• The Plant Pathology Journal
• /
• v.34 no.2
• /
• pp.104-112
• /
• 2018

Accurate and rapid detection of bacterial plant pathogen is the first step toward disease management and prevention of pathogen spread. Bacterial plant pathogens Clavibacter michiganensis subsp. nebraskensis (Cmn), Pantoea stewartii subsp. stewartii (Pss), and Rathayibacter tritici (Rt) cause Goss's bacterial wilt and blight of maize, Stewart's wilt of maize and spike blight of wheat and barley, respectively. The bacterial diseases are not globally distributed and not present in Korea. This study adopted comparative genomics approach and aimed to develop specific primer pairs to detect these three bacterial pathogens. Genome comparison among target pathogens and their closely related bacterial species generated 15-20 candidate primer pairs per bacterial pathogen. The primer pairs were assessed by a conventional PCR for specificity against 33 species of Clavibacter, Pantoea, Rathayibacter, Pectobacterium, Curtobacterium. The investigation for specificity and sensitivity of the primer pairs allowed final selection of one or two primer pairs per bacterial pathogens. In our assay condition, a detection limit of Pss and Cmn was $2pg/{\mu}l$ of genomic DNA per PCR reaction, while the detection limit for Rt primers was higher. The selected primers could also detect bacterial cells up to $8.8{\times}10^3cfu$ to $7.84{\times}10^4cfu$ per gram of grain seeds artificially infected with corresponding bacterial pathogens. The primer pairs and PCR assay developed in this study provide an accurate and rapid detection method for three bacterial pathogens of grains, which can be used to investigate bacteria contamination in grain seeds and to ultimately prevent pathogen dissemination over countries.

• The Plant Pathology Journal
• /
• v.22 no.4
• /
• pp.314-317
• /
• 2006

A phage technique for detection of Xanthomonas axonopodis pv. citri, a causal bacterium of canker on Sastuma mandarin fruits was developed. Phage and ELISA techniques were compared for their sensitivity for detection of Xanthomonas axonopodis pv. citri on orange fruits. Both of techniques revealed a similar efficiency for the bacterial detection; the pathogenic bacteria were observed in pellet from the fruits with over one canker spot with below 2 mm in diameter. In field assays, the increase of phage population(120%) on surface of the fruits related to the disease development one month later indicated that the bacterial pathogens inhabit on the surface. The procedure will be effectively used for detection of only living bacterial pathogen on fruit surfaces of Satsuma mandarin and for the disease forecasting.

• Korean Journal of Food Science and Technology
• /
• v.26 no.6
• /
• pp.764-769
• /
• 1994

This study was performed to obtain fast, consistant and reliable estimation system of bacterial counts of raw milk, which effectively related to the quality of sanitaion and the condition of production at the farm. This study compared regression equation and correlation coefficient relationship between standard plate counts and data of Malthus conductance method for the detection time of total, psychrotrophs, coliform bacterial counts in raw milk. Regression equation (RE) between conductance detection time (Y) and total bacterial log counts (X) was Y=18.27651 - 2.07550X, with correlation coefficient -0.95(n=201). In coliform, RE was Y=9.320848 - 1.15598X with correlation coefficient -0.90 (n=207). Psychrotrophs had the RE of Y=29.96008-3.02487 with correlation coeffecient -0.9 (n=201). This conductance method gave results more quickly and was less labor-intensive than traditional standard plate count method.

• Journal of Microbiology and Biotechnology
• /
• v.33 no.7
• /
• pp.964-972
• /
• 2023

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and an enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of Enterococcus faecalis using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the Escherichia coli expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes were determined using the bacterial colony counting from the magnetic separations after the binding reaction between bacteria and CBD-eMB complexes. E. faecalis could bind to CBD-eMB complexes, but other bacteria (such as Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Streptococcus mutans, and Porphyromonas gingivalis) could not. E. faecalis cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable E. faecalis cells were recovered. The E. faecalis recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for E. faecalis was >17 CFU/ml. We developed a simple method for the specific detection of E. faecalis using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for E. faecalis among other bacterial species.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.135.1-135
• /
• 2003

A technique for detection of Xanthomonas axonopodis pv. citri, a causal bacterium of canker on Unshiu orange fruits, was developed using bacteriophage. Procedure for the detection was designed on the basis of the previous reports that one group(CPI) of X. axonopodis pv. citri bacteriophage and corresponding two Iysotypes distributed in Korea. First, fruit surface was washed with sterile distilled water and pellet was obtained from centrifugation. The pellet was resuspended in Wakimoto's potato semi-synthetic broth medium and divided equally into two parts. One part was heated in boiling water to kill bacterial cells. Bacteriophages(CP$_1$) were respectively added into two parts and 0.1 ml from each part was mixed with soft agar medium. After incubation for 18 hrs at 25$^{\circ}C$, the causal bacterium of canker was determined based on plaques formed on the medium. This procedure can be effectively used for detection of living bacterial pathogen on fruit surfaces of Unshiu orange.

