• Research in Plant Disease
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• v.17 no.3
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• pp.376-380
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• 2011

The bacterial speck of tomato caused by Pseudomonas syringae pv. tomato leads to serious economic losses especially on fruits of susceptible genotype. Thus, Pseudomonas syringae pv. tomato is a plant quarantine bacterium in many countries including Korea. In this study, we developed specific PCR assays for detection of the bacterium from tomato seeds. A specific primer set is designed from the hrpZ gene for specific detection of Pseudomonas syringae pv. tomato. A 501 bp PCR product corresponding to hrpZ gene was amplified only form Pseudomonas syringae pv. tomato strains, but no PCR product was amplified from other tomato bacterial pathogens, such as Pseudomonas syringae pv. glycinea, P. syringae pv. maculicola, P. syringae pv. atropurpurea, P. syringae pv. morsprunorum, and from other P. syringae pathovar strains. The nested-PCR primer set corresponding to an internal fragment of the 501 bp sequence (hrpZ) gine was used to specific detection of Pseudomonas syringae pv. tomato in tomato seed. A 119 bp PCR product using nested PCR primer was highly specific and sensitive to detect low level of Pseudomonas syrigae pv. tomato in tomato seeds. We believe that the PCR assays developed in this study is very useful to detect Pseudomonas syringae pv. tomato from the tomato seeds.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.989-992
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• 2002

Sweet persimmon (Diospyros kaki Thunb.) is widely cultivated in the southern part of Korea and its cultivation is increasing. However, anthracnose disease caused by Colletotricuhum species is one of the major hinderances to the cultivation and production of sweet persimmon. Therefore, in the current study, PCR was used to specifically detect Colletotrichum spp., based on the sequences of the ITS II regions in the rDNA. Using the sequence data, CO-1 was designated to detect Colletotrichum together the with ITS 4 primer. The result showed that a single segment of ca. 500 bp was observed only in Colletotrichum, but not in any other fungal and bacterial isolates. The annealing temperatures and template DNA quantites were also investigated to identify optimal conditions for detection. Using these species-specific primers, a unique band was obtained at annealing temperatures ranging from $55^{\circ}C\;and\;61^{\circ}C$ and template DNA levels from 10 pg- $10{\mu}g$.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.575-579
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• 2018

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.90-96
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• 2015

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.

• Research in Plant Disease
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• v.7 no.3
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• pp.123-133
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• 2001

Many pathovars of the species Pseudomonas syringae are known to produce different phytotoxins as secondary metabolites. Although phytotoxins generally enhance the virulence of P. syringae, they are not required for pathogenesis. Among the phytotoxins produced by P. syringae, lipodepsipeptides, coronatine, phaseolotoxin, and tabtoxin are the most well-known toxins which have been intensively studied for their structure, mode of action, biosynthesis, and regulation. A polymerase chain reaction (PCR) technique that amplifies a segment of the phytotoxin gene cluster using a primer set has been developed in recent years. This method offers the advantages of speed and sensitivity compared to the approaches based on physiological and biochemical methods. PCR detection of genes involved in the production of toxins could be exploited for early diagnosis of plant diseases caused by P. syringae pathovars.

• Korean Journal of Veterinary Research
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• v.64 no.3
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• pp.26.1-26.9
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• 2024

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA.

• The Plant Pathology Journal
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• v.32 no.5
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• pp.396-406
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• 2016

Continuous supplementation of mineral nutrients and salicylic acid (SA) as foliar application could improve efficacy in controlling basal stem rot (BSR) disease in oil palm seedling. It is revealed from the results that the highest disease severity index (58.3%) was recorded in T8 treatments at 9 months after inoculation. The best disease control was achieved by T7 treatments (calcium/copper/SA [Ca/Cu/SA]) (5.0%) followed by T1 (5.5%), T5 (5.8%), T3 (8.3%), T6 (8.3%), T4 (13.3%), and T2 (15.8%) treatments. Continuous supplementation of Ca/Cu/SA was found to be the most effective in controlling the disease and the high performance liquid chromatography results showed the detection of ergosterol at very low concentration in the treated samples. Moreover, the transmission electron microscopy analysis results clearly indicated that T7 treatment was also enhancing lignification, which was responsible for the thickness of the secondary cell walls and middle lamella compared to untreated samples. It was therefore, concluded that continuous supplementation of minerals nutrients and SA could effectively suppress disease severity by reducing ergosterol activity and also improve the process of lignification in the treated plants. Furthermore, this treatment also managed to delay the onset of BSR symptoms and promote the growth of the seedlings and eventually suppress the BSR disease.

• The Plant Pathology Journal
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• v.32 no.5
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• pp.414-422
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• 2016

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

• Research in Plant Disease
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• v.27 no.2
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• pp.79-83
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• 2021

The aim of the present study was to develop a sensitive and specific detection method for the rapid detection of apple scar skin viroid (ASSVd) in apple leaves. The resulting reverse transcription recombinase polymerase amplification (RT-RPA) assay can be completed in 10 min at 42℃, is 10 times more sensitive than conventional reverse transcription polymerase chain reaction, and can specifically amplify ASSVd without any cross-reactivity with other common apple viruses, including apple stem grooving virus, apple stem pitting virus, and apple chlorotic leaf spot virus. The reliability of the RT-RPA assay was assessed, and the findings suggested that it can be successfully utilized to detect ASSVd in field-collected samples. The RT-RPA assay developed in the present study provides a potentially valuable means for improving the detection of ASSVd in viroid-free certification programs, especially in resource-limited conditions.

• Research in Plant Disease
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• v.23 no.1
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• pp.65-68
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• 2017

Barley yellow mosaic virus (BaYMV), which is transmitted by the root-inhabiting protist Polymyxa graminis, causes a soil-borne disease. In this study, we detected BaYMV from soil using two-step nested polymerase chain reaction (PCR). Specific primers based on a coat protein region of BaYMV segment RNA1 were used in the first round of amplification. Based on the sequenced amplicon, an inner primer was designed for the second round of amplification. A PCR product of 372 bp exhibited 98%-100% nucleotide sequence identity with the coat protein region of BaYMV segment RNA1. In this study, we propose an easy method for the detection of BaYMV from soil, may considerably assist in accurate fungus-transmitted virus diagnosis and subsequent disease forecasting. This is the first report on the detection of BaYMV from soil.