Genetic diversity among Curtobacterium flaccumfaciens pv. flaccumfaciens populations in the American high plains.

Curtobacterium flaccumfaciens pv. flaccumfaciens is a Gram-positive bacterium and has reemerged as an incitant of bacterial wilt in common (dry, edible) beans in western Nebraska, eastern Colorado, and southeastern Wyoming. Curtobacterium flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically and is represented by several different pathogen color variants. The population structure of 67 strains collected between 1957 and 2009, including some isolated from alternate hosts, was determined with 3 molecular typing techniques: amplified fragment length polymorphism (AFLP), repetitive extragenic palindromic polymerase chain reaction (rep-PCR), and pulsed-field gel electrophoresis (PFGE). All 3 typing techniques showed a great degree of population heterogeneity, but they were not congruent in cluster analysis of the C. flaccumfaciens pv. flaccumfaciens populations. Cluster analysis of a composite data set (AFLP, PFGE, and rep-PCR) using averages from all experiments yielded 2 distinct groups: cluster A included strains with colonies of yellow, orange, and pink pigments, and cluster B had strains of only yellow pigment. Strains producing purple extracellular pigment were assigned to both clusters. Thus, C. flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically.

Genetic diversity and population structure of Clavibacter michiganensis subsp. nebraskensis.

Clavibacter michiganensis subsp. nebraskensis (CMN) is a gram-positive bacterium and an incitant of Goss's bacterial wilt and leaf blight or "leaf freckles" in corn. A population structure of a wide temporal and geographic collection of CMN strains (n = 131), originating between 1969 and 2009, was determined using amplified fragment length polymorphism (AFLP) analysis and repetitive DNA sequence-based BOX-PCR. Analysis of the composite data set of AFLP and BOX-PCR fingerprints revealed two groups with a 60% cutoff similarity: a major group A (n = 118 strains) and a minor group B (n = 13 strains). The clustering in both groups was not correlated with strain pathogenicity. Group A contained two clusters, A1 (n = 78) and A2 (n = 40), with a linkage of 75%. Group A strains did not show any correlation with historical, geographical, morphological, or physiological properties of the strains. Group B was very heterogeneous and eight out of nine clusters were represented by a single strain. The mean similarity between clusters in group B varied from 13% to 63%. All strains in group B were isolated after 1999. The percentage of group B strains among all strains isolated after 1999 (n = 69) was 18.8%. Implications of the findings are discussed.

Differential Pathogenicity of Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans Strains on Bean Genotypes with Common Blight Resistance.

Both the common bacterial blight (CBB) pathogen (Xanthomonas campestris pv. phaseoli) and X. fuscans subsp. fuscans, agent of fuscous blight, cause indistinguishable symptoms in common bean, Phaseolus vulgaris. Yield losses can exceed 40%. Lack of information about the specificity between X. campestris pv. phaseoli strains and major quantitative trait loci (QTL) or alleles conferring resistance makes the task of identifying genetic changes in host-pathogen interactions and the grouping of bacterial strains difficult. This, in turn, affects the choice of pathogen isolates used for germplasm screening and complicates breeding for CBB resistance. Common bean host genotypes carrying various sources and levels of resistance to CBB were screened with 69 X. campestris pv. phaseoli and 15 X. fuscans subsp. fuscans strains from around the world. Differential pathogenicity of the CBB pathogen was identified on the 12 selected bean genotypes. The X. fuscans subsp. fuscans strains showed greater pathogenicity than X. campestris pv. phaseoli strains having the same origin. African strains were most pathogenic. The largest variation in pathogenicity came from X. campestris pv. phaseoli strains that originated in Caribbean and South American countries. Pathogenic variation was greater within X. campestris pv. phaseoli than within X. fuscans subsp. fuscans strains. Implications for breeding for CBB resistance are discussed.