• The Plant Pathology Journal
• /
• v.19 no.4
• /
• pp.205-209
• /
• 2003

Dispersal of citrus bacterial canker caused by Xanthomonas axonopodis pv. citri on Unshiu orange was investigated in naturally infested nursery plot at Seogwipo in Jeju island, Korea. Based on phage detection, over 2% of the bacterial pathogen over-wintered in canker lesions and started to multiply in late May. However, symptoms were first observed 1 month after the phage detection. The disease dispersed non-directionally to nearby plants possibly because of indirect dissemination of the bacterium by rain splashes. The disease increased from late June to late August and decreased thereafter. Population of phage increased constantly, however, disease occurrence somewhat fluctuated due to environmental factors. Disease incidence and severity were correlated with rainfall with wind that occurred 14-32 days earlier from late May to late August.

• Research in Plant Disease
• /
• v.24 no.4
• /
• pp.342-347
• /
• 2018

Bacterial canker caused by Clavibacter michiganensis is considered to be one of the most serious diseases, leading to economic damage to tomato worldwide. Diagnosis of the bacterial canker on tomato is known to be difficult because the causal pathogen is slow-growing on artificial media as well as causes latent infection in tomato. In this study, as a less time-consuming method, a specific primer set was newly designed for rapid detection of C. michiganensis. The method presented here is so simple, easy, and fast that it can be useful and practical in direct detection of the bacterial canker pathogen from tomato plants.

• International Journal of Oral Biology
• /
• v.38 no.4
• /
• pp.181-188
• /
• 2013

The presence of distinct bacterial species is found to be dependent on age, diet, and disease. We compared the detection rate of several oral bacterial strains in a cohort of 36 subjects including healthy volunteers, periodontal patients, and oral cancer patients. Gargling samples were obtained from these subjects from which DNA was then extracted. Specific primers for 29 bacterial species were used for PCR detection. In the oral cancer patients, Capnocytophaga ochracea, Gemella morbillorum, and Streptococcus salivarius were detected more frequently compared with the healthy volunteers and periodontitis patients. Fusobacterium nucleatum/ polymorphym and Prevotella nigrescens were significantly less prevalent in oral cancer patients than the other groups. In periodontitis patients, Porphyromonas gingivalis and Treponema denticola were more frequently found compared with the healthy volunteers. In the healthy volunteer group, Peptostreptococcus anaerobius was more frequently found than the other groups. The detection rate of several oral bacterial species was thus found to differ between healthy volunteers, periodontitis patients and oral cancer patients.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.6
• /
• pp.732-739
• /
• 2014

We describe the development of a polymerase chain reaction method for the rapid, precise, and specific detection of the Xanthomonas oryzae pv. oryzae (Xoo) K3a race, the bacterial blight pathogen of rice. The specific primer set was designed to amplify a genomic locus derived from an amplified fragment length polymorphism specific for the K3a race. The 1,024 bp amplicon was generated from the DNA of 13 isolates of Xoo K3a races out of 119 isolates of other races, pathovars, and Xanthomonas species. The assay does not require isolated bacterial cells or DNA extraction. Moreover, the pathogen was quickly detected in rice leaf 2 days after inoculation with bacteria and at a distance of 8 cm from the rice leaf 5 days later. The results suggest that this PCR-based assay will be a useful and powerful tool for the detection and identification of the Xoo K3a race in rice plants as well as for early diagnosis of infection in paddy fields.

• Korean Journal Plant Pathology
• /
• v.10 no.4
• /
• pp.297-304
• /
• 1994

This study was carried out to develop bioassay for detection of pesticide residues in agricultural products by using the soil microbial isolates sensitive to pesticides. One hundred bacterial isolates and eighty five fungal isolates were obtained from soil and their sensitivity to 10 ppm of several pesticides was examined in vitro. Five bacterial isolates and three fungal isolates were found sensitive to organochloride fungicide and two fungal isolates sensitive to organocopper fungicide. Among these isolates, B46, B93 and F67 were tested to find out the difference in sensitivity according to the methods of fungicide treatment. All of the isolates were found sensitive to 10 ppm of organochloride fungicides mixed directly in PDA. But they were found insensitive to the fungicide mixed in PDA after filtering through membrane filter. In case of organocopper fungicide, the isolates were found sensitive only when it was treated in PDA. And their sensitivity showed difference among various kinds of organochloride fungicides. B46 and B93 were employed to check the possibility as the agent for detection of the pesticidal residues in twenty eight agricultural products including rice. It was found that all samples had not residues because the samples did not inhibit the growth of isolates. When organochloride fungicides were applied to the above products, it was possible to detect the residues in fruits and vegetables at the concentration of 10 ppm, but not in starch-rich grains. B46 and B93 were identified as Bacillus sp. according to their bacterial characteristics in culture.