Partial 16S rDNA Sequencing Research Articles

Isolation and Characterization of Rhizobacteria from Composts That Suppress the Severity of Bacterial Leaf Spot of Radish

ABSTRACT Composts can induce systemic resistance in plants to disease. Unfortunately, the degree of resistance induced seems highly variable and the basis for this effect is not understood. In this work, only 1 of 79 potting mixes prepared with different batches of mature, stabilized composts produced from several different types of solid wastes suppressed the severity of bacterial leaf spot of radish caused by Xanthomonas campestris pv. armoraciae compared with disease on plants produced in a nonamended sphagnum peat mix. An additional batch of compost-amended mix that had been inoculated with Trichoderma hamatum 382 (T(382)), which is known to induce systemic resistance in plants, also suppressed the disease. A total of 11 out of 538 rhizobacterial strains isolated from roots of radish seedlings grown in these two compostamended mixes that suppressed bacterial leaf spot were able to significantly suppress the severity of this disease when used as inoculum in the compost-amended mixes. The most effective strains were identified as Bacillus sp. based on partial sequencing of 16S rDNA. These strains were significantly less effective in reducing the severity of this disease than T(382). A combined inoculum consisting of T(382) and the most effective rhizobacterial Bacillus strain was less effective than T(382) alone. A drench applied to the potting mix with the systemic acquired resistance-inducing chemical acibenzolar-S-methyl was significantly more effective than T(382) in several, but not all tests. We conclude that systemic suppression of foliar diseases induced by compost amendments is a rare phenomenon. Furthermore, inoculation of compost-amended potting mixes with biocontrol agents such as T(382) that induce systemic resistance in plants can significantly increase the frequency of systemic disease control obtained with natural compost amendments.

Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts: characterization, direct quantification, and in situ location of transconjugant cells.

The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells per donor cell for pKJK5::gfp and TOL::gfp, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera Pseudomonas and ERWINIA: The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from P. putida in the absence of selective pressure that could favor the presence of the investigated plasmids.

PCR-based genetic evidence for occurrence of Helicobacter pylori and novel Helicobacter species in the canine gastric mucosa

The canine gastric mucosa is known to be a habitat for various Helicobacter species. So far, five Helicobacter species have been described from the canine gastric mucosa, but histological studies have demonstrated a greater variety. In order to gain more information on diversity of canine gastric mucosa colonising helicobacters, biopsy samples of four pet dogs were examined by DNA-based techniques. PCR with a primer pair binding specifically to the 16S rDNA of the species of the genus Helicobacter and generating a fragment of approximately 400 bp indicated the presence of Helicobacter strains in the stomachs of the four dogs. PCR products were cloned into Escherichia coli DH10B™ and PCR-re-amplified 16S rDNA fragments were subjected to amplified ribosomal DNA restriction analysis (ARDRA) employing restriction enzyme HhaI. Restriction profiles indicated the presence of at least two different Helicobacter species in two dogs. Partial sequences of 16S rDNA of six clones were compared with sequences available in the EMBL data bank. Two sequences obtained from different dogs were identical with the corresponding sequences of Helicobacter pylori strains. Three sequences showed highest but moderate similarity values to H. pylori (96.6–98.0%) and one sequence to Helicobacter salomonis (97.3%). In contrast to previous reports our data implicate that the gastric mucosa of dogs may be colonised by strains of H. pylori or a very closely related species but they also confirm indications for the presence of so far uncultivated species of Helicobacter.

Inhibition of Listeria monocytogenes in Cold-Smoked Salmon by Carnobacterium piscicola CS526 Isolated from Frozen Surimi

Strain CS526 was isolated from frozen surimi and identified as a bacteriocin producer that had strong inhibitory activity against Listeria monocytogenes. Strain CS526 was identified as Carnobacterium piscicola by partial 16S rDNA sequence similarity. The ability of this bacteriocinogenic strain and nonbacteriocinogenic C. piscicola JCM5348 to inhibit the growth of L. monocytogenes was examined in culture broth incubated at 12°C and cold-smoked salmon stored at 4, 12, and 20°C. L. monocytogenes viable counts in the culture broth rapidly declined from 106 colony-forming units per ml to less than 10 colony-forming units per ml within 1 day at 12°C in the presence of C. piscicola CS526. At 4 and 12°C, inhibition of L. monocytogenes on salmon depended on the initial inoculum level of C. piscicola CS526. However, C. piscicola CS526 was bactericidal to L. monocytogenes within 21 and 12 days at 4 and 12°C in cold-smoked salmon, respectively, even when the initial inoculum levels were low. C. piscicola CS526 suppressed the maximum cell number of L. monocytogenes by two and three log cycles, even at 20°C. However, C. piscicola JCM5348 did not prevent the growth of the pathogen, except at 4°C. Bacteriocin was detected in the samples coinoculated with C. piscicola CS526. The study shows that C. piscicola CS526 might have potential for biopreservation of refrigerated foods against L. monocytogenes.

