrus tatter leaf virus, and citrus leaf blotch virus (CLBV), this study also uncovered several viruses and viroids previously unreported in Korean citrus. Phylogenetic analysis revealed that majority of identified viruses exhibited the closest affilations with isolates from China or Japan. However, CLBV and citrus viroid-I-LSS displayed diverse phylogenetic positions, reflecting their regional origins. This study advances our understanding of citrus virome diversity and regional dynamics through HTS, emphasizing its potential in unraveling intricate viral pathogens in agriculture. Consequently, it significantly contributes to disease management strategies, ensuring the resilience of the citrus industry.

Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/µL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.

Identification of an Isolate of Citrus Tristeza Virus by Nanopore Sequencing in Korea and Development of a CRISPR/Cas12a-based Assay for Rapid Visual Detection of the Virus.

Citrus tristeza virus (CTV) is a highly destructive viral pathogen posing a significant threat to citrus crops worldwide. The disease management and crop protection strategies necessitate the development of rapid and accurate detection methods. In this study, we employed Oxford Nanopore sequencing (ONT) to detect CTV in Citrus unshiu samples. Subsequently, we developed a specific and sensitive detection assay combining CRISPR/Cas12a with reverse transcription-recombinase polymerase amplification. The CRISPR-Cas12a assay exhibited exceptional specificity for CTV, surpassing conventional RT-PCR by at least 10-fold in sensitivity. Remarkably, the developed assay detected CTV in field samples, with zero false negatives. This diagnostic approach is user-friendly, cost-effective, and offers tremendous potential for rapid on-site detection of CTV. Therefore, the CRISPR-Cas12a assay plays a significant role in managing and preserving citrus trees that are free from viruses in the industry.

DNA Damage Triggers the Activation of Immune Response to Viral Pathogens via Salicylic Acid in Plants.

Plants are challenged by various pathogens throughout their lives, such as bacteria, viruses, fungi, and insects; consequently, they have evolved several defense mechanisms. In addition, plants have developed localized and systematic immune responses due to biotic and abiotic stress exposure. Animals are known to activate DNA damage responses (DDRs) and DNA damage sensor immune signals in response to stress, and the process is well studied in animal systems. However, the links between stress perception and immune response through DDRs remain largely unknown in plants. To determine whether DDRs induce plant resistance to pathogens, Arabidopsis plants were treated with bleomycin, a DNA damage-inducing agent, and the replication levels of viral pathogens and growth of bacterial pathogens were determined. We observed that DDR-mediated resistance was specifically activated against viral pathogens, including turnip crinkle virus (TCV). DDR increased the expression level of pathogenesis-related (PR) genes and the total salicylic acid (SA) content and promoted mitogen-activated protein kinase signaling cascades, including the WRKY signaling pathway in Arabidopsis. Transcriptome analysis further revealed that defense- and SA-related genes were upregulated by DDR. The atm-2atr-2 double mutants were susceptible to TCV, indicating that the main DDR signaling pathway sensors play an important role in plant immune responses. In conclusion, DDRs activated basal immune responses to viral pathogens.

Bioplastic (poly-3-hydroxybutyrate)-producing Massilia endophytica sp. nov., isolated from Cannabis sativa L. 'Cheungsam'.

A rod-shaped, motile, Gram-negative bacterial strain named DM-R-R2A-13T was isolated from the plant Cannabis sativa L. 'Cheungsam'. The phylogenetic analysis of the 16S rRNA gene sequence revealed that strain DM-R-R2A-13T belongs to the family Oxalobacteraceae and is closely related to members of the genus Massilia, with Massilia flava (97.58% sequence similarity) and Massilia armeniaca (97.37% sequence similarity) being the closest members. The digital DNA-DNA hybridization (dDDH) values between strain DM-R-R2A-13T and Massilia flava CGMCC 1.10685T and Massilia armeniaca ZMN-3Twere 22.2% and 23.3%, while the average nucleotide identity (ANI) values were 78.85% and 79.63%, respectively. The DNA G+C content was measured to be 64.6 mol%. Moreover, the bacterium was found to contain polyhydroxyalkanoate (PHA) granules based on transmission electron microscopy, indicating its potential to produce bioplastic. Genome annotation revealed the presence of PHA synthase genes (phaC, phaR, phaP, and phaZ), and the biopolymer was identified as poly-3-hydroxybutyrate (PHB) based on nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) analyses. Using maltose as a carbon source, the strain produced PHB of up to 58.06% of its dry cell weight. Based on the phenotypic, chemotaxonomic, and phylogenetic characteristics, it has been determined that DM-R-R2A-13T represents a novel species belonging to the genus Massilia. As such, the name Massilia endophytica sp. nov. is proposed for this newly identified species. The type strain is DM-R-R2A-13T (= KCTC 92072T = GDMCC 1.2920T).

