Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa.

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.

Genetic characteristics of Borrelia coriaceae isolates from the soft tick Ornithodoros coriaceus (Acari: Argasidae).

Two Borrelia isolates (CA434 and CA435) cultured from the soft tick Ornithodoros coriaceus were analyzed by contour-clamped homogeneous electric field gel electrophoresis of unrestricted and ApaI-restricted DNA, standard electrophoresis of BamHI- and HindIII-restricted DNA, Southern hybridization, restriction fragment length polymorphism and sequencing of the 16S rRNA gene, and amplification of the 5S-23S intergenic spacer region. These isolates were compared with Borrelia coriaceae type strain Co53, B. burgdorferi sensu stricto strain CA4, and the relapsing-fever spirochete B. parkeri (undesignated). The 16S rRNA region of CA434 and CA435 differed from that of B. coriaceae type strain Co53 by the presence of 1 base (C) at position 367 (GenBank accession no. U42286). The linear plasmid profile of CA434 was similar to that of Co53, and the ApaI, BamHI, and HindIII restriction fingerprints of the total cellular DNA of CA434 and Co53 were similar. In contrast, CA435 differed somewhat from CA434 and Co53, which demonstrates that B. coriaceae is genetically diverse. Southern hybridization showed that the DNAs of CA434 and CA435 hybridized strongly with the digoxigenin-labeled DNA of Co53. Low homology was found between the DNA of Co53 and that of B. parkeri. The 16S rRNA sequence of B. parkeri was identical to previously published results for B. parkeri strain M3001 (GenBank accession number U42296). CA434 and CA435 represent only the second and third isolates of B. coriaceae obtained from any source since its initial isolation from an O. coriaceus tick in 1985. All three B. coriaceae isolates were derived from adult ticks collected from the same locality in northwestern California. Difficulties encountered in detecting B. coriaceae in, and isolating this spirochete from, the tissues of O. coriaceus are discussed. The lack of concordance between different detection or isolation methods suggests that reliance upon a single technique may grossly underestimate the true prevalence of spirochetal infection in wild-caught O. coriaceus ticks.

Life history of Ixodes (Ixodes) jellisoni (Acari: Ixodidae) and its vector competence for Borrelia burgdorferi sensu lato.

Ixodes (Ixodes) jellisoni Cooley & Kohls, a nonhuman biting and little known tick, is one of 4 members of the I. ricinus complex in the United States. A localized population of I. jellisoni inhabiting a grassland biotope in Mendocino County, CA, was studied from 1993 to 1997. Rodent trapping in all seasons revealed that the only host of both immature and adult I. jellisoni was the heteromyid rodent Dipodomys californicus Merriam. Field investigations suggested that I. jellisoni is nidicolous in habit, and laboratory findings demonstrated that it reproduces parthenogenetically. Known parthenogenetic females (n = 4) produced an average of 530 eggs of which 74% hatched, which was comparable to the fecundity and fertility of wild-caught females (n = 8). After the transstadial molt, 57 F1 or F2 nymphs derived from 2 wild-caught or 4 laboratory-reared, unmated females produced only females. Ixodes jellisoni males were not found on 112 wild-caught D. californicus individuals that were captured an average of 2 times. Collectively, these findings suggest that I. jellisoni may be obligatorily parthenogenetic. Borrelial isolates were obtained from 85% of 58 D. californicus and 33% of 21 I. jellisoni females removed from this rodent. None of the 7 infected female ticks passed borreliae ovarially to its F1 larval progeny. Eight D. californicus and 5 I. jellisoni-derived isolates that were genetically characterized belonged to 2 restriction pattern groups of Borrelia burgdorferi s.l. Neither restriction pattern group has been assigned to a particular genospecies yet. After placement on naturally infected D. californicus, noninfected larval ticks acquired and transstadially passed spirochetes as efficiently as (group 1 borreliae) or 6 times more efficiently (group 2 borreliae) than Ixodes pacificus Cooley & Kohls. As few as 1-4 infected I. jellisoni nymphs were capable of transmitting group 1 or group 2 borreliae to naive D. californicus. We conclude that I. jellisoni is a competent vector of both restriction fragment groups when D. californicus is used as the animal model.

