rep-PCR Genomic Fingerprinting Research Articles

Isolation, Characterization and Phylogenetic Analysis of <i>Acidiphilium</i>-Like Bacteria from Acid Mine Drainage

The acidophilic heterotrophic bacteria Acidiphilium spp. were considered as a ministrant of Leptospirillum spp., Acidithiobacillus spp. and other autotrophic bacteria in acid mine drainage (AMD). 25 Acidiphilium-like bacterial strains were isolated from eight different mines in China and their physiology was characterized. Their marmatite-bioleaching ability has been investigated and compared with that of a mixed culture of Acidiphilium sp. and Acidithiobacillus ferrooxidans. The results indicated that some Acidiphilium-like strains exhibited a high marmatite-bioleaching ability, which was even higher than that of Acidithiobacillus ferrooxidans. The leaching efficiency was not higher in mixed than in pure culture. The phylogenetic relationship of the 25 Acidiphilium-like strains was analyzed by 16S rRNA sequencing, GyrB sequencing and rep-PCR genomic fingerprinting. The 25 strains and other eight species of Acidiphilium spp. were clustered into three groups.

Phenotypic and genotypic diversity of Bacillus cereus isolates recovered from honey

Of 433 honey samples collected in Argentina, 114 (27%) yielded Bacillus cereus isolates (27%) and 60 (14%) yielded other species of Bacillus. The Argentinian B. cereus isolates were compared with isolates recovered from honey from other countries ( n = 16) and with strain ATCC 11718. The 133 B. cereus isolates and the ATCC strain were characterized using rep-PCR fingerprinting with primers BOX, REP and ERIC, restriction fragment length polymorphism analysis of a 16S rRNA gene fragment (16S rRNA PCR/RFLP), and morphological and biochemical tests. There was a high degree of diversity, both phenotypic and genotypic among the isolates of B. cereus. Our results demonstrate the usefulness of rep-PCR genomic fingerprinting for characterizing populations of B. cereus. According to our knowledge, this is the first report of phenotypic and genotypic characterization of B. cereus isolates from honey.

Classification and Identification of Xanthomonas translucens Isolates, Including Those Pathogenic to Ornamental Asparagus

ABSTRACT In order to confirm and refine the current classification scheme of Xanthomonas translucens and to identify novel strains from ornamental asparagus, a collection of field and reference strains was analyzed. Rep-polymerase chain reaction (PCR) genomic fingerprint profiles were generated from 33 isolates pathogenic to asparagus as well as 61 X. trans-lucens reference strains pathogenic to cereals and grasses. Amplified ribo-somal gene restriction analysis profiles were obtained from most of these and 29 additional Xanthomonas reference strains. Rep-PCR genomic fingerprint profiles of all strains were compared with those in a large Xanthomonas database using computer-assisted analysis. Rep-PCR ge-nomic fingerprinting facilitated the characterization and discrimination of X. translucens, including the pathovars arrhenatheri, graminis, phlei, phleipratensis, and poae, as well as a number of strains received as X. translucens pv. cerealis. Strains received as pathovars hordei, secalis, translucens, undulosa, and other cerealis strains were grouped in two subclusters that correspond to the recently redefined pathovars X. trans-lucens pvs. undulosa and translucens. All 33 novel isolates from ornamental asparagus (tree fern; Asparagus virgatus) were identified as X. translucens pv. undulosa. Moreover, a unique amplified small subunit ribosomal gene MspI/AluI restriction profile specific for all X. translucens strains tested, including those pathogenic to asparagus, allowed discrimination from all other Xanthomonas spp. Although phage tests were inconclusive, the classification of the asparagus strains within the X. translucens complex was supported by pathogenicity assays in which all the isolates from ornamental asparagus induced watersoaking on wheat. Surprisingly, several X. translucens reference strains affected asparagus tree fern as well. That the novel asparagus isolates belong to X. translucens pv. undulosa is extraordinary because all hosts of X. translucens pathovars described to date belong only to the families Gramineae and Poaceae, whereas asparagus belongs to the phylogenetically distant family Liliaceae.

Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben)

The bacterial diversity occurring in traditional Moroccan soft white cheese, produced in eight different regions in Morocco, was studied. A total of 164 lactic acid bacteria were isolated, purified and identified by whole-cell protein fingerprinting and rep-PCR genomic fingerprinting. The majority of the strains belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Enterococcus. Sixteen species were identified: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, Lactococcus garvieae, Lactococcus raffinolactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc citreum, Eterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus saccharominimus and Streptococcus sp.

Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation

A combination of population genetics and phylogenetic inference methods was used to delineate Bradyrhizobium species and to uncover the evolutionary forces acting at the population-species interface of this bacterial genus. Maximum-likelihood gene trees for atpD, glnII, recA, and nifH loci were estimated for diverse strains from all but one of the named Bradyrhizobium species, and three unnamed “genospecies,” including photosynthetic isolates. Topological congruence and split decomposition analyses of the three housekeeping loci are consistent with a model of frequent homologous recombination within but not across lineages, whereas strong evidence was found for the consistent lateral gene transfer across lineages of the symbiotic (auxiliary) nifH locus, which grouped strains according to their hosts and not by their species assignation. A well resolved Bayesian species phylogeny was estimated from partially congruent glnII + recA sequences, which is highly consistent with the actual taxonomic scheme of the genus. Population-level analyses of isolates from endemic Canarian genistoid legumes based on REP-PCR genomic fingerprints, allozyme and DNA polymorphism analyses revealed a non-clonal and slightly epidemic population structure for B. canariense isolates of Canarian and Moroccan origin, uncovered recombination and migration as significant evolutionary forces providing the species with internal cohesiveness, and demonstrated its significant genetic differentiation from B. japonicum, its sister species, despite their sympatry and partially overlapped ecological niches. This finding provides strong evidence for the existence of well delineated species in the bacterial world. The results and approaches used herein are discussed in the context of bacterial species concepts and the evolutionary ecology of (brady)rhizobia.

Identification and Characterisation of Xanthomonas campestris pv. campestris Strains from Tanzania by Pathogenicity Tests, Biolog, rep-PCR and Fatty Acid Methyl Ester Analysis

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a major disease constraint to cabbage production by smallholder farmers in Africa. Variability exists within the pathogen, and yet differentiation of Xcc strains from other closely-related xanthomonads attacking crucifers is often difficult. The Biolog system, fatty acid methyl ester analysis using microbial identification system (MIS), rep-PCR and pathogenicity tests were used to identify and characterise Xcc strains from Tanzania. Great diversity was observed among Xcc strains in their Biolog and rep-PCR profiles. Specific rep-PCR genomic fingerprints were linked to some geographical areas in the country. Most of the Xcc strains were clustered in two groups based on their fatty acid profiles and symptom expression in cabbage although some deviant strains were found. Each of the methods allowed a degree of identification from species, pathovar to the strain level. Biolog and MIS identified all Xcc strains at least to the genus level. Additionally, Biolog identified 47% of Xcc strains to the pathovar and 43% to strain level, whereas MIS identified 43% of the strains to pathovar level. In the absence of a database, the utility of rep-PCR for routine diagnosis of strains was limited, although the procedure was good for delineation of Xcc to the strain level. These findings indicate the existence of Xcc strains in Tanzania that are distinct from those included in Biolog and MIS databases. The limitations noticed warrant continued improvement of databases and inclusion of pathogenicity testing, using universally susceptible cultivars, as an integral part of strain identification.

