Angular Leaf Spot of Cucurbits is Associated With Genetically Diverse Pseudomonas syringae Strains.

Angular leaf spot of cucurbits is generally considered to be caused by Pseudomonas syringae pv. lachrymans. It has a worldwide distribution and has been observed to emerge sporadically under humid and wet conditions. Reports of multiple P. syringae pathovars associated with the disease and lack of molecular analysis has left the true diversity of populations in the United States unclear. In this study, we collected 27 P. syringae strains causing foliar lesions and blighting on watermelon, cantaloupe, and squash in Florida, Georgia, and California over several years. Strains were fluorescent on King's medium B agar and displayed the typical phenotypic and biochemical characteristics of P. syringae. P. syringae pv. lachrymans is a member of genomospecies 2. However, the genetic profiles obtained through both MLSA (gyrB, rpoD, gapA, and gltA) and BOX-PCR (BOXA1R) identified 26 of the P. syringae strains to be distributed among three clades within genomospecies 1, and phylogenetically distinct from genomospecies 2 member P. syringae pv. lachrymans. A novel MLSA haplotype of the pathogen common to all states and cucurbit hosts was identified. Considerable genetic diversity among P. syringae strains infecting cucurbits is associated with the same disease, and reflects the larger ecological diversity of P. syringae populations from genomospecies 1.

Agrobacterium larrymoorei sp. nov., a pathogen isolated from aerial tumours of Ficus benjamina.

Tumorigenic Agrobacterium strains isolated from tumours growing on pruned branches of Ficus benjamina have previously been shown to have unique opine metabolism and sufficient 16S rRNA sequence differences to suggest that they belong to a new species. DNA-DNA hybridization results confirmed that these strains represent a new species and Agrobacterium larrymoorei sp. nov. (type strain ATCC 51759T = CFBP 5473T = NCPPB 4096T) is proposed as the name for the species.

A New Bacterial Leaf Spot Disease of Broccolini, Caused by Pseudomonas syringae pathovar maculicola, in California.

In 1998, a new disease was detected on 3-week-old commercial broccolini (Brassica oleracea L. var. botrytis × B. alboglabra) transplants in a Salinas Valley, Monterey County, CA greenhouse. Initial symptoms were small (2 to 4 mm diameter) circular to angular, water-soaked spots. As the disease progressed, spots remained relatively small, but turned tan to brown. When diseased tissues were macerated and streaked on King's medium B, a blue-green fluorescent pseudomonad was consistently isolated. Strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices, but induced a hypersensitive reaction on tobacco (Nicotiana tabacum L. 'Turk'). Fatty acid methyl ester analysis (MIS-TSBA, version 4.10, MIDI Inc., Newark, DE) indicated that strains had a high similarity index (0.82 or higher) to Pseudomonas syringae, and GN (version 3.50, Biolog, Inc., Hayward, CA) profiles also identified strains as P. syringae. The bacterium associated with the disease, therefore, was identified as P. syringae van Hall. Pathogenicity was demonstrated by growing inoculum in nutrient broth shake cultures for 48 h, misting the broth cultures (1×106 CFU/ml) onto broccolini (cv. Aspabrock), and subjecting the plants to 48 h of high humidity. Control plants were misted with sterile nutrient broth. After 4 to 5 days in a greenhouse, leaf spot symptoms developed on all inoculated broccolini plants, and reisolated strains were characterized and found to be P. syringae. Control plants remained symptomless. The results of two sets of pathogenicity tests were the same. Repetitive sequence-based polymerase chain reaction using the BOXA1R primer resulted in identical banding patterns for the broccolini pathogen and for known isolates of P. syringae pv. maculicola from crucifers. In host range testing, P. syringae pv. maculicolawas pathogenic to broccolini plants. The broccolini isolates and P. syringae pv. maculicola isolates had the same pathogenicity results when crucifers and tomatoes were tested as hosts; broccoli and cauliflower (B. oleracea var. botrytis) were infected, and tomato results were variable. These tests suggest that the broccolini pathogen is the bacterial leaf spot pathogen, Pseudomonas syringae pv. maculicola, that occurs on broccoli and cauliflower transplants (1). To our knowledge, this is the first report of this pathogen causing a disease on commercially grown broccolini. Reference: (1) S. T. Koike et al. Plant Dis. 82:727, 1998.

Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions.

