Putida Biovar Research Articles

Identification of endophytic bacterial strain MGP1 selected from papaya and its biocontrol effects on pathogens infecting harvested papaya fruit

Traditional approaches to the control of diseases of papaya fruit rely on the use of synthetic chemicals, which can cause serious human health and environmental problems. Endophytes might be used as an alternative to chemicals to effectively control diseases of harvested papaya fruit. MGP1 was one of the biological control agents that was selected from the pericarp of papaya and identified as Pseudomonas putida biovar A. The bacterium was able to colonise in the lamina, leafstalk, pericarp and pulp of papaya and strongly inhibit ten kinds of phytopathogen. Positive control effects were achieved when fruits were challenged with Phytophthora nicotianae at 24 and 48 h after MGP1 treatment. The control effect of MGP1 on anthracnose of harvested papaya fruit reached 54%. The application of MGP1 at five preharvest stages of papaya significantly reduced the disease index of anthracnose, with the best control effect reaching 63% after application at the florescence stage. However, the rate of latent infection of Colletotrichum gloeosporioides was significantly reduced only after application at the fluorescence stage. The results indicated the powerful ability of MGP1 as a biological agent.

Effect of a Nickel-Tolerant ACC Deaminase-Producing Pseudomonas Strain on Growth of Nontransformed and Transgenic Canola Plants

Four bacterial strains were isolated from soils at nickel-contaminated sites based on their ability to utilize 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen. The four isolates were all identified as Pseudomonas putida Biovar B, and subsequent testing revealed that they all exhibited traits previously associated with plant growth promotion (i.e., indoleacetic acid and siderophore production and ACC deaminase activity). These four strains were also tolerant of nickel concentrations of up to 13.2 mM in the culture medium. The strain, HS-2, selected for further characterization, was used in pot experiments to inoculate both nontransformed and transgenic canola plants (expressing a bacterial ACC deaminase gene in its roots). Plants inoculated with the HS-2 strain produced an increase in plant biomass as well as in nickel (Ni) uptake by shoots and roots. The results suggest that this strain is a potential candidate to be used as an inoculant in both phytoremediation protocols and in plant growth promotion.

Utilization of Phenylpropanoids by Pseudomonas putida Soil Isolates and Its Probable Taxonomic Significance

Twenty seven soil bacterial isolates able to grow on cinnamic acid or ferulic acid as a sole carbon and energy source were examined for the ability to utilize other phenylpropanoids and phylogenetic interrelationships. A 16S rRNA gene sequence analysis revealed that 26 of the isolates were closely related to Pseudomonas putida biovar A and classified into 6 OTUs: cinnamic acid utilization-positive (OTUs, CN-1 to CN-3) and -negative groups (FR-1 to FR-3). P. putida NBRC 14164(T) and JCM 13061 belonged to FR-3. A probable reclassification of P. putida was discussed in relation to the utilization of phenylpropanoids.

Taxonomic heterogeneity, as shown by siderotyping, of strains primarily identified as Pseudomonas putida

One hundred and forty-four fluorescent pseudomonad strains isolated from various environments (soil, water, plant rhizosphere, hospital) and received as Pseudomonas putida (83 strains), P. putida biovar A (49 strains), P. putida biovar B (10 strains) and P. putida biovar C (2 strains), were analysed by the pyoverdine-isoelectrofocusing and pyoverdine-mediated iron uptake methods of siderotyping. Both methods demonstrated a great diversity among these strains, which could be subdivided into 35 siderovars. Some siderovars specifically included strains that have subsequently been transferred to well-defined Pseudomonas species, e.g. Pseudomonas monteilii or Pseudomonas mosselii, or which could be related by their siderotype to Pseudomonas jessenii or Pseudomonas mandelii. Other siderovars included strains sharing a high level of DNA-DNA relatedness (>70%), thus demonstrating that siderotyping could easily circumscribe strains at the species level. However, a group of seven strains, including the type strain, P. putida ATCC 12633T, were allocated into four siderovars, despite sharing DNA-DNA relatedness values of higher than 70 %. Interestingly, the strong genomic relationships between these seven strains were supported by the structural relationships among their pyoverdines, thus reflecting their phylogenetic affinities. These results strongly support the view that pyoverdine-based siderotyping could be used as a powerful tool in Pseudomonas taxonomy.

Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes.

Phylogenetic analysis of the genus Pseudomonas: was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas: (a total of 125 strains). Pseudomonas: strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the 'P: aeruginosa complex', which included P: aeruginosa, P: alcaligenes, P: citronellolis, P: mendocina, P: oleovorans and P: pseudoalcaligenes, and the 'P: stutzeri complex', which included P: balearica and P: stutzeri. IGC II was further split into three subclusters that were designated the 'P: putida complex', the 'P: syringae complex' and the 'P: fluorescens complex'. The 'P: putida complex' included P: putida and P: fulva. The 'P: syringae complex' was the cluster of phytopathogens including P: amygdali, P: caricapapayae, P: cichorii, P: ficuserectae, P: viridiflava and the pathovars of P. savastanoi and P. syringae. The 'P. fluorescens complex' was further divided into two subpopulations, the 'P. fluorescens lineage' and the 'P. chlororaphis lineage'. The 'P. fluorescens lineage' contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the 'P. chlororaphis lineage' included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the 'P. fluorescens complex'.

Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis.

A new Pseudomonas species, for which the name Pseudomonas plecoglossicida is proposed, was isolated from cultured ayu (Plecoglossus altivelis) with bacterial haemorrhagic ascites. The causative agent was similar to Pseudomonas putida biovar A in its phenotypic characteristics and on the basis of 16S rRNA gene sequence analysis, but it reduced nitrate to nitrite. Furthermore, it was distinguished phenotypically from Pseudomonas putida biovar A by utilization of D-malate, L-(+)-tartrate, m-tartrate and nicotinate. The levels of DNA-DNA hybridization between the isolate strain FPC 951T and other reference strains of Pseudomonas species, including Pseudomonas putida, were less than 50%. The G+C content of the DNA of FPC 951T was 62.8 mol%. Strain FPC 951T (= ATCC 700383T) is designated the type strain of the new species.

Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India

Phenol is an industrially important compound which has a wide range of applications. Being highly soluble in water, it appears as the major pollutant in waste waters arising from both phenol manufacturing and from industrial units that utilise phenol. Because of its toxicity, bioremediation of phenol is necessary. Since some of the phenol-bearing industrial waste waters are alkaline in nature, use of alkaliphilic bacteria for bioremediation of phenol was investigated. Alkaliphilic bacteria were isolated from sediments of an alkaline lake in Lonar, Dist. Buldhana, Maharashtra State, India, by phenol enrichment at pH 10.0 and phenol concentration of 500 mg/l. The lake (lat. 19°58'45", long. 76°34') is known to be a unique inland saline lake in Asia. It has a circular periphery and diameter of 2 km around the top of the banks and 1.2 km at the bottom. The lake has a high saline level (∼ 2649 mg/l sodium chloride) and a high level of alkalinity (∼ 2605 mg/l calcium carbonate). Alkaliphilic strains of Arthrobacter spp., Bacillus cereus, Citrobacter freundii, Micrococcus agilis and Pseudomonas putida biovar B were capable of removing phenol from waste waters arising from industries manufacturing methyl violet (using phenol as one of the major raw materials) and cumene-phenol. The waste waters from both these units were alkaline in nature (pH ∼ 9.95-10.1) and had a high phenol content (368-660 mg/l). The alkaliphilic bacteria being studied removed 100% of the phenol from the industrial waste waters within 48 h of incubation under shake culture conditions and at an ambient temperature of 28 ± 2 °C. Bioremediation of phenol by alkaliphilic strains of Arthrobacter spp., B. cereus, C. freundii and M. agilis seems to be the first report.

Phylogenetic relationships of Pseudomonas putida strains deduced from the nucleotide sequences of gyrB, rpoD and 16S rRNA genes.

