DOI QR코드

DOI QR Code

Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains

  • Baek, Kwang Yeol (Department of Applied Bioscience, Dong-A University) ;
  • Lee, Hyun-Hee (Department of Microbiology, Pusan National University) ;
  • Son, Geun Ju (Department of Applied Bioscience, Dong-A University) ;
  • Lee, Pyeong An (Department of Applied Bioscience, Dong-A University) ;
  • Roy, Nazish (Department of Applied Bioscience, Dong-A University) ;
  • Seo, Young-Su (Department of Microbiology, Pusan National University) ;
  • Lee, Seon-Woo (Department of Applied Bioscience, Dong-A University)
  • Received : 2017.11.24
  • Accepted : 2018.01.11
  • Published : 2018.04.01

Abstract

Accurate and rapid detection of bacterial plant pathogen is the first step toward disease management and prevention of pathogen spread. Bacterial plant pathogens Clavibacter michiganensis subsp. nebraskensis (Cmn), Pantoea stewartii subsp. stewartii (Pss), and Rathayibacter tritici (Rt) cause Goss's bacterial wilt and blight of maize, Stewart's wilt of maize and spike blight of wheat and barley, respectively. The bacterial diseases are not globally distributed and not present in Korea. This study adopted comparative genomics approach and aimed to develop specific primer pairs to detect these three bacterial pathogens. Genome comparison among target pathogens and their closely related bacterial species generated 15-20 candidate primer pairs per bacterial pathogen. The primer pairs were assessed by a conventional PCR for specificity against 33 species of Clavibacter, Pantoea, Rathayibacter, Pectobacterium, Curtobacterium. The investigation for specificity and sensitivity of the primer pairs allowed final selection of one or two primer pairs per bacterial pathogens. In our assay condition, a detection limit of Pss and Cmn was $2pg/{\mu}l$ of genomic DNA per PCR reaction, while the detection limit for Rt primers was higher. The selected primers could also detect bacterial cells up to $8.8{\times}10^3cfu$ to $7.84{\times}10^4cfu$ per gram of grain seeds artificially infected with corresponding bacterial pathogens. The primer pairs and PCR assay developed in this study provide an accurate and rapid detection method for three bacterial pathogens of grains, which can be used to investigate bacteria contamination in grain seeds and to ultimately prevent pathogen dissemination over countries.