Analysis of Wheat Virome in Korea Using Illumina and Oxford Nanopore Sequencing Platforms.

Wheat (Triticum aestivum L.) is one of the most important staple crops in the world, along with maize and rice. More than 50 plant viruses are known to infect wheat worldwide. To date, there are no studies on the identification of viruses infecting wheat in Korea. Therefore, we investigated virome in wheat from three different geographical regions where wheat is mainly cultivated in Korea using Oxford Nanopore Technology (ONT) sequencing and Illumina sequencing. Five viral species, including those known to infect wheat, were identified using high-throughput sequencing strategies. Of these, barley virus G (BVG) and Hordeum vulgare endornavirus (HvEV) were consistently present in all libraries. Sugarcane yellow leaf virus (SCYLV) and wheat leaf yellowing-associated virus (WLYaV) were first identified in Korean wheat samples. The viruses identified by ONT and Illumina sequencing were compared using a heatmap. Though the ONT sequencing approach is less sensitive, the analysis results were similar to those of Illumina sequencing in our study. Both platforms served as reliable and powerful tools for detecting and identifying wheat viruses, achieving a balance between practicality and performance. The findings of this study will provide deeper insights into the wheat virosphere and further help improve disease management strategies.

Rapid Identification of Jasmine Virus H Infecting Ixora coccinea by Nanopore Metatranscriptomics.

The global climate change and international trade have facilitated the movement of plants across borders, increasing the risk of introducing novel plant viruses in new territories. Ixora coccinea exhibited virus-like foliar symptoms, including mosaic and mild mottle. An Oxford Nanopore Technologies-based compact and portable MinION platform was used to identify the causal viral pathogen. The complete genome sequence of jasmine virus H (JaVH; 3867 nt, JaVH-CNU) was determined and found to share 88.4-90.3% nucleotide identity with that of Jasminum sambac JaVH isolate in China. Phylogenetic analysis based on the complete amino acid sequences of RNA-dependent RNA polymerase and coat protein revealed that JaVH-CNU was grouped separately with other JaVH isolates. This is the first report of a natural JaVH infection of >i<I. coccinea. The application of rapid nanopore sequencing for plant virus identification was demonstrated and is expected to provide accurate and rapid diagnosis for virus surveillance.

Application of Rapid and Reliable Detection of Cymbidium Mosaic Virus by Reverse Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Immunoassay.

Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39°C) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.

Sphingomonas cannabina sp. nov., isolated from Cannabis sativa L. 'Cheungsam'.

A Gram-negative, aerobic, rod-shaped bacterium, designated DM2-R-LB4T was isolated from Cannabis sativa L. 'Cheungsam' in Andong, Republic of Korea. The strain DM2-R-LB4T grew at temperatures of 15-45 °C (optimum, 30-37 °C), pH of 5.5-9 (optimum, 8.0), and 0-2 % (w/v) NaCl concentration (optimum, 0%). Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strain DM2-R-LB4T is related to species of the genus Sphingomonas, and shared 97.8 and 97.5% similarity to Sphingomonas kyenggiensis KCTC 42244T and Sphingomonas leidyi DSM 4733T, respectively. The DNA G+C content was 67.9 mol% and genome analysis of the strain DM2-R-LB4T revealed that the genome size was 4 386 171 bp and contained 4 009 predicted protein-coding genes. The average nucleotide identity (ANI) values between strain DM2-R-LB4T and S. kyenggiensis KCTC 42244T, and S. leidyi DSM 4733T was 76.8 and 76.7 %, respectively, while the values of digital DNA-DNA hybridization (dDDH) were 20.7 and 20.6 %, respectively. C14 : 0 2-OH, C16 : 0, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) were the major fatty acids (>10 %) in the strain DM2-R-LB4T. The polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), sphingoglycolipid (SGL), glycolipid (GL), phospholipid (PL), and two unidentified polar lipids (L1 and L2). Ubiquinone-10 (Q-10) was the only respiratory quinone. The polyamine pattern was found to contain homospermidine, putrescine, and spermidine. The results of phylogenetic anlayses, polyphasic studies, revealed that strain DM2-R-LB4T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cannabina sp. nov., is proposed. The type strain is DM2-R-LB4T (=KCTC 92075T = GDMCC 1.3018T).

