Yeong-Hoon Lee

Characterization of SMV resistance of soybean produced by genetic transformation of SMV-CP gene in RNAi

Soybean mosaic virus (SMV), a species of the Potyvirus genus in the Potyviridae family, is one of the most typical viral diseases and results in yield and quality loss of cultivated soybean. Due to the depletion of genetic resources for resistance breeding, a trial of genetic transformation to improve disease resistance has been performed by introducing the SMV-CP gene by the RNA interference (RNAi) method via Agrobacterium-mediated transformation. Among 30 transgenic plants produced, 7 lines with enough seeds were infected with SMV and two lines (3 and 4) showed viral resistance to SMV infection. In genomic Southern blot analysis, all the lines tested contained at least one T-DNA insertion. Subsequent investigation confirmed that no viral CP gene expression was detected in two SMV-resistant lines after artificial inoculation of SMV, while non-transgenic control and other transgenic lines expressed substantial amounts of the viral gene. Viral symptoms affected seed morphology, and clean seeds were harvested from the resistant lines. Also, strong viral gene expression was detected from the seeds of susceptible lines. In further generations, the same phenotypic appearance was maintained among non-transgenic and transgenic plants. Finally, the presence of helper component-proteinase (HC-Pro), known as a suppressor of gene silencing apparatus, was checked among transgenic lines. No expression of HC-Pro in resistant lines indicated that the viral CP-RNAi transformation into soybean somehow created a functional gene silencing system and resulted in a viral-resistant phenotype.

Incidence and Distribution of Virus Diseases on Paprika (Capsicum annuum var. grossum) in Jeonnam Province of Korea

The incidence and occurrence of virus infecting paprika (Capsicum annuum var. grossum) in Jeonnam province, the main areas of cultivation in Korea is undetermined. In this study, a total of 1,020 samples with virus-like symptoms were collected in Jeonnam province during summer season for 3 consecutive years (2002-2005) and were tested using enzyme linked immunosorbent assay (ELISA). Results showed that Pepper mottle virus (PepMoV), Broad bean wilt virus (BBWV), and Cucumber mosaic virus (CMV) were found to be the most prevalent viruses with a 3-year average percent incidence of 41.3, 19.8, and 4.4 respectively. Mixed infection with more than two viruses was also found with 3.5%, 17.0%, and 8.3%, respectively. Symptoms of these virus diseases were not evident at the seedling stage but slowly appeared at the transplanting stage and increased to the middle stage (4-5 months after transplanting) during the 3-year cultivation periods. Symptom appearance of infected plants however varied largely with transplanting time. Those plants transplanted from November to January were found to be infected with viruses in June, whereas symptoms appeared with in a month those plants transplanted from June to August. There were differences in the virus incidence from primary factor such as district, type of green house and variety, but these were not statistically significant (data not shown). Recommended control measures of paprika against these viruses is also discussed in this paper.

First Report of Cobweb Disease Caused by Cladobotryum mycophilum on the Edible Mushroom Pleurotus eryngii in Korea