The taxonomy and evolutionary relationships among members of the genus Xanthomonas associated with tomato and pepper have been a matter of considerable controversy since their original description in 1921. These bacteria, which are a major affliction of tomato and pepper crops in warm and humid regions, were originally described as a single species, but subsequent research has shown the existence of at least two genetic groups differentiated by physiological, biochemical and pathological characteristics. This work synthesizes the findings from several approaches, including pathogenicity tests, enzymic activity, restriction fragment analysis of the entire genome, DNA-DNA hybridization and RNA sequence comparisons based on a 2097 base sequence comprising the 16S rRNA gene, the intergenic spacer located between the 16S and 23S rRNA genes and a small region of the 23S rRNA gene. Within the group of xanthomonads pathogenic on pepper and tomato four distinct phenotypic groups exist, of which three form distinct genomic species. These include Xanthomonas axonopodis pv. vesicatoria (A and C group), Xanthomonas vesicatoria (B group) and Xanthomonas gardneri (D group). On the basis of phenotypic and genotypic differences between A- and C-group strains, the C strains should be considered as a subspecies within Xanthomonas axonopodis pv. vesicatoria.

Stem Blight of Eustoma grandiflorum Caused by Sclerotium rolfsii.

During a 4-week period in May through June 1996, 15% of 50 mature lisianthus (Eustoma grandiflorum) 'Maurine Blue' and 'Maurine White' plants exhibited stem blight in a landscape planting in west-central Florida. Initial disease symptoms included stem necrosis at the soil line, and yellowing and tan discoloration of leaves. As blighting of the stem progressed, infected plants wilted and died. Symptomatic stem sections from three plants were surface-disinfested in 0.5% NaOCl and placed on acidified 25% potato dextrose agar (APDA). Sclerotium rolfsii Sacc. was isolated from all three diseased stems. Pathogenicity of each of three S. rolfsii isolates was confirmed using two lisianthus 'Flamenco Blue' plants grown in 10.2-cm-diameter plastic pots containing a peat-based medium. Sclerotia produced on APDA were sprinkled on the soil surface around each plant base; 50, 100, and 5 sclerotia from isolates A, B, and C, respectively, were used (isolate C grew more slowly and produced fewer sclerotia than either A or B). Two noninoculated lisianthus served as controls. Plants were maintained in a greenhouse at minimum and maximum temperatures of ≈24 and 35°C, respectively. Plants inoculated with sclerotia from isolates A and B developed blight symptoms within 6 days. One of two plants inoculated with isolate C developed blight symptoms within 17 days, and the other remained symptomless, as did the control plants. Infection by S. rolfsii was confirmed by reisolation from symptomatic tissue. This is the first report of stem blight of lisianthus caused by S. rolfsii.

Multiphasic analysis of xanthomonads causing bacterial spot disease on tomato and pepper in the Caribbean and central america: evidence for common lineages within and between countries.

ABSTRACT Four hundred thirty-three xanthomonad strains isolated from tomato or pepper plants from 32 different fields in four Caribbean and Central American countries were screened for the ability to hydrolyze starch and sodium polypectate and for resistance to copper and streptomycin. Of these, 95 representative strains were further characterized by various phnetic tests, and 63 of these strains were then analyzed by genomic fingerprinting. Most of the strains (>90%) were tolerant to copper. However, there was much more variability in sensitivity to streptomycin. All strains in Guadeloupe and 93% of the strains in Barbados were sensitive to streptomycin. The majority of strains were typical Xanthomonas campestris pv. vesicatoria group A strains. In Barbados, however, a unique group of strains was identified that was serologically similar to group A strains but was amylolytic. These strains were designated A1. The occurrence of X. campestris pv. vesicatoria group B strains in Central America was found to be limited to two fields in Costa Rica and one in Guatemala. No group B strains were identified in the Caribbean, in contrast to common occurrence in the central United States and in South America. T3 strains were not found in this study, despite the recent increase of such strains in Florida and Mexico. Unique strains from Costa Rica belonging to the X. gardneri group were identified. Little linkage was found among phenotypic and rep-polymerase chain reaction (rep-PCR) genomic fingerprinting profiles of the pathogens except at the species/pathovar level; strains displaying virtually identical fingerprint profiles were found to correspond to distinct races and vice versa. The rep-PCR genomic fingerprinting analyses suggest that certain lineages may have evolved or predominated in specific regions or specific countries.