Phylogenetic analysis of 20 Pseudomonas strains (Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas chlororaphis) was conducted by using the nucleotide sequences of the genes for 16S RNA, DNA gyrase B subunit (gyrB) and RNA polymerase delta 70 factor (rpoD), which have been determined by the direct sequencing of PCR-amplified fragments. On the basis of gyrB and rpoD sequences, these strains were split into two major clusters: one including the type strain of P. putida and all biovar A strains and the other including all P. putida biovar B strains, P. fluorescens stains and the P. chlororaphis strain. In the phylogenetic tree reconstructed from the 16S rRNA sequences included variable regions, P. Putida biovar A and B strains were not separated into two independent clusters, whereas in the phylogenetic tree reconstructed from the 16S rRNA sequences excluding the variable region sequences, these strains were separated into P. putida biovar A and biovar B clusters. The pairwise distances estimated from the variable regions of 16S rRNA correlated poorly with the synonymous distances estimated from the gyrB and rpoD genes. On the other hand, a highly significant correlation was observed between the pairwise distances estimated from the non-variable regions of 16S rRNA and the synonymous distances from gyrB and rpoD genes. Consequently, only the 16S rRNA sequences in the non-variable regions should be used for the phylogenetic analysis. The gyrB and rpoD analyses showed the necessity for the reclassification of P. putida biovar B strains.

Lithocholic acid side-chain cleavage to produce 17-keto or 22-aldehyde steroids by Pseudomonas putida strain ST-491 grown in the presence of an organic solvent, diphenyl ether.

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.

Pseudomonas monteilii sp. nov., isolated from clinical specimens.

We propose the name Pseudomonas monteilii for a new species of gram-negative, rod-shaped, motile bacteria that were nonhemolytic on blood agar and were isolated from clinical sources. The 10 strains of P. monteilii were incapable of liquefing gelatin. They grew at 10 degrees C but not at 41 degrees C, produced fluorescent pigments, catalase, and cytochrome oxidase, and possessed the arginine dihydrolase system. They were capable of respiratory but not fermentative metabolism. They did not hydrolyze esculin or starch and were able to use benzylamine, alpha-aminobutyrate, D-ribose, L-arabinose, butyrate, valerate, isovalerate, isobutyrate, inositol, phenylacetate, D-alanine, and amylamine. They possessed L-phenylalanine arylamidase, L-lysine arylamidase, L-alanine arylamidase, gamma-glutamyl-transferase, glycyl-phenylalanine arylamidase, L-tryptophan arylamidase, glycyl-L-alanine arylamidase, esterase C4, esterase C6, esterase C8, esterase C9, esterase C10, and esterase C18. DNA relatedness studies revealed that P. monteilii strains formed a homogeneous DNA hybridization group. A total of 57 strains representing previously described or partially characterized taxa belonging to the genus Pseudomonas were 6 to 54% related to P. monteilii. The highest hybridization values were obtained with strains belonging to or related to Pseudomonas putida biovar A. The average G+C content of the DNA was 60.5 +/- 0.5 mol% for four of the P. monteilii strains studied. The type strain of P. monteilii is CFML 90-60 (= CIP 104883); it was isolated from bronchial aspirate and has a G+C content of 60 mol%. The clinical significance of these organisms is not known.

Taxonomic Diversity of Pseudomonads Revealed by Computer-interpretation of Ribotyping Data

Summary In the framework of a European project entitled “High Resolution Automated Microbial Identification”, the ability of rRNA gene restriction pattern determination (ribotyping) to uncover the taxonomic diversity of a collection of bacteria was tested on 226 strains of Pseudomonas sensu lato. Improved ribotyping methodology included automated DNA extraction and purification, low voltage electrophoresis, vacuum transfer of restricted, DNA fragments to nylon membranes, hybridization with a chemically labeled probe in a hybridization incubator, computer capture of restriction patterns with automatic migration measurements, fragment size interpolation, and comparison of patterns using programs of the Taxotron package (Institut Pasteur, Paris, France). Two endonucleases were used (SmaI and HincII). The strains, belonging to more than 40 nomenspecies, displayed 169 unique rRNA gene restriction patterns with SmaI, and 159 unique rRNA gene restriction patterns with HincII. A combined analysis of both restriction data yielded 79 ribogroups or isolated ribotypes. Most (92.7%) ribogroups were homogeneous with respects to nomenspecies. Some nomenspecies were split in several ribogroups (e.g. P. putida, P. fluorescens, P. marginalis, P. pseudoalcaligenes). Nomenspecies represented by single strains gave isolated ribotypes. Biovars of P. fluorescens and P. putida formed distinct ribogroups. However, P. marginalis pathovars were not separated by ribotyping. When the ribotyping data (ribogroups) were compared to published phenotypic data (phenons) obtained with Biotype-100 strips (BioMerieux, La Balme-les-Grottes, France) and Biolog GN Microplates (Biolog, Hayward, CA), an exclusive phenon-ribogroup correspondence was observed for 13 Biotype-100 phenonslribogroups and for 14 Biolog phenonslribogroups. Other Biotype-100 or Biolog phenons showed partial correspondence with ribogroups or were split into several ribogroups. Most unclustered strains in phenotypic analyses were also unclustered in the ribotyping study. DNA-DNA hybridization was used to verify some species delineation. Ribogroup R38 which contained most of P. fluorescens biovar I strains was found to constitute a DNA hybridization group distinct from P fluorescens strains in other ribogroups or biovars. The type strain of P fluorescens, which was unclustered by ribotyping, was found isolated by DNA relatedness. Ribogroup R1 which contained most of P. putida biovar A strains was found to constitute a DNA relatedness group distinct from biovar B strains. A total of 30 isolates from polluted environment were included in the study and 9 of them were identified to six known taxons. Thirteen other isolates constituted five ribogroups not represented by a reference strain and seven isolates gave isolated ribotypes. This work showed the identification otential of ribotyping and the need to extend the ribotype database. It is now clear that the ribotype carries taxonomic information in addition to typing information.

Bioremediation of colour of methyl violet and phenol from a dye‐industry waste effluent using Pseudomonas spp. isolated from factory soil

Methyl violet, a basic dye, is manufactured using phenol and dimethylaniline as the raw materials. It is, therefore likely that the waste effluent arising from such units may contain the dye and unused chemicals. Since such pollutants may be toxic, their removal becomes necessary. The studies were therefore aimed at their bioremediation using microbial species.Four species of Pseudomonas, namely Ps. alcaligenes, Ps. mendocina, Ps. putida biovar B and Ps. stutzeri, were isolated from cattle dung enrichments and the soil samples in the premises of the factory manufacturing methyl violet, nearby Pune.All the four species of Pseudomonas were able to remove phenol and methyl violet with simultaneous reduction in chemical oxygen demand (COD), total organic carbon (TOC) and ammoniacal nitrogen from the waste effluent and the removal was found to be optimum at the original alkaline pH (range 7.45–10.6) of the waste effluent, at ambient temperature (28 ± 2°C under aerated culture condition, at inoculum density of 1.5 × 108 cells ml−1 of the waste effluent and incubation period of 24–48 h. Thus, Pseudomonas spp. isolated from the environment of the dye industry itself and thus naturally adapted could be used for bioremediation of these pollutants.

Effect of Two Plant Species, Flax (Linum usitatissinum L.) and Tomato (Lycopersicon esculentum Mill.), on the Diversity of Soilborne Populations of Fluorescent Pseudomonads

Suppression of soilborne disease by fluorescent pseudomonads may be inconsistent. Inefficient root colonization by the introduced bacteria is often responsible for this inconsistency. To better understand the bacterial traits involved in root colonization, the effect of two plant species, flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations was assessed. Fluorescent pseudomonads were isolated from an uncultivated soil and from rhizosphere, rhizoplane, and root tissue of flax and tomato cultivated in the same soil. Species and biovars were identified by classical biochemical and physiological tests. The ability of bacterial isolates to assimilate 147 different organic compounds and to show three different enzyme activities was assessed to determine their intraspecific phenotypic diversity. Numerical analysis of these characteristics allowed the clustering of isolates showing a high level (87.8%) of similarity. On the whole, the populations isolated from soil were different from those isolated from plants with respect to their phenotypic characteristics. The difference in bacteria isolated from uncultivated soil and from root tissue of flax was particularly marked. The intensity of plant selection was more strongly expressed with flax than with tomato plants. The selection was, at least partly, plant specific. The use of 10 different substrates allowed us to discriminate between flax and tomato isolates. Pseudomonas fluorescens biovars II, III, and V and Pseudomonas putida biovar A and intermediate type were well distributed among the isolates from soil, rhizosphere, and rhizoplane. Most isolates from root tissue of flax and tomato belonged to P. putida bv. A and to P. fluorescens bv. II, respectively. Phenotypic characterization of bacterial isolates was well correlated with genotypic characterization based on repetitive extragenic palindromic PCR fingerprinting.