Emended phenotypic characterization of Prototheca zopfii: a proposal for three biotypes and standards for their identification.

A representative selection of Prototheca zopfii strains isolated from different environmental habitats or clinical cases was characterized in a polyphasic approach in order to assess their intraspecies taxonomic position. Recently, the recognition of distinct phenotypic clusters has been reported as the assignment of 'variants'. In the present study, 11 strains were compared by a number of phenotypic and genetic criteria, including growth characteristics, biochemical reactions and serotyping results. Based on emended standards for biotype identification, P. zopfii strains showed auxanographic differences and distinct assimilation patterns with respect to utilization of amino acids and glycerol. Serotyping by means of immunoblotting revealed that all isolates of variant II obtained from clinical cases, i.e. isolates from bovine mastitis or from human enteropathia, showed specific antigen patterns. They were found to be different from strains assigned to the other two variants with respect to their immunogenic antigens. Furthermore, comparison of partial 18S rDNA sequences confirmed distinct differences between the former variants. Based on these results, it is proposed that P. zopfii merits classification as a species comprising three biotypes.

Seven novel species of Acinetobacter isolated from activated sludge.

Thirteen isolates of Acinetobacter were obtained from activated sludge plants in Victoria, Australia. Earlier 16S-23S rDNA genomic fingerprinting and partial 16S rDNA sequence data had suggested that these isolates might contain previously undescribed species. This view was confirmed here. A polyphasic taxonomic approach involving phenotypic characterization, near-complete 16S rDNA sequence data and DNA-DNA hybridization analyses support the view that seven novel genomic species can be differentiated in this group of isolates. However, when fluorescence in situ hybridization (FISH) studies were performed with a 16S-rRNA-targeted probe specific for the genus Acinetobacter, used to identify Acinetobacter in activated sludge plants, all these strains responded positively. This suggests that these isolates would not have been missed in earlier FISH studies where their role as polyphosphate-accumulating bacteria has been questioned. This report describes these isolates and proposes that they be named Acinetobacter baylyi (type strain B2T = DSM 14961T = CIP 107474T), Acinetobacter bouvetii (type strain 4B02T = DSM 14964T = CIP 107468T), Acinetobacter grimontii (type strain 17A04T = DSM 14968T = CIP 107470T), Acinetobacter tjernbergiae (type strain 7N16T = DSM 14971T = CIP 107465T), Acinetobacter towneri (type strain AB1110T = DSM 14962T = CIP 107472T), Acinetobacter tandoii (type strain 4N13T = DSM 14670T = CIP 107469T) and Acinetobacter gerneri (type strain 9A01T = DSM 14967T = CIP 107464T).

A deep-sea slant on the molecular phylogeny of the Scleractinia

Lophelia pertusa and Madrepora oculata are azooxanthellate corals with nearly cosmopolitan distributions. They form cold-water reefs in the upper bathyal zone on continental margins and offshore banks [A.D. Rogers, Int. Rev. Hydrobiol. 84 (1999) 315]. Lophelia is classified in the family Caryophylliidae and Madrepora in the family Oculinidae, both on the basis of skeletal morphology. Recent molecular studies of the scleractinians have given a new insight into the evolutionary history of this group. This study was aimed at clarifying the phylogenetic relationships of Lophelia and Madrepora, through the analysis of partial sequences of the mitochondrial 16S rDNA. Sequences were obtained for samples of L. pertusa collected in the northeast Atlantic and off Brazil, M. oculata, four other deep-sea and eight tropical coral species from the Réunion island in the Indian Ocean. The sequences were aligned with 69 homologous sequences of Scleractinia. Maximum parsimony and Bayesian analyses support previously published molecular topologies. The two specimens of L. pertusa grouped with two caryophylliids, confirming the existing classification of the species, but the large genetic distance between the two Lophelia samples suggests that these populations are genetically isolated from one another. M. oculata did not cluster with oculinids, but formed a monotypic clade lying between the families Pocilloporidae and Caryophyliidae. Phylogenetic analysis also suggested cryptic speciation within the tropical taxa Pocillopora meandriana and possibly Acropora humilis.