Causal Role of Xylella fastidiosa in Oleander Leaf Scorch Disease.

ABSTRACT A lethal leaf scorch disease of oleander (Nerium oleander) appeared in southern California in 1993. A bacterium, Xylella fastidiosa, was detected by culturing, enzyme-linked immunoassay, and polymerase chain reaction in most symptomatic plants but not in symptomless plants or negative controls. Inoculating oleanders mechanically with X. fastidiosa cultures from diseased oleanders caused oleander leaf scorch (OLS) disease. The bacterium was reisolated from inoculated plants that became diseased. Three species of xylem sap-feeding leafhoppers transmitted the bacterium from oleander to oleander. The bacterium multiplied, moved systemically, and caused wilting in Madagascar periwinkle (Catharanthus rosea) and leaf scorch in periwinkle (Vinca major) in a greenhouse after inoculation with needle puncture. No bacterium was reisolated from grapevine (Vitis vinifera), peach (Prunus persica), olive (Olea europaea), California blackberry (Rubus ursinus), or valley oak (Quercus lobata) mechanically inoculated with OLS strains of X. fastidiosa. A 500-bp sequence of the 16S-23S ribosomal intergenic region of oleander strains showed 99.2% identity with Pierce's disease strains, 98.4% identity with oak leaf scorch strains, and 98.6% identity with phony peach, plum leaf scald, and almond leaf scorch strains.

Expanded diversity among Californian borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127).

Up to now, the only species in the complex Borrelia burgdorferi sensu lato known to cause Lyme borreliosis in the United States has been B. burgdorferi sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of B. burgdorferi sensu lato group DN127, we analyzed the nucleotide sequences of the rrf-rrl intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American B. burgdorferi sensu stricto strains (6 from California). The spacer region sequences from the entire B. burgdorferi sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B. andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from B. burgdorferi sensu stricto. Within this cluster, the deep branches expressing the distances between the rrf-rrl sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by B. burgdorferi sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the rrs sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American B. burgdorferi sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species, B. bissettii sp. nov., and that other related but distinct strains, which require further characterization, be referred to as Borrelia spp.

Analysis of Expression of a Phenazine Biosynthesis Locus of Pseudomonas aureofaciens PGS12 on Seeds with a Mutant Carrying a Phenazine Biosynthesis Locus-Ice Nucleation Reporter Gene Fusion.

A derivative of Pseudomonas aureofaciens PGS12 expressing a promoterless ice nucleation gene under the control of a phenazine biosynthesis locus was used to study the expression of a phenazine antibiotic locus (Phz) during bacterial seed colonization. Seeds of various plants were inoculated with wild-type PGS12 and a PGS12 ice nucleation-active phz:inaZ marker exchange derivative and planted in soil, and the expression of the reporter gene was monitored at different intervals for 48 h during seed germination. phz gene expression was first detected 12 h after planting, and the expression increased during the next 36-h period. Significant differences in expression of bacterial populations on different seeds were measured at 48 h. The highest expression level was recorded for wheat seeds (one ice nucleus per 4,000 cells), and the lowest expression level was recorded for cotton seeds (one ice nucleus per 12,000,000 cells). These values indicate that a small proportion of bacteria in a seed population expressed phenazine biosynthesis. Reporter gene expression levels and populations on individual seeds in a sample were lognormally distributed. There was greater variability in reporter gene expression than in population size among individual seeds in a sample. Expression on sugar beet and radish seeds was not affected by different inoculum levels or soil matric potentials of -10 and -40 J/kg; only small differences in expression on wheat and sugar beet seeds were detected when the seeds were planted in various soils. It is suggested that the nutrient level in seed exudates is the primary reason for the differences observed among seeds. The lognormal distribution of phenazine expression on seeds and the timing and difference in expression of phenazine biosynthesis on seeds have implications for the potential efficacy of biocontrol microorganisms against plant pathogens.

