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Genomics-based Sensitive and Specific Novel Primers for Simultaneous Detection of Burkholderia glumae and Burkholderia gladioli in Rice Seeds

  • Lee, Chaeyeong (Department of Microbiology, Pusan National University) ;
  • Lee, Hyun-Hee (Department of Microbiology, Pusan National University) ;
  • Mannaa, Mohamed (Department of Microbiology, Pusan National University) ;
  • Kim, Namgyu (Department of Microbiology, Pusan National University) ;
  • Park, Jungwook (Department of Microbiology, Pusan National University) ;
  • Kim, Juyun (Department of Microbiology, Pusan National University) ;
  • Seo, Young-Su (Department of Microbiology, Pusan National University)
  • Received : 2018.07.26
  • Accepted : 2018.08.28
  • Published : 2018.12.01

Abstract

Panicle blight and seed rot disease caused mainly by Burkholderia glumae and Burkholderia gladioli is threatening rice cultivation worldwide. The bacteria have been reported as seed-borne pathogens from rice. Accurate detection of both pathogens on the seeds is very important for limiting the disease dissemination. Novel primer pairs targeting specific molecular markers were developed for the robust detection of B. glumae and B. gladioli. The designed primers were specific in detecting the target species with no apparent cross-reactions with other related Burkholderia species at the expected product size. Both primer pairs displayed a high degree of sensitivity for detection of B. glumae and B. gladioli separately in monoplex PCR or simultaneously in duplex PCR from both extracted gDNA and directly preheated bacterial cell suspensions. Limit of detection was as low as 0.1 ng of gDNA of both species and $3.86{\times}10^2cells$ for B. glumae and $5.85{\times}10^2cells$ for B. gladioli. On inoculated rice seeds, the designed primers could separately or simultaneously detect B. glumae and B. gladioli with a detection limit as low as $1.86{\times}10^3cells$ per rice seed for B. glumae and $1.04{\times}10^4cells$ per rice seed of B. gladioli. The novel primers maybe valuable as a more sensitive, specific, and robust tool for the efficient simultaneous detection of B. glumae and B. gladioli on rice seeds, which is important in combating rice panicle blight and seed rot by early detection and confirmation of the dissemination of pathogen-free rice seeds.

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Fig. 1. Amplification of the expected PCR products (A) Products from bacterial genomic DNA. (B) Products from suspensions of five strains of Burkholderia glumae (Lane 1-5) amplified using Bglu3-f and Bglu3-r primers and six strains of Burkholderia gladioli (Lane 6-11) amplified using Bgla9-f and Bgla9-r primers. Lane 12 is a negative control. Specific bands were clearly visible on the 2% agarose gel at 174 bp for B. glumae and 289 bp for B. gladioli. M denotes the 1 kb DNA ladder.

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Fig. 2. Specificity assay for the designed primer pairs (Bglu3 and Bgla9), by amplification of the expected PCR products (A) Products from bacterial genomic DNA. (B) Products from cell suspensions. Lane 1, Burkholderia glumae BGR1; lane 2, B. gladioli BSR3; lane 3, B. cepacia KACC 10189; lane 4, B. cepacia KACC 10190; lane 5, B. cepacia KACC 10337; lane 6, B. cepacia KACC 12679; lane 7, B. cepacia KACC 15010; lane 8, B. kururiensis KACC 12038; lane 9, Burkholderia sp. KJ006; lane 10, B. megalochromosomata KACC 17925; lane 11, B. phymatum KACC 12032; lane 12, B. phytofirmans KACC 12042; lane 13, B. pyrrocinia KACC 17914; lane 14, B. stabilis KACC 12028. Specific bands were clearly visible on the 2% agarose gel at 174 bp for B. glumae BGR1 and 289 bp for B. gladioli BSR3. No bands were observed with the other tested Burkholderia species. M denotes the 1 kb DNA ladder.

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Fig. 3. Sensitivity assay for the designed primer pairs (Bglu3 and Bgla9), by amplification of the expected PCR product from genomic DNA (gDNA) from (A) Burkholderia glumae BGR1, (B) B. gladioli BSR3 and (C) mixed sample (1:1) of B. glumae BGR1 and B. gladioli BSR3 in a duplex PCR assay Lane 1, 100 ng gDNA; lane 2, 10 ng gDNA; lane 3, 1 ng gDNA; lane 4, 0.1 ng gDNA; lane 5, 0.01 ng gDNA, lane 6, 1 pg gDNA, lane 7, 0.1 pg; lane 8, no gDNA template. Specific bands were clearly visible on the 2% agarose gel at 174 bp for B. glumae and 289 bp for B. gladioli up to the 0.1 ng gDNA template. M denotes the 1 kb DNA ladder.

E1PPBG_2018_v34n6_490_f0004.png 이미지

Fig. 4. Sensitivity assay for the designed primer pairs (Bglu3 and Bgla9), by amplification of the expected PCR product from bacterial cell suspensions of (A) Burkholderia glumae BGR1, (B) Burkholderia gladioli BSR3 and (C) mixed sample of B. glumae BGR1 and B. gladioli BSR3 in the multiplex PCR assay Lane 1, Overnight cultures; lane 2-10, serially diluted cultures (10-1 to 10-9); lane 11, no cells. Clear specific bands were visible on the 2% agarose gel at 174 bp for B. glumae BGR1 and 289 bp for B. gladioli BSR3 up to the 10-3 dilution (approximately 300 cells for B. glumae and approximately 500 cells for B. gladioli). M denotes the 1 kb DNA ladder.

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Fig. 5. Sensitivity assay for the designed primer pairs (Bglu3 and Bgla9), by amplification of the expected PCR product from rice seeds inoculated with different dilutions of Burkholderia glumae BGR1, Burkholderia gladioli BSR3 and mixed co-inoculation Suspensions from rice seeds inoculated with different dilutions of the tested strains or their mix were collected using sterile distilled water containing 0.03% Tween 20 and used as templates for the PCR. Lane 1, 100 ng gDNA from B. glumae BGR1; lane 2, 100 ng gDNA from B. gladioli BSR3; lane 3, cell suspension of overnight culture from B. glumae; lane, 4-7, 10-1 to 10-4 dilutions; lane 8, cell suspension of overnight culture from B. gladioli; lane 9-12, 10-1 to 10-4 dilutions; lane 13, 1:1 cell suspension mix from overnight culture from B. glumae and B. gladioli; lane 14-17, 10-1 to 10-4 dilutions. M, 1 kb DNA ladder; lane 18, un-inoculated surface sterilized rice seeds as a negative control.

Table 1. List of bacterial strains or genomic DNA used in this study

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Table 2. Genome sequences of bacterial strain used in this study

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Acknowledgement

Supported by : National Research Foundation of Korea (NRF), Ministry of Agriculture, Food and Rural Affairs

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