Genetic Diversity in Populations of Xanthomonas campestris pv. campestris in Cruciferous Weeds in Central Coastal California.

ABSTRACT Xanthomonas campestris pv. campestris (X. campestris) infects a large number of cruciferous plants, including weeds. California has one of the largest and most diverse populations of wild cruciferous plants in the world. Although considerable information is available on the genetic diversity of X. campestris in commercial crop plants, nothing is known about the diversity in strains infecting weeds. To assess the genetic diversity among strains of X. campestris in weeds in noncultivated and cultivated areas, strains of the pathogen were isolated from populations of cruciferous weeds growing in coastal valley crop-production sites and from remote nonproduction sites along the California central coast. Results of fingerprinting over 68 strains using amplified fragment length polymorphism along with representative strains by sequence analysis showed the presence of seven genotypes. Genotypes A and B were limited to coastal sites; genotypes C, D, and E were from inland cultivated sites; and genotypes F and G were present in both coastal noncultivated and inland cultivated sites. Crop strains were grouped outside any weed strain group and were separated from the weed strains and other pathovars of X. campestris. These results revealed, for the first time, that strains of X. campestris present in noncultivated coastal weed populations generally were unique to a site and genetically distinct from strains present in populations of weeds in crop-production areas located nearby.

An assessment model for rating high-threat crop pathogens.

ABSTRACT Natural, accidental, and deliberate introductions of nonindigenous crop pathogens have become increasingly recognized as threats to the U.S. economy. Given the large number of pathogens that could be introduced, development of rapid detection methods and control strategies for every potential agent would be extremely difficult and costly. Thus, to ensure the most effective direction of resources a list of high-threat pathogens is needed. We address development of a pathogen threat assessment model based on the analytic hierarchy process (AHP) that can be applied world-wide, using the United States as an illustrative example. Previously, the AHP has been shown to work well for strategic planning and risk assessment. Using the collective knowledge of subject matter expert panels incorporated into commercial decision-making software, 17 biological and economic criteria were determined and given weights for assessing the threat of accidental or deliberately introduced pathogens. The rating model can be applied by experts on particular crops to develop threat lists, especially those of high priority, based on the current knowledge of individual diseases.

Genetic Characterization and Diversity of Rathayibacter toxicus.

ABSTRACT Rathayibacter toxicus is a nematode-vectored gram-positive bacterium responsible for a gumming disease of grasses and production of a highly potent animal and human toxin that is often fatal to livestock and has a history of occurring in unexpected circumstances. DNA of 22 strains of R. toxicus from Australia were characterized using amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE). AFLP analysis grouped the 22 strains into three genetic clusters that correspond to their geographic origin. The mean similarity between the three clusters was 85 to 86%. PFGE analysis generated three different banding patterns that enabled typing the strains into three genotypic groups corresponding to the same AFLP clusters. The similarity coefficient was 63 to 81% for XbaI and 79 to 84% for SpeI. AFLP and PFGE analyses exhibited an analogous level of discriminatory power and produced congruent results. PFGE analysis indicated that the R. toxicus genome was represented by a single linear chromosome, estimated to be 2.214 to 2.301 Mb. No plasmids were detected.

New Outbreaks of Bacterial Wilt of Dry Bean in Nebraska Observed from Field Infections.

Bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens was one of the more problematic diseases of dry bean (Phaseolus vulgaris L.) throughout the irrigated High Plains (Colorado, Nebraska, and Wyoming) in the 1960s and early 1970s, but has not been observed since that time. However, in August of 2003, plants exhibiting wilting and irregular, interveinal necrotic foliar lesions surrounded by a bright yellow border were found in three dry bean fields (market class Great Northern) in Scotts Bluff County, Nebraska. During 2004, plants exhibiting identical symptoms were additionally found occurring in more than 40 dry bean fields in western Nebraska. Affected fields were planted with dry bean from multiple market classes and seed sources, including yellow bean, Great Northern bean, and pinto bean, and incidence varied from trace levels to 80 to 90%. Isolations were made from leaf and stem tissues and seeds collected after harvest from infected plants, and all yielded slow-growing, creamy yellow or orange, fluidal colonies on nutrient broth-yeast extract medium. The bacterium was identified as C. flaccumfaciens pv. flaccumfaciens based on cell morphology (coryneformshaped motile rods), positive Gram stain and KOH reactions, fatty acid profiles, and BIOLOG (Hayward, CA) identifications. Great Northern (cv. Orion) plants were inoculated by bacterial suspensions (5 × 107 CFU/ml) injected into leaf axils adjacent to the first fully expanded trifoliolate and were incubated in the greenhouse under ambient conditions fluctuating between 24 and 35°C. Wilting symptoms developed 7 days after inoculation with foliar necrosis and yellowing symptoms appearing after 24 days. Identical bacterial colonies were reisolated from inoculated tissues, completing Koch's postulates. Although recent reports of wilt have been made in North Dakota (2) and western Canada (1) in 1995 and 2002, respectively, they were based only on the presence of discolored seeds observed in dockage from processing plants after harvest. To our knowledge, this report represents the first widespread observations of bacterial wilt from field infections in Nebraska in more than 30 years. References: (1) J. R. Venette et al. Plant Dis. 79:966, 1995. (2) T. F. Hsieh et al. Plant Dis: 86:1275, 2002.

Evaluation of proposed amended names of several pseudomonads and xanthomonads and recommendations.

ABSTRACT In 1980, over 90% of all plant-pathogenic pseudomonads and xanthomonads were lumped into Pseudomonas syringae and Xanthomonas campestris, respectively, as pathovars. The term "pathovar" was created to preserve the name of plant pathogens, but has no official standing in nomenclature. Proposals to elevate and rename several pathovars of the genera Pseudomonas and Xanthomonas to the rank of species has caused great confusion in the literature. We believe the following changes have merit and expect to adopt them for publication in a future American Phytopathological Society Laboratory Guide for Identification of Plant Pathogenic Bacteria. Upon review of published data and the Rules of The International Code of Nomenclature of Bacteria, we make the following recommendations. We reject the proposal to change the name of P. syringae pvs. phaseolicola and glycinea to P. savastanoi pvs. phaseolicola and glycinea, respectively, because both pathogens are easily differentiated phenotypically from pv. savastanoi and convincing genetic data to support such a change are lacking. We accept the elevation of P. syringae pv. savastanoi to the rank of species. We accept the reinstatement of X. oryzae to the rank of species with the inclusion of X. oryzicola as a pathovar of X. oryzae and we accept the species X. populi. We agree with the elevation of the pvs. cassavae, cucurbitae, hyacinthi, pisi, and translucens to the rank of species but not pvs. melonis, theicola, and vesicatoria type B. We recommend that all type A X. vesicatoria be retained as X. campestris pv. vesicatoria and all type B X. vesicatoria be named X. exitiosa. We reject the newly proposed epithets arboricola, bromi, codiaei (poinsettiicola type B), hortorum, sacchari, and vasicola and the transfer of many pathovars of X. campestris to X. axonopodis. The proposed pathovars of X. axonopodis should be retained as pathovars of X. campestris.

Expression of Human Lactoferrin cDNA Confers Resistance to Ralstonia solanacearum in Transgenic Tobacco Plants.

ABSTRACT A construct containing a human lactoferrin cDNA was used to transform tobacco (Nicotiana tabacum) using an Agrobacterium-mediated DNA-transfer system to express this human protein in transgenic plants. Transformants were analyzed by Southern, Northern, and Western blots to determine integration of the cDNA into the plant genome and lactoferrin gene expression levels. Most transgenic plants demonstrated significant delays of bacterial wilt symptoms when inoculated with the bacterial pathogen Ralstonia solanacearum. Quantification of the expressed lactoferrin protein by enzyme-linked immunosorbent assay in transgenic plants indicated a significant positive relationship between lactoferrin gene expression levels and levels of disease resistance. Incorporation of the lactoferrin gene into crop plants may enhance resistance to other phytopathogenic bacteria as well.

