First report of konjac mosaic virus in Spuriopimpinella brachycarpa in Korea.

Spuriopimpinella brachycarpa Nakai (Common name, Chamnamul; family Apiaceae) is a plant whose leaves are consumed as a vegetable and used as a folk medicine in Korea (Kim et al., 2020). In February 2020, seven samples of S. brachycarpa leaf showing virus symptoms including yellowing, vein chlorosis, chlorotic lesions, and severe mottling were collected from a greenhouse in Busan, South Korea, to diagnose the potential disease (Fig. S1a, b). The disease incidence rate in the greenhouse was >10% (2,970 m2). To identify the causal virus, we analyzed leaf dip preparation and thin sections of the symptomatic leaves by transmission electron microscopy. Filamentous virus particles and pinwheel structures were observed, indicating the presence of a potyvirus (Fig. S1c, d). To confirm these results, the symptomatic leaf samples were further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using potyvirus universal primers (Table S2) and direct sequencing of the PCR products. All samples were positive for konjac mosaic virus (KoMV). To exclude the possibility of infection by multiple viruses, we performed high-throughput sequencing (HTS) on an Illumina NovaSeq 6000 system (Macrogen Inc., Seoul, South Korea). There were two contigs (9,267 and 2,851 nt) mapping to KoMV sequences. A large contig (9,267 nt; 705,967 mapped reads; mean read coverage of 11,351.4x) showed about 80% identity (93% coverage) with KoMV-F (GenBank accession no. NC_007913) isolated from Amorphophallus konjac in Japan (Nishiguchi et al., 2006). To isolate KoMV from S. brachycarpa, we mechanically inoculated leaf extracts from symptomatic samples onto Chenopodium quinoa as an assay host via three single-lesion passages, followed by propagation in Nicotiana benthamiana. In a bioassay of the KoMV isolate (KoMV-BS), we mechanically inoculated sap from infected N. benthamiana onto 31 indicator plants including Cryptotaenia japonica (Apiaceae), which is similar to S. brachycarpa (Table S3). KoMV-BS systemically induced vein chlorosis and/or leaf mottling in four Nicotiana species and C. japonica, and chlorotic local lesions in upper leaves of C. quinoa; no symptoms were observed in 25 other indicator plants. These results were confirmed by RT-PCR. Next, we obtained the complete genome sequence of KoMV-BS using HTS and 5' and 3' rapid amplification of cDNA ends, with newly designed primers (Table S2). The assembled full-length KoMV-BS genome sequence was 9,392 nt in length, excluding the poly(A) tail, and encoded a polyprotein composed of 3,060 amino acids. The sequence was deposited in GenBank (accession no. OR001914). BLAST analysis showed 84~88% and 90~98% identities at CP nucleotide and amino acid levels, respectively with the reported KoMV isolates, confirming the virus to be an isolate of KoMV (synonym; Japanese hornwort mosaic virus, zantedeschia mosaic virus) (Adams et al., 2005; Nishiguchi et al., 2006). KoMV infection was first reported in A. konjac from Japan (Shimoyama et al. 1992) and has been spread worldwide as one of the major causal agents of viral diseases in calla lily (Liao et al., 2020). To the best of our knowledge, this is the first report of KoMV infection in S. brachycarpa. To date, cucumber mosaic virus and tobacco mosaic virus have been reported to infect S. brachycarpa in Korea (Yoon et al., 2016; 2017). Our findings will be helpful for developing virus-management strategies to prevent yield and quality loss in S. brachycarpa.

First report of beet western yellows virus in radish in Korea.