Differentiation of Ascosphaera apis isolates by rep-PCR fingerprinting and determination of chalkbrood incidence in Argentinean honey samples

SUMMARYAscosphaera apis, the causative agent of chalkbrood disease in honey bee larvae occurs throughout the world and is found in many beekeeping areas of Argentina. Microbiological analysis of 394 honey samples from Buenos Aires province from three years of sampling (1999–2001) yielded 51 positive cases (13% incidence). Eighty-four isolates of A. apis from Argentina and Chile isolated from diseased larvae and/or honey samples were characterized on the basis of DNA fingerprints using the repetitive-sequence-based polymerase chain reaction technique (rep-PCR) with BOX, REP, and ERIC sequence-specific primers. Computer-assisted analysis of combined fingerprints distinguished six groups of patterns, designated A, B, C, D, E and F. Pattern C was the most prevalent and suggests a limited diversity in the populations of A. apis from Argentina and Chile. The results demonstrated the usefulness of rep-PCR genomic fingerprinting to characterize populations of A. apis. In addition, a simple and efficient protocol for the extraction of total fungal genomic DNA was developed that was appropriate for simultaneous and cost-efficient processing of many samples.

Characterization of Pseudoalteromonas citrea and P. nigrifaciens Isolated from Different Ecological Habitats Based on REP-PCR Genomic Fingerprints

DNA primers corresponding to conserved repetitive interspersed genomic motifs and PCR were used to show that REP, ERIC and BOX-like DNA sequences are present in marine, oxidative, gram-negative Pseudoalteromonas strains. REP, ERIC and BOX-PCR were used for rapid molecular characterization of both the type species of the genus and environmental strains isolated from samples collected in different geographical areas. PCR-generated genomic fingerprint patterns were found to be both complex and strain specific. Analysis of the genotypic structure of phenotypically diverse P. citrea revealed a geographic clustering of Far Eastern brown-pigmented, agar-digesting strains of this species. Marine isolates of P. nigrifaciens with 67-70% DNA relatedness generated genomic patterns different from those of the type strain and formed a separate cluster. It is concluded that REP, ERIC and BOX-PCR are effective in generating strain specific patterns that can be used to elucidate geographic distribution, with these genomic patterns providing a valuable biogeographic criterion.

Use of a monoclonal antibody and genomic fingerprinting by repetitive-sequence-based polymerase chain reaction to identify Xanthomonas populi pathovars

Diagnostic tools to facilitate the rapid and specific identification of Xanthomonas populi pv. populi, the cause of bacterial canker of poplar, were evaluated. A monoclonal antibody, MAb 13E7, that reacted in immunofluorescence tests with representative strains of physiological races of X. populi pv. populi as well as with the related X. populi pv. salicis and showed less cross-reaction with other xanthomonads than did a polyclonal antibody, was produced. The Xanthomonas strains were also characterized by rep-PCR (repetitive-sequence-based polymerase chain reaction) genomic fingerprinting, using three DNA primer sets (BOX, ERIC, and REP). Computer-assisted phylogenetic analysis of the linearly combined fingerprints clearly distinguished the strains of X. populi pv. populi. Fingerprint clusters of the two X. populi pathovars had ~60% similarity while the X. populi group had <20% similarity to the other xanthomonads. Des outils diagnostiques visant à faciliter l'identification rapide et spécifique du Xanthomonas populi pv. populi, agent causal du chancre bactérien du peuplier, ont été évalués. L'anticorps monoclonal MAb 13E7 qui a été produit a réagi, au cours des épreuves d'immunofluorescence, avec des souches représentatives des races physiologiques du X. populi pv. populi ainsi qu'avec le pathovar apparenté X. populi pv. salicis. De plus, il a montré une réaction croisée moins intensive qu'un anticorps polyclonal avec les autres xanthomonades. Les souches de Xanthomonas ont également été caractérisées par empreinte génomique par réaction en chaîne de la polymérase basée sur une séquence répétitive à l'aide de trois ensemble d'amorces d'ADN (BOX, ERIC et REP). L'analyse phylogénique assistée par ordinateur des empreintes linéairement combinées a permis de distinguer clairement les différentes souches du X. populi pv. populi. La similarité des groupes d'empreintes des deux pathovars du X. populi était d'environ 60%, tandis que celle du groupe du X. populi était de moins de 20% par rapport aux autres xanthomonades.

