Sclerotium cepivorum Berk. in onion (Allium cepa L.) crops: isolation and characterization of bacteria antagonistic to the fungus in Queensland

Bacteria antagonistic to the fungal pathogen Sclerotium cepivorum Berk., which causes onion white rot disease, were isolated and characterized. Eighty percent of the bacterial antagonists isolated from soil samples and sclerotia of S. cepivorum recovered from soil were identified as belonging to the genus Bacillus. The other antagonists were coryneforms and Pseudomonas spp. In contrast, 90% of the antagonists isolated from onion roots were mostly Pseudomonas spp. and the remainder were Erwinia spp. The significance of these antagonistic bacteria is discussed in relation to the control of white rot disease of onion.     

Extracellular enzymes produced by microorganisms isolated from maritime Antarctica

Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.     

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland

AIMS: To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent-treated soil. METHODS AND RESULTS: A Most Probable Number (MPN) method was developed to assess the levels of Arcobacter spp. in seven pig effluent ponds and six effluent-treated soils, immediately after effluent irrigation. Arcobacter spp. levels in the effluent ponds varied from 6.5 x 10(5) to 1.1 x 10(8) MPN 100 ml(-1) and in freshly irrigated soils from 9.5 x 10(2) to 2.8 x 10(4) MPN g(-1) in all piggery environments tested. Eighty-three Arcobacter isolates were subjected to an abbreviated phenotypic test scheme and examined using a multiplex polymerase chain reaction (PCR). The PCR identified 35% of these isolates as Arcobacter butzleri, 49% as Arcobacter cryaerophilus while 16% gave no band. All 13 nonreactive isolates were subjected to partial 16S rDNA sequencing and showed a high similarity (>99%) to Arcobacter cibarius. CONCLUSIONS: A. butzleri, A. cryaerophilus and A. cibarius were isolated from both piggery effluent and effluent-irrigated soil, at levels suggestive of good survival in the effluent pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide quantitative information on Arcobacter spp. levels in piggery effluent and to associate A. cibarius with pigs and piggery effluent environments.     

Use of different nitrogen sources by the edible ectomycorrhizal mushroom Cantharellus cibarius

The growth of three strains of Cantharellus cibarius on liquid media containing ammonium, nitrate and bovine serum albumin (BSA) in different combinations was determined. The most readily utilisable source of N was ammonium. BSA utilisation was limited compared with media containing ammonium. Growth on nitrate was also poor, suggesting a limited capacity of C. cibarius to metabolise this nitrogen source. There was some indication of considerable intraspecific variation within C. cibarius in the utilisation of nitrogen sources. Possible links between atmospheric nitrogen deposition and the observed decrease of sporocarp formation by C. cibarius in Europe are discussed. We highlight the potential ecological significance of bacteria associated with C. cibarius which may circumvent the need for fungal extracellular enzymes to access complex nitrogen sources.     

Development and utilisation of a medium to isolate phenanthrene-degrading Pseudomonas spp.

In this study, we isolated phenanthrene degraders belonging to Pseudomonas spp. by combining the selective force of two previously described media. The two compounds, sodium lauryl sarcosine and trimethoprim, from the Gould S1 medium, were added to minimal agar plates sprayed with phenanthrene. Pseudomonas spp. that could produce clearing zones were isolated in one step from the rhizosphere without first selecting for Pseudomonas spp. and subsequently screening for degraders or vice versa. Enumeration and isolation of Pseudomonas spp. attached to the rhizosphere showed clear differences between two types of soil. Rhizosphere-attached phenanthrene degraders (from Pseudomonas spp.) were isolated from a former coal gasification site, but were absent in an agricultural soil subjected to organic farming. We isolated 23 phenanthrene degraders producing clearing zones from the rhizosphere of barley roots. All of these 23 isolates (of which 16 were fluorescent in UV light) proved to be members of the Pseudomonas RNA homology group I, on the basis of results of the analytical profile index (API) test system and classic taxonomic tests.     