Nanopore Metagenomics Sequencing for Rapid Diagnosis and Characterization of Lily Viruses.

Lilies (Lilium spp.) are one of the most important ornamental flower crops grown in Korea. Most viral diseases in lilies are transmitted by infected bulbs, which cause serious economic losses due to reduced yields. Various diagnostic techniques and high-throughput sequencing methods have been used to detect lily viruses. According to Oxford Nanopore Technologies (ONT), MinION is a compact and portable sequencing device. In this study, three plant viruses, lily mottle, lily symptomless, and plantago asiatica mosaic virus, were detected in lily samples using the ONT platform. As a result of genome assembly of reads obtained through ONT, 100% coverage and 90.3-93.4% identity were obtained. Thus, we show that the ONT platform is a promising tool for the diagnosis and characterization of viruses that infect crops.

Detection and Quantification of Apple Stem Grooving Virus in Micropropagated Apple Plantlets Using Reverse-Transcription Droplet Digital PCR.

Apple stem grooving virus (ASGV) is a destructive viral pathogen of pome fruit trees that causes significant losses to fruit production worldwide. Obtaining ASGV-free propagation materials is essential to reduce economic losses, and accurate and sensitive detection methods to screen ASGV-free plantlets during in vitro propagation are urgently necessary. In this study, ASGV was sensitively and accurately quantified from in vitro propagated apple plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The optimized RT-ddPCR assay was specific to other apple viruses, and was at least 10-times more sensitive than RT-real-time quantitative PCR assay. Furthermore, the optimized RT-ddPCR assay was validated for the detection and quantification of ASGV using micropropagated apple plantlet samples. This RT-ddPCR assay can be utilized for the accurate quantitative detection of ASGV infection in ASGV-free certification programs, and can thus contribute to the production of ASGV-free apple trees.

Metatranscriptomic Analysis of Plant Viruses in Imported Pear and Kiwifruit Pollen.

Pollen is a vector for viral transmission. Pollen-mediated viruses cause serious economic losses in the fruit industry. Despite the commercial importance of pollen-associated viruses, the diversity of such viruses is yet to be fully explored. In this study, we performed metatranscriptomic analyses using RNA sequencing to investigate the viral diversity in imported apple and kiwifruit pollen. We identified 665 virus-associated contigs, which corresponded to four different virus species. We identified one virus, the apple stem grooving virus, from pear pollen and three viruses, including citrus leaf blotch virus, cucumber mosaic virus, and lychnis mottle virus in kiwifruit pollen. The assembled viral genome sequences were analyzed to determine phylogenetic relationships. These findings will expand our knowledge of the virosphere in fruit pollen and lead to appropriate management of international pollen trade. However, the pathogenic mechanisms of pollen-associated viruses in fruit trees should be further investigated.

Rapid and Visual Detection of Barley Yellow Dwarf Virus by Reverse Transcription Recombinase Polymerase Amplification with Lateral Flow Strips.

Barley yellow dwarf virus (BYDV) has been a major viral pathogen causing significant losses of cereal crops including oats worldwide. It spreads naturally through aphids, and a rapid, specific, and reliable diagnostic method is imperative for disease monitoring and management. Here, we established a rapid and reliable method for isothermal reverse transcription recombinase polymerase amplification (RT-RPA) combined with a lateral flow strips (LFS) assay for the detection of BYDV-infected oat samples based on the conserved sequences of the BYDV coat protein gene. Specific primers and a probe for RT-RPA reacted and optimally incubated at 42 o C for 10 min, and the end-labeled amplification products were visualized on LFS within 10 min. The RT-RPA-LFS assay showed no cross-reactivity with other major cereal viruses, including barley mild mosaic virus, barley yellow mosaic virus, and rice black streaked dwarf virus, indicating high specificity of the assay. The sensitivity of the RT-RPA-LFS assay was similar to that of reverse transcription polymerase chain reaction, and it was successfully validated to detect BYDV in oat samples from six different regions and in individual aphids. These results confirm the out-standing potential of the RT-RPA-LFS assay for rapid detection of BYDV.

