Infection, Survival, and Growth of Clavibacter nebraskensis on Crop, Weed, and Prairie Plant Species.

Clavibacter nebraskensis is the causal agent of Goss's leaf blight and wilt, an important disease of maize in the United States and Canada. The epidemiology and ecology of this bacterial pathogen are poorly understood. Infested maize residue is often considered to be the primary source of inoculum for maize; however, the potential for many other plant species to be infected and serve as inoculum sources is unknown. The goal of this study was to determine if C. nebraskensis could infect, survive, and grow on common weed, crop, and grass species. Seedling leaves of 18 plant species that grow in maize production areas in the United States were inoculated with this pathogen in a controlled environment and in the field. Lesion development, bacterial streaming, and pathogen population size on leaves were then determined and used as criteria to evaluate host-pathogen interactions. Woolly cupgrass (Eriochloa villosa) and the native prairie grasses big bluestem (Andropogon gerardii) and little bluestem (Schizachyrium scoparium) developed lesions and bacterial streaming, identifying them as hosts and susceptible to infection. To our knowledge, this is the first report of these grass species being hosts of C. nebraskensis. Ten other grass species, including wheat and oats, were identified as potential sustaining hosts that maintained epiphytic or endophytic pathogen populations >106 colony forming units per leaf sample but displayed no evidence of infection. Five broadleaf species tested were nonhosts based on the three criteria. This study suggests that multiple plant species support infection and growth of C. nebraskensis and further elucidates the ecology of this pathogen and the epidemiology of Goss's wilt.

Goss's Bacterial Blight and Wilt of Corn Caused by Clavibacter michiganensis subsp. nebraskensis Occurs in Minnesota.

Goss's bacterial wilt and blight caused by Clavibacter michiganensis subsp. nebraskensis (Vidaver and Mandel) Davis et al. can be an economically significant disease of corn (Zea mays L.) (1). Corn hybrids with typical leaf and wilt symptoms of Goss's bacterial blight were observed in two western Minnesota fields in Chippewa and Stephens counties in August 2009. Disease incidence was estimated at 40% in one field and 90% in the other. Symptoms consisted of large, tan-to-gray, linear lesions with irregular margins parallel to the veins, with up to 50% of the leaf area symptomatic. Irregular, dark green-to-black, water-soaked spots occurred in the lesions and dried bacterial exudate was present on the lesions. Bacterial streaming from the cut edge of lesions was visible with light microscopy. Fungal structures were not observed in the lesions. Bacteria were isolated from infected leaves collected in both fields. Sections were cut from the margins of the lesions and placed in 0.02 mM phosphate buffer (PB). Bacterial suspensions were spread onto yeast glucose medium (YGM) (3) and incubated for 5 days at 22°C. All colonies were orange and similar in appearance to C. michiganensis subsp. nebraskensis reference strain CIC016 (= CN313.0). Single colonies were subcultured onto YGM and CNS media. Two gram-positive strains, CIC251 and CIC252, were orange, circular, and convex on CNS medium and used to demonstrate Koch's postulates on corn (2). Bacterial suspensions containing 2 × 108 CFU/ml were prepared in PB from 5-day-old cultures grown on YGM. For each of strains CIC251 and CIC252, six plants of the hybrid DKC51-45 were inoculated at the V3 growth stage by swabbing inoculum over the second and third youngest leaves with Carborundum. Three control plants were treated similarly with sterile PB. Plants were incubated in a greenhouse at 20 to 24°C. Linear, water-soaked lesions typical of Goss's wilt began to develop on all inoculated leaves 7 days after inoculation. No symptoms developed on control plants. Two leaf samples with lesions were collected per plant and bacteria isolated as described above. Colonies with characteristics of C. michiganensis subsp. nebraskensis were isolated from all lesions. Presumptive identification of strains CIC251 and CIC252 as C. michiganensis subsp. nebraskensis, as well as colonies isolated from inoculated plants, was validated by rDNA sequencing. Genomic DNA was extracted from 3-day-old colonies on YGM and the 16S region was amplified (~1,480 bp) by PCR assay using primers F27 and r1492 (4). Forward and reverse sequences were aligned and base calls confirmed using Sequencher 4.9. Consensus sequences for each strain were compared with the nucleotide database with BLAST to confirm a 99% match to C. michiganensis subsp. nebraskensis (NCBI GenBank AM410697.1 and U09763.1). This confirms, for the first time (to our knowledge), that Goss's bacterial leaf blight and wilt of corn occurs in Minnesota and could be a production and phytosanitary concern in that state. References: (1) M. Davis et al. Int. J. Syst. Bacteriol. 34:107, 1984. (2) M. Davis and A. Vidaver. Page 221 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) S. DeBoer and R. Copeman, Am. Potato J. 57:457, 1980. (4) S. Giovannoni. Page 177 in: Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt and M. Goodfellow, eds. John Wiley & Sons, Chichester, New York, 1991.