Radish (Raphanus sativus L.) is an important root vegetable widely consumed in kimchi in Korea. In October 2021, radish leaves with virus-like symptoms of mosaic and yellowing were collected in three fields around Naju, Korea (Fig. S1). A pooled sample (n = 24) was screened for causal viruses by high-throughput sequencing (HTS), with detection confirmed by reverse transcription (RT) PCR. Total RNA was extracted from symptomatic leaves using the Plant RNA Prep kit (Biocube System, Korea), and a cDNA library was constructed and sequenced on an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo transcriptome assembly yielded 63,708 contigs, which were analyzed against the viral reference genome database in GenBank by BLASTn and BLASTx searches. Two large contigs were clearly of viral origin. BLASTn analysis showed that a 9,842-bp contig (4,481,600 mapped reads, mean read coverage 68,758.6×) had 99% identity (99% coverage) with isolate CCLB of turnip mosaic virus (TuMV) from radish in China (KR153038). A second contig of 5,711 bp (7,185 mapped reads, mean read coverage 189.9×) had 97% identity (99% coverage) with isolate SDJN16 of beet western yellows virus (BWYV) from Capsicum annuum in China (MK307779). To confirm the presence of these viruses, total RNA extracted from 24 leaf samples was subjected to RT-PCR using primers specific for TuMV (N60_5'-ACATTGAAAAGCGTAACCA-3' and C30_5'-TCCCATAAGCGAGAATACTAACGA-3', amplicon 356 bp) and BWYV (95F_5'-CGAATCTTGAACACAGCAGAG-3' and 784R_5'-TGTGGG ATCTTGAAGGATAGG-3', amplicon 690 bp) for virus detection. Of the 24 samples, 22 were positive for TuMV and 7 were co-infected with BWYV. Single infection of BWYV was not detected. Infection with TuMV, the predominant virus in radish in Korea, was previously reported (Choi and Choi, 1992; Chung et al., 2015). To determine the complete genomic sequence of the BWYV isolate (BWYV-NJ22) from radish, RT-PCR was conducted using eight overlapping primer pairs designed according to the alignment of previously reported BWYV sequences (Table S2). Terminal sequences of the viral genome were analyzed by 5' and 3' rapid amplification of cDNA ends (RACE; Thermo Fisher Scientific Corp.). The assembled complete genome sequence of BWYV-NJ22 was 5,694 nt long and was deposited in GenBank (accession no. OQ625515). The Sanger sequences shared 96% nt identity with the HTS-derived sequence. BLASTn analysis showed that BWYV-NJ22 had high nucleotide identity (98%) at the complete genome level with a BWYV isolate (OL449448) from C. annuum in Korea. BWYV (genus Polerovirus, family Solemoviridae), is an aphid-borne virus with a host range that includes > 150 plant species and is one of the most important viruses causing yellowing and stunting of vegetable crops (Brunt et al., 1996; Duffus 1973). In Korea, BWYV was first reported to infect paprika, followed by pepper, motherwort, and figwort (Jeon et al., 2021; Kwon et al., 2016; 2018; Park et al., 2018). During fall and winter 2021, 675 radish plants with virus-like symptoms of mosaic, yellowing, and chlorosis were collected from 129 farms in major cultivation areas in Korea and analyzed by RT-PCR using the BWYV detection primers. The incidence of BWYV in radish plants was 4.7%, and all infections were mixed infections with TuMV. To our knowledge, this is the first report of BWYV infecting radish in Korea. The symptoms of single BWYV infection are unclear, as radish is a new host plant of BWYV in Korea. Further research on the pathogenicity and impact of this virus in radish is therefore needed.

Complete Genome Sequence of a Novel Monopartite Mastrevirus, Soybean Geminivirus B, Isolated from Soybean (Glycine max (L.) Merrill).

Soybean is one of the most important crops in Korea. To identify the viruses infecting soybean, we conducted RNA sequencing with samples displaying symptoms of viral disease. A contig displaying sequence similarity to the known Geminivirus was identified. A polymerase chain reaction (PCR) using two different pairs of back-to-back primers and rolling circle amplification (RCA) confirmed the complete genome of a novel virus named soybean geminivirus B (SGVB), consisting of a circular monopartite DNA genome measuring 2616 nucleotides (nt) in length. SGVB contains four open reading frames (ORFs) and three intergenic regions (IRs). IR1 includes a nonanucleotide origin of replication in the stem-loop structure. Phylogenetic and BLAST analyses demonstrated that SGVB could be a novel virus belonging to the genus Mastrevirus in the family Geminiviridae. We generated infectious clones for SGVB by adding a copy of the IR1 region of SGVB, comparing the V-ori in addition to the full-length genome of SGVB. Using the infectious clones, we observed chlorosis and leaf curling with a latent infection in the inoculated Nicotiana benthamiana plants, while none of the inoculated soybean plants showed any visible symptoms of disease. This study provides the complete genome sequence and infectious clones of a novel Mastrevirus referred to as SGVB from soybean in Korea.

Development of a transcranial direct current stimulation (tDCS) device based on polarity interchangeable electrodes.

BACKGROUND: Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that can help modulate cortical excitability by transmitting direct current (DC) between a pair of scalp electrodes. To date, most studies on tDCS have been relatively short-lived, and the DC stimulations only lasted a few minutes. Conventional tDCS devices usually have some problems such as needing a lot of patches and lead lines. OBJECTIVE: Since conventional tDCS devices are unsuitable for use in long-term stimulations, we developed a new tDCS which can easily be used by unskilled persons. METHODS: We developed a new tDCS device that does not have lead lines for tDCS electrodes and has a simple structure. RESULT: This device can achieve stimulation with polarity interchangeable DC without physically swapping the anode and cathode. The performance of the proposed device was verified through an experiment. CONCLUSION: The developed tDCS device can contribute to long-term research as it uses neuroelectric stimulation.

The Development of Simple Methods for the Maintenance and Quantification of Polymyxa graminis.