Pleurotus eryngii is one of the most commercially important mushrooms in Korea. In May 2009, unusual symptoms were observed in P. eryngii grown in mushroom farms in Changnyeong and Hapcheon, in Gyeong-nam Province, Korea. One of the main symptoms was cobweb-like growth of fungal mycelia over the mushroom surface. Colonies on the surface rapidly overwhelmed the mushrooms, which turned pale brown or yellow. Mushrooms eventually turned dark brown and became rotten. Colonies of the isolates on potato dextrose agar (PDA) were yellowish, and a reddish or orange color was evident in the agar. The colonies grew 20 to 30 mm per day on PDA. Large spores with a single septum were produced on vertically branched conidiophores bearing two to four, mostly three to four, sporogenous cells, ranging from 17.2 to 20.5 μm long and 8.0 to 10.2 μm thick. The shape of the conidia was ellipsoid and obovoid. These morphological characteristics are consistent with descriptions of Cladobotryum mycophilum, a causal agent of cobweb disease in Agaricus bisporus (1,4). To identify the isolated fungal pathogen, the ITS region was amplified with ITS1 and ITS4 primers and sequenced. The sequence data from the isolate was deposited in GenBank (Accession No. JF693809). A BLAST search showed that the isolated strain belonged to a species of Cladobotryum. The highest similarity (99.5%) was to the ITS sequence of C. mycophilum (teleomorph Hypomyces odoratus) (GenBank Accession Nos. JF505112 and Y17096) (3,4). The strain that was tested for pathogenicity was grown on PDA at 25°C for 72 h. The inoculum was prepared by flooding the agar surface with 10 ml of sterilized double distilled water and scraping it with a spatula. The resulting spore suspension was filtered through three layers of cheesecloth. Conidial concentration was adjusted with a hemacytometer to 1 × 106 conidia ml-1. A conidia suspension was inoculated onto each of several stages of mushroom cultivation with a pipette. The control was spotted with double distilled water. In the case of infection during the inoculation and spawn running stages, the fungal mycelia colonized the media and hampered development of the mycelium of P. eryngii. In the regeneration and primordia formation stages of the host, the mycelium of the pathogen covered the surface of the plastic bottle containing the substrates and developed many spores. In the growing and harvesting stages, the surface of mushroom was overwhelmed by the mycelium of the fungal pathogen and turned pale or dark brown, accompanied by cracking of the stipe surface and finally rotting with a foul odor. These symptoms were similar to the observation from natural infection. The symptoms of the cobweb-like disease in A. bisporus (1,2) were observed within 5 to 7 days of inoculation with conidia suspensions of C. mycophilum. Fungi isolated from inoculated mushrooms were shown to be identical, based on phenotypic characteristic, to the inoculated strain used in these pathogenicity tests. No symptoms were observed on controls. To our knowledge, this is the first report on the occurrence of C. mycophilum on the edible mushroom P. eryngii in Korea. Based on the pathogenicity test results, the pathogen could attack P. eryngii in any cultivation stage, making it a potentially serious fungal pathogen in P. eryngii. References: (1) C. G. Back et al. J. Gen. Plant Pathol. 76:232, 2010. (2) R. H. Gaze. Mushroom J. 546:23, 1995. (3) F. J. Gea et al. Plant Dis. 95:1030, 2011. (4) H. M. Grogan and R. H. Gaze. Mycol. Res. 104:357, 2000.

Development of Multiplex RT-PCR for Simultaneous Detection of Garlic Viruses and the Incidence of Garlic Viral Disease in Garlic Genetic Resources

Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic.

First Report of the Virus Diseases in Victory Onion (Allium victorialis var. platyphyllum)

In 2005, a survey was conducted to identify virus diseases on victory onion, Allium victorialis var. platyphyllum grown in Ulleung island located in the East Sea. A total of 61 samples were collected from victory onion in the neighborhood of Seonginbong. The identification of viruses from the samples were carried out by electron microscopy and RT-PCR using primers species specific to GCLV, LYSV, SLV, OYDV and genus specific to Allexivirus, respectively. From sixty-one samples, filamentous rod particles (600-900 nm) were detected from four victory onion samples in EM, three samples containing SLV and one sample containing both SLV and Allexivirus in RT-PCR analysis, respectively. Victory onions naturally infected by the viruses were asymptomatic apparently. The viruses detected by RT-PCR were further characterized by the nucleotide sequence analysis of the coat protein region. Three isolates of SLV showed approximately 99% identities in the nucleotide and amino acid sequences, suggesting that they were likely to be the same strain. On the other hand, they showed approximately 75.7~83.7% identities in the nucleotide and 89.2~97.0% in amino acid sequences compared with the previously reported SLV isolates in Allium. The CP gene of the Allexivirus showed approximately 99.2% nucleotide identities and 98.8% amino acid identities with Garlic virus A. However, there was relatively low homology ranging from 60.6% to 81.5% compared with other Allexiviruses (GarV-C, GarV-E, GarV-X, GMbMV, and Shal-X). These data suggested that two viruses, SLV and GarV-A identified from victory onion, are named SLV-Ulleungdo and GarV-A-Ulleungdo, respectively. This is the first report of viruses infecting victory onion.

First Report of Soybean Dwarf Virus on Soybean(Glycine max) in Korea

In year 2003, a soybean(Glycine max) sample showing severe dwarfing symptom was collected from a farmers` field in Cheongsong in Korea. The results from the diagnosis of the sample by RT-PCR revealed that it was infected by Soybean dwarf virus(SbDV), SbDV-L81. This study could be the first report of the occurrence of the virus in Korea. To further characterize the virus, the partial nucleotide sequence of the genomic RNA of SbDV-L81 was determined by RT-PCR using species-specific primers. The sequences were analyzed and subsequently compared to previously characterized strains of SbDV based on the pattern of symptom expression and vector specificities. The intergenic region between ORF 2 and 3 and the coding regions of ORF 2, 3 and 4 were relatively similar to those of dwarfing strains(SbDV-DS and DP) rather than those of yellowing strains(SbDV-YS and YP). Likewise, the result from the analysis of 5`-half of the coding region of ORF5 indicated that SbDV-L81 was closely related to strains(SbDV-YP and DP) transmitted by Acyrthosiphon pisum. These data from the natural symptom and the comparisons of five regions of nucleotide sequences of SbDV suggested that SbDV-L81 might be closely related SbDV-DP.