Plant pathogen forensics: capabilities, needs, and recommendations.

A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

Purification of catechol siderophores by boronate affinity chromatography: identification of chrysobactin from Erwinia carotovora subsp. carotovora.

Catechols are co-planar cis-diols known to form stable, isolable complexes with borate under weakly basic conditions. We exploited this chemistry and developed a boronate affinity chromatography for isolating catechol siderophores. The method was applied to the isolation of chrysobactin, enterobactin, and an unknown catechol siderophore produce by Erwinia carotovora subsp. carotovora W3C105. Yields of chrysobactin and enterobactin purified by boronate affinity chromatography were at least two-fold greater than those achieved through alternate methods. The unknown catechol produced by E. carotovora subsp. carotovora W3C105 was isolated by boronate affinity chromatography and shown to be identical to chrysobactin. Boronate affinity chromatography enabled separation of catechol from its rust-colored decomposition products, and simultaneous isolation of catechol and hydroxamate siderophores. Boronate affinity chromatography is a rapid and efficient method for purifying catechol siderophores from bacterial culture supernatants.

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis.

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.

Clavibacter michiganensis subsp. Sepedonicus Elicits a Hypersensitive Response in Tobacco and Secretes Hypersensitive Response-Inducing Protein(s).

ABSTRACT Strains of Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot of potato, showed marked differences in virulence on host plants. When infiltrated into tobacco leaves, virulent strains caused a rapid localized necrotic response (within 24 to 48 h) characteristic of the hypersensitive response (HR), whereas nonpathogenic strains did not. Concentrated cell-free culture supernatants (CCS) from virulent strains caused a necrotic reaction on tobacco, whereas CCS from nonpathogenic strains did not. The necrosis-inducing activity was heat stable and protease sensitive. Inhibitors of eukaryotic metabolism suppressed the necrotic reaction of tobacco to CCS. No necrotic response was observed when host plants were infiltrated with either cells or CCS from virulent strains. HR-inducing protein(s) from a virulent strain separated from the majority of other proteins on DEAE cellulose at 250 to 300 mM NaCl. Ammonium sulfate-precipitated proteins from a virulent strain produced a necrotic reaction at a total protein concentration of 18 mug/ml, whereas those from a nonpathogenic strain did not, even at a concentration of 180 mug/ml. We conclude that virulent strains of C. michiganensis subsp. sepedonicus elicit a typical HR in tobacco and secrete proteinaceous elicitor(s) of the nonhost HR.

Two Genomic Regions Involved in Catechol Siderophore Production by Erwinia carotovora.

