Development of a Bio-PCR Protocol for the Detection of Xanthomonas arboricola pv. pruni.

A real-time SYBR Green I assay was developed and evaluated as a biological and enzymatic polymerase chain reaction (Bio-PCR) protocol for the detection of Xanthomonas arboricola pv. pruni. Suppression subtractive hybridization was used to generate a X. arboricola pv. pruni-specific subtracted DNA library, using X. arboricola pv. corylina as the driver strain. Primer pair 29F/R, designed from cloned sequence, showed no homology to GenBank sequences and amplified a 344-bp product in all X. arboricola pv. pruni isolates. Compared with other published X. arboricola pv. pruni primers, this primer pair was shown to be the only one capable of differentiating X. arboricola pv. pruni from all other X. arboricola pathovars. A real-time assay was developed and shown to be capable of detecting less than 10 CFU and 0.1 pg of DNA. Epiphytic bacteria isolated from plum tissue was used to further evaluate the specificity of the assay. A Bio-PCR protocol, developed for field evaluation, confirmed X. arboricola pv. pruni isolation from asymptomatic and symptomatic plum tissue over a 9-week period between host flowering and the first appearance of leaf and fruit symptoms in an orchard. Dilution plating enabled X. arboricola pv. pruni numbers to be quantified, providing supportive evidence for the usefulness of the Bio-PCR protocol in plant pathology and quarantine surveillance.

Stenotrophomonas and Lysobacter: ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology.

The exploration of new source materials and the use of alternative isolation and identification methods have led to rapid expansion in the knowledge of diversity; in Lysobacter, 11 new species having been described since 2005, and in Stenotrophomonas with six new species since 2000. The new species of Lysobacter, isolated by dilution and direct plating on standard media, differ in several key phenotypic properties from those obtained by enrichment on complex polysaccharides in the original description of the genus. Revision of the definition of the genus will be required. Both culture-dependent and culture-independent methods to assess community structure, in a variety of host and nonhost environments, have established that some species of Lysobacter are a dominant component of the microflora, where previously their presence had not been suspected. Culture-independent studies have generally not added new information on the occurrence and distribution of Stenotrophomonas maltophilia and other members of the genus, which are readily isolated on standard media from source materials. Lysobacter enzymogenes and Sten. maltophilia produce similar antibiotics and share some enzyme activities which, subject to safety considerations, may make them attractive candidates for use in biological control of plant diseases and of nematodes.

Plant-pathogenic bacteria as biological weapons - real threats?

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

Determination of self-absorption corrections for gamma analysis of environmental samples: comparing gamma-absorption curves and spiked matrix-matched samples.

Accurate determination of the massic activity of gamma-emitting radionuclides in environmental samples, particularly sediments and soils, cannot be achieved without taking into account sample self-absorption. The extent of self-absorption in the sample will depend on a number of factors including sample composition, density, sample size and gamma-ray energy. The preferred method for correcting for this effect is to use spiked or natural matrix reference materials that match each sample type to be analysed. However, for laboratories that must measure a wide variety of sample matrices this method is too costly and time-consuming. Another technique commonly used is to make direct gamma-ray transmission measurements for each sample. This method, while more practical, still requires a minimum of three measurements to be made for each sample analysed. A more convenient method is to prepare sets of gamma-absorption curves. This approach involves making a series of direct transmission measurements for samples of varying densities but similar type. Sets of matching samples, both spiked and unspiked, were prepared and density correction factors determined using the direct transmission method and the spiked sample approach. It was found that, for soil and sediment samples, these two methods typically differed by 5-9% for higher energy gamma rays and by 12-15% for the 59.54 keV 241Am peak. Gamma-absorption curves were also derived and, for the admittedly limited dataset, 95% confidence intervals of +/-7% for the curve generated using the spiked samples method were obtained.

Structure data entry using graphical input: recording symptoms for multiple sclerosis.

Structured Data Entry provides flexible and efficient input to the computer-based patient record. We have utilized the SDE approach to build a prototype interface for recording common symptoms associated with Multiple Sclerosis (MS). The software provides both graphical input and output, to facilitate efficient data entry and monitoring of data. Graphical input is transformed to textual information, which is stored in a database in a hierarchical tree structure. Pain management in MS may be achieved by careful monitoring of the symptom in response to treatment. Pain location is selected on a body image and severity and other attributes represented using a graphical visual analog scale, leading to more convenient input and a less ambiguous coding than is achievable with narrative text alone. This approach could provide an objective means of monitoring the progress of the disease and management of the symptoms. The intuitive interface may also facilitate self-monitoring.

Use of ribotyping and random amplified polymorphic DNA to differentiate isolates of Burkholderia andropogonis.

AIMS: The aim of this study was to determine the genetic diversity among isolates of Burkholderia andropogonis from various host plant species and geographic locations. METHODS AND RESULTS: Both random amplified polymorphic DNA (RAPD) and ribotyping analyses were used to assess the diversity of B. andropogonis isolates and compare these results with pathogenicity assays carried out on a number of common hosts of the organism. CONCLUSIONS: Both RAPD and ribotyping analyses revealed a high level of genetic diversity between isolates of B. andropogonis. Both methods demonstrated a similar clustering of isolates. However, there was no strict correlation between the genetic diversity revealed and the original host, geographic location or pathogenicity of the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the genetic diversity of isolates of B. andropogonis. The great degree of diversity revealed in this study contrasts with the lack of phenotypic diversity within this species.

Technetium-99 in the Irish marine environment.

Technetium-99 activity concentrations in seawater and biota from Irish coastal waters are presented. Time series measurements of 99Tc in seawater and Fucus vesiculosus from the western Irish Sea show that activity concentrations have increased in line with the increase in discharges of 99Tc from Sellafield. The peak in activity concentrations in both seawater and Fucus vesiculosus occurred in 1997 approximately two years after the peak in 99Tc discharges. The highest activity concentration recorded in Fucus vesiculosus showed a 29-fold increase over the mean concentration for the period 1988-1993. Technetium-99 activity concentrations were measured in fish, lobsters, prawns, mussels and oysters landed at major fishing ports on the east and northeast coasts of Ireland between 1996 and 1998. Concentration factors for 99Tc in the brown seaweed Fucus vesiculosus and certain species of fish, crustaceans and molluscs from the Irish Sea were estimated. In general, these concentration factors were higher than those in the literature which were derived from laboratory studies, but agreed well with values which were based on field studies. The mean committed effective doses to Irish typical and heavy seafood consumers due to 99Tc in the period 1996-1998 were 0.061 and 0.24 microSv, respectively.

Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR.

Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G. sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested. This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G. sacchari from environmental samples and will allow these tools to be used in further ecological research.

Partial sequencing of the hrpB and endoglucanase genes confirms and expands the known diversity within the Ralstonia solanacearum species complex.

We determined partial hrpB and endoglucanase genes sequences for 30 strains of Ralstonia solanacearum and one strain of the blood disease bacterium (BDB), a close relative of Ralstonia solanacearum. Sequence comparisons showed high levels of variability within these two regions of the genome involved in pathogenicity. Phylogenetic analysis based upon sequence comparisons of these two regions revealed three major clusters comprising all Ralstonia solanacearum isolates, the BDB strain constituted a phylogenetically distinct entity. Cluster 1 and cluster 2 corresponded to the previously defined divisions 1 and 2 of Ralstonia solanacearum. Moreover, two subclusters could be identified within cluster 2. The last cluster, designated cluster 3 in this study, included biovar 1 and N2 strains originating from Africa. This recently described group of strains was confirmed to be clearly different from the other strains suggesting a separate evolution from those of both divisions 1 and 2.

Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug.

A new species of the genus Gluconacetobacter, for which the name Gluconacetobacter sacchari sp. nov. is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia. The nearest phylogenetic relatives in the alpha-subclass of the Proteobacteria are Gluconacetobacter liquefaciens and Gluconacetobacter diazotrophicus, which have 98.8-99.3% and 97.9-98.5% 16S rDNA sequence similarity, respectively, to members of Gluconacetobacter sacchari. On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q10 ubiquinone, this new species was assigned to the genus Gluconacetobacter. No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0.01% malachite green, growth on 30% glucose, an inability to fix nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source. The type strain of this species is strain SRI 1794T (= DSM 12717T).

Phylogenetic position of Chitinophaga pinensis in the Flexibacter-Bacteroides-Cytophaga phylum.

Comparison of the 16S rRNA gene sequence determined for Chitinophaga pinensis showed that this species is most closely related to Flexibacter filiformis in the Flexibacter-Bacteroides-Cytophaga phylum. These two chitinolytic bacteria, which are characterized by transformation into spherical bodies on ageing, belong to a strongly supported lineage that also includes Cytophaga arvensicola, Flavobacterium ferrugineum and Flexibacter sancti. The lineage is distinct from the microcyst-forming species Sporocytophaga myxococcoides.

Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov.

The phylogenetic relationships among members of the family Comamonadaceae and several unclassified strains were studied by direct sequencing of their PCR-amplified 16S rRNA genes. Based on the 16S rRNA gene sequence analysis, members of the family formed a coherent group. The closest relatives are species of the Rubrivivax sub-group: Leptothrix discophora, Ideonella dechloratans and Rubrivivax gelatinosus. The genus Hydrogenophaga formed two subclusters, as did the species of Acidovorax, whereas the five species of the genus [Aquaspirillum] were polyphyletic. Comamonas acidovorans was phylogenetically distant from the type species of Comamonas, Comamonas terrigena. On the basis of this work and previous studies, Comamonas acidovorans is removed from the genus Comamonas and renamed as Delftia acidovorans gen. nov., comb. nov. Descriptions of the new genus Delftia and of the type species Delftia acidovorans, for which the type strain is ATCC 15668T, are presented.

Phylogenetic heterogeneity within the genus Herpetosiphon: transfer of the marine species Herpetosiphon cohaerens, Herpetosiphon nigricans and Herpetosiphon persicus to the genus Lewinella gen. nov. in the Flexibacter-Bacteroides-Cytophaga phylum.

Analysis of the 16S rDNA sequences of species currently assigned to the genus Herpetosiphon revealed intrageneric phylogenetic heterogeneity. The thermotolerant freshwater species Herpetosiphon geysericola is most closely related to the type species Herpetosiphon aurantiacus in the Chloroflexus subdivision of the green non-sulfur bacteria. The marine species Herpetosiphon cohaerens, Herpetosiphon nigricans and Herpetosiphon persicus, on the other hand, were found to form a cluster with sheathed bacterium Haliscomenobacter hydrossis in the Saprospira group of the Flexibacter-Bacteroides-Cytophaga (FBC) phylum. A proposal is made to transfer these marine species to the genus Lewinella gen. nov. as Lewinella cohaerens comb. nov., Lewinella nigricans comb. nov. and Lewinella persica comb. nov. The marine sheathed gliding bacterium Flexithrix dorotheae was also found to be a member of the FBC phylum but on a separate phylogenetic line to the marine herpetosiphons now assigned to the genus Lewinella.

Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus.

The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers. The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense. The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences. Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet. diazotrophicus from environmental samples with nifH gene-based primers.

Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii, and the blood disease bacterium of banana based on 16S rRNA gene sequences.

We determined nearly complete 16S rRNA gene sequences for 19 isolates of Burkholderia solanacearum, three isolates of the blood disease bacterium of bananas, and two isolates of Pseudomonas syzygii, the cause of Sumatra disease of cloves. The dendrogram produced by comparing all of these sequences revealed that there were two divisions, which corresponded to the results obtained previously in a restriction fragment length polymorphism analysis (D. Cook, E. Barlow, and L. Sequeira, Mol. Plant Microbe Interact. 2:113-121, 1989) and a total 16S ribosomal DNA (rDNA) sequence analysis of four isolates representing four biovars of B. solanacearum (X. Li, M. Dorsch, T. Del Dot, L. I. Sly, E. Stackebrandt, and A. C. Hayward, J. Appl. Bacteriol. 74:324-329, 1993). Division 1 comprised biovars 3, 4, and 5 and an aberrant biovar 2 isolate (strain ACH0732), and division 2 included biovars 1, 2, and N2, the blood disease bacterium, and P. syzygii. Specific nucleotides at positions 458 to 460 (UUC) and 474 (A) characterized division 2, whereas in division 1 the nucleotides at these positions were ACU and U, respectively. However, strain ACH0732 had a U at position 458, as did division 2 isolates, and G instead of U at position 474. Division 2 consisted of two subdivisions; one subdivision contained two B. solanacearum isolates that originated from Indonesia, P. syzygii strains, and blood disease bacterium strains, and the other subdivision contained all of the other division 2 isolates. Within division 1, the level of 16S rDNA sequence similarity ranged from 99.8 to 100%, and within division 2, the levels of 16S rDNA sequence similarity ranged from 99.1 to 100%. The division 1 isolates exhibited an average level of 16S rDNA sequence similarity to division 2 isolates of 99.3% (range, 99.1 to 99.5%). The occurrence of consistent polymorphisms in the 16S rDNA sequences of B. solanacearum strains, in particular unique 16S rDNA sequence differences in aberrant biovar 2 isolate ACH0732, and the occurrence of the Indonesian subdivision of division 2 suggest that this group is a rapidly evolving (tachytelic) group.