Effects of transgenic potatoes with an altered starch composition on the diversity of soil and rhizosphere bacteria and fungi

The aim of this study was to investigate potential effects on the composition of the bacterial and fungal diversity in rhizosphere and soil of a transgenic potato line (SIBU S1) which was modified in its starch composition by RNA anisensing, compared to the non-transgenic parental cultivar (SIBU) at the flowering stage in 2000. Furthermore a second non-transgenic cultivar (SOLANA) was included in the study. To avoid artefacts derived from cultivation depending approaches, molecular techniques based on 16S-(bacteria) and 18S-(fungi) rDNA respectively were used to describe the microbial community structure. Comparing 16S- and 18S-rDNA DGGE fingerprints from the different bulk soil samples, it could be shown that no significant differences between the two cultivars and the transgenic line were found. Similar results were obtained for the rhizosphere samples using the eubacterial, α-and β-proteobacterial and fungal specific primers with the exception of, the eubacterial DGGE patterns obtained for the rhizosphere of SOLANA. These patterns revealed that the relative abundance of one band was enhanced compared with the patterns of SIBU and SIBU S1 and the sequence of the differentiating band showed the highest similarity with Enterobacter amnigenus. When Pseudomonas specific primers were used, relevant differences were found between the rhizosphere patterns of the transgenic potato line (SIBU S1) and the parental cultivar (SIBU). However, clear effects of the cultivar SOLANA on the structure of the Pseudomonas community compared to SIBU were also detected.     

Distribution of bacteria and fungi in the earthworm Libyodrillus violaceous (Annelida: Oligochaeta), a native earthworm from Nigeria

Earthworms are soil invertebrates that play a key role in recycling organic matter in soils. In Nigeria, earthworms include Libyodrillus violaceous. Aerobic and anaerobic bacterial counts, as well as fungal counts of viable microorganisms in soils and gut sections, were made on twenty L. violaceous collected from different sites on the campus of the University of Agriculture, Abeokuta, Nigeria. The samples were collected between April and November, 2002. Numbers of microorganisms were higher in castings and gut sections than in un-ingested soil samples. The guts and their contents also had higher moisture and total nitrogen contents than the un-ingested soils. Bacteria and fungi isolated from the samples were identified by standard microbiological procedures on the bases of their morphological and biochemical characteristics. Isolated bacteria were identified as Staphylococcus, Bacillus spp., Pseudomonas aeruginosa, Streptococcus mutans, Clostridium, Spirocheata spp., Azotobacter spp., Micrococcus lylae, Acinetobacter spp., Halobacterium for bacteria. Yeast isolates were identified as Candida spp., Zygosaccharomyces spp., Pichia spp., and Saccharomyces spp while molds were identified as, Aspergillus spp., Pytium spp., Penicillium spp., Fusarium spp and Rhizopus spp. Of the five locations examined, the refuse dump area had the highest numbers of both aerobic and anaerobic organisms, followed by the arboretum while the cultivated land area recorded the lowest counts. The higher numbers of microorganisms observed in the gut sections and casts of the earthworms examined in this work reinforce the general concept that the gut and casts of earthworms show higher microbial diversity and activity than the surrounding soil.     

Polyhydroxyalkanoate production by antarctic soil bacteria isolated from Casey Station and Signy Island

Polyhydroxyalkanoate (PHA) is a family of biopolymers produced by some bacteria and is accumulated intracellularly as carbon and energy storage material. Fifteen PHA-producing bacterial strains were identified from bacteria isolated from Antarctic soils collected around Casey Station (66°17'S, 110°32'E) and Signy Island (60°45'S, 45°36'W). Screening for PHA production was carried out by incubating the isolates in PHA production medium supplemented with 0.5% (w/v) sodium octanoate or glucose. 16S rRNA gene sequence analysis revealed that the isolated PHA-producing strains were mainly Pseudomonas spp. and a few were Janthinobacterium spp. All the isolated Pseudomonas strains were able to produce medium-chain-length (mcl) PHA using fatty acids as carbon source, while some could also produce mcl-PHA by using glucose. The Janthinobacterium strains could only utilize glucose to produce polyhydroxybutyrate (PHB). A Pseudomonas isolate, UMAB-40, accumulated PHA up to 48% cell dry mass when utilizing fatty acids as carbon source. This high accumulation occurred at between 5°C and 20°C, then decreased with increasing temperatures. Highly unsaturated mcl-PHA was produced by UMAB-40 from glucose. Such characteristics may be associated with the ability of UMAB-40 to survive in the cold.     