Phylogenetic identification of bacteria with antimicrobial activities isolated from Mediterranean sponges

Bacteria were isolated from Mediterranean sponges and specifically selected according to their antimicrobial activity. They were classified on the basis of partial 16S rDNA sequences as members of distinct clusters of α-Proteobacteria and γ-Proteobacteria. The action spectrum of antimicrobial activities against target organisms was related to the phylogenetic position of the isolates.

Species boundaries within the Acropora humilis species group (Cnidaria; Scleractinia): a morphological and molecular interpretation of evolution

Species boundaries remain unresolved in many scleractinian corals. In this study, we examine evolutionary boundaries of species in the Acropora humilis species group. Five morphologically discrete units are recognized using principal components and hierarchical cluster analyses of quantitative and qualitative characters, respectively. Maximum parsimony and likelihood analyses of partial 28S rDNA sequences suggest that these morphological units diverged to form two evolutionarily distinct lineages, with A. humilis and A. gemmifera in one lineage and A. digitifera and two morphological types of A. monticulosa in the other. Low levels of sequence divergence but distinct morphologies of A. humilis and A. gemmifera within the former lineage suggest recent divergence or ongoing hybridization between these species. Substantially higher levels of divergence within and between A. digitifera and A. monticulosa suggest a more ancient divergence between these species, with sequence types being shared through occasional introgression without disrupting morphological boundaries. These results suggest that morphology has evolved more rapidly than the 28S rDNA marker, and demonstrate the utility of using morphological and molecular characters as complementary tools for interpreting species boundaries in corals.

The synthesis of the UV‐screening pigment, scytonemin, and photosynthetic performance in isolates from closely related natural populations of cyanobacteria (Calothrix sp.)

Two populations of the cyanobacterium Calothrix sp. found in Yellowstone thermal spring outflows differ greatly in their contents of scytonemin, a UV-screening pigment, and in their photosynthetic carbon assimilation rates. Clonal isolates from both populations were used to investigate these phenotypic differences. Identical partial 16S rDNA sequences ( approximately 900 bp) suggest a very close relationship between the two Calothrix populations and indicate that environmental differences may, in part, explain the field observations. The effects of native spring water on scytonemin synthesis and photosynthesis were tested during experiments using plated cells. Results show differences in the spring water environment were at least partly responsible for the differences in scytonemin content observed in the field. Furthermore, spring water effects on photosynthetic performance suggest adaptation in these strains to their spring of origin. Controlled experiments performed using cultures grown in artificial liquid medium showed no significant difference in photosynthetic carbon uptake between strains. However, significant differences were detected in their ability to synthesize scytonemin indicating genetic differences between populations. These findings suggest that both genetic and environmental differences are responsible for the naturally occurring variation in scytonemin content and photosynthetic ability in these two closely related populations.

Jannaschia helgolandensis gen. nov., sp. nov., a novel abundant member of the marine Roseobacter clade from the North Sea.

Heterotrophic bacteria were isolated from a water sample taken from the North Sea, 2 km off the coast of the island of Helgoland, by direct plating of the serially diluted sample on complex marine media. Sixteen of 80 strains from the highest sample dilution belonged to the 'Roseobacter-Sulfitobacter-Silicibacter' group within the 2-subclass of the Proteobacteria on the basis of partial 16S rDNA sequence analysis. Phylogenetic analysis of nearly complete 16S rDNA sequences showed that the closest relative of two strains, Hel 10(T) and Hel 26, was Ketogulonicigenium vulgare (94.4% similarity). These strains were Gram-negative, non-motile rods, obligate aerobes, required sodium ions and 1-7% sea salts for growth and did not produce bacteriochlorophyll. Their optimal growth temperature was 25-30 degrees C. The strains had Q-10 as the dominant respiratory quinone. Chemotaxonomic analysis showed a combination of ester-linked 3-OH 10 : 0, 12 : 1 and amide-linked 3-oxo 14 : 0 (or 3-OH 14 : 1) and 3-OH 14 : 0 fatty acids, which appears to be a unique feature of strains Hel 10(T) and Hel 26 within this subsection of the 2-subclass of the Proteobacteria. Based on 16S rDNA sequence analysis and chemotaxonomic data, the strains are assigned to a new genus and species, Jannaschia helgolandensis gen. nov., sp. nov., with the type strain Hel 10(T) (= DSM 14858(T) = NCIMB 13941(T)).