Differential Effects of Lithium Chloride on In Vitro Growth of Clavibacter michiganense subsp. nebraskense Depending upon Inoculum Source.

The bacterium Clavibacter michiganense subsp. nebraskense (Corynebacterium michiganense subsp. nebraskense) was grown in broth cultures and inoculated into corn plants. The plating efficiency of cells from broth cultures was essentially the same on nutrient broth-yeast extract and the semiselective medium for this bacterium, CNS. However, when cells were isolated from Goss bacterial wilt- and blight-infected corn, very few were recovered on CNS compared with the amount recovered on nutrient broth-yeast extract agar. When lithium chloride was omitted from the CNS, recoveries from infected corn were nearly the same as on nutrient broth-yeast extract agar. No other ingredient of CNS was inhibitory, nor did substitution of other salts for lithium chloride cause equal inhibition. The amount of inhibition was proportional to lithium chloride concentration. The inhibition by lithium chloride occurred with several strains of the bacterium isolated from one corn cultivar and with one of the strains recovered from three different cultivars of infected corn.

Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains.

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.

Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora.

Erwinia amylovora infected with bacteriophage ERA103 produced an enzyme which degraded the extracellular polysaccharide of noninfected cells. The depolymerase enzyme was purified 15-fold by a procedure which included ammonium sulfate precipitation, ultracentrifugation, CM-Sephadex batchwise separation, Sephadex G-50 column chromatography, and Sephacryl S-200 column chromatography. The enzyme had a molecular weight of approximately 21,000 and a pH optimum of 6.0. Activity was enhanced by supplements of 2-mercaptoethanol or dithiothreitol.

Conjugative Plasmid in Corynebacterium flaccumfaciens subsp. oortii That Confers Resistance to Arsenite, Arsenate, and Antimony(III).

Gene transfer systems for phytopathogenic corynebacteria have not been reported previously. In this paper we describe a conjugative 46-megadalton plasmid (pDG101) found in Corynebacterium flaccumfaciens subsp. oortii CO101 that mediates resistance to arsenite, arsenate, and antimony(III). Transfer of the plasmid from CO101 to four other strains from the C. flaccumfaciens group occurred between cells immobilized on nitrocellulose filters or on agar surfaces. Transconjugant strains expressed the same levels of metal resistance as the donor strain and were able to act as donor strains in subsequent matings. The physical presence of the plasmid was detected by agarose gel electrophoresis. Arsenite-sensitive derivatives of the donor and transconjugant strains were obtained after heat treatment; these were cured of pDG101.

Bacteriocin production by Pseudomonas syringae PsW-1 in plant tissue.

The production and activity of syringacin W-1, a particulate bacteriocin made by Pseudomonas syringae PsW-1, was studied in plant tissue. The bacteriocin is rod shaped, approximately 20 nm wide and 75 nm long, and composed of an outer sheath and inner core. Both the producing strain, PsW-1, and a sensitive strain, 16, grew within red kidney bean stems. Strains PsW-1 and 16, or mutants derived from them, were injected into bean stems singly or in mixtures. All singly inoculated strains grew well. However, when the bacteriocin-producing strain was co-inoculated with the sensitive strain, the latter grew poorly, if at all. This was not due to competition for available nutrients, since the sensitive strain grew as well in the presence of a bacteriocin-nonproducing mutant as it did alone. Also, a bacteriocin-resistant mutant grew as well in the presence of a bacteriocin-nonproducing mutant as it did alone. Also, a bacteriocin-resistant mutant grew as well in the presence of the producing strain as it did alone. Bacteriocin activity and particles were recovered from infected plant tissue.