Identification and incidence of fungal strains in chronic rhinosinusitis patients

The fungal revolution taking place in otorhinology inspired us to study the frequency of occurrence of fungi in the nasal mucus of chronic rhinosinusitis (CRS) patients (with or without polyposis) in order to evaluate the incidence of eosinophilic fungal sinusitis in CRS patients. Ninety-six samples were examined from patients with CRS. In 74 cases mucus was collected non-invasively, and in 22 cases during operation. The Gram-stained direct smears of all samples were also evaluated. Bacteria and fungi colonizing in the mucus were detected by culturing method. The control group consisted of 50 healthy volunteers. Typical aerobic pathogenic bacteria could be isolated from 34 patients. Fifty-seven aerobic bacteria were isolated, i.e. 1.6 bacteria/positive patient with a maximum of 3 different bacteria/sample. The most frequently isolated bacteria were Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, and Haemophilus influenzae. Yeasts and moulds could be detected from 79 patients (83%): Candida albicans, Candida spp., Aspergillus spp., Cladosporium spp, and Penicillium spp. were isolated most frequently. Altogether 237 yeasts and moulds were isolated, i.e. 3.0 different fungi/positive patient, with a maximum of 5 different fungi/sample. In the control group aerobic pathogens were not isolated, only apathogenic species. Fungi were isolated from 22 healthy patients (44%). These data indicate that fungi are frequently involved in the aetiology of CRS. IgE-medicated hypersensitivity to fungal allergens could not be proven in our patients.     

Estimation of root nodule development by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. Using hydroponic culture

A nitrogen-fixing bacterium isolated from the root nodules of a cultivated leguminous plant, soybean (Glycine max L.), was cultivable and was identified as Rhizobium sp. Bacterial species isolated from root nodules of wild leguminous plants including -bush clover, white dutch clover, wisteria, and false acacia were identified as Burkholderia cepacia, Pseudomonas migulae, Pseudomonas putida, and Flavobacterium sp, respectively, all of which are heterotrophic bacteria that grow in the rhizosphere. Temperature gradient gel electrophoresis (TGGE) 16S-rDNA bands extracted directly from the bacterial population within the root nodules of the wild leguminous plants were identified as Rhizobium sp, Mesorhizobium sp, and Bradyrhizobium sp. none were cultivable. Rhizobium sp. isolated from soybean root nodule generated approximately 48 and 19 mg/L of ammonium in glucose- and starch-defined medium, respectively, during 8 days of growth. The growth rate of Rhizobium sp. was increased by the addition of yeast extract but not by the addition of ammonium. K _m and V _max for starch saccharification measured with the extracellular crude enzyme of Rhizobium sp. were 0.7556 mg/L and 0.1785 mg/L/min, respectively. The inoculation of Rhizobium sp. culture into a hydroponic soybean plant culture activated root nodule development and soybean plant growth. The inoculated Rhizobium sp. survived for at least 4 weeks, based on the TGGE pattern of 16S-rDNA. The 16S-rDNA of Rhizobium sp. isolated from newly developed root nodules was homologous with the inoculated species.     

A 13C-NMR study of exudation and storage of carbohydrates and amino acids in the ectomycorrhizal edible mushroom Cantharellus cibarius

(13)C-NMR analyses of Cantharellus cibarius growth media were performed. We found exudation of trehalose and mannitol, which may explain the phenomenon of reproducing Pseudomonas bacteria observed inside fruit bodies. Exudation varied with strain and environment. NMR analyses of stored (13)C was also performed. Trehalose, mannitol, and arginine were revealed. The mannitol pathway seems to play an important role for trehalose production in this species. This is the first study of the fate of the photosynthetically derived carbon in the highly appreciated edible ectomycorrhizal mushroom Cantharellus cibarius.     