Flavobacterium endoglycinae sp. nov., an endophytic bacterium isolated from soybean (Glycine max L. cv. Gwangan) stems.

A Gram-stain-negative, facultatively anaerobic, motile by gliding, rod-shaped, oxidase- and catalase-positive bacterial strain, designated BB8T, was isolated from the stems of a Korean soybean cultivar (Glycine max L. cv. Gwangan). The strain produced a yellow pigment on tryptic soy agar. Growth of strain BB8T occurred at pH 5.0-8.0 (optimum, pH 7.0), at 10-35 °C (optimum, 25-30 °C) and in the presence of 0-1 % (w/v) NaCl (optimum, 0.5%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BB8T formed a lineage within the genus Flavobacterium and was most closely related to Flavobacterium artemisiae SYP-B1015T (96.9 % 16S rRNA gene sequence similarity) and Flavobacterium ustbae T13T (96.8%). The complete genome sequence of strain BB8T was 5 513 159 bp long with a G+C content of 34.1 mol%. The major fatty acids (>10 %) of strain BB8T were iso-C15 : 0 (21 %), summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c, 20.3%) and iso-C16 : 0 3-OH (13.7%). The predominant polar lipids were phosphatidylethanolamine and unidentified aminolipids, and the major respiratory quinone was menaquinone-6. Based on these phenotypic, genotypic and chemotaxonomic characteristics, strain BB8T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium endoglycinae sp. nov. is proposed. The type strain is BB8T (=KCTC 82167T=CCTCC AB 2020070T).

Identification of Viruses Infecting Oats in Korea by Metatranscriptomics.

Controlling infectious plant viruses presents a constant challenge in agriculture. As a source of valuable nutrients for human health, the cultivation of oats (Avena sativa L.) has recently been increased in Korea. To date, however, few studies have been undertaken to identify the viruses infecting oats in this country. In this study, we carried out RNA-sequencing followed by bioinformatics analyses to understand the virosphere in six different geographical locations in Korea where oats are cultivated. We identified three different virus species, namely, barley yellow dwarf virus (BYDV) (BYDV-PAV and BYDV-PAS), cereal yellow dwarf virus (CYDV) (CYDV-RPS and CYDV-RPV), and rice black-streaked dwarf virus (RBSDV). Based on the number of virus-associated reads and contigs, BYDV-PAV was a dominant virus infecting winter oats in Korea. Interestingly, RBSDV was identified in only a single region, and this is the first report of this virus infecting oats in Korea. Single nucleotide polymorphisms analyses indicated that most BYDV, CYDV, and RBSDV isolates show considerable genetic variations. Phylogenetic analyses indicated that BYDVs and CYDVs were largely grouped in isolates from Asia and USA, whereas RBSDV was genetically similar to isolates from China. Overall, the findings of this study provide a preliminary characterization of the types of plant viruses infecting oats in six geographical regions of Korea.

First report of zucchini yellow mosaic virus infecting bitter melon (Momordica charantia) in South Korea.

Bitter melon (Momordica charantia L., family Cucurbitaceae) is used in traditional medicine for diabetes, cancer, and inflammation-associated diseases due to bioactive compounds in Asia and tropical Africa (Bortolotti et al. 2019). In July 2021, approximately 10% of bitter melon plants in the field showed symptoms such as mosaic, yellowing, and leaf deformation on the leaves, in Samchcuk, South Korea. Cucumber and zucchini plants growing in the same field exhibited symptoms like those of bitter melon plants (Ali et al. 2012). To investigate the causative virus, leaf dip preparations from three symptomatic bitter melon leaf samples with symptoms were analyzed by transmission electron microscopy (TEM). Potyvirus-like particles (approximately 680-730 nm in length and 11-13 nm in diameter) were observed in all samples. To further identify the causal viral pathogens, leaf extracts from five symptomatic bitter melon plants were tested by DAS-ELISA using specific antibodies (Agdia, Elkhart, IN, USA) against cucumber mosaic virus, zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus, and papaya ring spot virus. Positive controls from commercial kits and negative controls from healthy bitter melon plants were included in ELISA assay. The serological assay revealed that all five symptomatic samples positively reacted with the antiserum against ZYMV, but not for other viruses. Total RNA extracted from the five ELISA-positive samples and two healthy bitter melon plants (as negative controls), using Clear-S Total RNA extraction kit (InVirusTech Co., Gwangju, Korea), was tested by RT-PCR with ZYMV-specific primers as previously described (Cho et al. 2011). All amplicons of the expected size (~822 bp) were individually cloned into the pGEM-T Easy Vector (Promega, Madison, WI), and sequenced in both orientations. Thereafter, all the sequenced clones shared 100% nucleotide identity. The sequence of ZYMV-MC1 isolated from bitter melon was deposited in the GenBank (accession no. LC652434). Pairwise comparison of the nucleotide sequence with that of ZYMV isolates in the GenBank revealed 99% sequence identity with ZYMV-chk (MG020559) from Korea, 98% with ZYMV-14-HY-SCS (KU743321) from China, 97% with ZYMV-Y21 (MW345249) from Turkey, 96% with ZYMV-AUIKTPK (KR261951) from Pakstan. Leaf saps from the ZYMV-positive bitter melon samples, prepared in 10 mM phosphate buffer (pH 7.0), were mechanically inoculated in five young, healthy bitter melon plants to fulfil Koch's postulates. ZYMV-MC1 isolate caused mosaic and leaf deformation on bitter melon plants 10 days post-inoculation. The presence of ZYMV in the symptomatic leaves was confirmed by RT-PCR using the mentioned above primers mentioned above followed by nucleotide sequencing of the amplicons. Several cotton aphids (Aphis gossypii) were observed in the bitter melon field, which indicated that they might transmit the virus from ZYMV-infected cucumber or zucchini plants. ZYMV is one of the economically important viruses of cucurbits worldwide and has been recently reported from various crops as natural hosts, including Chayote (Yoon et al. 2018) and balloon flowers (Kim et al. 2021). To the best of our knowledge, this is the first report of ZYMV naturally infecting bitter melon in South Korea. Further large -scale surveys are required to determine its incidence, yield losses, and management in bitter melon in Korea.

Development and application of reverse transcription droplet digital PCR assay for sensitive detection of apple scar skin viroid during in vitro propagation of apple plantlets.

Apple scar skin viroid (ASSVd), of the genus Apscaviroid, causes serious pome fruit diseases, such as apple scar skin, dapple apple, pear rusty skin, pear fruit crinkle, and pear dimple fruit. This study aimed at establishing a sensitive and accurate method for quantification of ASSVd in apple leaves and plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity was analyzed using other apple viruses, and the negative amplification of the cross-reaction assay demonstrated the high specificity of RT-ddPCR. The detection limit of ASSVd by RT-ddPCR was 1.75 × 102 copies/µL (0.14 concentration), and the sensitivity was ten-fold higher than that of RT-qPCR. Similarly, positive detection in apple plantlet samples by RT-ddPCR was higher than that by RT-qPCR. The RT-ddPCR assay represents a promising alternative for accurate quantitative detection and diagnosis of ASSVd infection in ASSVd-free certification programs.

Identification of Plant Viruses Infecting Pear Using RNA Sequencing.

Asian pear (Pyrus pyrifolia) is a widely cultivated and commercially important fruit crop, which is occasionally subject to severe economic losses due to latent viral infections. Thus, the aim of the present study was to examine and provide a comprehensive overview of virus populations infecting a major pear cultivar ('Singo') in Korea. From June 2017 to October 2019, leaf samples (n = 110) of pear trees from 35 orchards in five major pear-producing regions were collected and subjected to RNA sequencing. Most virus-associated contigs matched the sequences of known viruses, including apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV). However, some contigs matched the sequences of apple green crinkle-associated virus and cucumber mosaic virus. In addition, three complete or nearly complete genomes were constructed based on transcriptome data and subjected to phylogenetic analyses. Based on the number of virus-associated reads, ASGV and ASPV were identified as the dominant viruses of 'Singo.' The present study describes the virome of a major pear cultivar in Korea, and looks into the diversity of viral communities in this cultivar. This study can provide valuable information on the complexity of genetic variability of viruses infecting pear trees.