Fig. 1. Clavibacter michiganensis-specific PCR using the CmmF/CmmR primer pair. PCR was carried out under condition of 95oC, 3 min predenaturation / 95 oC, 5 sec; 66 oC, 10 sec; 72 oC, 10 sec (35 cycles) / 72 oC, 5 min final extension. Ten out of ten strains of C. michiganensis (lane 1 to lane 10) were amplified. Lane M: 100 bp plus DNA ladder, Lane 1: C. michiganensis 12-019, Lane 2: C. michiganensis 12-031, Lane 3: C. michiganensis 13-065, Lane 4: C. michiganensis 14-132, Lane 5: C. michiganensis 14-135, Lane 6: C. michiganensis 14-310, Lane 7: C. michiganensis 15-079, Lane 8: C. michiganensis 16-043, Lane 9: C. michiganensis 17-156, Lane 10: C. michiganensis 17-338, Lane 11: C. tessellarius NCPPB 3664, Lane 12: C. nebraskensis NCPPB 2581, Lane 13: C. insidiosus NCPPB 1109, Lane 14: C. capsici KACC 18448, Lane 15: Tomato DNA, Lane 16: Tomato leaf sap.
Fig. 2. Species-specific PCR detection of Clavibacter michiganensis using the CmmF/CmmR primer pair. No amplification was detected except C. michiganensis (lane 1, positive control). Lane M: 100 bp plus DNA ladder, Lane 1: Clavibacter michiganensis, Lane 2: Acidovorax citrulli, Lane 3: Acidovorax konjaci, Lane 4: Agrobacterium tumefaciens, Lane 5: Pectobacterium carotovorum, Lane 6: Pseudomonas corrugata, Lane 7: Pseudomonas syringae, Lane 8: Pseudomonas viridiflava, Lane 9: Xanthomonas arboricola, Lane 10: Xanthomonas perforans.
Fig. 3. Sensitivity of PCR detection from bacterial cells of Clavibacter michiganensis using the CmmF/CmmR primer pair in a 10-fold dilution series. Amplified products were obtained from lane 1 (109 cfu/ ml) to lane 6 (104 cfu/ml). Lane 6 showed a weak PCR product band. Lane M: 100bp plus DNA ladder, Lane 1: 109 cfu/ml, Lane 2: 108 cfu/ ml, Lane 3: 107 cfu/ml, Lane 4: 106 cfu/ml, Lane 5: 105 cfu/ml, Lane 6: 104 cfu/ml, Lane 7: 103 cfu/ml, Lane 8: 102 cfu/ml, Lane 9: 101 cfu/ml, Lane 10: 100 cfu/ml, Lane 11: 10-1 cfu/ml, Lane 12: distilled water.
Fig. 4. Specific detection of Clavibacter michiganensis from different parts of artificially infected plant (A) using the CmmF/CmmR primer pair by direct PCR (B). Lane 1 to lane 9 showed PCR amplification of genomic DNA of C. michiganensis directly from various parts of the diseased plant. Lane M: 100bp plus DNA ladder, Lane C: uninfected control plant, Lane 1: leaf showing wilting symptom, Lane 2: leaf showing wilting symptom, Lane 3: petiole of leaf showing wilting symptom, Lane 4: shoots with no symptoms, Lane 5: petiole of leaf showing wilting symptom, Lane 6: middle part of stem, Lane 7: lower part of stem, Lane 8: root, Lane 9: discolored xylem vessel.
Table 1. List of bacterial strains used in this study
Table 2. Primer sequences of PCR-detection for Clavibacter michiganensis
Table 3. Detection of Clavibacter michiganensis in the artificially inoculated tomato seedling by plating undiluted (1/1) and serially diluted (1/10-1/106) leaf extract on selective BCT medium and PCR assay
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