Relatedness of strains of xanthomonas fragariae by restriction fragment length polymorphism, DNA-DNA reassociation, and fatty acid analyses

The levels of relatedness of strains of Xanthomonas fragariae collected over several years from locations in Canada and the United States were compared by determining fatty acid methyl ester profiles, restriction fragment length polymorphisms (RFLP) based on pulsed-field gel electrophoresis (PFGE) analysis, and DNA-DNA reassociation values. Based on qualitative and quantitative differences in fatty acid profiles, the strains were divided into nine groups and four groups by the MIDI "10% rule" and unweighted pair analysis, respectively. Restriction analysis of genomic DNA by PFGE with two endonucleases (XbaI and SpeI) revealed four distinct profiles. When a third endonuclease (VspI) was used, one group was divided into three subgroups. The profile of the American Type Culture Collection type strain differed from the profile of every other strain of X. fragariae. Considerable diversity was observed within X. fragariae, although the majority of the strains represented a clonal population. The four groups based on fatty acid profiles were similar to the four groups based on RFLP, but neither method related groups to the geographic origins of the strains. The DNA-DNA reassociation values were high for representative strains, providing evidence that all of the strains belong to the same species.

Diversity among xanthomonads pathogenic on pepper and tomato.

Xanthomonas campestris pv. vesicatoria, causal agent of bacterial spot of tomato and pepper, had been considered for nearly 70 years to be a relatively homogeneous organism. However, in the past decade this bacterium was determined to be composed of two genetically and phenotypically distinct groups. The two groups, designated A and B, were distinguished based on amylolytic activity, expression of unique protein bands, reaction on differential hosts (tomato races T1 and T2), reaction patterns with monoclonal antibodies, DNA restriction profiles, and DNA:DNA hybridization. The A and B groups were placed into X. axonopodis pv. vesicatoria and X. vesicatoria, respectively. A third group, designated C, was pathogenically (race T3) and serologically distinct from A and B strains, and formed unique DNA restriction profiles. DNA:DNA hybridization data suggest that C is distinct but related to A strains and may represent a subspecies of A. A final group, designated D, consisted of X. gardneri, an organism identified in Yugoslavia in 1957, and also found in Costa Rica. Group D was determined to be genetically distinct from strains within the other two groups; it represents a third Xanthomonas species pathogenic on tomato and pepper.

Evidence for the Preemptive Nature of Tomato Race 3 of Xanthomonas campestris pv. vesicatoria in Florida.

ABSTRACT Until recently, tomato race 1 (T1) of Xanthomonas campestris pv. vesicatoria was the only race causing bacterial spot of tomato in Florida. In 1991, tomato race 3 (T3) was first identified in 3 of 13 tomato production fields surveyed. By 1994, T3 was observed in 21 of 28 fields and was the only race identified in 14 fields. In field studies, tomato genotypes with resistance to either T1 or T3 or susceptibility to both were co-inoculated with strains of both races. Lesions on 10 plants in each of three replications for each genotype were sampled three times during the experiment; bacterial isolations were made from each lesion, and tomato race identifications were made for each strain. At the third sampling date, T3 was isolated from 97% of the lesions on the susceptible genotype Walter and the T1-resistant genotype Hawaii 7998, while T3 was isolated from 23% of the lesions and T1 from the remaining 77% on the T3-resistant genotypes PI 128216 and PI 126932. In surface population studies done in growth rooms, suspensions of T1 and T3 were applied alone and in combination to the leaf surfaces of susceptible and resistant genotypes. T1 populations were reduced more than 10-fold when applied in combination with T3, compared with populations that developed when T1 was applied alone. T3 populations were not affected when applied in combination with a T1 strain. In greenhouse studies with the T3-resistant genotype Hawaii 7981, disease was significantly reduced in plants inoculated with T3 in combination with T1, compared with plants inoculated with T1 alone. These results clearly demonstrate the competitive nature of T3 in the presence of T1 and help explain the emergence of T3 as a prevalent race in Florida.

Novel Ti plasmids in Agrobacterium strains isolated from fig tree and chrysanthemum tumors and their opinelike molecules.

Galls naturally induced on Fig and chrysanthemum plants by strains of Agrobacterium contained, in addition to other well-characterized opines such as nopaline, three tumor-specific opinelike molecules. These molecules were identified as deoxy-fructosyl-glutamine (dfg), deoxy-fructosyl-5-oxo-proline (dfop), and chrysopine (Chilton et al., unpublished). Strains isolated from Fig tree and chrysanthemum tumors harbored different and unrelated Ti plasmids as judged by hybridization with various vir and T-DNA probes. They also exhibited different opine-catabolic properties. The strains isolated from chrysanthemum plants (Chry strains) and Fig trees degraded chrysopine, but only the Chry strains used dfg and dfop. Remarkably, other strains of Agrobacterium catabolized these two molecules: dfg was degraded by most pathogenic and nonpathogenic Agrobacterium strains, and dfop by all Agrobacterium strains degrading the opine agropinic acid. These results have strong ecological and evolutionary inferences which fit previous speculation on the origin of opine-related functions.

