Infection of Maize by Clavibacter michiganensis subsp. nebraskensis Does Not Require Severe Wounding.

Goss's bacterial wilt and leaf blight of maize is caused by Clavibacter michiganensis subsp. nebraskensis. Infested residue is the primary source of inoculum and infection occurs via wounds caused by sand blasting, hail, or wind damage. The pathogen survives as an epiphyte on maize leaves and, because the disease has been observed on plants in the field with no obvious wounding, we wondered whether infection by epiphytic C. michiganensis subsp. nebraskensis and disease development could occur in the absence of severe wounding. Consequently, greenhouse experiments were done to evaluate disease development in the absence of wounding in ambient and increased humidity conditions. Maize plants at the V4 to V5 crop development stage were spray inoculated with a suspension of C. michiganensis subsp. nebraskensis (108 cells ml-1). Leaf blight incidence was assessed on whole plants and individual leaves; epiphytic populations of C. michiganensis subsp. nebraskensis were monitored by dilution plating of leaf washes; and epiphytic C. michiganensis subsp. nebraskensis colonization was visualized using scanning electron microscopy (SEM). Goss's leaf blight symptoms were observed on nonwounded plants in ambient (37.0% plant incidence) and increased humidity conditions (60.0% plant incidence). Populations of epiphytic C. michiganensis subsp. nebraskensis survived and increased on maize leaves, particularly at increased humidity. We observed C. michiganensis subsp. nebraskensis colonizing maize leaves in localized sites that included epidermal junctions, cuticle depressions, in and around stomata, and at the base of trichomes. Single cells and aggregates of C. michiganensis subsp. nebraskensis were observed within substomatal chambers using SEM. These data indicate that severe wounding is not necessary for C. michiganensis subsp. nebraskensis infection of maize, and stomata or trichomes may serve as entry points for the bacterium.

Characterization and Comparison of Clavibacter michiganensis subsp. nebraskensis Strains Recovered from Epiphytic and Symptomatic Infections of Maize in Iowa.

Clavibacter michiganensis subsp. nebraskensis (Cmn), the causal organism of Goss's wilt and leaf blight of maize, can be detected in the phyllosphere of its host prior to disease development. We compared the morphology and pathogenicity of 37 putative isolates of Cmn recovered from asymptomatic and symptomatic maize leaves. Thirty-three of the isolates produced mucoid orange colonies, irrespective of the source of isolation and all but four of these isolates were pathogenic on maize. The remaining 4 isolates recovered from asymptomatic leaves had large fluidal yellow colonies, and were non-pathogenic on maize. Isolates varied in their aggressiveness on a susceptible hybrid of maize but no significant differences in aggressiveness were detected between epiphytic isolates and those recovered from diseased maize tissues. The genomics of Cmn is poorly understood; therefore as a first step to determining what genes may play a role in virulence, we compared 33 putative virulence gene sequences from 6 pathogenic and a non-pathogenic isolate recovered from the phyllosphere. Sequence polymorphisms were detected in 5 genes, cellulase A, two endoglucanases, xylanase B and a pectate lyase but there was no relationship with pathogenicity. Further research is needed to determine what genes play a role in virulence of Cmn. Our data show however, that the virulence factors in Cmn likely differ from those reported for the closely related subspecies michiganensis and sepedonicus.

Phylogeny of Fusarium oxysporum f. sp. lactucae Inferred from Mitochondrial Small Subunit, Elongation Factor 1-alpha, and Nuclear Ribosomal Intergenic Spacer Sequence Data.

ABSTRACT Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathogen recently reported in Arizona. Sequence analysis of the mitochondrial small subunit (mtSSU), translation elongation factor 1-alpha (EF-1alpha) gene, and the nuclear ribosomal DNA intergenic spacer (IGS) region was conducted to resolve relationships among f. sp. lactucae isolates, F. oxysporum isolates from other hosts, and local non-pathogenic isolates. Analysis of mtSSU sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from 13 other F. oxysporum isolates. Analysis of EF-1alpha sequences resulted in moderate resolution, grouping seven formae speciales with the lactucae isolates. Analysis of the IGS region revealed numerous sequence polymorphisms among F. oxysporum formae speciales consisting of insertions, deletions, and single nucleotide transitions and substitutions. Repeat sequence analysis revealed several duplicated subrepeat units that were distributed across much of the region. Based on analysis of the IGS sequence data, lactucae race 1 isolates resolved as a monophyletic group with three other formae speciales of F. oxysporum. In all analyses, lactucae race 2 isolates composed a separate lineage that was phylo-genetically distinct and distantly related to the lactucae race 1 isolates.