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

다국어 초록 (Multilingual 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 crossreactions 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 × 102 cells for B. glumae and 5.85 × 102 cells 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 × 103 cells per rice seed for B. glumae and 1.04 × 104 cells 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.

참고문헌 (Reference)

1 Jeong, Y., "Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops" 87 : 890-895, 2003

2 Stanier, R. Y., "The aerobic pseudomonads: a taxonomic study" 43 : 159-271, 1966

3 Stackebrandt, E., "Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology" 44 : 846-849, 1994

4 Furuya, N., "Specific oligonucleotide primers based on sequences of the 16S-23S rDNA spacer region for the detection of Burkholderia gladioli by PCR" 68 : 220-224, 2002

5 Takeuchi, T., "Specific detection of Burkholderia plantarii and B. glumae" 63 : 455-462, 1997

6 백광열, "Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains" 한국식물병리학회 34 (34): 104-112, 2018

7 Yu, S., "Species-specific PCR detection of the food-borne pathogen Vibrio parahaemolyticus using the irgB gene identified by comparative genomic analysis" 307 : 65-71, 2010

8 Nzula, S., "Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages" 28 : 307-312, 2000

9 Tsushima, S., "Selective medium for detecting Pseudomonas glumae Jurita et Tabei, the causal bacterium of grain rot of rice" 52 : 253-259, 1986

10 Yabuuchi, E., "Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov" 36 : 1251-1275, 1992

11 Rozen, S., "Primer3 on the WWW for general users and for biologist programmers" 132 : 365-386, 2000

12 Maeda, Y., "Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences" 56 : 1031-1038, 2006

13 Cottyn, B., "Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control" 107 : 885-897, 2009

14 Trung, H. M., "Occurrence of rice grain rot disease in Vietnam" 18 : 30-, 1993

15 Lee, C. J., "Occurrence of bacterial soft rot of onion plants caused by Burkholderia gladioli pv. alliicola in Korea" 34 : 287-292, 2005

16 Lee, C. J., "Occurrence of bacterial soft rot of Pleurotus ostreatus caused by Burkholderia gladioli pv. agaricicola in Korea" 92 : 235-240, 2010

17 Zaid, A. M., "OEM-A new medium for rapid isolation of onion-pathogenic and onionassociated bacteria" 91 : 520-526, 2012

18 Kawaradani, M., "New selective medium for isolation of Burkholderia glumae from rice seeds" 66 : 234-237, 2000

19 Kawanishi, T., "New detection systems of bacteria using highly selective media designed by SMART: Selective Medium-Design Algorithm Restricted by Two Constraints" 6 : e16512-, 2011

20 Goto, K., "New bacterial diseases of rice (brown stripe and grain rot)" 21 : 46-47, 1956

21 Cui, Z., "Multiplex PCR assay for simultaneous detection of six major bacterial pathogens of rice" 120 : 1357-1367, 2016

22 Salles, J. F., "Molecular method to assess the diversity of Burkholderia species in environmental samples" 68 : 1595-1603, 2002

23 Tao, T., "Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach" 31 : 1955-1966, 2015

24 이광열, "Isolation and Identification of Burkholderia pyrrocinia CH-67 to Control Tomato Leaf Mold and Damping-off on Crisphead Lettuce and Tomato" 한국식물병리학회 27 (27): 59-67, 2011

25 Cho, H. S., "Interference of quorum sensing and virulence of the rice pathogen Burkholderia glumae by an engineered endophytic bacterium" 60 : 14-23, 2007

26 Hikichi, Y., "Immunofluorescent antibody technique for detecting Pseudomonas glumae on rice plants" 59 : 477-480, 1993

27 Vandamme, P., "Identification and population structure of Burkholderia stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV)" 38 : 1042-1047, 2000

28 FAO, "Feeding the world, eradicating hunger: executive summary. World summit on food security 2009. FAO Database. Version: 2009"

29 Nandakumar, R., "Evidence for the soilborne nature of the rice sheath rot and panicle blight pathogen, Burkholderia gladioli 1" 30 : 148-154, 2008

30 Jung, B., "Cooperative interactions between seedborne bacterial and air-borne fungal pathogens on rice" 9 : 31-, 2018

31 Seo, Y. S., "Complete genome sequence of Burkholderia gladioli BSR3" 193 : 3149-, 2011

32 Sessitsch, A., "Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plantbeneficial properties" 55 : 1187-1192, 2005

33 Baek, I., "Burkholderia megalochromosomata sp. nov., isolated from grassland soil" 65 : 959-964, 2015

34 Zhang, H., "Burkholderia kururiensis sp. nov., a trichloroethylene (TCE)-degrading bacterium isolated from an aquifer polluted with TCE" 50 : 743-749, 2000

35 Ham, J. H., "Burkholderia glumae: next major pathogen of rice?" 12 : 329-339, 2011

36 Nandakumar, R., "Burkholderia glumae and B. gladioli cause bacterial panicle blight in rice in the southern United States" 93 : 896-905, 2009

37 Ura, H., "Burkholderia gladioli associated with symptoms of bacterial grain rot and leaf-sheath browning of rice plants" 72 : 98-103, 2006

38 Cottyn, B., "Bacterial populations associated with rice seed in the tropical environment" 91 : 282-292, 2001

39 Cottyn, B., "Bacterial disease of rice. II. Characterization of pathogenic bacteria associated with sheath rot complex and grain discoloration of rice in the Philippines" 80 : 438-445, 1996

40 Awika, J. M., "Advances in cereal science: Implications to food processing and health promotion" American Chemical Society 1-13, 2011

41 Papaiakovou, M., "A novel, species-specific, real-time PCR assay for the detection of the emerging zoonotic parasite Ancylostoma ceylanicum in human stool" 11 : e0005734-, 2017