Two regions involved in catechol biosynthesis (cbs) of Erwinia carotovora W3C105 were cloned by functional complementation of Escherichia coli mutants that were deficient in the biosynthesis of the catechol siderophore enterobactin (ent). A 4.3-kb region of genomic DNA of E. carotovora complemented the entB402 mutation of E. coli. A second genomic region of 12.8 kb complemented entD, entC147, entE405, and entA403 mutations of E. coli. Although functions encoded by catechol biosynthesis genes (cbsA, cbsB, cbsC, cbsD, and cbsE) of E. carotovora were interchangeable with those encoded by corresponding enterobactin biosynthesis genes (entA, entB, entC, entD, and entE), only cbsE hybridized to its functional counterpart (entE) in E. coli. The cbsEA region of E. carotovora W3C105 hybridized to genomic DNA of 21 diverse strains of E. carotovora but did not hybridize to that of a chrysobactin-producing strain of Erwinia chrysanthemi. Strains of E. carotovora fell into nine groups on the basis of sizes of restriction fragments that hybridized to the cbsEA region, indicating that catechol biosynthesis genes were highly polymorphic among strains of E. carotovora.

Cloning and Characterization of Aerobactin Biosynthesis Genes of the Biological Control Agent Enterobacter cloacae.

Five strains of Enterobacter cloacae that are biological control agents of Pythium damping-off diseases produced the hydroxamate siderophore aerobactin under iron-limiting conditions. Genes determining aerobactin biosynthesis of the biocontrol strain E. cloacae EcCT-501 were localized to a 12.3-kb region, which conferred aerobactin production to Escherichia coli DH5alpha. The aerobactin biosynthesis genes of E. cloacae hybridized to those of the pColV-K30 plasmid of E. coli, but restriction patterns of the aerobactin regions of pColV-K30 and E. cloacae differed. A derivative strain with a deletion in the aerobactin biosynthesis locus was as effective as strain EcCT-501 in biological control of Pythium damping-off of cucumber. Thus, aerobactin production did not contribute significantly to the biological control activity of EcCT-501 under the conditions of this study.

High-affinity iron uptake systems present in Erwinia carotovora subsp. carotovora include the hydroxamate siderophore aerobactin.

The phytopathogenic bacterium Erwinia carotovora subsp. carotovora W3C105 produced the hydroxamate siderophore aerobactin under iron-limiting conditions. A survey of 22 diverse strains of E. carotovora revealed that strain W3C105 alone produced aerobactin. The ferric-aerobactin receptor of strain W3C105 was an 80-kDa protein, identified by immunoblots of Sarkosyl-soluble proteins obtained from E. carotovora cells grown in iron-depleted medium and probed with antiserum raised against the 74-kDa ferric-aerobactin receptor encoded by the pColV-K30 plasmid of Escherichia coli. Genes determining aerobactin biosynthesis and uptake were localized to an 11.3-kb EcoRI-HindIII chromosomal fragment of strain W3C105. A 10-kb subclone of the fragment conferred on E. coli DH5 alpha both aerobactin biosynthesis and uptake, determined by cloacin DF13 sensitivity, the presence of the 80-kDa receptor protein, and iron-independent growth of E. coli clones. The aerobactin biosynthesis genes of E. carotovora W3C105 hybridized to those of the pColV-K30 plasmid of E. coli, but the restriction patterns of the aerobactin regions of E. coli and E. carotovora differed. Although the aerobactin region of enteric bacteria is commonly flanked by IS1-like sequences, IS1 sequences were not detected in the genomic DNA or the cloned aerobactin region of E. carotovora. E. coli DH5 alpha cells harboring cloned aerobactin biosynthesis genes from E. carotovora W3C105 produced greater quantities of aerobactin and the 80-kDa ferric-aerobactin receptor when grown in iron-limited than in iron-replete medium. Strain W3C105 grew on an iron-limited medium, whereas derivatives that lacked a functional aerobactin iron acquisition system did not grow on the medium. These results provide evidence for the occurrence and heterogeneity of aerobactin as a high-affinity iron uptake system of both clinical and phytopathogenic species of the Enterobacteriaceae. Although future studies may reveal a role for aerobactin in the virulence or ecology of strain W3C105, a functional aerobactin iron acquisition system is not necessary for the pathogenicity of E. carotovora.