Bacterial community structure and physiological state in a biofilm reactor degrading 4-chloroaniline.

Degradation of 4-chloroaniline in the presence of aniline by a microbial community in a laboratory-scale biofilm reactor was evaluated. The starter inoculum was isolated and reconstructed from a percolating column enrichment of Indonesian agricultural soil. The capacity to mineralise and detoxify 4-chloroaniline in the presence of aniline was demonstrated by the biofilm reactor when operated at high hydraulic retention time (HRT; 0.87 h). At low HRT (0.23 h and 0.39 h) 4-chlorocatechol accumulated in the effluent, accompanied by a decrease in dechlorination and detoxification. When returned to high HRT (2.14 h), the accumulation of 4-chlorocatechol stopped and the extent of dechlorination and detoxification increased. Bacteria other than the original inoculum appeared in the reactor when the operating mode was switched from closed cycle to open cycle. One of these bacteria, identified as Pseudomonas putida R1 by partial 16S rDNA sequencing, subsequently dominated the reactor at every HRT imposed. PCR-based single-strand conformational polymorphism of 16 s rDNA and traditional cultivation procedures indicated that the bacterial composition in the reactor shifted in response to applied HRT. The relationship between the bacterial abundance and the degradation capacity of the reactor is discussed.

Seasonal shifts in population structure of Vibrio vulnificus in an estuarine environment as revealed by partial 16S ribosomal DNA sequencing

The partial sequence (600 bp) containing the most variable region of Vibrio vulnificus 16S ribosomal DNA (rDNA) was determined for 208 randomly selected V. vulnificus strains isolated from Galveston Bay, TX, USA between June 2000 and June 2001. A comparative analysis of the determined partial 16S rDNA sequences revealed the existence of two different partial 16S rDNA sequences (type A and type B, 1.3% base substitutions) among the 208 V. vulnificus isolates. A higher proportion of 16S rDNA type A strains was isolated in June and July while a considerably higher proportion of type B strains was isolated in September. In addition, after no V. vulnificus strains were detected during the winter months (December–February), only type A strains were isolated during the following months (March–May). The results suggest that the relative abundance of type A and type B V. vulnificus strains in Galveston Bay varies with the season and that the differences between the two 16S rDNA types may affect the viability of these organisms in the natural environment.

Shifts in diversity and microscale distribution of the adapted bacterial phenotypes due to Hg(II) spiking in soil.

In a previous experiment [Ranjard et al. (2000) FEMS Microbiol Ecol 31:107-115], the spatial heterogeneity of a mercury impact on soil bacterial community was revealed by an increase of mercury-resistant (HgR) bacterial numbers in the outer fraction and the sand fractions when compared to those in the silt fractions. The objectives of the present study were (i) to investigate whether mercury exposure affects the diversity and the distribution within the various fractions of the HgR populations and (ii) to evaluate the contribution of the HgR populations to the overall community adaptation. A total of 236 strains isolated before (104 isolates) and 30 days (132 isolates) after spiking were characterized by an amplified ribosomal DNA restriction analysis (ARDRA) and grouped into 30 different genotypes. Whereas almost the same numbers of different genotypes were observed at both sampling times when considering all microenvironments, important changes in their evenness were observed. At the microscale level, we also noticed a heterogeneous distribution of the genotypes. Partial 16S rDNA sequences of each genotype were determined to permit phylogenetic affiliation. Whereas Pseudomonas-like species were dominant in all microenvironments at T = 0, genotypes not detected before spiking such as genotypes closely related to Xanthomonas, Pseudoaminobacter, and Sphingomonas-like species became dominant at T = 30. Similarly, several genotypes close to Bacillus, Streptomyces, and Rhodococcus species were only detected in the sand fractions at T = 0 and could not be detected in these fractions at T = 30. Probes defined within the intergenic spacer between the rrs and rrl genes were designed for the dominant genotypes and were hybridized toward the RISA (Ribosomal Intergenic Spacer Analysis) profiles derived from the T = 0 and T = 30 bacterial communities associated to the unfractionated soil. Results showed that the culturable dominant HgR genotypes partially contributed to the adaptation of the whole bacterial community.