The symbiotic bacteria Alcaligenes faecalis of the entomopathogenic nematodes Oscheius spp. exhibit potential biocontrol of plant- and entomopathogenic fungi

Soil-dwelling entomopathogenic nematodes (EPNs) kill arthropod hosts by injecting their symbiotic bacteria into the host hemolymph and feed on the bacteria and the tissue of the dying host for several generations cycles until the arthropod cadaver is completely depleted. The EPN–bacteria–arthropod cadaver complex represents a rich energy source for the surrounding opportunistic soil fungal biota and other competitors. We hypothesized that EPNs need to protect their food source until depletion and that the EPN symbiotic bacteria produce volatile and non-volatile exudations that deter different soil fungal groups in the soil. We isolated the symbiotic bacteria species (Alcaligenes faecalis) from the EPN Oscheius spp. and ran infectivity bioassays against entomopathogenic fungi (EPF) as well as against plant pathogenic fungi (PPF). We found that both volatile and non-volatile symbiotic bacterial exudations had negative effects on both EPF and PPF. Such deterrent function on functionally different fungal strains suggests a common mode of action of A. faecalis bacterial exudates, which has the potential to influence the structure of soil microbial communities, and could be integrated into pest management programs for increasing crop protection against fungal pathogens.     

Heat stable lipase activity of thermotolerant bacteria from hot springs at Orissa, India

Thermotolerant bacteria (35 in toto) isolated from three hot springs (Atri, Taptapani and Deuljhari, Orissa), were screened for lipase activities. Of these, nine strains of Bacillus spp. and three strains of Pseudomonas spp. showed heat stable lipase activity at 60 degrees C. The hydrolytic activity of these bacteria was tested using Tween-20 and Tween-80 as substrates at different temperatures using plate assay and titration techniques. The hydrolytic activity at different pH values and salt concentrations was investigated.     

Isolation and characterization of heterotrophic bacteria able to grow aerobically with quaternary ammonium alcohols as sole source of carbon and nitrogen

The quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are hydrolysis products of their parent esterquat surfactants, which are widely used as softeners in fabric care. We isolated several bacteria growing with QAAs as the sole source of carbon and nitrogen. The strains were compared with a previously isolated TM-degrading bacterium, which was identified as a representative of the species Pseudomonas putida (Syst. Appl. Microbiol. 24 (2001) 252). Two bacteria were isolated with DM, referred to as strains DM 1 and DM 2, respectively. Based on 16S-rDNA analysis, they provided 97% (DM 1) and 98% (DM 2) identities to the closest related strain Zoogloea ramigera Itzigsohn 1868AL. Both strains were long, slim, motile rods but only DM 1 showed the floc forming activity, which is typical for representatives of the genus Zoogloea. Using MM we isolated a Gram-negative, non-motile rod referred to as strain MM 1. The 16S-rDNA sequence of the isolated bacterium revealed 94% identities (best match) to Rhodobacter sphaeroides only. The strains MM 1 and DM 1 exclusively grew with the QAA which was used for their isolation. DM 2 was also utilizing TM as sole source of carbon and nitrogen. However, all of the isolated bacteria were growing with the natural and structurally related compound choline.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

Culturable bacterial diversity associated with cysts of <Emphasis Type="Italic">Eurhizococcus brasiliensis</Emphasis> (Hempel) (Hemiptera: Margarodidae)

The ground-pearl Eurhizococcus brasiliensis is an important insect pest of grapes. Nowadays, its biology is still barely known and studies related to its secondary symbionts are virtually non-existent. Our main goal was to evaluate the bacterial diversity associated with cysts of E. brasiliensis using a culture-dependent approach. Six different isolation media were used and shown to be suitable for culturing bacteria associated with E. brasiliensis. A total of 39 bacteria strains were isolated and classified into 10 different morphotypes. The ISP-4 medium was the most suitable for bacteria isolation from cysts of the ground-pearl, providing the highest number of morphotypes. 16S rDNA gene analysis demonstrated a high diversity of bacteria associated with cysts, with six Pseudomonas chlororaphis isolates (the most predominant morphotype) and three different morphotypes of Bacillus spp. as the most representative genera. The phylogenetic analysis showed close affinity between the isolated morphotypes and bacterial strains usually isolated from plant and soil samples. Sphingopyxis and Stenotrophomonas were isolated for the first time from arthropods. Although it was not possible to determine the primary source of infection by these bacteria, our data suggests that these microorganisms may be transitory and acquired from the environment.     