Construction of species-specific primers for Pseudomonas andropogonis based on 16S rDNA sequences.

Approximately 94% of the total 16S rDNA of Pseudomonas andropogonis strain ACH 01053A was sequenced and compared with that of strain ATCC 23061 obtained from the GenBank database. The two sequences were highly homologous with 1.3% difference. Alignment of sequences with those from closely related bacteria revealed two possible regions for design of a specific primer suitable for detection of Ps. andropogonis. Using primers designed to these variable regions in a PCR test, an amplification product of approximately 410 bp was specifically produced by 40 strains of Ps. andropogonis. No other bacterial species showed an amplification product under optimized PCR conditions. As few as 1000 cells per reaction were detected.

Antimicrobial sensitivity testing of Australian isolates of Bordetella avium and the Bordetella avium-like organism.

The in-vitro sensitivity of 16 Australian isolates of Bordetella avium and 15 isolates of B avium-like organism to 11 antimicrobial agents or combinations of agents was determined using a microtitre plate system to establish minimal inhibitory concentrations. All the B avium isolates were sensitive to ampicillin but resistant to erythromycin, lincomycin, spectinomycin, sulphamethoxazole, trimethoprim, and lincomycin + spectinomycin. Most of the B avium isolates were sensitive to tetracycline and resistant to streptomycin and sulphadiazine. All the B avium-like isolates were resistant to ampicillin, erythromycin, lincomycin, spectinomycin, streptomycin, tetracycline, trimethoprim, and lincomycin+spectinomycin. Most B avium-like isolates were sensitive to sulphadiazine, sulphamethoxazole and trimethoprim-sulphamethoxazole.

Random amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae.

Metarhizium anisopliae isolates from several insect hosts and from various sugar cane growing areas of Queensland, Australia, were examined for genetic diversity using random amplified polymorphic DNA (RAPD) markers. Thirty isolates of M. anisopliae var. anisopliae and one isolate of M. anisopliae var. majus were examined. Ten randomly chosen 10mer or 11mer primers were used and RAPD banding patterns were compared. Thirty distinct genotypes could be distinguished amongst the 31 isolates tested on the basis of RAPD patterns. Six of the isolates classified as M. anisopliae var. anisopliae exhibited closer similarity to the M. anisopliae var. majus isolate than to other anisopliae strains tested. Isolates exhibiting similar (> 80% similarity) RAPD profiles tended to be isolated from the same geographic area and evidence for the persistence of particular fungal genotypes in specific geographical localities was obtained. Pathogenicity assays suggested that, in some instances, RAPD groupings may also indicate insect host range. The mean similarity amongst isolates measured by band sharing in all pairwise comparisons was 41% and the most distinct pair of isolates shared only 9% of their RAPD bands. We conclude that the isolates tested belonging to the species M. anisopliae, as assessed on morphological grounds, represent a very diverse genetic group. The results also suggest that RAPD markers may be useful for the tracking of specific biocontrol strains in the field.

Heterogeneity, persistence, and distribution of Pseudomonas aeruginosa genotypes in cystic fibrosis patients.

A collection of 222 isolates of Pseudomonas aeruginosa was obtained from the respiratory tract of 16 patients with cystic fibrosis over a 4- to 9-month period. Fourteen of these patients were unrelated, while the remaining two were siblings. Isolates were typed by conventional pyocin typing and also by the use of a DNA probe containing 741 bp immediately upstream of the exotoxin A structural gene and the initial 732 bp of the exotoxin A structural gene. By pyocin typing, 69% (11 of 16) of the patients were shown to harbor a single type that persisted in the lung throughout the study. By genotyping (DNA probe typing), all but three patients (13 of 16, 81%) harbored a single persistent genotype in their lungs. Six patients other than the sibling pair (6 of 14, 43%) shared a common genotype in their lungs as judged by DNA probing, and the pyocin type of these isolates was also identical. In four of these six patients, the shared genotype was also the persistent genotype. The sibling pair studied also carried a common genotype in their lungs as indicated by DNA probing, even though the pyocin type of these isolates varied. Results presented suggest that the majority of patients harbor a persistent strain in their lungs and that cross-colonization may occur.