Fourier-transform infrared (FT–IR) spectroscopy is a promising tool for monitoring the population dynamics of microorganisms in food stuff

The routine identification and enumeration of large numbers of bacteria in complex microbial consortia is expensive and time consuming. In the present study, FT–IR spectroscopy has been used for the first time to rapidly monitor the population dynamics of bacteria in a food system. The bacterial cheese surface flora from two different batches was examined throughout the production process, starting with the inoculum. The ripening process was monitored over 12 or 29 days. One hundred bacterial isolates were investigated at each sampling point resulting in a total of 1307 isolates. Classical microbiological methods and Fourier–transform infrared (FT–IR) spectroscopy, partly supplemented by partial 16S rDNA sequencing, were used for identification. In the smear brine of cheese A, Staphylococcus equorum was found to dominate, while corynebacteria dominated the smear liquid of cheese B. The flora consisted mainly of the coryneform bacteria Corynebacterium casei and C. variabile, which made up 80–90% of the aerobic surface flora after 5–6 days of ripening and stayed in that order of magnitude until the end of ripening. The other flora components consisted of staphylococci, gram-negative bacteria, catalase-negative bacteria, and aerobic sporeformers. Only 1% of the coryneform bacteria could not be identified by FT–IR spectroscopy. The results demonstrate that FT–IR spectroscopy is a suitable method for the rapid monitoring of the population dynamics of coryneform bacteria on the surface of smeared cheeses. Generally, application of this method will enable a food producer to obtain a detailed overview on the microbiological status of a product down to the species level, which allows for correction measures to be taken early in the process.

Detection of Euryarchaeota and Crenarchaeota in an oxic basalt aquifer

Groundwater from an oxic, fractured basalt aquifer was examined for the presence of Archaea. DNA was extracted from cells concentrated from groundwater collected from five wells penetrating the eastern Snake River Plain Aquifer (Idaho, USA). Polymerase chain reaction (PCR) amplification of 16S rDNA was performed with Archaea-specific primers using both nested (ca. 200-bp product) and direct (ca. 600-bp product) PCR approaches. Estimates of the archaeal diversity were made by separating PCR products from all five wells by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis of partial 16S rDNA sequences from two wells was performed following cloning procedures. Archaea were detected in all wells and the number of DGGE bands per well ranged from two to nine and varied according to PCR approach. There were 30 unique clonal 16S rDNA partial sequences (ca. 600 bp) within a total of 100 clones that were screened from two wells. Twenty-two of the 16S rDNA fragments recovered from the aquifer were related to the Crenarchaeota and Euryarchaeota kingdoms (one large clade of clones in the former and six smaller clades in the latter), with sequences ranging from 23.7 to 95.4% similar to those found in other investigations. The presence of potentially thermophilic or methanogenic Archaea in this fully oxic aquifer may be related to deep thermal sources or elevated dissolved methane concentrations. Many sequences were similar to those that represent non-thermophilic Crenarchaeota of which there are no known cultured members and therefore no putative function.

Bacterial activities in the sediment of Lake Velencei, Hungary

Lake Velencei (Hungary) is one of the westernmost shallow soda lakes, extending from Eastern Europe to the Carpatian basin. The spatial and temporal distribution of the sediment microbiota, the metabolic potential of bacterial communities and the species composition of the genera Bacillus and Clostridium, as well as sulphate-reducing bacteria (SRB) were investigated regarding the close interactions between the lake sediment and the overlaying water column, the special water chemical parameters, and the extensive reed coverage of the lake. Aerobic microbial activities were tested with community-level physiological profiling (CLPP) using BIOLOG microplates. The quantification of the anaerobic fermentative and sulphate-reducing bacteria was done by the MPN (Most Probable Number) method. The cultivation of bacteria adapted to the special physico-chemical characteristics of the lake was carried out employing selective media. Multivariate analysis of CLPP data indicated that the microbial communities of the sediment separated from that of the water and showed seasonal variations of the utilised carbon sources. The results of the MPN demonstrated that the counts of the fermentative and sulphate-reducing bacteria in the reed rhizosphere were about one order higher than in the sediment. Among the isolated bacterial strains, a large number were characterised as facultative or obligate alkaliphilic and also moderately halophilic. The partial sequencing of 16S rDNA of the selected representatives resulted in species of aerobic bacteria, such as Bacillus pseudofirmus, B. halmapalus, B. cohnii, B. (Marinibacillus) marinus, and anaerobes, such as Clostridium putrificum – sporogenes, C. scatologenes, C. bifermentans, Desulfotomaculum guttoideum, Desulfovibrio alcoholivorans, and Desulfovibrio burkinensis.

