Su-Heon Lee

One-step multiplex reverse transcription-polymerase chain reaction for the simultaneous detection of three rice viruses.

Rice stripe virus (RSV), Rice black-streaked dwarf virus (RBSDV), and Rice dwarf virus (RDV) are major rice-infecting viruses in Korea that can cause serious crop losses. A one-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for the simultaneous detection of these rice viruses. Three sets of specific primers targeted to the capsid protein coding genes of RSV, RBSDV, and RDV were used to amplify fragments that were 703 bp, 485 bp, and 252 bp, respectively. The one-step mRT-PCR assay proved to be a sensitive and rapid method for detecting the three rice viruses. This method could be used to facilitate better control of rice viruses.

Complete Genome Sequences of Grapevine Yellow Speckle Viroid 1 and Hop Stunt Viroid Assembled from the Transcriptome of Ixeridium dentatum Plants.

ABSTRACT Here, we report complete genome sequences of grapevine yellow speckle viroid 1 (GYSVd1) and hop stunt viroid (HSVd), members of the family Pospiviroidae, assembled from the transcriptome data generated from Ixeridium dentatum plants. To our knowledge, this is the first report of GYSVd1 and HSVd in I. dentatum.

A novel set of polyvalent primers that detect members of the genera Bromovirus and Cucumovirus.

Rapid detection and diagnosis of plant virus infection is one of the most important steps in preventing damages caused by viral diseases. Bromoviruses and cucumoviruses belong to the family Bromoviridae, which is one of the most important families of plant viruses, and infect a broad range of host plants including various economically important crops. In this study, an RT-PCR assay was developed for the universal detection of bromoviruses and cucumoviruses using a set of primers designed to target the conserved sequences in viral RNA1. The assay detected three species of Cucumovirus (Cucumber mosaic virus (CMV), Peanut stunt virus (PSV) and Tomato aspermy virus (TAV)) and two species of Bromovirus (Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV)) with high specificity and sensitivity. The assay developed in this study is predicted to have the potential to detect all major members of the genera Bromovirus and Cucumovirus and to be used as a routine diagnostic assay.

Complete genome sequence and construction of infectious full-length cDNA clones of tobacco ringspot Nepovirus, a viral pathogen causing bud blight in soybean

Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host–TRSV interactions.

Complete Genome Sequence of Chinese Yam Necrotic Mosaic Virus from Dioscorea opposita in the Republic of Korea

ABSTRACT The complete genome sequence of Chinese yam necrotic mosaic virus (ChYNMV) consisting of 8,213 nucleotides containing one open reading frame was determined by the transcriptome data generated from Discorea opposita. This is the first report of the complete nucleotide sequence of ChYNMV from Dioscorea opposita in the Republic of Korea.

First Report of Frosty Mildew Caused by Mycopappus alni on Asian Pear in Korea

Asian pear (Pyrus pyrifolia Nakai), also known as Japanese or Korean pear, is widely cultivated in East Asia. On September 2011, irregularly shaped necrotic lesions were observed on leaves of cv. Shinheung growing in an orchard in Gangneung City, Korea. At 40× magnification under a microscope, the white to cream colored propagules were epiphyllous, conical, scattered to aggregated, and composed of stroma-like bases, globose to subglobose, 55 to 100 μm wide and 35 to 75 μm high with filamentous and claviform hyphae. The filamentous hyphae were cylindrical, 125 to 425 × 3.5 to 6 μm, 2- to 8-septate, and obtuse to subobtuse at the apex. The claviform hyphae were clavate to cylindrical, 35 to 125 × 5 to 12.5 μm, aseptate to 3-septate, and obtuse at the apex. The fungus was isolated from leaf lesions and cultured on potato dextrose agar (PDA). The colonies consisted of thin mycelia colored whitish at first and then pale brown on PDA. Sclerotia were produced on PDA after 2 weeks incubation at 15°C, but conidia were not observed in culture. An isolate from KUS-F26196 was deposited in the Korean Agricultural Culture Collection (Accession No. 25 KACC46693). These morphological and cultural characteristics were consistent with Mycopappus alni (Dearn. & Barthol.) Redhead & G.P. White (1,3,4). Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting sequence product of 520 bp was deposited in GenBank (Accession No. JX458815). A BLAST search in GenBank revealed that the sequence was 99% similar to M. alni (AB254190, AB254177, AB254189). To determine the pathogenicity of the fungus, propagules were detached from lesions on the naturally infected leaves using fine needles. Each propagule was transferred individually onto five places of six detached healthy leaves. Control treatment comprised placing small agar blocks onto five places of six detached healthy leaves. The plants were incubated in a humid chamber at RH 100% and 18°C. Symptoms were observed after 2 days on all inoculated leaves. The pathogen was reisolated from lesions on the inoculated leaves, confirming Kochs postulates. No symptoms were observed on control leaves. The fungus has been associated with frosty mildew on Alnus spp., Betula spp., Crataegus spp., and Pyrus spp. in North America, Turkey, Russia, and Japan (1,2,4). To our knowledge, this is the first report of frosty mildew on P. pyrifolia caused by M. alni globally as well as in Korea. Since the infections may be limited to the mountainous area with low night temperature and high humidity, economic losses seem to be negligible. However, the disease could be a potential threat to the safe production of Korean pears in case of prolonged period of cool and moist weather. References: (1) U. Braun et al. Mikologiya i Fitopatologiya 34(6):1, 2000. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology & Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , August 2, 2012. (3) S. A. Redhead and G. P. White. Can. J. Bot. 63:1429, 1985. (4) Y. Takahashi et al. Mycoscience 47:388, 2006.