Keywords

References

  1. Agarkova, I. V., Lambrecht, P. A. and Vidaver, A. K. 2011. Genetic diversity and population structure of Clavibacter michiganesis subsp. nebraskensis. Can. J. Microbiol. 57:366-374. https://doi.org/10.1139/w11-016
  2. Ahmed, N., Dobrindt, U., Hacker, J. and Hasnain, S. E. 2008. Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention. Nat. Rev. Microbiol. 6:387-394. https://doi.org/10.1038/nrmicro1889
  3. An, J. H., Noh, Y.-H., Kim, Y.-E., Lee, H.-I. and Cha, J.-S. 2015. Development of PCR and TaqMan PCR assays to detect Pseudomonas coronafaciens, a causal agent of halo blight of oats. Plant Pathol. J. 31:25-32. https://doi.org/10.5423/PPJ.OA.09.2014.0096
  4. Baker, G. C., Smith, J. J. and Cowan, D. A. 2003. Review and reanalysis of domain-specific 16S primers. J. Microbiol. Methods 55:541-555. https://doi.org/10.1016/j.mimet.2003.08.009
  5. Castillo, J. A. and Plata, G. 2016. The expansion of brown rot disease throughout Bolivia: possible role of climate change. Can. J. Microbiol. 62:442-448. https://doi.org/10.1139/cjm-2015-0665
  6. Cho, M. S., Kang, M. J., Kim, C. K., Seol, Y., Hahn, J. H., Park, S. C., Hwang, D. J., Ahn, T., Park, D. H., Lim, C. K. and Park, D. S. 2011. Sensitive and specific detection of Xanthomonas oryzae pv. oryzae by real-time bio-PCR using pathovar-specific primers based on an rhs family gene. Plant Dis. 95:589-594. https://doi.org/10.1094/PDIS-06-10-0399
  7. Coplin, D. L., Majerczak, D. R., Zhang, Y. X., Kim, W. S., Jock, S. and Geider, K. 2002. Identification of Pantoea stewartii subsp. Stewartii by PCR and strain differentiation by PFGE. Plant Dis. 86:304-311. https://doi.org/10.1094/PDIS.2002.86.3.304
  8. De Boer, S., Wieczorek, A. and Kummer, A. 1988. An ELISA test for bacterial ring rot of potato with a new monoclonal antibody. Plant Dis. 72:874-878. https://doi.org/10.1094/PD-72-0874
  9. Demkin, V. V., Koshechkin, S. I. and Slesarev, A. 2017. A novel real-time PCR assay for highly specific detection and quantification of vaginal lactobacilli. Mol. Cell. Probes 32:33-39. https://doi.org/10.1016/j.mcp.2016.11.006
  10. Eggenberger, S., Diaz-Arias, M. M., Gougherty, A. V., Nutter, F. W., Jr., Sernett, J. and Robertson, A. E. 2016. Dissemination of Goss's wilt of corn and epiphytic Clavibacter michiganesis subsp. nebraskensis from inoculum point sources. Plant Dis. 100:686-695. https://doi.org/10.1094/PDIS-04-15-0486-RE
  11. Gudmestad, N. C., Mallik, I., Pasche, J. S., Anderson, N. R. and Kinzer, K. 2009. A real-time PCR assay for the detection of Clavibacter michiganensis subsp. sepedonicus based on the cellulase A gene sequence. Plant Dis. 93:649-659. https://doi.org/10.1094/PDIS-93-6-0649
  12. Hayward, A. C. 1964. Characteristics of Pseudomonas solanacearum. J. Appl. Bacteriol. 27:265-277. https://doi.org/10.1111/j.1365-2672.1964.tb04912.x
  13. Henriques, A., Cereija, T., Machado, A. and Cerca, N. 2012. In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp. BMC Res. Notes 5:637. https://doi.org/10.1186/1756-0500-5-637
  14. Kang, I. J., Kang, M.-H., Noh, T.-H., Shim, H.-K., Shin, D. B. and Heu, S. 2016. Simultaneous detection of three bacterial seed-borne diseases in rice using multiplex polymerase chain reaction. Plant Pathol. J. 32:575-579. https://doi.org/10.5423/PPJ.NT.05.2016.0118
  15. Lamka, G. L., Hill, J. H., Mcgee, D. C. and Braun, E. J. 1991. Development of an immunosorbent-assay for seed-borne Erwinia stewartii in corn seeds. Phytopathology 81:839-846. https://doi.org/10.1094/Phyto-81-839
  16. Lee, J., Park, J., Kim, S., Park, I. and Seo, Y. S. 2016. Differential regulation of toxoflavin production and its role in the enhanced virulence of Burkholderia gladioli. Mol. Plant. Pathol. 17:65-75. https://doi.org/10.1111/mpp.12262
  17. Mergaert, J., Verdonck, L. and Kersters, K. 1993. Transfer of Erwinia ananas (synonym, Erwinia uredovora) and Erwinia stewartii to the genus Pantoea emend. as Pantoea ananas (Serrano 1928) comb. nov. and Pantoea stewartii (Smith 1898) comb. nov., respectively, and description of Pantoea stewartii subsp. indologenes subsp. nov. Int. J. Syst. Evol. Microbiol. 43:162-173.
  18. Michener, P. M., Pataky, J. K. and White, D. G. 2002. Rate of transmitting Erwinia stewartii from seed to seedlings of a sweet corn hybrid susceptible to Stewart's wilt. Plant Dis. 86:1031-1035. https://doi.org/10.1094/PDIS.2002.86.9.1031
  19. Murray, T. D., Schroeder, B. K., Schneider, W. L., Luster, D. G., Sechler, A., Rogers, E. E. and Subbotin, S. A. 2017. Rathayibacter toxicus, other Rathayibacter species inducing bacterial head blight of grasses, and the potential for livestock poisonings. Phytopathology 107:804-815. https://doi.org/10.1094/PHYTO-02-17-0047-RVW
  20. Nutter, F. W., Jr. and Madden, L. V. 2009. Plant pathogens as biological weapons against agriculture. In: Beyond anthrax: the weaponization of infectious disease, eds. by L. I. Lutwick and S. M. Lutwick, pp. 335-363. Springer, New York, USA.
  21. Oh, E.-J., Bae, C., Lee, H.-B., Hwang, I. S., Lee, H. I., Yea, M. C., Yim, K.-O., Lee, S., Heu, S., Cha, J.-S. and Oh, C.-S. 2016. Clavibacter michiganensis subsp. capsici subsp. nov., causing bacterial canker disease in pepper. Int. J. Syst. Evol. Microbiol. 66:4065-4070. https://doi.org/10.1099/ijsem.0.001311
  22. Papaiakovou, M., Pilotte, N., Grant, J. R., Traub, R. J., Llewellyn, S., McCarthy, J. S., Krolewiecki, A. J., Cimino, R., Mejia, R. and Williams, S. A. 2017. A novel, species-specific, real-time PCR assay for the detection of the emerging zoonotic parasite Ancylostoma ceylanicum in human stool. PLoS Negl. Trop. Dis. 11:e0005734. https://doi.org/10.1371/journal.pntd.0005734
  23. Park, J., Lee, P. A., Lee, H.-H., Choi, K., Lee, S.-W. and Seo, Y.-S. 2017. Comparative genome analysis of Rathayibacter tritici NCPPB 1953 with Rathayibacter toxicus strains can facilitate studies on mechanisms of nematode association and host infection. Plant Pathol. J. 33:370-381. https://doi.org/10.5423/PPJ.OA.01.2017.0017
  24. Paruthi, I. J. and Bhatti, D. S. 1985. Estimation of loss in yield and incidence of Anguina tritici on wheat in Haryana (India). Int. Nematol. Network Newsl. 2:13-16.
  25. Paruthi, I. J. and Gupta, D. C. 1987. Incidence of 'tundu' in barley and kanki in wheat field infested with Anguina tritici. Haryana Agric. Univ. J. Res. 17:78-79.
  26. Pataky, J. and Ikin, R. 2003. Pest risk analysis: the risk of introducing Erwinia stewartii in maize seeds. The International Seed Federation, Nyon.
  27. Harwood, T. D., Moslonka-Lefebvre, M. and Pellis, L. 2010. Disease spread in small-size directed trade networks: the role of hierarchical categories. J. Appl. Ecol. 47:1300-1309. https://doi.org/10.1111/j.1365-2664.2010.01884.x
  28. Rane, K. K. and Latin, R. X. 1992. Bacterial fruit blotch of watermelon: association of the pathogen with seed. Plant. Dis. 76:509-512. https://doi.org/10.1094/PD-76-0509
  29. Rozen, S. and Skaletsky, H. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132:365-386.
  30. Smidt, M. and Vidaver, A. K. 1986. Population dynamics of Clavibacter michiganesis subsp. nebraskensis in field-grown dent corn and popcorn. Plant Dis. 70:1031-1036. https://doi.org/10.1094/PD-70-1031
  31. Tambong, J. T., Mwange, K. N., Bergeron, M., Ding, T., Mandy, F., Reid, M. and Zhu, X. 2008. Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan real-time PCR assay targeting the cpsD gene. J. Appl. Microbiol. 104:1525-1537. https://doi.org/10.1111/j.1365-2672.2007.03674.x
  32. Tancos, M. A., Chalupowicz, L., Barash, I., Manulis-Sasson, S. and Smart, C. D. 2013. Tomato fruit and seed colonization by Clavibacter michiganensis subsp. michiganensis through external and internal routes. Appl. Environ. Microbiol. 79:6948-6957. https://doi.org/10.1128/AEM.02495-13
  33. Tao, T., Chen, Q., Bie, X., Lu, F. and Lu, Z. 2015. Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach. World J. Microbiol. Biotechnol. 31:1955-1966. https://doi.org/10.1007/s11274-015-1942-y
  34. Vidaver, A. K. 1967. Synthetic and complex media for the rapid detection of fluorescence of phytopathogenic Pseudomonas: effect of carbon source. Appl. Microbiol. 15:1523-1524.
  35. Vidaver, A. K. and Mandel, M. 1974. Corynebacterium nebraskense a new, orange-pigmented phytopathogenic species. Int. J. Syst. Bacteriol. 24:482-485. https://doi.org/10.1099/00207713-24-4-482
  36. Vincelli, P. and Tisserat, N. 2008. Nucleic acid-based pathogen detection in applied plant pathology. Plant Dis. 92:660-669. https://doi.org/10.1094/PDIS-92-5-0660
  37. Wensing, G. A., Gernold, M., Wiedermann, W., Coplin, D. L. and Geider, K. 2014. Molecular differentiation of Pantoea stewartii subsp. indologenes from subspeces stewartii and identification of new isolates from maize seeds. J. Appl. Microbiol. 116:1553-1562. https://doi.org/10.1111/jam.12467
  38. Wensing, A., Zimmermann, S. and Geider, K. 2010. Identification of the corn pathogen Pantoea stewartii by mass spectrometry of whole-cell extracts and its detection with novel PCR primers. Appl. Environ. Microbiol. 76:6248-6256. https://doi.org/10.1128/AEM.01032-10
  39. Yim, K.-O., Lee, H.-I., Kim, J.-H., Lee, S.-D., Cho, J.-H. and Cha, J.-S. 2012. Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum. Eur. J. Plant Pathol. 133:559-575. https://doi.org/10.1007/s10658-011-9927-7
  40. Yu, S., Chen, W., Wang, D., He, X., Zhu, X. and Shi, X. 2010. Species-specific PCR detection of the food-borne pathogen Vibrio parahaemolyticus using the irgB gene identified by comparative genomic analysis. FEMS Microbiol. Lett. 307:65-71. https://doi.org/10.1111/j.1574-6968.2010.01952.x
  41. Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N. and Kalakoutskii, L. V. 1993. Rathayibacter gen. nov., including the species Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., Rathayibacter iranicus comb. nov., and six strains from annual grasses. Int. J. Syst. Bacteriol. 43:143-149. https://doi.org/10.1099/00207713-43-1-143