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PCR-Based Assay for Rapid and Specific Detection of the New Xanthomonas oryzae pv. oryzae K3a Race Using an AFLP-Derived Marker
PCR-Based Assay for Rapid and Specific Detection of the New Xanthomonas oryzae pv. oryzae K3a Race Using an AFLP-Derived Marker
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 732-739
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : November 05, 2013
  • Accepted : February 27, 2014
  • Published : June 30, 2014
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About the Authors
Eun-Sung Song
National Academy of Agricultural Science, Rural Development Administration, Suwon 441-857, Republic of Korea
Song-Yi Kim
Department of Horticulture, Kong-Ju National University, Yesan 340-802, Republic of Korea
Tae-Hwan Noh
National Institute of Crop Science, Rural Development Administration, Iksan 570-880, Republic of Korea
Heejung Cho
National Academy of Agricultural Science, Rural Development Administration, Suwon 441-857, Republic of Korea
Soo-Cheon Chae
Department of Horticulture, Kong-Ju National University, Yesan 340-802, Republic of Korea
Byoung-Moo Lee
National Academy of Agricultural Science, Rural Development Administration, Suwon 441-857, Republic of Korea
lbmoo@korea.kr

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.
Keywords
Introduction
Xanthomonas oryzae pv. oryzae (Xoo) is a gram-negative bacterium that causes bacterial blight (BB) of rice ( Oryza sativa L.). BB is one of the most serious diseases of rice in Asian countries. The bacteria enter the rice plant through the hydathodes or wounded leaves and multiply in the vascular system. The disease can cause yield losses as high as 50% in severely infected plants in the field [9] .
Some bactericides have been developed and used to control BB disease, but none of them is highly effective and economical in the case of outbreaks. The most effective and economical way to control the disease is to use resistant cultivars. However, the large-scale and long-term cultivation of varieties carrying a single resistance gene results in the evolution of new pathogenic races. Indeed, the introduction of resistance genes into rice was correlated with a change in the pathogenic diversity of Xoo [10] .
Studies conducted in Asian countries on the pathotypic and genetic diversity of Xoo are based on differential interactions with rice cultivars containing different resistance genes. These studies employed repetitive DNA elements as probes to detect restriction fragment length polymorphisms and PCR-based DNA fingerprinting [2 - 4 , 6 - 8 , 13] . Unfortunately, these methods are lengthy and labor intensive. As an alternative, PCR-based methods have been developed for the detection and identification of Xoo at the pathovar and species levels [1 , 5 , 14] . An available triplex PCR specifically detects the race of Xoo strains [15] , but is unable to effectively distinguish Xoo K3a races from the other races of Korean Xoo. A race-specific primer, which can be used for identifying a particular Xoo race, is still lacking. Therefore, a PCR-based rapid assay that can detect and discriminate the Xoo K3a race is required.
In the present study, we describe the development of a race-specific marker derived from an amplified fragment length polymorphism (AFLP) for the detection and identification of the Xoo K3a race in rice plants. PCR amplification using a race-specific primer validated the specificity of this primer set for detecting the Xoo K3a race and should be a reliable technique for the detection and diagnosis of this pathogen.
Materials and Methods
- Bacterial Strains, Culture Conditions, and DNA Isolation
All bacterial strains used in this study are listed in Table 1 . The Xoo and other xanthomonad strains were obtained from the Korean Agricultural Culture Collection (KACC, Korea), the Ministry of Agriculture, Forestry and Fisheries (MAFF, Japan), the Belgian Co-ordinated Collections of Micro-organisms (BCCM, Belgium), and from Dr. T. H. Noh at the National Institute of Crop Science (RDA, Korea). All Xanthomonas strains were cultured on YDC medium (2% D-glucose, 2% CaCO 3 , 1% yeast extract, and 1.5% agar) at 28℃ for 48 h. The Escherichia coli strain was grown at 37°C for 24 h in Luria-Bertani (LB) medium containing 200 μg/ml of ampicillin. Genomic DNA was isolated using a genomic DNA extraction kit (MagExtractor Genome, Japan) according to the manufacturer’s instructions.
- Development of a Race Marker using AFLP Analysis
AFLP was performed using a published method [16] , with some modification. Genomic DNA (600 ng) was digested with 1 μl of FastDigest Eco RI and Mse I (Thermo Scientific) at 37℃ for 30 min, and the restriction fragments were ligated to EcoR I and Mse I double-stranded adapters ( Table 2 ) at 16℃ for 5 h. The preselective PCR was performed using AccuPower PCR Premix (Bioneer, Korea) in a 25 μl reaction mixture containing 1 μl of DNA (50 ng/μl), 10 pmol of E0 primer, and 10 pmol of M0 primer ( Table 2 ). The preselective PCR amplification was carried out in a T100 Thermal Cycler (Bio-Rad, USA) according to the program as follows: initial denaturation at 94℃ for 5 min followed by 25 cycles at 94℃ for 30 sec, 57℃ for 1 min, and 72℃ for 1 min; and a final extension for 5 min at 72℃. Pre-amplification PCR products were diluted 3-fold with sterile distilled water and used as templates. The AFLP reaction with primers having two selective bases ( Table 2 ) was performed for 35 cycles under the conditions as follows: denaturation for 30 sec at 94℃, annealing for 20 sec at 67℃ followed by lowering the temperature (0.7℃) in the next 12 cycles, and then at 57℃ for the remaining 23 cycles; and a final extension for 1 min at 72℃. The amplified products were separated on a 1.2% agarose gel, stained with ethidium bromide, and visualized using a UV transilluminator.