Comparison of Ribotyping Analysis and Numerical Taxonomy Studies of Pseudomonas putida Biovar A

Numerical phenotypic analysis, using the unweighted pair group average linkage method and Dice similarity coefficient, was performed on 39 strains of clinical origin belonging or related to Pseudomonas putida biovar A and 19 collection strains belonging to the fluorescent members of Pseudomonas rRNA group I (Palleroni, 1984). Five main phenotypic clusters (I to V) were obtained. Phenotypic cluster II (37 strains) was identified as Pseudomonas putida biovar A and it could be subdivided into 4 phenotypic subclusters (IIa to IId). The 37 strains of phenotypic cluster II were then characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns, after digestion of whole-cell DNA by Pvu II. A pattern of hybridized fragments was obtained from each DNA studied. Numerical analysis was then performed and seven ribotypic clusters (A to G) were described in phenotypic cluster II. We noted the total correlation between phenotypic subclusters IIa and IId and ribotypic clusters A and B respectively. Phenotypic subclusters IIb and IIc could be divided into 3 (E, F and G) and 2 (C and D) ribotypic clusters respectively.

Analysis of fluorescent pseudomonads based on 23S ribosomal DNA sequences

The regions from positions 1391 to 1545 and 1620 to 1865 (Escherichia coli numbers) of the 23S ribosomal DNA sequences have been analyzed for a number of Pseudomonas fluorescens and P. putida isolates. Variability was observed only in three smaller regions from positions 1484 to 1508, 1531 to 1542, and 1714 to 1748, corresponding to helices 58, 59, and 63, respectively, where up to 53% dissimilarity was found. Sequence analysis did not allow clear distinctions among P. fluorescens biovars, P. chlororaphis, and P. putida biovar B.

Microbial metabolism of quinoline and related compounds. XIX. Degradation of 4-methylquinoline and quinoline by Pseudomonas putida K1.

A bacterial strain, designated K1, which utilizes 4-methylquinoline and quinoline as sole source of carbon, nitrogen and energy was isolated from soil. Based on its morphological and physiological characteristics, it was classified as Pseudomonas putida biovar B. Four metabolites of 4-methylquinoline degradation were isolated from the culture supernatant and identified as 4-methyl-2-oxo-1,2-dihydroquinoline, 8-hydroxy-4-methyl-2-oxo-1,2-dihydroquinoline, 7,8-dihydroxy-4-methyl-2-oxo-1,2-dihydroquinoline and 6-hydroxy-5-(2-carboxyethenyl)-4-methyl-1H-2-pyridone. Formation of the latter compound is suggested to proceed by decarbonylation of a putative meta-cleavage product of the 7,8-dihydroxy derivative. During growth on quinoline four compounds were released into the culture fluid, too. Upon isolation they were identified as 2-oxo-1,2-dihydroquinoline, 6-hydroxy-2-oxo-1,2-dihydroquinoline, 5-hydroxy-6-(3-carboxy-3-oxopropenyl)-1H-2-pyridone and 2H-pyran-2-on-[3,2b]-5H-6-pyridone. Thus it is proved, that Pseudomonas putida possesses two different catabolic pathways for various quinoline derivatives, which are induced selectively depending on the growth substrate.

Classification of the spoilage flora offish, with special reference toShewanella putrefaciens

One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes, were phenotypically classified using 124 unit characters. Data were processed by the Simple Matching (SSM) and Jaccard (SJ) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% SJ similarity level which correspond to the SSM level of 86%. SJ-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps. lundensis (cluster 2; 2% of the field strains), Ps. fluorescens biovar III (cluster 4; 4% of the field strains), Ps. putida biovar A (cluster 5; 3% of the field strains), Ps. fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer. sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish). Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10(8) cfu/g. Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071T and reference strains from sources other than fish, did not group in any of the clusters. The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.

Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage.

A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks.

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4 degrees C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.