Genetic and metabolic diversity of streptomycetes in pulp and paper mill effluent treated crop fields

Irrigation of farm field with water mixed with pulp and paper mill effluent from Century pulp and paper mill in Uttrakhand state of India for over last 25 years in succession increased streptomycetes population (120 × 10⁵) compared to the fresh water irrigated fields (48 × 10³ in WIF). Denaturing gradient gel electrophoresis, amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, BIOLOG™ substrate usage, production of extracellular enzymes (xylanase and cellulase) and plant growth promoting attributes were applied to monitor changes in genetic and metabolic diversity of streptomycetes. Significant variation was observed for production of extracellular enzymes, Indolic compounds, siderophore and P-solubilisation among isolates. Metabolic substrate usage of Streptomyces isolates was evaluated using the BIOLOG™ GP2 plates and unique carbon substrate usage profiles were observed. Based on 16S rRNA gene sequencing, the isolates were identified as Streptomyces variabilis, Streptomyces spp. S. glaucescens, S. viridochromogenes, S. cinnabarinus, S. aburaviensis, S. viridis, S. xylophagus, S. macrosporeus, S. thermocarboxydus, and S. albogriseolus. The diversity index parameters like Shannon index, reciprocal of Simpson’s index (1/D), and Pielou index of evenness based on ARDRA revealed that streptomycetes community in effluent irrigated field (EIF) was more diverse. DGGE profiles of Streptomyces specific 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar in both soils.     

Influence of Long Term Irrigation with Pulp and Paper Mill Effluent on the Bacterial Community Structure and Catabolic Function in Soil

Microbial communities play a vital role in maintaining soil health. A multiphasic approach to assess the effect of pulp and paper mill effluent on both the structure and function of microbial soil communities is taken. Bacterial communities from agricultural soils irrigated with pulp and paper mill effluent were compared to communities form soils irrigated with well water. Samples were taken from fields in the state of Uttarakhand, India, where pulp and paper mill effluent has been used for irrigation for over 25 years. Comparisons of bacterial community structure were conducted using sequencing of the 16S rRNA gene from both isolates and clone libraries attained from the soil. Community-level physiological profiling was used to characterize the functional diversity and catabolic profile of the bacterial communities. The multiphasic approach using both physiological and molecular techniques proved to be a powerful tool in evaluating the soil bacterial community population and population differences therein. A significant and consistent difference in the population structure and function was found for the bacterial communities from soil irrigated with effluent in comparison to fields irrigated with well water. The diversity index parameters indicated that the microbial community in pulp and paper mill effluent irrigated fields were more diverse in both structure and function. This suggests that the pulp and paper mill effluent is not having a negative effect on the soil microbial community, but in fact may have a positive influence. In terms of soil health, this finding supports the continued use of pulp and paper mill effluent for irrigation. This is however only one aspect of soil health which was evaluated. Further studies on soil resistance and robustness could be undertaken to holistically evaluate soil health in this situation.     

Characterization of bacterial pectinolytic strains involved in the water retting process

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.     

Enhanced growth and nodulation of pigeon pea by co-inoculation of Bacillus strains with Rhizobium spp

Endophytic bacteria which are known to reside in plant tissues have often been shown to promote plant growth. Present study deals with the isolation of putative endophytes from the surface sterilized root nodules of pigeon pea (Cajanus cajan) designated as non-rhizobial (NR) isolates. Three of these non-rhizobial isolates called NR2, NR4 and NR6 showed plant growth promotion with respect to increase in plant fresh weight, chlorophyll content, nodule number and nodule fresh weight when co-inoculated with the rhizobial bioinoculant strain IC3123. The three isolates were neither able to nodulate C. cajan nor did they show significant plant growth promotion when inoculated alone without Rhizobium spp. IC3123. All the three isolates were gram positive rods with NR2 and NR4 showing endospore formation and formed one single cluster in Amplified Ribosomal DNA Restriction Analysis (ARDRA). Partial sequences of 16S rRNA genes of NR4 and NR6 showed 97% similarity to Bacillus megaterium. The Bacillus strains NR4 and NR6 were able to produce siderophores which the rhizobial bioinoculant IC3123 was able to cross-utilize. Under iron starved conditions IC3123 showed enhanced growth in the presence of the Bacillus isolates indicating that siderophore mediated interactions may be underlying mechanism of beneficial effect of the NR isolates on nodulation by IC3123.     

