PVX-tolerant potato development using a nucleic acid-hydrolyzing recombinant antibody.

3D8 scFv, a catalytic recombinant antibody developed in the MRL mouse, exhibits nucleic acid-hydrolyzing activity. Previous studies have demonstrated that tobacco plants harboring 3D8 scFv antibodies showed broad-spectrum resistance to infection by both DNA and RNA viruses. In this study, potatoes were transformed with the 3D8 scFv gene and screened by potato virus X (PVX) challenge. Starting with the T0 and T1 potato lines, PVX-tolerant T1 potatoes were identified in the field and characterized by ELISA and RT-PCR analysis. T2 potatoes were propagated for T3 generation and additional virus challenges in the field, and 44% of the 3D8 scFv T3 transgenic potatoes grown in GMO fields were found to be tolerant to PVX infection. Tubers from PVX-tolerant T3 lines were 60% bigger and 24% heavier, compared with tubers from PVX-susceptible transgenic lines and wild-type potatoes. Three-step virus challenge experiments and molecular characterization techniques were used for plants grown in growth chambers or fields to identify 3D8 scFv-transgenic, PVX-tolerant potatoes. These studies also revealed that the viral tolerance enabled by 3D8 scFv persisted during asexual propagation.

Differential identification of three species of Curtovirus using loop-mediated isothermal amplification.

Rapid and sensitive detection methods for three species of Curtovirus were developed using a loop-mediated isothermal amplification (LAMP) technique. A universal primer set for detecting the three main species of Curtovirus at the same time, and three kinds of species-specific primer sets were designed and used for LAMP reactions. Results from the LAMP reactions were visualized both by color changes after adding SYBR Green I staining dye and by DNA laddering on agarose gel electrophoresis. The optimal conditions for the curtovirus LAMP reaction were confirmed at 60°C for the universal primers and at 62°C for the three species-specific primer sets. Amplification of curtoviruses by LAMP reaction was ten-fold more sensitive than that by polymerase chain reaction. Primers designed for curtovirus detection in this study did not anneal to or amplify DNA from other DNA or RNA viruses (tomato yellow leaf curl virus, tomato spotted wilt virus, and potato virus Y). Taken together, the primer sets and reaction conditions developed in this study show that the LAMP technique could be a useful tool to detect the three species of Curtovirus simultaneously and distinguish them in the laboratory and the field.

First Report of Chickpea chlorotic dwarf virus Infecting Hot Pepper in India.

Hot pepper (Capsicum annuum) cultivated in India has been identified as a host of geminiviruses causing leaf curl disease such as Chilli leaf curl virus and Pepper leaf curl virus, leading to serious crop losses (3). In June 2013, hot pepper plants growing in Bangalore showed stunting and upward leaf curling. Viral DNA was extracted from a hot pepper with a Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and amplified by rolling circle amplification using the illustra TempliPhi 100 Amplification Kit (GE Healthcare, Uppsala, Sweden) (2). Amplified products were digested by restriction enzyme KpnI (Takara Bio, Shiga, Japan), cloned, and sequenced (Macrogen, Seoul, Korea). Based on a BLAST search, a 2.6-kb DNA obtained from one plant sample was identified as Chickpea chlorotic dwarf virus (CpCDV), belonging to the genus Mastrevirus (family Geminiviridae) (GenBank Accession No. KF632712). The CpCDV-Bangalore isolate is 2,585 bases in length and exhibits 85.9 to 98.5% identity to previously reported CpCDV isolates. To our knowledge, this is the first report of CpCDV infecting hot pepper in India. CpCDV was recently reported from pepper plants in Oman (KF111683) (1), but it shared the lowest sequence identity (85.9%) with CpCDV-Bangalore isolate. References: (1) S. Akhtar et al. Plant Dis. 98:286, 2014. (2) E.-J. Kil et al. Arch. Virol. 159:2387. (3) D. M. J. B. Senanayake et al. Plant Pathol. 56:343, 2007.

First Report of Tomato yellow leaf curl virus Infecting Eustoma (Eustoma grandiflorum) in Korea.

