An Inexpensive System for Rapid and Accurate On-site Detection of Garlic-Infected Viruses by Agarose Gel Electrophoresis Followed by Array Assay.

Garlic can be infected by a variety of viruses, but mixed infections with leek yellow stripe virus, onion yellow dwarf virus, and allexiviruses are the most damaging, so an easy, inexpensive on-site method to simultaneously detect at least these three viruses with a certain degree of accuracy is needed to produce virus-free plants. The most common laboratory method for diagnosis is multiplex reverse transcription polymerase chain reaction (RT-PCR). However, allexiviruses are highly diverse even within the same species, making it difficult to design universal PCR primers for all garlic-growing regions in the world. To solve this problem, we developed an inexpensive on-site detection system for the three garlic viruses that uses a commercial mobile PCR device and a compact electrophoresis system with a blue light. In this system, virus-specific bands generated by electrophoresis can be identified by eye in real time because the PCR products are labeled with a fluorescent dye, FITC. Because the electrophoresis step might eventually be replaced with a lateral flow assay (LFA), we also demonstrated that a uniplex LFA can be used for virus detection; however, multiplexing and a significant cost reduction are needed before it can be used for on-site detection.

Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization.

One of the major problems in strawberry production is difficulty in diagnosis of anthracnose caused by Colletotrichum acutatum or Glomerella cingulata in latent infection stage. We here developed a diagnostic tool for the latent infection consisting of initial culturing of fungi, DNA extraction, synthesis of PCR-amplified probes and microtube hybridization (MTH) using a macroarray. The initial culturing step is convenient to lure the fungi out of the plant tissues, and to extract PCR-inhibitor-free DNA directly from fungal hyphae. For specific detection of the fungi, PCR primers were designed to amplify the fungal MAT1-2 gene. The subsequent MTH step using the PCR products as probes can replace the laborious electrophoresis step providing us sequence information and high-throughput screening. Using this method, we have conducted a survey for a few thousands nursery plants every year for three consecutive years, and finally succeeded in eliminating latent infection in the third year of challenge.

Detection of plant viruses in mixed infection by a macroarray-assisted method.

The protocol for a simple, sensitive, and specific method using a cDNA macroarray to detect multiple viruses is provided. The method can be used even at the production sites for crops, which need a reliable routine diagnosis for mixed infection of plant viruses. The method consists of three steps: RNA extraction, duplex RT-PCR, and "microtube hybridization" (MTH). Biotinylated cDNA probes are prepared using RT-PCR and used to hybridize a nylon membrane containing target viral cDNAs by MTH. Positive signals can be visualized by colorimetric reaction and judged by eyes. We here demonstrate this method to detect asparagus viruses (Asparagus virus 1 and Asparagus virus 2) from latently infected asparagus plants.

Production of biologically active Atlantic salmon interferon in transgenic potato and rice plants.

Interferons (IFNs) are cytokines that induce an antiviral state in vertebrate cells. The Atlantic salmon (Salmo salar) IFN gene (SasaIFN-alpha1) was introduced in potato and rice plants by Agrobacterium-mediated transformation to produce a biologically active fish IFN in these plants. The transgenes and their transcripts were detected by PCR and Northern blot analysis. Western blot analysis showed the existence of SasaIFN-alpha1in transgenic plants. The antiviral activity of the SasaIFN-alpha1 protein expressed in these plants was determined by the survival rates of pre-treated cultured fish cells against pancreatic necrosis virus infection. The survival rate of cells pre-treated with transgenic samples was up to 95% but was reduced to 30-47% when cells were pre-treated with non-transgenic samples. These results demonstrated an antiviral effect of the SasaIFN-alpha1 protein derived from transgenic plants. Plant-derived IFNs may be suitable as components of functional feeds because such IFNs are free of animal pathogens and can be produced at a lower cost compared with those from transgenic mammalian and bacterial cells. This is the first study describing the production of a biologically active fish IFN using transgenic plants.