Table 1. Morphological and genetic characterization of 37 putative strains of Clavibacter michiganensis subsp. nebraskensis collected from Goss’s leaf blight symptomatic and asymptomatic maize leaves compared with two strains of Clavibacter michiganensis subsp. michiganensis.
| Strain ID | Field, County, State* | Source † | Year | Morphology and color ‡ | Pathogenic | Groups ¶ | PCR-RFLP groups** | Identity †† | Plasmid | |
|---|---|---|---|---|---|---|---|---|---|---|
| recA | rpoD | |||||||||
| C4 | 1, Iowa, IA | Epiphytic | 2012 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| C5 | 1, Iowa, IA | Epiphytic | 2012 | Small mucoid, orange | No | 2 | III | II | Cm | - |
| C12 | 1, Iowa, IA | Epiphytic | 2012 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| FN | 2, Boone, IA | Diseased | 2013 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| HF2 | 3, Story, IA | Epiphytic | 2012 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 11–5 | 4, Story, IA | Epiphytic | 2013 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| NE2 | 5, Grant, NE | Epiphytic | 2012 | Small mucoid, orange | Yes | 1 | I | I | Cmn | + |
| CL1 | 6, Carroll, IA | Diseased | 2013 | Small mucoid, orange | Yes | 1 | I | I | Cmn | + |
| C10 | 1, Iowa, IA | Epiphytic | 2012 | Large mucoid, orange | No | 4 | III | II | Cm | - |
| HF4 | 3, Story, IA | Epiphytic | 2012 | Large mucoid, orange | No | 4 | I | I | Cmn | - |
| NE1 | 5, Grant, NE | Epiphytic | 2012 | Large mucoid, orange | No | 4 | III | II | Cm | + |
| HI 4–5 | 4, Story, IA | Epiphytic | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 6–5 | 4, Story, IA | Epiphytic | 2013 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| CL4 | 6, Carroll, IA | Diseased | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| GIL1 | 7, Story, IA | Diseased | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| GIL3 | 7, Story, IA | Diseased | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| NE6 | 5, Grant, NE | Epiphytic | 2011 | Large fluidal, yellow | No | 5 | II | na | Cm | - |
| BR2 | 8, Boone, IA | Epiphytic | 2011 | Large fluidal, yellow | No | 5 | II | na | Cm | - |
| BS2 | 9, Boone, IA | Epiphytic | 2011 | Large fluidal, yellow | No | 5 | II | na | Cm | - |
| C8 | 1, Iowa, IA | Epiphytic | 2012 | Small mucoid, orange | No | 2 | IV | na | Cm | - |
| G1 | 10, Story, IA | Epiphytic | 2012 | Large fluidal, yellow | No | 5 | II | na | Cm | - |
| C2 | 1, Iowa, IA | Epiphytic | 2012 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| C3 | 1, Iowa, IA | Epiphytic | 2012 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| C7 | 1, Iowa, IA | Epiphytic | 2012 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HF1 | 3, Story, IA | Epiphytic | 2012 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 2-AN | 4, Story, IA | Epiphytic | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 2-BS | 4, Story, IA | Epiphytic | 2013 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| HI 2–4 | 4, Story, IA | Epiphytic | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 11–7 | 4, Story, IA | Epiphytic | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 11–8 | 4, Story, IA | Epiphytic | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 6–4 | 4, Story, IA | Epiphytic | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| HI 7–7 | 4, Story, IA | Epiphytic | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| NE3 | 5, Grant, NE | Epiphytic | 2012 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| NE4 | 5, Grant, NE | Epiphytic | 2012 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| CL2 | 6, Carroll, IA | Diseased | 2013 | Small mucoid, orange | Yes | 1 | I | I | Cmn | - |
| CL3 | 6, Carroll, IA | Diseased | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| GIL2 | 7, Story, IA | Diseased | 2013 | Large mucoid, orange | Yes | 3 | I | I | Cmn | - |
| BR-4 (Cmm ‡‡ ) | - | Large mucoid, yellow | No | 6 | nr | III | Cmm | + | ||
| DR-60 (Cmm) | - | Large mucoid, yellow | No | 6 | V | III | Cmm | + | ||
Pathogenicity tests were done on corn plants that were inoculated with each isolate at the V3–V4 crop developmental stage. Foliar blight severity was rated as the proportion of leaf area affected six days after inoculation.
* GPS co-ordinates of nearest town to which fields were located are provided in S1 Table.
† Epiphytic, strain recovered from asymptomatic maize leaves; diseased, strain recovered from maize leaves with symptoms of Goss’s leaf blight
‡ Colony size was measured after 5 days growth on CNS medium [12] using a ruler and divided into three groups as A, small mucoid orange, (2–3 mm); B, large mucoid orange (3–4 mm) and C, large fluidal yellow (3–4 mm or larger in diameter) (see Fig 1).
¶ Groups based on colony morphology after 5 days’ growth on NBY medium and pathogenicity on maize
** PCR-RFLP of housekeeping genes recA and rpoD [24]. Numbers in columns represent fragment patterns; na, not amplified; nr, not restricted.
††Strains identified as Clavibacter michiganensis subsp. nebraskensis based on PCR-RFLP pattern of recA and rpoD genes [24], all belonged to Groups 1 and 3; Cm stands for Clavibacter michiganensis.
‡‡ Clavibacter michiganensis subsp. Michiganensis