First Report of Bacterial Black Spot Disease in Watermelon Caused by Acidovorax valerianellae in Korea

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), an important member of the Cucurbitaceae family, is cultivated on 21,000 ha that produces 850,000 t in Korea. In April 2011, we received grafted watermelon with necrotic leaf spots from a commercial watermelon grower in Andong, Korea. Black spots were observed on cotyledons of the plants in seedbeds, and approximately 9% of watermelon plants were infected with the disease. Initial symptoms on the seedling were black, greasy spots sometimes surrounded by a halo of discoloration. Younger leaves usually showed symptoms later than cotyledons. Bacteria isolated from the infected plants were gram-negative, motile, straight rods with a single flagellum and 0.84 to 0.89 μm wide and 1.54 to 1.69 μm long. They formed rough colonies with a white-cream color after 48 h of incubation on Luria-Bertani (LB) agar at 28°C. Colonies of isolates were nonfluorescent, smooth, and white on Kings medium B. On YBGA (7 g of yeast extract, 7 g of bactopeptone, 7 g of glucose, 15 g of agar, 1,000 ml of distilled water; pH 7.2) colonies are circular, raised with an entire margin, and white to cream. Pathogenicity tests were conducted with potted, greenhouse-grown watermelon plants. Bacterial colonies grown on LB medium for 48 h at 28°C were suspended in sterile distilled water, and the suspension (1.0 × 108 CFU/ml) was infiltrated into mesophyll of watermelon leaves with a syringe as previously described (2). Inoculated plants were maintained at 28°C and 90% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. Five plants were used for inoculation. Sterilized distilled water was used as a control. The bacterial isolates induced necrosis in the infiltrated area within 3 to 5 days. Typical water-soaked spots appeared after 3 days of incubation and became gray to black after 6 days. The bacterium was successfully reisolated from the diseased lesions, thus completing Kochs postulates. A cell suspension (50 μl of 1 × 106 CFU/ml) was infiltrated with a syringe into the intercellular spaces of tobacco leaves to determine the hypersensitive reaction (HR). A typical HR developed 20 h after leaf infiltration. The 16S rDNA region of the isolates, amplified by using universal PCR primers, shared 99% sequence identity with an Acidovorax valerianellae strain (GenBank Accession No. AJ431731) (1). The resulting sequences of 1,424 bp were deposited in GenBank (Accession No. JN983471). The isolates we obtained in this study clustered with A. valerianellae on a phylogenetic tree generated by the neighbor-joining method implemented in MEGA Version 4.1. In the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), all isolates were 63 to 77% similar with a match probability of 100% to A. konjaci. Fatty acid composition analysis of isolates based on the MIDI Library version TSBA 5.0 and Library Generation system software version 5.0 showed that the isolates were 52 and 72% similar to an Acidovorax sp., respectively. To our knowledge, this is the first report of bacterial black spot disease in watermelon caused by A. valerianellae in Korea. A. valerianellae is a causal agent of bacterial black spot in corn salad and is transmitted by inoculated seeds (3). Further studies are required to determine whether it is seed transmitted in watermelon. References: (1) L. Gardan et al. Int. J. Syst. Evol. Microbiol. 53:795, 2003. (2) C. Grondeau et al. Plant Pathol. 56:302, 2007. (3) C. Grondeau et al. Plant Pathol. 58:846, 2009.

Natural Hosts and Disease Cycle of Soybean yellow mottle mosaic virus

In surveys of weed occurrence undertaken from 2006 to 2007, near to the Daegu experimental fields of theNational Institute of Crop Science, plants belonging to 31 families, 74 genera and 96 species were found. Forthe investigation of the natural or alternative hosts of Soybean yellow mottle mosaic virus (SYMMV), 495 plantsamples belonging to 26 families 84 species were subjected to RT-PCR. SYMMV was detected only fromlegume plants such as Glycine soja, Vigna angularis var. nipponensis, Trifolium repens, and Lespedeza cuneata.Among legume plants tested, more than a third of G. soja (wild soybean) contained SYMMV, indicating thatthe wild soybean played an important role as a reservoir of SYMMV. Wild soybeans may be infected withSYMMV as early as mid-July. Considering the results of early infection and the high infection rate of seedand seed transmission of SYMMV in G. soja , wild soybeans may have played an important role in thecompletion of disease cycle of the virus.Keywords : Disease cycle, Natural hosts, Soybean, Soybean yellow mottle mosaic virus , Wild soybean

Incidence of Viral Diseases and Occurrence of Three Unreported Viruses in Yams in Korea

United Nations (FAO), yams (Dioscorea species, Dioscoreaceae) are the most important food tuber crop, a class of foods including potatoes, sweet potatoes, and cassava (FAO, 2012). Approximately 600 species of yams are distributed throughout the world, and only 10 of these species are used as edible crops (Mambole et al., 2014). Yams are an important traditional crop and staple food and provide income to small farmers in West Africa (Asiedu and Sartie, 2010). Moreover, yams are commonly used as medicines and vegetables (Kwon et al., 2016a), and more than 4,000 tons are produced in Korea each year. Yams are propagated through seeds or tubers, resulting in accumulation of various pathogens, such as viruses, fungi, and bacteria. Of the many pathogens that affect these plants, viral infections in particular, can cause significant reductions in the yield and quality of yam crops (Mantell and Haque, 1978). Approximately eight viruses (Chinese yam necrotic mosaic virus [ChYNMV], Dioscorea alata badnavirus [DaBV], D. alata virus [DAV], Dioscorea dumetorum virus [DDV], Dioscorea esculenta virus [DEV], Yam mosaic virus [YMV], Yam mild mosaic virus [YMMV], and Japanese yam mosaic virus [JYMV]) have been reported in Asia (Kenyon et al., 2001; Wang et al., 2015), whereas only two viruses (Broad bean wilt virus 2 [BBWV2] and ChYNMV)

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...