• The Plant Pathology Journal
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• v.15 no.3
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• pp.137-143
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• 1999

A novel chromosomal gene tagging technique using a specific fragment of the fatty acid desaturase-like open reading frame (des-like ORF) from the tox-argK gene cluster of Pseudomonas syringae pv. phaseolicola was developed to identify Xanthomonas spp.released into the environment as biocontrol agents. X. campestris pv. convolvuli FB-635, a pathogen of Convolvulus arvensis L., (bindweed), was chosen as the organism in which to develop and test the system. A 0.52 kb DES fragment amplified from P. syringae pv. phaseolicola C-199 was inserted into pGX15, a cosmid clone containing a 10.3 kb Eco RI-HindIII fragment derived from the xanthomonadin biosynthetic gene cluster contained in plasmid pIG102, to create a pigG::DES insertion. The 10.8 kb EcoRI-BamHI fragment carrying the pigG:: DES insertion was cloned into pLAFR3 to generate pLXP22. pLXP22 was then conjugated into X. campestris pv. convolvuli FB-635 and the pigG::DES insertion integrated into the bacterial chromosome by marker exchange. Rifampicin resistant, tetracycline sensitive, starch hydrolyzing, white colonies were used to differentiate the marked strain from yellow pigmented wild-type ones. PCR primers specific for the unique DES fragment were used for direct detection of the marked strain. Result showed the marked strain could be detected at very low levels even in the presence of high levels of other closely related or competitive bacteria. This PCR-based DES-tagging system provides a rapid and specific tool for directly monitoring the dispersal and persistence of Xanthomonas spp.released into the environment.

• Research in Plant Disease
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• v.15 no.1
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• pp.8-12
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• 2009

Virus disease surveys of strawberries cultivated and preserved as germplasm resources in Korea was conducted during 2007-2008. Virus detection was conducted by RT-PCR using total RNAs extracted from strawberry samples. We detected the infection with Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV), Strawberry vein banding virus (SVBV) and Strawberry pallidosis associated virus (SPaV) while no infection with Strawberry crinkle virus (SCV), Strawberry necrotic shock virus (SNSV), Strawberry latent ring spot virus (SLRSV) and Arabis mosaic virus (ArMV) was observed. The infection rate of virus disease on 4 cultivars including Seolhyang, Maehyang, Gumhyang, and Dahong, bred in Korea, was 0.1, 1.9, 0, and 0%, respectively. Surprisingly, however, cultivar Red Peal introduced from Japan in 1997 revealed 48.3% virus infection rate. SMYEV, SMoV and SPaV were also identified in strawberries growing in the farm fields of Korea. In the field, however, SMYEV was the most predominant virus (97.4%) among those 3 identified viruses. SVBV was detected only in strawberry kept as a germplasm.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

• The Plant Pathology Journal
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• v.30 no.4
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• pp.407-415
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• 2014

Viral diseases have been a major limiting factor threating sustainable potato (Solanum tuberosum L.) production in Pakistan. Surveys were conducted to serologically quantify the incidence of RNA viruses infecting potato; Potato virus X (PVX), Potato virus Y (PVY), Potato virus S (PVS), Potato virus A (PVA), Potato virus M (PVM) and Potato leaf roll virus (PLRV) in two major potato cultivars (Desiree and Cardinal). The results suggest the prevalence of multiple viruses in all surveyed areas with PVY, PVS and PVX dominantly widespread with infection levels of up to 50% in some regions. Co-infections were detected with the highest incidence (15.5%) for PVX and PVS. Additionally the data showed a positive correlation between co-infecting viruses with significant increase in absorbance value (virus titre) for at least one of the virus in an infected plant and suggested a synergistic interaction. To test this hypothesis, glasshouse grown potato plants were challenged with multiple viruses and analyzed for systemic infections and symptomology studies. The results obtained conclude that multiple viral infections dramatically increase disease epidemics as compared to single infection and an effective resistance strategy in targeting multiple RNA viruses is required to save potato crop.

• The Plant Pathology Journal
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• v.39 no.1
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• pp.149-157
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• 2023

Phytoplasmas were discovered in diseased Elaeocarpus sylvestris trees growing on Jeju Island that showed symptoms of yellowing and darkening in the leaves. Leaf samples from 14 symptomatic plants in Jeju-si and Seogwipo-si were collected and phytoplasma 16S rRNA was successfully amplified by nested polymerase chain reaction using universal primers. The sequence analysis detected two phytoplasmas, which showed 99.5% identity to 'Candidatus Phytoplasma asteris' and 'Ca. P. malaysianum' affiliated to 16SrI and 16SrXXXII groups, respectively. Through polymerase chain reaction-restriction fragment length polymorphism (RFLP) analyses using the AfaI (RsaI) restriction enzyme, the presence of two phytoplasmas strains as well as cases of mixed infection of these strains was detected. In a virtual RFLP analysis with 17 restriction enzymes, the 16S rRNA sequence of the 'Ca. P. asteris' strain was found to match the pattern of the 16SrI-B subgroup. In addition, the phytoplasmas in the mixed-infection cases could be distinguished using specific primer sets. In conclusion, this study confirmed mixed infection of two phytoplasmas in one E. sylvestris plant, and also the presence of two phytoplasmas (of the 16SrI and 16SrXXXII groups) in Jeju Island (Republic of Korea).