Cloning of a phenazine biosynthetic locus of Pseudomonas aureofaciens PGS12 and analysis of its expression in vitro with the ice nucleation reporter gene.

Pseudomonas aureofaciens PGS12 produces three phenazine antibiotics, in addition to siderophores, hydrogen cyanide, pyrrolnitrin, and indoleacetic acid. Tn5-259.7 transposon mutagenesis was carried out to identify and clone a chromosomal locus involved in phenazine biosynthesis. Three classes of mutants were obtained: mutants deficient in phenazine production (Phz-), mutants deficient in hydrogen cyanide production (HCN-), and mutants deficient in the production of both compounds. EcoRI DNA fragments that contained the transposon and flanking regions were cloned from three mutants with single-transposon insertions, one from each phenotypic class. Phenazine and hydrogen cyanide production was restored by complementation of Phz- or HCN- mutants with selected cosmids from a PGS12 genomic library. No cosmids that complemented the doubly deficient Phz-HCN- mutant were obtained. A promoterless ice nucleation reporter gene was inserted in a phenazine biosynthetic locus by Tn3-spice transposon mutagenesis of a cosmid which complemented a phenazine-minus mutant. Reporter gene fusions that expressed the ice nucleation phenotype and no longer complemented phenazine production were introduced into the PGS12 chromosome by marker exchange. The expression of this locus was then monitored under different culture conditions. Expression decreased at pH levels below 7, and it was not affected by iron. Shikimic acid and phenylalanine favored higher expression levels. Expression was reduced in media with low substrate concentrations, indicating the importance of nutrient availability.

Pseudomonas flavescens sp. nov., isolated from walnut blight cankers.

Two pseudomonad strains that produce a yellow cellular pigment, in addition to a diffusible fluorescent pigment on Kings medium B, were isolated from cankers on walnut trees. Biochemical properties, such as a positive oxidase reaction, a negative arginine dihydrolase reaction, and the production of a fluorescent pigment, in addition to the results of an extensive nutritional characterization study and DNA-DNA hybridization experiments, indicated that these strains belong to a new Pseudomonas rRNA group I species. This conclusion was supported by the results of a determination of the sequence of the PCR-amplified 16S rRNA gene and a comparison with the 16S rRNA genes of other bacterial species. The genomic DNAs of the strains had a base composition of 63 mol% G+C. The name Pseudomonas flavescens sp. nov. is proposed. Strain B62 (= NCPPB 3063) is the type strain of the species.

Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase.

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 of X. campestris pv. juglandis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains of X. campestris pv. juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA, CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.

Use of an Exotic Carbon Source To Selectively Increase Metabolic Activity and Growth of Pseudomonas putida in Soil.

Respiration and growth of Pseudomonas putida PpG7, containing catabolic plasmid NAH7, was determined in three agricultural field soils amended with the carbon source salicylate. The addition of salicylate to soil significantly increased the population of PpG7. However, there was a lack of relationship between microbial numbers and activity as determined by evolution of CO(2). In soils containing 30 to 1,500 mug of salicylate per g, metabolic activities of PpG7 peaked between 18 and 42 h and population densities increased approximately 10-to 10-fold. However, the metabolic activity of PpG7 rapidly declined after salicylate was utilized, whereas peak population densities were maintained for the duration of the experiments (5 to 7 days). Thus, elevated population densities of PpG7 were represented by inactive cells. Soil type had only minor effects on respiration rates or growth curves of PpG7 when amended with comparable concentrations of salicylate. Respiration and growth rates were optimal at concentrations between 300 and 1,000 mug of salicylate per g in the test soils. At 1,500 to 2,500 mug/g, respiration and growth of PpG7 were initially suppressed, but after a short lag time both attained levels similar to or greater than those resulting from the use of lower concentrations of salicylate. The culturing of PpG7 on a salicylate-amended medium to induce salicylate-degradative enzymes did not affect the lag time before utilization of salicylate in soil. Although PpG7 competed well with fungi for the substrate, suppression of fungal populations with cycloheximide resulted in significantly increased population densities of PpG7 in two of three soils amended with salicylate. The beneficial activities of bacteria in soil are discussed in relation to population density, population metabolic activity, and selective carbon source utilization.

Enhancement of Population Densities of Pseudomonas putida PpG7 in Agricultural Ecosystems by Selective Feeding with the Carbon Source Salicylate.

Sodium salicylate (1,000 mug/ml) was delivered through a drip irrigation system to agricultural field soils planted to tomato and infested with Pseudomonas putida PpG7, the host of the salicylate catabolic plasmid NAH7. In nonfumigated soils infested with approximately 10 CFU of PpG7 per g in the top 30 cm, population densities were increased up to 112-fold within 14 days of the initial application of salicylate compared with the densities in the respective nonamended soils. Mean season-long population densities of PpG7 in the top 30 cm of soil were significantly increased (P < 0.01) from 216 CFU/g in nonamended soils to 1,370 CFU/g in salicylate-amended soils. In the respective rhizosphere soils, mean population densities of PpG7 were significantly increased (P < 0.01) from 92 to 2,066 CFU/cm of root. Soil fumigation interacted (P < 0.01) with salicylate amendment and further increased the mean population densities of PpG7 in nonrhizosphere soil by an additional 5,689 CFU/g of soil. This fumigation effect was not detected in rhizosphere soils. The effect of salicylate in increasing population densities of PpG7 in soil also was affected by inoculum level, field site, and soil depth. Proportionate differences were greater in soils infested with approximately 10 CFU of PpG7 per g than in comparable soils infested with 10 CFU/g. In low-inoculum soils, increases from salicylate amendments were 26- and 29-fold in rhizosphere and nonrhizosphere soils, respectively, and in high-inoculum soils, the respective increases were 5.6- and 5-fold. No increases of fungi able to utilize salicylate were detected in soils amended with salicylate. However, soil fumigation with metham-sodium significantly reduced (P < 0.01) population densities of fungal salicylate utilizers in rhizosphere and nonrhizosphere soils.

Enhanced growth and activity of a biocontrol bacterium genetically engineered to utilize salicylate.

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 mug/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 x 10 CFU/g to approximately 3 x 10 CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 x 10 CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 mug of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10 CFU/g of soil by 48 h of incubation, with the fastest increase at 100 mug/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 mug/g; at 1,000 mug/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 mug of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.

Broad-Host-Range Agrocin of Agrobacterium tumefaciens.

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.

Mutagenesis by insertion of drug resistance transposon Tn7 into a vibrio species.

A halotolerant, collagenolytic strain of Vibrio sp. was conjugated with an Escherichia coli strain carrying plasmid RP4. The plasmid was transferred to and maintained in the Vibrio and could be subsequently transferred in matings to suitably marked stains of the same species. After conjugation with an E. coli carrying the cointegrate plasmid RP4::Mu cts61::Tn7, Vibrio transconjugants were selected that carried Tn7 inserted into the bacterial chromosome. A large proportion of these transconjugants were auxotrophic, showing that plasmid suicide by Mu can be used to isolate Tn7-derived mutants in Vibrio. Approximately half of the auxotrophs isolate Tn7-derived mutants in Vibrio. Approximately half of the auxotrophs isolated were ilv mutants, all of which exhibited the same phenotype. Thus, although Tn7 insertion can induce auxotrophy, including trp, thy, his and ura, in Vibrio, there does appear to be a hot spot for integration in the ilv operon.