Ecology of cycloheximide-resistant fungi in field soils receiving raw city wastewater or normal irrigation water

The effect of raw city wastewater irrigation on biodiversity and population densities of a cycloheximide-resistant (CH) fungal community was studied in 13 field soils receiving either raw city wastewater or normal irrigation, and in raw city wastewater in the Nablus area, using the hair baiting technique (HBT) and a surface soil dilution plating (SSDP) technique. Three of these fields [one had been receiving raw city wastewater for more than ten years and was designated a heavily polluted field, and the other 2 were cultivated for the first time and were either irrigated with raw city wastewater (newly polluted field) or normal irrigation water (nonpolluted)], were sampled 4–7 times over a 9-month period. The other ten fields, which had been under raw city wastewater irrigation for more than 10 years, were sampled only once. Fifty-seven CH-resistant species belonging to 18 genera were recovered, of which 49 species were recovered from soil habitats and 28 species from raw city wastewater. The HBT had shown to be more efficient in the isolation of pathogenic and potentially pathogenic fungi including dermatophytes. A higher percentage of this group of fungi was recovered from the three main field soils studied using HBT (70% of all isolates), than the SSDP (35.5%); no dermatophytes were recovered by the SSDP method. Two dermatophytes (Microsporum gypseum, and Trichophyton ajelloi), and five more fungi (Arthroderma cuniculi, A. curreyi, Chrysosporium keratinophilum, C. tropicum, and C. pannorum), were recovered from these habitats. Wastewater irrigation seemed to have affected the fungal population densities, with the highest population densities being found in the heavily polluted field soil, while lower population densities were found in the nonpolluted field soil. Increases in organic matter were also observed as a result of sewage effluent irrigation. However, basic similarities in the biodiversity of CH-resistant fungal communities existed in nonpolluted and polluted field soils, and raw city wastewater. Comparable numbers of fungal species were recovered from the three main field soils. The species most commonly found in those habitats included: Alternaria alternata, Aspergillus candidus, Geotrichum candidum, and Paecilomyces lilacinus. Field soils receiving either raw city wastewater or normal irrigation water, were found to be rich in pathogenic and potentially pathogenic CH-resistant fungi, including dermatophytes, with raw city wastewater yielding the highest percentage (81%), followed by the newly wastewater irrigated field (77.7%), the nonpolluted field (67%), and the heavily polluted field (63.4%). Hygienic measures should therefore be taken to control the spread of these fungi in the environment of human communities, and to avoid mycotic infections among farmers. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation of plant-growth-promoting Bacillus strains from soybean root nodules

Endophytic bacteria reside within plant tissues and have often been found to promote plant growth. Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max. (L.) Merr.) root nodules. These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB). Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B. japonicum alone. In the absence of B. japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth. All three isolates were Gram-positive spore-forming rods. While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species. Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.     

Identification of thermophilic and marine bacilli from shallow thermal vents by restriction analysis of their amplified 16S rDNA

AIMS: In order to identify 73 thermophilic isolates from shallow, marine thermal vents of Eolian Islands, we compared their restriction patterns of amplified 16S rDNA with those of nine well described Bacillus species and eight Eolian Bacillus strains. METHODS AND RESULTS: This study allowed to assign 57 (78%) isolates to different Bacillus species. Nineteen field strains were recognised as representatives of four described species, namely B. thermodenitrificans, "B. caldolyticus", B. vulcani and B. stearothermophilus. The profiles of 38 isolates matched instead, those of seven Eolian strains (B. thermodenitrificans strain A2, B. licheniformis strain B3-15, and five novel species, represented by Bacillus strain 1bw, Bacillus strain 4-1, Bacillus strain 5-2, Bacillus strain 10-1, Bacillus strain 1as). Among the 16 unidentified isolates, seven restriction patterns were recognised. CONCLUSIONS: This study showed that restriction analysis of amplified 16S rDNA is useful for a rapid and reliable identification of strains belonging to described species as well as for recognition of new species. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed a high taxonomic diversity among the thermophilic bacilli isolated from Eolian Islands and a distinct distribution of the species within the Eolian hydrothermal vent system.     

内蒙古海拉尔地区碱湖嗜碱细菌的多样性

从内蒙古海拉尔地区碱湖样品中分离到53株嗜碱细菌,其形态与生理生化等特征表现出丰富的多样性。其中28株细菌的16S rRNA基因的限制性内切酶图谱显示出分离菌株之间具有较大差异。根据其中20株菌的16S rRNA基因序列所进行的系统发育分析表明,革兰氏阴性菌均属于Proteobacteria的gamma亚群。革兰氏阳性菌表现出更广泛的多样性,但大部分属于芽孢杆菌谱系（Bacillus spectrum)。     

Restriction fragment length polymorphism analysis of 16S rDNA and low molecular weight RNA profiling of rhizobial isolates from shrubby legumes endemic to the Canary islands