The specific AFLP fragments were excised from the gel and DNA was eluted using a DNA fragment purification kit (MagExtractor PCR & Gel Clean up; Toyobo, Japan) according to the manufacturer’s instructions. Eluted DNA was directly cloned into the pGEM-T Easy Vector (Promega, USA) and used to transform competent cells (DH5α, RBC) according to the supplier’s information. The sequencing reaction and sequence data analysis were performed using an ABI Prism 3100 Automatic DNA Sequencer (Applied Biosystems).
- Primer Design and PCR Specificity
The race-specific primers, K3aF (5’-TCTGATTCGCAACGCTTTTGAGGAC-3’) and K3aR (5’-CTTCCTAATCAATAGTCACCTTGAA-3’), were designed according to the sequences of the Xoo K3a race. The specificity of the race-specific primer set was tested against Xoo K3a races, other races, pathovars, and Xanthomonas species. The PCR was performed using a premixed type polymerase (Taq PreMix, TNT Research, Korea) in a 20 μl reaction mixture containing 50 ng of template DNA and 10 pmol of each race-specific primer. PCR amplification was carried out in a T100 Thermal Cycler (Bio-Rad, USA) according to the program as follows: initial denaturation at 96℃ for 5 min followed by 25 cycles at 96℃ for 15 sec, 62℃ for 15 sec, and 72℃ for 30 sec; and a final 5 min extension at 72℃. Subsequently, 5 μl of each reaction mixture was subjected to electrophoresis through a 1.2% agarose gel, stained with ethidium bromide, and visualized using a UV transilluminator.
- PCR Assay for Detection of the Pathogen in Rice Plants
The leaves of 50-day-old susceptible rice cultivar IR24 were inoculated by clipping the leaf tips with sterile scissors and then dipping them into a saturated culture (10 9 cells/ml) of Xoo strain HB01001 [15] . The plants were grown in a greenhouse at 25–30℃, with a relative humidity of 60%. To evaluate the ability of the assay to directly detect the pathogen in infected rice plants, leaf samples were collected 1, 2, 3, 4, 5, 6, 7, and 8 cm from the lesion site 5 days after inoculation. Artificially infected rice leaves were harvested 1, 2, 3, 4, and 5 days after inoculation (1 cm from the lesion site). Each sample was soaked in 200 μl of sterile distilled water for 20 min. After soaking, 10 μl of the exudates was used as a template for the PCR assay as described above.
Results
- Specificity of a Race Marker Using AFLP Analysis
To develop a marker for the specific detection of the Xoo K3a race, 64 AFLP primer combinations were tested for their ability to identify five K3a races and 17 other races of Korean Xoo (data not shown). From these primer combinations, a race-specific 1,062 bp DNA fragment for K3a was selected, cloned, and sequenced. Based on this sequence, the K3aF/K3aR primer set was designed to amplify a 1,024 bp fragment that specifically detects K3a races of Korean Xoo ( Fig. 1 ).
The specificity of the putative race-specific primer was evaluated using genomic DNA extracted from the Xoo K3a race and the other Xanthomonas strains listed in Table 1 . The PCR product was only produced from 13 isolates of Xoo K3a races among 39 isolates of other species and strains of Xanthomonas ( Fig. 2 ). These results indicate that the primer set is highly specific for detecting the Xoo K3a race.
List of bacterial strains used in this study.
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aKACC, Korean Agricultural Culture Collection, Korea (http://www.genebank.go.kr/); MAFF, Ministry of Agriculture, Forestry and Fisheries of Japan; LMG, The Belgian Co-ordinated Collections of Microorganisms (BCCM), Belgium; HB, Department of Rice and Winter Cereal Crop, National Institute of Crop Science, Korea. ‘–’ unknown.
Oligonucleotide adapters and primers used for AFLP analysis.
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Oligonucleotide adapters and primers used for AFLP analysis.
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Agarose gel electrophoresis of PCR products amplified from X. oryzae pv. oryzae strains using the race-specific K3aF/K3aR primer set. Lane M: size marker (1 kb ladder; TNT Research, Korea); lanes 1–93: Xoo strains (numbers 1–93, respectively, in Table 1).
