• Korean Journal of Ecology and Environment
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• v.56 no.1
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• pp.36-44
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• 2023

Environmental DNA (eDNA) can exist in both intracellular and extracellular forms in natural ecosystems. When targeting harmful cyanobacteria, extracellular eDNA indicates the presence of traces of cyanobacteria, while intracellular eDNA indicates the potential for cyanobacteria to occur. However, identifying the "actual" potential for harmful cyanobacteria to occur is difficult using the existing sediment eDNA analysis method, which uses silica beads and cannot distinguish between these two forms of eDNA. This study analyzes the applicability of a density gradient centrifugation method (Ludox method) that can selectively analyze intracellular eDNA in sediment to overcome the limitations of conventional sediment eDNA analysis. PCR was used to amplify the extracted eDNA based on the two different methods, and the relative amount of gene amplification was compared using electrophoresis and Image J application. While the conventional bead beating method uses sediment as it is to extract eDNA, it is unknown whether the mic gene amplified from eDNA exists in the cyanobacterial cell or only outside of the cell. However, since the Ludox method concentrates the intracellular eDNA of the sediment through filtration and density gradient, only the mic gene present in the cyanobacteria cells could be amplified. Furthermore, the bead beating method can analyze up to 1 g of sediment at a time, whereas the Ludox method can analyze 5 g to 30 g at a time. This gram of sediments makes it possible to search for even a small amount of mic gene that cannot be searched by conventional bead beating method. In this study, the Ludox method secured sufficient intracellular gene concentration and clearly distinguished intracellular and extracellular eDNA, enabling more accurate and detailed potential analysis. By using the Ludox method for environmental RNA expression and next-generation sequencing (NGS) of harmful cyanobacteria in the sediment, it will be possible to analyze the potential more realistically.

• Horticultural Science & Technology
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• v.33 no.2
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• pp.210-218
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• 2015

Melon leaves showing yellowing symptoms were analyzed using electron microscopy and RT-PCR for major cucurbit-infecting-viruses (CMV, MNSV, CGMMV, SqMV, WMV, KGMMV, PRSV and ZYMV) reported in Korea, but these viruses were not detected. As the result of further analysis by next-generation sequencing (NGS), the virus was identified as Cucurbit aphid-borne yellows virus (CABYV), and then confirmed by RT-PCR using CABYV-specific primers. When photosynthetic capacity was measured based on chlorophyll fluorescence yield (ChlFY), the leaves of the diseased plants showed $4.09{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, which was one-third of the readings observed for unaffected normal plants ($12.36{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$). The root functions of plants affected by leaf yellowing symptoms (LYS) was $0.28mg{\cdot}g^{-1}$, about half that measured for the normal unaffected plants ($0.48mg{\cdot}g^{-1}$). Cytological observations revealed that there were no morphological differences in the palisade parenchyma and mesophyll spongy cells of the leaves between the diseased and the normal plants. However, the same leaf cells of the affected plants contained more starch granules compared to those of the normal, unaffected plants. We conclude that the LYS of muskmelon is not merely a physiological disorder but a viral disease caused by CABYV and spread by aphids.