Thirty-six strains of slow-growing rhizobia isolated from nodules of four woody legumes endemic to the Canary islands were characterised by 16S rDNA PCR-RFLP analyses (ARDRA) and LMW RNA profiling, and compared with reference strains representing Bradyrhizobium japonicum, B. elkanii, B. liaoningense, and two unclassified Bradyrhizobium sp. (Lupinus) strains. Both techniques showed similar results, indicating the existence of three genotypes among the Canarian isolates. Analysis of the combined RFLP patterns obtained with four endonucleases, showed the existence of predominant genotype comprising 75% of the Canarian isolates (BTA-1 group) and the Bradyrhizobium sp. (Lupinus) strains. A second genotype was shared by nine Canarian isolates (BGA-1 group) and the B. japonicum and B. liaoningense reference strains. The BES-5 strain formed an independent group, as also did the B. elkanii reference strains. LMW RNA profile analysis consistently resolved the same three genotypes detected by 16S ARDRA among the Canarian isolates, and suggested that all these isolates are genotypically more related to B. japonicum than to B. elkanii or B. liaoningense. Cluster analysis of the combined 16S ARDRA and LMW RNA profiles resolved the BTA-1 group with the Bradyrhizobium sp. (Lupinus) strains, and the BES-5 isolate, as a well separated sub-branch of the B. japonicum cluster. Thus, the two types of analyses indicated that the isolates related to BTA-1 conform a group of bradyrhizobial strains that can be clearly distinguishable from representatives of the tree currently described Bradyrhizobium species. No correlation between genotypes, host legumes, and geographic location was found.     

Application of several molecular techniques to study numerically predominant Bifidobacterium spp. and Bacteroidales order strains in the feces of healthy children

Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.     

Species composition of Bacteroidales order bacteria in the feces of healthy people of various ages

A study of species distribution of numerically predominant Bacteroidales order isolates in feces of healthy people aged 1-33 years was accomplished using a combination of amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA (rDNA) sequencing. It was found that the majority of isolates in all age groups belonged to species B. xylanisolvens, B. vulgatus, and B. uniformis. Members of genera Alistipes, Parabacteroides, Odoribacter, Barnesiella, and Prevotella were also detected frequently.     

Diversity of culturable heterotrophic aerobic bacteria in pristine stream bed sediments

More than 900 culturable, heterotrophic aerobic isolates were obtained from the sediments of a forested, pristine stream and analyzed using three classical microbiological tests: API 20E, amplified ribosomal DNA restriction analysis (ARDRA), and fatty acid analysis. Gram-negative bacteria comprised most of the heterotrophic aerobic isolates (66.7%), similar to other oligotrophic environments. The isolates were assigned to the genus level as Pseudomonas, Flavobacterium, Micrococcus, Bacillus, Chromobacterium, Acinetobacter, Alcaligenes, Aeromonas, Methylobacterium, Enterobacter, Corynebacterium, and Sporolactobacillus. Genotypic analysis by ARDRA facilitated the comparison among strains within Pseudomonas, Bacillus, and Enterobacter groups. Temperature and predation may influence the survival of bacteria during seasons, as shown previously by others. Our results showed that the number of heterotrophic aerobic bacteria, especially Enterobacter, Alcaligenes, and Aeromonas, and Gram-positive bacteria, decreased in winter compared to summer conditions.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.     

Biomass production and nutrient cycling in Eucalyptus short rotation energy forests in New Zealand. I: Biomass and nutrient accumulation

Accumulation of biomass and nutrients (N, P, K, Ca, Mg and Mn) was measured during the first 3-year rotation of three Eucalyptus short rotation forest species (E. botryoides, E. globulus and E. ovata) irrigated with meatworks effluent compared with no irrigation. E. globulus had the highest biomass and nutrient accumulation either irrigated with effluent or without irrigation. After 3-year growth, E. globulus stands irrigated with effluent accumulated 72 oven dry t/ha of above-ground total biomass with a total of 651 kg N, 55 kg P, 393 kg K, 251 kg Ca, 35 kg Mg and 67 kg Mn. Effluent irrigation increased the accumulation of biomass, N, P, K and Mn, but tended to reduce the leaf area index and leaf biomass, and decreased the accumulation of Ca and Mg.     

Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species.

Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers. A spacer region of 144 bp was determined for all of 6 B. thuringiensis strains, 7 B. thuringiensis subspecies, and 11 B. thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis. Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B. thuringiensis subsp. kurstaki HD-1 when compared with ISR sequences from other subspecies. The same differences were identified between the ISR of B. thuringiensis strains and the ISR of B. cereus and B. anthracis. These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe.     

Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with Hae III revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, Apa I and Not I digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with Hae III.     

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Genetic diversity and biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.