Development of Molecular Markers for Low Raffinose and Stachyose in Korean Soybean Cultivars

A novel allele of the putative soybean raffinose synthase gene, RS2, was discovered in PI200508 that is associated with the low raffinose and stachyose content. Soybean line PI200508 was identified as expressing reduced levels of raffinose and stachyose as well as elevated levels of sucrose. The RS2 mutant gene shows three base pairs InDel with the normal gene. Based on InDel region we developed novel co-dominant and dominant marker. The aim of this study was to develop Korean soybean cultivars, Daewon, Cheongja, and Danmiput, containing low levels of raffinose and stachyose. A specific markers assay for the PI200508 RS2 allele was developed to allow direct selection of the low raffinose and stachyose phenotype. Our findings highlight the efficiency of allele-specific markers in selection, which is evident in the matching genotype and results of the HPLC in the F2 generations of Daewon×PI200508 population.

Complete genome sequence of peanut virus C, a putative novel ilarvirus

We determined the complete genome sequence of a putative novel ilarvirus, tentatively named “peanut virus C” (PVC), identified in peanut (Arachis hypogaea). The three segmented genomic RNA molecules of PVC were 3474 (RNA1), 2925 (RNA2), and 2160 (RNA3) nucleotides in length, with five predicted open reading frames containing conserved domains and motifs that are typical features of ilarviruses. The three genomic RNAs shared nucleotide sequence similarity (74% identity and 93% query coverage for RNA1, 75% identity and 85% query coverage for RNA2, and 72% identity and 70% query coverage for RNA3) with the most closely related ilarvirus, parietaria mottle virus. These results suggest that PVC is a novel member of the genus Ilarvirus in the family Bromoviridae.

First Report of Tomato spotted wilt virus Infecting Soybean in Korea

The soybean (Glycine max (L.) Merr.) is a legume with worldwide popularity which is native to East Asia. Soybeans are grown for their edible beans and used to create soy vegetable oil and fermented soy foods. To date, a large number of viruses that can infect soybeans have been identified (Hill et al. 2014). Tomato spotted wilt virus (TSWV) is a member of the genus Tospovirus in the family Bunyaviridae. TSWV causes serious diseases in many economically important plants including dicots and monocots. TSWV has been identified in soybeans in Iran (Golnaraghi et al. 2001) and Georgia (Nischwitz et al. 2006). In 2016, we collected soybean leaf samples displaying viral disease symptoms such as chlorotic, mosaic, mottling, stunting, and yellowing. 168 samples were collected and categorized by the region that they were collected in. Eight different libraries representing individual provinces were prepared for RNA sequencing using the TruSeq RNA Library Prep Kit v2 in accordance with the manufacturers instruction...

First report of Maize yellow mosaic virus infecting Panicum miliaceum and Sorghum bicolor in South Korea

Maize yellow mosaic virus (MaYMV) is a tentative new Polerovirus, which was recently identified from maize (Zea mays) in China (Chen et al. 2016). MaYMV has also been reported to infect sugarcane (Saccharum spp.) and itch grass (Rottboellia cochinchinensis), and it has been reported in Asia, Africa and South America (Goncalves et al. 2017; Palanga et al. 2017; Yahaya et al. 2017). In this study, MaYMV was detected in Panicum miliaceum and Sorghum bicolor using Illumina HiSeq2500 system by Theragen Etex Bio Institute (Suwon, Korea) and SG-VIPdb by SeqGenesis (Daejeon, Korea), as described by Lim et al. (2015). Twenty samples of P. miliaceum and sixty three samples of S. bicolor were collected from July to September and from June to October, 2016, respectively, in South Korea. The high-throughput RNA sequencing of the samples mixed into one pool resulted in a single large contig (5606-nt) with nearly complete MaYMV genome coverage. The contig was assembled from a total of 234,537 reads; the maximum, minimum...