A new Agrobacterium strain isolated from aerial tumors on Ficus benjamina L.

Crown gall tumors, collected from branches of 1-year-old weeping fig (Ficus benjamina L.) trees, yielded both tumorigenic and nonpathogenic agrobacteria. On the basis of classical diagnostic tests, the nonpathogenic strains were identified as Agrobacterium tumefaciens, whereas the tumorigenic strains could not be assigned to any of the known terrestrial Agrobacterium spp. The tumorigenic strains also differed from other members of the genus by producing more acid from mannitol. According to cluster analysis of carbon substrate oxidation (GN Microplate; Biolog, Inc.) and fatty acid content, the tumorigenic fig strains were distinct from strains of A. tumefaciens, Agrobacterium rhizogenes, Agrobacterium vitis, and Agrobacterium rubi. Furthermore, they had unusual opine metabolism, inducing tumors that synthesized nopaline and three recently discovered opines: chrysopine (d-lactone of N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-5-oxo-L-proline. The nonpathogenic A. tumefaciens strains present in the same tumors were unable to degrade any of the opines tested. The phylogenetic position of the tumorigenic fig strain AF3.10 was inferred from comparing its rrs (i.e., 16S rRNA gene) sequence with those from the type strains of Agrobacterium and Rhizobium species. The analysis showed that strain AF3.10 clustered with A. tumefaciens and A. rubi but not with A. vitis and was far removed from A. rhizogenes. However, the sequence was significantly different from those of A. tumefaciens and A. rubi to suggest that the tumorigenic fig strain may be a new Agrobacterium species that is as different from A. tumefaciens and A. rubi as these two species are from one another.

Correlative Association between Resident Plasmids and the Host Chromosome in a Diverse Agrobacterium Soil Population.

Soil samples collected from a fallow field which had not been cultivated for 5 years harbored a population of Agrobacterium spp. estimated at 3 x 10 CFU/g. Characterization of 72 strains selected from four different isolation media showed the presence of biovar 1 (56%) and bv. 2 (44%) strains. Pathogenicity assays on five different test plants revealed a high proportion (33%) of tumorigenic strains in the resident population. All tumorigenic strains belonged to bv. 1. Differentiation of the strains by restriction fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins, and utilization patterns of 95 carbon substrates (Biolog GN microplate) revealed a diversified bv. 1 population, composed of five distinct chromosomal backgrounds (chr A, C, D, E, and F), and a homogeneous bv. 2 population (chr B). chr A, B, C, and D were detected at similar levels throughout the study site. According to opine metabolism, pathogenicity, and agrocin sensitivity, chr A strains carried a nopaline Ti plasmid (pTi), whereas chr C strains had an octopine pTi. In addition, four of six nontumorigenic bv. 1 strains (two chr D, one chr E, and one chr F) had distinct and unusual opine catabolism patterns. chr B (bv. 2) strains were nonpathogenic and catabolized nopaline. Although agrocin sensitivity is a pTi-borne trait, 14 chr B strains were sensitive to agrocin 84, apparently harboring a defective nopaline pTi similar to pAtK84b. The other two chr B strains were agrocin resistant. The present analysis of chromosomal and plasmid phenotypes suggests that in this Agrobacterium soil population, there is a preferential association between the resident plasmids and their bacterial host.

Complementary methodologies to identify specific agrobacterium strains.

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.

Isolation of different agrobacterium biovars from a natural oak savanna and tallgrass prairie.

Populations of agrobacteria in excess of 10 CFU/g were recovered from 12 soil and root samples obtained from the Allison Savanna, Minn., a natural oak savanna and tallgrass prairie which has never been disturbed agriculturally. Of 126 strains picked randomly from selective media, 54 were identified as Agrobacterium spp. Biovar 2 strains predominated (35 of 54), but these strains were distributed into three phenotypically distinct subgroups. Of the remaining Agrobacterium strains, four were biovar 1-2, one was biovar 1, and none were biovar 3. The last 14 Agrobacterium strains formed a homogeneous group which differed biochemically from the hitherto reported biovars. Opine utilization (coded for by genes on the tumor-inducing plasmid in pathogenic Agrobacterium spp.) by these agrobacteria was limited to two biovar 2 strains. In contrast, 10 nonfluorescent gram-negative strains utilized either nopaline or octopine as the sole carbon and nitrogen source. There may be a need to reexamine the source and role of opines in the terrestrial environment because (i) all of these opine utilizers were isolated from an environment free of crown gall, the only known terrestrial source of opines, and (ii) 83% of the opine utilizers were not Agrobacterium spp.