Antifungal activity of rhizospheric bacteria

Fluorescent Pseudomonad spp. were isolated from the rhizosphere of potato plants (Algeria) by serial dilutions of rhizosphere soils on Kings B medium and were tested for their antifungal activity. The antifungal activity of the Pseudomonas isolated from Potatoes rhizosphere was tested against Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum in dual culture with bacteria on PDA. The Petri dish was divided into tow, on one the bacteria was spread and on the opposite side fungal plugs were inoculated and incubated for one week. Fourteen bacteria were isolated; only one isolate inhibited the growth of Pythium ultimum, Rhizoctonia solani, Fusarium solani; Fusarium oxysporum f.sp. albedinis and Fusarium oxysporum f. sp. Lycopersici with inhibition zones of 39.9, 33.7, 30.8, 19.9 and 22.5 mm respectively.     

Isolation and characterization of heavy polycyclic aromatic hydrocarbon-degrading bacteria adapted to electrokinetic conditions

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria capable of growing under electrokinetic conditions were isolated using an adjusted acclimation and enrichment procedure based on soil contaminated with heavy PAHs in the presence of an electric field. Their ability to degrade heavy PAHs under an electric field was individually investigated in artificially contaminated soils. The results showed that strains PB4 (Pseudomonas fluorescens) and FB6 (Kocuria sp.) were the most efficient heavy PAH degraders under electrokinetic conditions. They were re-inoculated into a polluted soil from an industrial site with a PAH concentration of 184.95 mg kg^?1. Compared to the experiments without an electric field, the degradation capability of Pseudomonas fluorescens and Kocuria sp. was enhanced in the industrially polluted soil under electrokinetic conditions. The degradation extents of total PAHs were increased by 15.4 and 14.0 % in the electrokinetic PB4 and FB6 experiments (PB4 + EK and FB6 + EK) relative to the PB4 and FB6 experiments without electrokinetic conditions (PB4 and FB6), respectively. These results indicated that P. fluorescens and Kocuria sp. could efficiently degrade heavy PAHs under electrokinetic conditions and have the potential to be used for the electro-bioremediation of PAH-contaminated soil, especially if the soil is contaminated with heavy PAHs.     

Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.     

Conservation of the response regulator gene gacA in Pseudomonas species

The response regulator gene gacA influences the production of several secondary metabolites in both pathogenic and beneficial Pseudomonas spp. In this study, we developed primers and a probe for the gacA gene of Pseudomonas species and sequenced a 425 bp fragment of gacA from ten Pseudomonas strains isolated from different plant-associated environments. Polymerase chain reaction analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas: multiple strains of different Pseudomonas species all responded positively to the probe, whereas no response was obtained from 18 other strains representing 14 species that belong to eight different genera of Gram-negative bacteria other than Pseudomonas. Furthermore, from a total of approximately 550 indigenous bacterial isolates obtained from the rhizosphere of wheat, all isolates that hybridized with the gacA probe were classified as Pseudomonas spp. by group-specific primers. Isolates that did not respond with the gacA probe and primers were identified as bacterial genera other than Pseudomonas, including Stenotrophomonas, Cryseomonas and Comamonas spp. These results indicate that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA fragments and the sequences of gacA homologues present in the databases, showed six distinct clusters that correspond to the following bacterial families: Pseudomonaceae, Enterobacteriaceae, Alteromonadaceae, Vibrionaceae, Burkholderia and Xanthomonas. Within the Pseudomonadaceae and Enterobacteriaceae, polymorphisms within gacA and its homologues allowed identification of six and five subclusters respectively. Comparison of the gacA gene and GacA protein-based trees with the tree inferred from 16S rDNA sequences yielded a similar overall clustering. These results suggest that gacA and its homologues may provide complementary markers for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas.