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. 2018 Apr;34(2):104-112.
doi: 10.5423/PPJ.OA.11.2017.0250. Epub 2018 Apr 1.

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

Kwang Yeol Baek  1 , Hyun-Hee Lee  2 , Geun Ju Son  1 , Pyeong An Lee  1 , Nazish Roy  1 , Young-Su Seo  2 , Seon-Woo Lee  1
Affiliations

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

Kwang Yeol Baek et al. Plant Pathol J. 2018 Apr.

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 2 pg/μl 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 × 103 cfu to 7.84 × 104 cfu 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: Clavibacter michiganensis subsp. nebraskensis; PCR detection; Pantoea stewartii subsp. stewartii; Rathayibacter tritici; comparative genomics.

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Figures

Fig. 1
Fig. 1
Amplification of expected PCR product from Clavibacter michiganensis subsp. nebraskensis (Cmn) using two primer pairs. (A) Amplification by Cmn-3, (B) amplification by Cmn-4. Lane 1, DNA size standard (1 kb ladder); lane 2, no template DNA; lane 3, Cmn NCPPB 2581; lane 4, Cmn NCPPB 2582; lane 5, Rathayibacter tritici (Rt) NCPPB 255; lane 6, Rt NCPPB 471; lane 7, R. toxicus NCPPB 3552; lane 8, R. rathayi NCPPB 2980; lane 9, R. iranicus NCPPB 2253; lane10, C. michiganensis subsp. insidiosus NCPPB 1109; lane 11, C. michiganensis subsp. tesselarius NCPPB 3664; lane 12, Rt NCPPB 1857; lane 13, Rt NCPPB 1953; lane 14, C. michiganensis subsp. michiganensis (Cmm) NCPPB LMG7333; lane 15, Cmm PPP1; lane 16, C. michiganensis subsp. capsici (Cmc) PF003; lane 17, Cmc PF005; lane 18, Cmc PS006; lane 19, Cmc PF008. The white arrow represents the expected PCR product.
Fig. 2
Fig. 2
Specific detection of Pantoea stewartii subsp. stewartii (Pss) using two primer pairs. (A) Amplification by Pss2-4, (B) amplification by Pss2-9. Lane 1, DNA size standard (1 kb ladder); lane 2, no template DNA; lane3, P. stewartii subsp. indologens (Psi) NCPPB 2280; lane 4, Pss NCPPG 1553; lane 5, Pss NCPPB 3253; lane 6, P. ananatis pv. ananatis (Paa) NCPPB 3619; lane 7, Psi NCPPB 228; lane 8, P. agglomerans (Pa) NCPPB 10054; lane 9, Pa NCPPB 15275; lane 10, Pectobacterium carotovorum subsp. carotovorum (Pcc) KACC 10057; lane 11, Pcc KACC 10225; lane 12, Pcc KACC 10408; lane 13, Pcc KACC 10421; lane 14, Pcc KACC 10458; lane 15, Pss NCPPB 2295; lane 16, Pss NCPPB 3379. The arrow on the right represents the expected PCR product.
Fig. 3
Fig. 3
Specific amplification of PCR product from Rathayibacter tritici (Rt) using selected two primer pairs. (A) Amplification by Rt-4, (B) amplification by Rt-5. Lane 1, DNA size standard (1 kb ladder); lane 2, no template DNA; lane 3, Rt NCPPB 1857; lane 4, Rt NCPPB 1953; lane 5, Rt NCPPB 255; lane 6, Rt NCPPB 471; lane 7, C. michiganensis subsp. nebraskensis (Cmn) NCPPB 2581; lane 8, Cmn NCPPB 2582; lane 9, R. toxicus NCPPB 3552; lane 10, R. rathayi NCPPB 2980; lane 11, R. iranicus NCPPB 2253; lane 12, C. michiganensis subsp. insidiosus NCPPB 1109; lane 13, C. michiganensis subsp. tesselarius NCPPB 3664; lane 14, C. michiganensis subsp. michiganensis (Cmm) LMG 7333; lane 15, Cmm PPP1; lane 16, C. michiganensis subsp. capsici (Cmc) PF003; lane 17, Cmc PF005; lane 18, Cmc PS006; lane 19, Cmc PF008. The white arrow represents the expected PCR product.
Fig. 4
Fig. 4
Primer sensitivity assay using serially diluted genomic DNA of plant pathogenic bacteria. (A) Sensitive detection of Clavibacter michiganensis subsp. nebraskensis (Cmn), (B) sensitive detection of Pantoea stewartii subsp. stewartii (Pss), (C) sensitive detection of Rathayibacter tritici (Rt). Primer pairs and used bacterial strains of were indicated. Lane 1: DNA size standard; lane 2, no genomic DNA; lane 3, genomic DNA 150 ng; lane 4, genomic DNA 100 ng; lane 5, genomic DNA 50 ng; lane 6, genomic DNA 10 ng; lane 7, genomic DNA 1 ng; lane 8, genomic DNA 0.1 ng.
Fig. 5
Fig. 5
Specific detection of bacteria from the artificially inoculated seeds. (A) Detection of Rathayibacter tritici by Rt-4 on wheat seeds, (B) detection of Clavibacter michiganensis subsp. nebraskensis by Cmn-3 on corn seeds, (C) detection of Pantoea stewartii subsp. stewartii by Pss2-3 on corn seeds. Lane M, DNA size standard (1 kb ladder); S, seeds original; Nt, surface sterilized seeds; Sb, seeds infected with bacteria.
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References

    1. Agarkova IV, Lambrecht PA, Vidaver AK. Genetic diversity and population structure of Clavibacter michiganesis subsp. nebraskensis. Can J Microbiol. 2011;57:366–374. doi: 10.1139/w11-016. - DOI - PubMed
    1. Ahmed N, Dobrindt U, Hacker J, Hasnain SE. Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention. Nat Rev Microbiol. 2008;6:387–394. doi: 10.1038/nrmicro1889. - DOI - PubMed
    1. An JH, Noh Y-H, Kim Y-E, Lee H-I, Cha J-S. Development of PCR and Taq Man PCR assays to detect Pseudomonas coronafaciens, a causal agent of halo blight of oats. Plant Pathol J. 2015;31:25–32. doi: 10.5423/PPJ.OA.09.2014.0096. - DOI - PMC - PubMed
    1. Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain-specific 16S primers. J Microbiol Methods. 2003;55:541–555. doi: 10.1016/j.mimet.2003.08.009. - DOI - PubMed
    1. Castillo JA, Plata G. The expansion of brown rot disease throughout Bolivia: possible role of climate change. Can J Microbiol. 2016;62:442–448. doi: 10.1139/cjm-2015-0665. - DOI - PubMed

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