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Agarose gel electrophoresis of PCR products amplified from Xoo K3a races and other xanthomonads using the race-specific K3aF/K3aR primer set. Lane M: size marker (1 kb ladder); lanes 1–13: Xoo K3a races (numbers 73–85, respectively, in Table 1); lanes 14–39, other xanthomonads (numbers 94–119, respectively, in Table 1).
- Direct PCR Assay for Detecting the Pathogen in Rice Plants
To evaluate the ability of the direct PCR assay to detect the pathogen in rice plants, artificially infected rice leaves (1 cm sections at the lesion site and at 2, 3, 4, 5, 6, 7, and 8 cm from the lesion) were soaked in sterile distilled water, and the exudates were used without further treatment as templates for the PCR assay. Amplicons were detected in all eight samples ( Fig. 3 ).
To verify the ability of the PCR assay to predict disease outbreaks in paddy fields, artificially infected rice leaves were sampled at 0, 1, 2, 3, 4, and 5 days after inoculation, and the exudates were used as templates for the direct PCR assay. The expected 1,024 bp amplicon was detected in samples taken 2–5 days after inoculation ( Fig. 4 ). These results indicate that the PCR-based assay can be used directly to detect and identify Xoo K3a in infected rice leaf samples without isolating the bacteria and can be applied to predict disease outbreaks in paddy fields.
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Direct PCR detection of X. oryzae pv. oryzae K3a race in rice leaves. (A) An artificially infected rice leaf 5 days after inoculation. (B) PCR assay for detection of the pathogen from different regions of the rice leaf sample shown in (A). Lane M: size marker (1 kb ladder); C, control Xoo HB01001 gDNA (50 ng/μl); lanes 1–8: samples (1–8 cm, respectively, from the lesion).
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PCR assay for pathogen detection in rice leaf samples up to 5 days after inoculation. Lane M: size marker (1 kb ladder); C, control Xoo HB01001 gDNA (50 ng/μl); lanes 0–5: leaf samples, 0–5 days after infection.
Discussion
The major objective of this study was to develop a rapid, precise, and specific PCR-based assay that can be used directly to identify the Xoo K3a race in infected rice plants, and to discriminate Xoo K3a from other races, pathovars, and Xanthomonas strains.
New races of Xoo are continuously being reported in rice-growing Asian countries [2 , 8 , 10 , 11] . For example, K3a, which is pathogenic for rice cultivars containing Xa3 resistance genes, was isolated from the southwestern coastal areas of Korea [12] . Xoo strains are classified into several races according to their interactions with rice cultivars containing different resistance genes. Nine pathogenic races of Xoo were isolated in Nepal [3] and nine from China [8] . The Korean Xoo strains are classified into five races (K1 to K5) using five rice cultivars to distinguish among them [17] . However, such methods are too expensive, labor intensive, and lengthy to be useful for testing large numbers of samples and identifying new races of the pathogen. To address this problem, we applied the AFLP technique to identify DNA fragments specific for the new K3a race of Xoo. Polymorphic amplicons that were generated only from isolates of the K3a race were cloned and sequenced. The sequence data were used to design a race-specific primer that unambiguously distinguished isolates of the K3a race from other Korean Xoo races ( Fig. 1 ).
Specific primers and DNA probes derived from the 16S– 23S rDNA spacer region and repeated elements can detect and identify Xoo strains [1 , 14] ; however, none of them discriminated X. oryzae pv. oryzae from X. oryzae pv. oryzcola. Cho et al. [5] recently described the Bio-PCR method based on the amplification of a member of the rhs family gene for detecting Xoo at the pathovar and species levels. We also described a triplex PCR method for the detection and identification of Xoo races that discriminated between K1, including some K2 and K4 races, and the K3 and K 5 races [15] , but d id not d iscriminate b etween K3a and K3 or K5 races. In contrast, the PCR-based assay described in the present study unambiguously discriminated the Xoo K3a race from other races of Korean Xoo as well as from other Xanthomonas strains ( Figs. 1 and 2 ). Interestingly, four strains that we previously classified as K3 and K5 races [15] were classified here as the K3a race. These results indicate that this race-specific primer set is highly specific for the Xoo K3a and can be used as a simple and rapid tool for identifying this pathogen.
Early diagnosis of the pathogen by rapid methods is important for assessing the health status of rice, because a latent infection can lead to a serious epidemic under favorable conditions. We show here that a PCR-based assay, which does not require isolating bacteria or extracting DNA, detected the pathogen in asymptomatic rice leaves 2 days after inoculation ( Fig. 4 ). Therefore, this assay can be used as a reliable and useful method for the detection and identification of the Xoo K3a race in rice plants, as well as for the diagnosis of latent and nonsymptomatic infections in paddy fields to prevent disease outbreaks.
Acknowledgements
This study was supported by the 2013 and 2014 Postdoctoral Fellowship Program (Project No. PJ0066312013 and PJ0100852014) of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.
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