Polymyxa graminis, a root endoparasite of several cereal species, is considered to be non-pathogenic but serves as a vector of various plant viruses belonging to the genera Bymovirus, Furovirus, and Pecluvirus. Specifically, it reduces barley productivity by transmitting the Barley Yellow Mosaic Virus (BaYMV). To date, due to its obligate biotrophic property, no artificial culturing of P. graminis was reported and its quantification was also technically challenging. Here, we developed a novel and simple method to infect P. graminis within sterile barley roots in contamination free by preparing nearly pure zoospore inoculum. Such artificial maintenance of P. graminis was verified based on the presence of various developmental stages in infected barley roots under microscope. In addition, the population of resting spores in host tissue was determined by establishing standard curve between manually counted number of spores and Ct values of 18S rDNA amplification using quantitative real-time PCR. Furthermore, it was validated that standard curve generated was also applicable to estimate the abundance of P. graminis in soil environments. In conclusion, the present study would help to generate a system to investigate the etiological causes as well as management of plant diseases caused by P. graminis and BaYMV in tissue and soil.

A systems approach for identifying resistance factors to Rice stripe virus.

Rice stripe virus (RSV) causes disease that can severely affect the productivity of rice (Oryza sativa). Several RSV-resistant cultivars have been developed. However, host factors conferring RSV resistance in these cultivars are still elusive. Here, we present a systems approach for identifying potential rice resistance factors. We developed two near-isogenic lines (NIL), RSV-resistant NIL22 and RSV-susceptible NIL37, and performed gene expression profiling of the two lines in RSV-infected and RSV-uninfected conditions. We identified 237 differentially expressed genes (DEG) between NIL22 and NIL37. By integrating with known quantitative trait loci (QTL), we selected 11 DEG located within the RSV resistance QTL as RSV resistance factor candidates. Furthermore, we identified 417 DEG between RSV-infected and RSV-uninfected conditions. Using an interaction network-based method, we selected 20 DEG highly interacting with the two sets of DEG as RSV resistance factor candidates. Among the 31 candidates, we selected the final set of 21 potential RSV resistance factors whose differential expression was confirmed in the independent samples using real-time reverse-transcription polymerase chain reaction. Finally, we reconstructed a network model delineating potential association of the 21 selected factors with resistance-related processes. In summary, our approach, based on gene expression profiling, revealed potential host resistance factors and a network model describing their relationships with resistance-related processes, which can be further validated in detailed experiments.

Suppression of NS3 and MP is important for the stable inheritance of RNAi-mediated rice stripe virus (RSV) resistance obtained by targeting the fully complementary RSV-CP gene.

Rice stripe virus (RSV) is a viral disease that seriously impacts rice production in East Asia, most notably in Korea, China, and Japan. Highly RSV-resistant transgenic japonica rice plants were generated using a dsRNAi construct designed to silence the entire sequence region of the RSV-CP gene. Transgenic rice plants were inoculated with a population of viruliferous insects, small brown planthoppers (SBPH), and their resistance was evaluated using ELISA and an infection rate assay. A correlation between the expression of the RSV-CP homologous small RNAs and the RSV resistance of the transgenic rice lines was discovered. These plants were also analyzed by comparing the expression pattern of invading viral genes, small RNA production and the stable transmission of the RSV resistance trait to the T3 generation. Furthermore, the agronomic trait was stably transmitted to the T4 generation of transgenic plants.

G7H, a New Soybean mosaic virus Strain: Its Virulence and Nucleotide Sequence of CI Gene.

A new Soybean mosaic virus (SMV) strain was isolated in Korea and designated as G7H. Its virulence on eight differentials and 42 Korean soybean cultivars was compared with existing SMV strains. G7H caused the same symptoms as G7 did on the eight differential cultivars. However, it caused different symptoms on the G7-immune Korean soybean cultivars; G7H caused necrosis in Suwon 97 (Hwangkeumkong) and Suwon 181 (Daewonkong), and a mosaic symptom in Miryang 41 (Duyoukong), while G7 caused only local lesions on those varieties. The nucleotide sequence of the cylindrical inclusion region of G7H was determined and compared with other SMV strains. G7H shared 96.3 and 91.3% nucleotide similarities with G2 and G7, respectively; whereas G7 shared 95.6% nucleotide similarity with G5H.

Molecular characterization of the gene encoding rice allene oxide synthase and its expression.

The gene encoding rice allene oxide synthase, OsAOS, was intronless and had nucleotide sequences with the high GC content of 67%. Deduced amino acid sequences had very high similarity with other AOS proteins, in particular 74% similarity to barley, characterized by the conserved motifs of P450 cytochrome of the CYP74A family. Purified recombinant rice AOS protein expressed in Escherichia coli converted 13-hydroperoxylinolenic acid to allene oxide. Several restriction enzyme digestions and Southern analysis showed that OsAOS was likely to have two copies in its genome. The basal level of OsAOS expression was detected in various tissues and the transcription level was increased by jasmonate treatment.