Relationships of in Vitro Pathogen Inhibition and Soil Colonization to Potato Scab Biocontrol by Antagonistic Streptomyces spp

Abstract Spontaneous mutants of two scab-suppressive streptomycetes that were defective in in vitro pathogen inhibition activity were isolated. Morphological characterization of these mutants by rep-PCR genomic DNA fingerprinting or by fatty acid analysis indicated that the mutants of each parent were closely related to one another and to their respective parent, though the mutants could be differentiated from the parent strains and from one another. Despite the reduced in vitro pathogen inhibition activity, most of the mutants demonstrated significant scab biocontrol activity against pathogenic Streptomyces scabies strains. These results suggest that pathogen inhibition activity detected in vitro may not be an accurate predictor of scab biocontrol. Colonization of the suppressive strain or its mutants was generally reduced in the presence versus in the absence of the pathogen. In addition, colonization assays showed no significant differences in pathogen population density among the suppressive strain and mutant strain treatments.

Differentiation of Tilletia Species by rep-PCR Genomic Fingerprinting.

The potential of repetitive-sequence-based polymerase chain reaction (rep-PCR) fingerprinting of fungal genomic DNA as a rapid and simple alternative to random amplified polymorphic DNA (RAPD) analysis in the study of phylogenetic relationships, and also as a diagnostic method, was investigated with species of Tilletia. DNA primers (BOX, ERIC, and REP) corresponding to conserved repetitive element motifs, originally described in prokaryotes, were used to generate genomic fingerprints of T. indica, T. walkeri, T. controversa, T. laevis, T. tritici, T. goloskokovii, T. barclayana, and members of the T. fusca complex. Computer-assisted analysis of the database of combined fingerprints clearly distinguished each taxon and indicated phylo-genetic relationships consistent with previously reported RAPD analyses. There were three main clusters with isolates showing 35 to 40% similarity. Group 1 included T. indica and T. walkeri; group 2 included members of the T. fusca complex, as well as T. controversa, T. laevis, T. tritici, and T. goloskokovii; and group 3 included only T. barclayana. If, as is likely, the conserved repetitive element motifs on which this technique is based are widespread or universal in fungal species, rep-PCR shows strong potential, not only as a simple generic taxonomic tool, but also as a diagnostic method.

High diversity in Curtobacterium flaccumfaciens pv. flaccumfaciens characterized by serology and rep-PCR genomic fingerprinting

The diversity found in culture collection strains of Curtobacterium flaccumfaciens pv. flaccumfaciens was characterized by serology and repetitive-sequence-based polymerase chain reaction (rep-PCR). Using strain NCPPB 559 as the reference antigen, immunofluorescence tests with polyclonal and monoclonal antibodies confirmed that no epitope appeared to be common to all strains of this pathovar. Screening of hybridoma cell lines againsl strains of this pathovar, as well as against other members of this genus and species, identified two distinct epitopes. Phylogenetic analysis of the rep-PCR genomic fingerprints of Curtobacter, Clavibacter, and Rathayibacter species, grouped all the C. flaccumfaciens strains and pathovars together, but the C. flaccumfaciens pv. flaccumfaciens strains did not form a single cluster. Rather, they dispersed into two clusters, along with strains of other pathovars of this species. Members of one cluster reacted with one of the monoclonal antibodies, while members of the other did not.

Genetic and Pathogenic Diversity of Pseudomonas avellanae Strains Isolated from Corylus avellana Trees in North-west of Italy, and Comparison with Strains from Other Regions

Forty strains of Pseudomonas avellanae isolated from hazelnut (Corylus avellana L.) trees in the Langhe district of Italy were compared with 15 strains collected from various geographic areas. All strains were assessed by rep-PCR genomic fingerprinting using ERIC, REP and BOX primer sets. Cluster analysis was performed by means of UPGMA. To check the possible differential virulence of the strains, pathogenicity tests were carried out by inoculating leaf scars of hazelnut trees in early autumn. Cluster analysis indicated that, during a four-year study, at least five groups of strains were isolated from different hazelnut orchards located in the small district of Langhe. Two groups were isolated from the same twig. Such strains showed around 20% similarity with other P. avellanae strains collected from northern Greece and central Italy. The strains from Langhe were less aggressive to hazelnut than strains from northern Greece and central Italy. These results and previous genomic characterizations indicate a possible correlation between genomic profile type and regional geographic distribution of P. avellanae strains. In addition, the genetic variability found in the strains from Langhe indicates that such populations are older than the more homogenous P. avellanae populations from other regions.