• The Plant Pathology Journal
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• v.38 no.6
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• pp.656-664
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• 2022

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferumspecific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.1
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• pp.46-52
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• 2013

After an outbreak of viral disease in an aquafarm, release of virus (es) from infected fish into environmental seawater has been suspected. In the present study, we utilized a negatively charged membrane (HA type) as an efficient method for concentration and detection of fish pathogenic viruses, specifically, megalocytivirus and viral hemorrhagic septicemia virus (VHSV) present in field-collected seawater samples or inoculated into seawater artificially. Positively charged viruses adsorbed onto the negatively charged membrane and were eluted with 1 mM NaOH (pH 10.5) following rinsing with 0.5 mM $H_2SO_4$ (pH 3.0). Megalocytivirus and VHSV particles isolated using anegatively charged HA membrane from seawater inoculated with each virus at a concentration of 10 viral particles/mL were of sufficient quantity to show positive results in atwo-step PCR (or RT two-step PCR); however, despite it being negatively charged, a cellulose acetate (CA) membraneshowed negative results. In quantitative PCR, the detection limits of the HA membrane for megalocytivirus and VHSV in seawater were 1.20E+00 viral particles/mL and 1.22E+01 viralparticles/mL, respectively. The calculated mean recovery yields from 1 L seawater spiked with known concentrations of megalocytivirus and VHSV particles were 28.11% and 23.00%, respectively. The concentrate of a 1-L sample of culturing seawater from the aquatank of flounder suffering from VHSV showed clear positive results in PCR when isolated with an HA, but not a CA, membrane. Thus, viral isolation using an HA membrane is a practical and reliable method for detection of fish pathogenic viruses in seawater.

• Research in Plant Disease
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• v.29 no.1
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• pp.52-59
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• 2023

Cucurbit chlorotic yellows virus (CCYV) is a plant virus that causes damage to cucurbit crops such as watermelon and cucumber, and is transmitted by an insect vector known as the whitefly. Since CCYV was first detected on cucumber in Chungbuk in 2018, it has been reported in other areas including Gyeongsang in Korea. In 2020, we performed field surveys of yellowing diseases in the greenhouses growing melon and watermelon in Chungbuk (Jincheon and Eumseong). Reverse transcription-polymerase chain reaction analysis of 79 collected samples including melon, watermelon, and weeds resulted in detection of CCYV in 4 samples: Three samples were singly infected with CCYV and one samples was mixed infected with CCYV, Cucurbit aphid borne yellows virus, and Watermelon mosaic virus. The complete genome sequences of the four collected CCYV melon isolates (ES 1-ES 4) were determined and genetically compared with those of previously reported CCYV isolates retrieved from GenBank. Phylogenetic analyses of RNA 1 and 2 sequences revealed that four ES isolates were clustered in one group and closely related to the CCYV isolates from China. The analysis also revealed very low genetic diversity among the CCYV ES isolates. In general, CCYV isolates showed little genetic diversity, regardless of host or geographic origins. CCYV has the potential to pose a serious threat to melon, watermelon, and cucumber production in Korea. Further studies are needed to examine the pathogenicity and transmissibility of CCYV in weeds and other cucurbits including watermelon.

• The Plant Pathology Journal
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• v.34 no.5
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• pp.381-392
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• 2018

Clavibacter michiganensis subsp. michiganesis (Cmm) is a quarantine-worthy pest in $M{\acute{e}}xico$. The implementation and validation of new technologies is necessary to reduce the time for bacterial detection in laboratory conditions and Raman spectroscopy is an ambitious technology that has all of the features needed to characterize and identify bacteria. Under controlled conditions a contagion process was induced with Cmm, the disease epidemiology was monitored. Micro-Raman spectroscopy ($532nm\;{\lambda}$ laser) technique was evaluated its performance at assisting on Cmm detection through its characteristic Raman spectrum fingerprint. Our experiment was conducted with tomato plants in a completely randomized block experimental design (13 plants ${\times}$ 4 rows). The Cmm infection was confirmed by 16S rDNA and plants showed symptoms from 48 to 72 h after inoculation, the evolution of the incidence and severity on plant population varied over time and it kept an aggregated spatial pattern. The contagion process reached 79% just 24 days after the epidemic was induced. Micro-Raman spectroscopy proved its speed, efficiency and usefulness as a non-destructive method for the preliminary detection of Cmm. Carotenoid specific bands with wavelengths at 1146 and $1510cm^{-1}$ were the distinguishable markers. Chemometric analyses showed the best performance by the implementation of PCA-LDA supervised classification algorithms applied over Raman spectrum data with 100% of performance in metrics of classifiers (sensitivity, specificity, accuracy, negative and positive predictive value) that allowed us to differentiate Cmm from other endophytic bacteria (Bacillus and Pantoea). The unsupervised KMeans algorithm showed good performance (100, 96, 98, 91 y 100%, respectively).

• Research in Plant Disease
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• v.23 no.2
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• pp.193-201
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• 2017

Major tomato viruses in Korea are Tomato chlorosis virus (ToCV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), and Tomato mosaic virus (ToMV). RT-PCR conditions for the viruses were examined, especially in primer set and RT-PCR mixture. Total 46 primer sets from the unique sequence of the viruses were tested for nonspecific background products in a RT-PCR mixture without template. Among them 16 primer sets were applied to healthy tomato RNA, resulting the compatibility between RT-PCR mixture and primer set influenced RT-PCR to reduce nonspecific background products. Based on the combinations among cDNA synthesis parameters and RT-PCR mixtures, two reaction mixtures were finally selected for ToCV detection. The condition allowed to determine more specific primer sets; C029 (ToCV), C072 (TSWV), C070 (CMV), C048 (PepMoV), and C065 (ToMV). These primer sets are expected to be of use to specific detection of the major viruses in tomato plants.