PCR-Based Assay for Rapid and Specific Detection of the New Xanthomonas oryzae pv. oryzae K3a Race Using an AFLP-Derived Marker

Abstract

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.

Preliminary genome-wide association mapping of rice bacterial leaf blight resistance loci using major Korean races of Xoo (Xanthomonas oryzae)

“…In 2003, a new race K3a emerged in Korea which proved to be an epidemic for rice crop (Noh et al, 2003). Among these BLB isolates, four races K1 (HB01013), K2 (HB01014), K3 (HB01015), and K3a (HB01009) (Song et al, 2014) that were maintained at -80 o C were revived on Peptone sucrose agar (PSA) plates at 28 o C for 48 h. Each bacterial colony was suspended with sterilized distilled water and adjusted to concentrations of approximately 10 9 cfu/ml (Fang et al, 1981). At the booting stage (approximately 40 days after transplanting), the uppermost fully expanded leaves of each plant were inoculated by clipping the scissors in bacterial suspension and by clipping off the leaves 2-3 cm from leaf tip (Kauffman et al, 1973).…”

PCR-based Assay for the Specific Detection of Pseudomonas syringae pv. tagetis using an AFLP-derived Marker

“…A pre-selective PCR reaction was performed with the AccuPower PCR Premix (Bioneer, Daejeon, Korea) in a 25 ml reaction mixture containing 1 ml of DNA (50 ng/ ml), 10 pmol of Eco0 (5'-GACTGCGTACCAATTC-3'), and 10 pmol of Ms0 (5'-GATGAGTCCTGAGTAA-3'). The pre-selective PCR and AFLP PCR amplification were conducted as described previously (Song et al, 2014). The amplified products were resolved in a 1.2% agarose gel with a 1-kb DNA ladder (TNT Research, Seoul, Korea) as a reference, stained with ethidium bromide, and visualized on a UV transilluminator.…”

“…The specific DNA fragment was eluted as described previously (Song et al, 2014). DNA was directly used in the ligation reaction with a pGEM-T Easy Vector (Promega, Madison, WI, USA) and was then transferred into competent DH5a (RBC Bioscience, Taipei, Taiwan) cells according to the supplier's instructions.…”

“…The bacterial strains that are listed in Table 1 were obtained from the Korean Agricultural Culture Collection (KACC) in Suwon, Korea, and the Belgian Co-ordinated Collections of Micro-organisms (BCCM) in Brussels, Belgium. The genomic DNA was isolated as described previously (Song et al, 2014).…”

“…The AFLP assay was performed using a previously described method (Song et al, 2014), with minor modification. Genomic DNA (approximately 300 ng) was (Table 2).…”