Identification of bacterial rep-PCR genomic fingerprints using a backpropagation neural network

A backpropagation neural network (BPN) was used to identify bacterial plant pathogens based on their genomic fingerprints. Genomic fingerprint data, comprised of complex DNA band patterns generated using BOX, enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP)-primers (rep-PCR), were used to train three independent BPNs. 10 Strains of the genus Xanthomonas, each with a characteristic host plant range, were identified correctly using the three trained BPNs. When tested with fingerprints of bacterial strains not present in the training sets, the rejection rate was 100%, using the three BPN classifiers combined. Thus, BPN protocols can be employed to generate a powerful computer-based system for the identification of pathogenic bacteria in the genus Xanthomonas.

Identification of bacterial rep-PCR genomic fingerprints using a backpropagation neural network

A backpropagation neural network (BPN) was used to identify bacterial plant pathogens based on their genomic fingerprints. Genomic fingerprint data, comprised of complex DNA band patterns generated using BOX, enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP)-primers (rep-PCR), were used to train three independent BPNs. 10 Strains of the genus Xanthomonas, each with a characteristic host plant range, were identified correctly using the three trained BPNs. When tested with fingerprints of bacterial strains not present in the training sets, the rejection rate was 100%, using the three BPN classifiers combined. Thus, BPN protocols can be employed to generate a powerful computer-based system for the identification of pathogenic bacteria in the genus Xanthomonas.

Rhizobia Isolated from Root Nodules of Tropical Leguminous Trees Characterized Using DNA-DNA dot-blot Hybridisation and rep-PCR Genomic Fingerprinting

Fifty-one fast growing rhizobial strains isolated from root nodules of Acacia Senegal and Prosopis chilensis in Sudan and Kenya were divided into DNA homology groups using non-radioactive DNA-DNA dot-blot hybridisation. Rhizobium leguminosarum, R. galegae, R. tropici, Mesorhizobium loti, Sinorhizobium fredii, S. meliloti used in numerical taxonomy were included in the hybridisation experiments as reference strains and, at a later stage S. saheli and S. terangae . Scores given to the intensities of dots detected in the hybridisation experiments were used in principal component analysis, which clustered the majority of the tree rhizobia in two separate DNA-homology groups. The 51 strains were also analysed by genomic fingerprinting using the repetitive sequence-based polymerase chain reaction (rep-PCR) method with REP, ERIC, BOX and GTG5 primers. The resulting genomic fingerprints were compared with strains representing 15 rhizobial species. The relationship of 17 Sudanese strains to established sinorhizobial species was examined using the optical renaturation rates method and the G+C content of nine strains was determined. Results from dot-blot hybridisation and rep-PCR experiments were found to be in close agreement with each other and with the results obtained from spectrophotometric reassociation analysis. We suggest that rep-PCR fingerprinting can be used as a first and dot-blot hybridisation as a second rapid and dependable genomic screening method to classify new rhizobial isolates of unknown taxonomic status and to choose the representative strains for the more laborious DNA-DNA reassociation experiments.

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.

Rep-PCR based genomic fingerprinting of isolates of Leptosphaeria maculans from Poland

Leptosphaeria maculans, the ascomycete fungus which causes blackleg disease of oilseed rape, has been considered for a long time as a single species divided into ‘aggressive’ and ‘non-aggressive’ pathogenicity groups which differ in their economic importance. However, the development of accurate biochemical and molecular characterisation methods has demonstrated that the world-wide L. maculans population actually comprises at least two species. The aim of this research was to assess the ability of rep (repetitive element based)-PCR genomic fingerprinting methods, initially developed for bacterial identification, to characterise a collection of 90 isolates of L. maculans from Poland, in comparison with reference isolates from the IBCN (International Blackleg of Crucifers Network) collection. REP (repetitive extragenic palindromic)-, ERIC (enterobacterial repetitive intergenic consensus)-, and BOX primers for rep-PCR genomic fingerprinting, and primers derived from LMR1, a L. maculans specific repeated element, were tested. Rep-PCR and LMR1-based analyses were able to discriminate the different components of the species complex and to evaluate the genetic diversity within each member of the complex. These analyses suggested that Polish populations of L. maculans mainly belong to the ‘non-aggressive’ species, rather than the ‘aggressive’ species which is prevalent in Western Europe, Canada and Australia.