Eustoma (Eustoma grandiflorum), also called lisianthus, belongs to the family Gentianaceae and is cultivated for flower production globally (1), including in Korea. At least 10 viruses can infect eustoma, including Cucumber mosaic virus (genus Cucumovirus), Tobacco mosaic virus (genus Tobamovirus), Tomato spotted wilt virus (genus Tospovirus), and Tomato yellow leaf curl virus (TYLCV, genus Begomovirus) (1,2). In December 2012, disease symptoms such as leaf curling and stunting were observed on eustoma plants grown in Gumi, Korea, where TYLCV outbreak was reported on tomato farms. In a eustoma greenhouse, about 5% of eustoma plants showed the leaf curling and stunting symptoms. Total DNA was isolated from 15 symptomatic eustoma plants with a Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and viral DNA was amplified by rolling circle amplification (TempliPhi Amplification Kit, GE Healthcare Life Sciences, Uppsala, Sweden) following the manufacturer's instructions. All amplicons were digested with the restriction enzyme SacI (TaKaRa Bio, Shiga, Japan) and 2.8-kb DNA fragments were verified on an agarose gel. Fifteen digested DNA fragments were purified from the gel, ligated into pGEM-T easy vector (Promega, Madison, WI), and sequenced (Macrogen, Seoul, Korea, GenBank Accession No. KF225312.1). A BLAST search exhibited a 99% identity to TYLCV previously reported in Korea (GenBank HM856911.1). This is the first report of TYLCV in eustoma plants in Korea. To identify the movement and replication of TYLCV in infected eustoma plants, PCR and Southern hybridization analysis were performed with samples from four organs (flower, leaf, stem, and root) of three individual TYLCV-infected plants. TYLCV TYL DNA from each organ sample was amplified using 2× Taq PCR MasterMix (Bioneer, Daejeon, Korea) with TYLCV-specific primers (TYLCV-F: 5'-ATATTACCGGATGGCCGCGCCT-3', CV-R: 5'-TCCACGGGGAACATCAGGGCTT-3'). Single-stranded as well as double-stranded TYLCV DNA were identified from all organs of symptomatic eustoma, indicating TYLCV can replicate and move systemically in eustoma plants. Whitefly (Bemisia tabaci)-mediated plant-to-plant viral transmission was performed with one TYLCV-infected eustoma plant and five healthy eustoma plants and revealed that 80% (4 of 5) of the eustoma plants were infected by whitefly-mediated transmission. These results indicate that TYLCV-infected eustoma plants could act as virus reservoirs to healthy eustoma plants as well as other potential TYLCV hosts, such as tomatoes. In Korea, TYLCV has been the most notorious plant virus since 2008 (3), but, until now, TYLCV infection in eustoma plants has not been reported in Korea. References: (1) C. C. Chen et al. Plant Dis. 84:506, 2000. (2) A. Kritzman et al. Plant Dis. 84:1185, 2000. (3) H. Lee et al. Mol. Cells 30:467, 2010.

First Report of Sweet potato golden vein associated virus Infecting Sweet Potato in Korea.

Sweet potato (Ipomoea batatas) is one of the most important crops in eastern Asia, including Korea. Consumption of sweet potato is increasing gradually because of its growing reputation as a health food. Recently, outbreaks of viruses infecting sweet potatoes have increased all over the world, probably because sweet potatoes are produced via vegetative propagation (1,2). In Korea, most sweet potatoes in fields have been infected by a begomovirus, Sweet potato leaf curl virus (SPLCV), and other viruses such as Sweet potato feathery mottle virus, Sweet potato virus G, and Sweet potato latent virus (3). Many countries have monitored sweet potato virus infections in fields as well as in germplasm collections to select virus-free stocks. In 2013, 20 sweet potato plants showing leaf roll symptoms in Muan, South Korea, were collected and analyzed. Total DNA was isolated from sweet potato leaves (Viral Gene-spin Viral DNA/RNA Extraction Kit, iNtRON Biotechnology, Seongnam, Korea) and viral DNA was amplified by rolling circle amplification (RCA, TempliPhi Amplification Kit, GE Healthcare Life Sciences, Uppsala, Sweden) following the manufacturer's instructions. Amplicons were digested by restriction enzyme SacI (TaKaRa Bio, Shiga, Japan) and products were run on a 1.5% agarose gel. A 2.8-kb DNA fragment was purified from a gel, ligated into a pGEM-T easy vector (Promega, Madison, WI), and sequenced (Macrogen, Seoul, Korea). Based on a BLAST search, most of the sequences (36/38) were identified as SPLCV, but two independent clones 2,824 nt in length from sweet potato cv. Sincheonmi were similar to Sweet potato golden vein associated virus (SPGVaV) isolate US:MS:1B-3 (94.38%, GenBank Accession No. HQ333143). The complete genome sequence of the SPGVaV-Korea isolate contained six ORFs, as expected for a typical monopartite begomovirus. The sequence was deposited in GenBank under accession number KF803170. SPGVaV is a whitefly (Bemisia tabaci)-transmitted virus (genus Begomovirus, family Geminiviridae). A phylogenetic analysis that included other begomoviruses that infect sweet potato showed SPGVaV-Korea to segregate with other SPGVaV isolates. SPGVaV has previously only been reported in Brazil and the United States (1). This is the first report of SPGVaV in sweet potato outside of the Americas. References: (1) L. C. Albuquerque et al. Virol. J. 9:241, 2012. (2) E. Choi et al. Acta Virol. 56:187, 2012. (3) H. R. Kwak et al. Plant Pathol. J. 22:239, 2006.