title: Rapid and Specific Detection of Acidovorax avenae subsp. citrulli Using SYBR Green-Based Real-Time PCR Amplification of the YD-Repeat Protein Gene.
abstract: The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10(0) fg/μl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.
authors.affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju 54874, Republic of Korea.|Department of Microbiology, Dankook University, Cheonan 31116, Republic of Korea.
authors.forename: Min Seok
authors.initials: MS
authors.lastname: Cho
authors.affiliation: Department of Applied Biology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea.
authors.forename: Duck Hwan
authors.initials: DH
authors.lastname: Park
authors.affiliation: Department of Microbiology, Dankook University, Cheonan 31116, Republic of Korea.
authors.forename: Tae-Young
authors.initials: TY
authors.lastname: Ahn
authors.affiliation: National Academy of Agricultural Science, Rural Development Administration, Jeonju 54874, Republic of Korea.
authors.forename: Dong Suk
authors.initials: DS
authors.lastname: Park
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D052160:Benzothiazoles; D042621:Comamonadaceae / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D009930:Organic Chemicals / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
keywords: Acidovorax avenae subsp. citrulli; Bacterial fruit blotch; Real-time PCR; YD repeat protein; detection