A Comparative Study of Malaysian and Japanese Actinomycetes Using a Simple Identification Method Based on Partial 16S rDNA Sequence

A comparative analysis was performed on the taxonomy of actinomycete isolates of Malaysia and Japan. The taxonomical positions of isolates were determined using a simple identification method based on 16S rDNA partial sequence homology. The 16S rDNA partial sequence determination was made by direct PCR and direct sequencing. Simple identification was attempted for each of the 1128 actinomycete isolates for each country. We succeeded in simple identification of 790 Malaysian and 981 Japanese isolates. Malaysian isolates belonged to 9 families, 23 genera, and 185 species, including one genus as yet undescribed. The Japanese isolates belonged to 9 families, 22 genera, and 207 species. There was little taxonomical overlap between Malaysian isolates and Japanese isolates. Only 14% of the species and only 50% of the genera occurred in both groups. These results indicate that a group of actinomycetes of greater taxonomic diversity can be obtained when gathered from both regions, rather than from one or the other. Among Malaysian isolates, two strains were found that apparently do not belong to any known genus. Phylogenetic analysis indicates that they belong to the suborder Sreptosporangineae. The existence of candidate strains of a novel genus suggests that Malaysian actinomycetes have not been thoroughly investigated and therefore has promising potential as a source for novel bioactive compounds.

Isolation and phylogenetic characterization of bacteria capable of inducing differentiation in the green alga Monostroma oxyspermum.

Many green algae cannot develop normally when they are grown under axenic conditions. Monostroma oxyspermum, for example, proliferates unicellularly in an aseptic culture, but develops into a normal foliaceous gametophyte in the presence of some marine bacteria. More than 1000 bacterial strains were isolated from marine algae and sponges and assayed for their ability to induce the morphogenesis of unicellular M. oxyspermum. Fifty bacterial strains exhibiting morphogenesis-inducing activity against unicellular M. oxyspermum were isolated. The partial gyrB (approximately 1.2 kbp) and 16S rDNA (approximately 1.4 kbp) sequences of about 40 active strains were determined, and their phylogenetic relationships were analysed. All these strains were located within the Cytophaga-Flavobacterium-Bacteroides (CFB) complex, and most of these strains were clustered in a clade comprising Zobellia uliginosa. On the other hand, these bacteria also exhibited morphogenetic activity against germ-free spores of Ulva pertusa, Ulva conglobata and Enteromorpha intestinalis. Moreover, these bacteria induced the release of spores from the leafy young gametophyte of M. oxyspermum. These results indicate that strains belonging to several groups in the CFB complex play an important role in the normal development of green algae in the marine coastal environment.

Phylogenetic relationships of the Dactylogyridae Bychowsky, 1933 (Monogenea: Dactylogyridea): the need for the systematic revision of the Ancyrocephalinae Bychowsky, 1937.

Phylogenetic analyses based on partial 18S rDNA sequences of polyonchoinean monogeneans were conducted in order to investigate the relationships between selected families and subfamilies of the Dactylogyrinea, mainly within the Dactylogyridae. We tested the status of the Ancyrocephalidae sensu Bychowsky & Nagibina (1978) and the Ancyrocephalinae sensu Kritsky & Boeger (1989). Within the Dactylogyrinea, the Diplectanidae and Dactylogyridae are well supported by maximum likelihood and maximum parsimony analyses, but their phylogenetic relationship with the Pseudomurraytrematidae remains unresolved. Phylogenetic relationships between the Pseudodactylogyrinae, Ancyrocephalinae, Ancylodiscoidinae and Dactylogyrinae indicate paraphyly of the Ancyrocephalidae sensu Bychowsky & Nagibina (1978). The group of species recently considered as the Dactylogyridae sensu Kritsky & Boeger (1989) comprises two sister groups. The first group includes the freshwater Ancyrocephalinae and the Ancylodiscoidinae. The second group includes the Pseudodactylogyrinae, Dactylogyrinae and the Ancyrocephalinae from the fish species Siganus doliatus and Tetraodon fluviatilis. The non-monophyly of the Ancyrocephalinae (i.e. the non-monophyly of the group of species recently considered as members of Ancyrocephalinae), previously suggested by Kritsky & Boeger (1989) using the morphological characters, indicates that classification of the Dactylogyridae needs to be revised.