Microbial biofilms and catabolic plasmid harbouring degradative fluorescent pseudomonads in Scots pine mycorrhizospheres developed on petroleum contaminated soil

Cellular interactions and catabolic activities of mycorrhizal root associated non-sporulating bacteria were investigated in a simplified phytoremediation simulation involving a woody plant species. Mycorrhizal Scots pine ( Pinus sylvestris) seedlings pre-colonised by Suillus bovinus or Paxillus involutus were grown in forest humus containing microcosms amended with petroleum hydrocarbon (PHC) contaminated soil. Fungal hyphae of both species, emanating from mycorrhizal roots, colonised the PHC contaminated soil over a 16-week period and dense long-lived patches of S. bovinus hyphae formed on the PHC contaminated soil. Transmission electron microscopy revealed a microbial biofilm at the PHC soil-fungal interface composed of differentiated pseudoparenchymous patch hyphae supporting a morphologically diverse bacterial population. Certain non-sporulating bacterial isolates closely associated with the S. bovinus patch hyphae or P. involutus `web' hyphae from the PHC soil harboured similar sized mega-plasmids (approx. 150 kb). Isolates of Pseudomonas fluorescens from the `patch' mycorrhizospheres represented different biovars, displayed similar REP-PCR genomic fingerprints, grew on e.g. m-toluate and m-xylene as sole carbon sources, cleaved catechol, and harboured plasmid-borne catabolic marker genes, xylE and xylMA, involved in degradation of mono-aromatics. The plasmids were transmissible in vitro, and Pseudomonas putida transconjugants retained a similar catabolic profile. The identification of microbial biofilms containing catabolic bacteria in the external mycorrhizosphere is discussed in relation to both phytoremediation mechanisms and normal efficient nutrient mobilisation from highly lignin-rich forest soils.

Microbial biofilms and catabolic plasmid harbouring degradative fluorescent pseudomonads in Scots pine mycorrhizospheres developed on petroleum contaminated soil

Abstract Cellular interactions and catabolic activities of mycorrhizal root associated non-sporulating bacteria were investigated in a simplified phytoremediation simulation involving a woody plant species. Mycorrhizal Scots pine ( Pinus sylvestris ) seedlings pre-colonised by Suillus bovinus or Paxillus involutus were grown in forest humus containing microcosms amended with petroleum hydrocarbon (PHC) contaminated soil. Fungal hyphae of both species, emanating from mycorrhizal roots, colonised the PHC contaminated soil over a 16-week period and dense long-lived patches of S. bovinus hyphae formed on the PHC contaminated soil. Transmission electron microscopy revealed a microbial biofilm at the PHC soil-fungal interface composed of differentiated pseudoparenchymous patch hyphae supporting a morphologically diverse bacterial population. Certain non-sporulating bacterial isolates closely associated with the S. bovinus patch hyphae or P. involutus `web' hyphae from the PHC soil harboured similar sized mega-plasmids (approx. 150 kb). Isolates of Pseudomonas fluorescens from the `patch' mycorrhizospheres represented different biovars, displayed similar REP-PCR genomic fingerprints, grew on e.g. m -toluate and m -xylene as sole carbon sources, cleaved catechol, and harboured plasmid-borne catabolic marker genes, xylE and xylMA , involved in degradation of mono-aromatics. The plasmids were transmissible in vitro, and Pseudomonas putida transconjugants retained a similar catabolic profile. The identification of microbial biofilms containing catabolic bacteria in the external mycorrhizosphere is discussed in relation to both phytoremediation mechanisms and normal efficient nutrient mobilisation from highly lignin-rich forest soils.