16S rDNA Gene Sequence Research Articles

Diversity of auxin‐producing bacteria associated to Pseudomonas savastanoi ‐induced olive knots

Forty three strains were isolated from knots induced by Pseudomonas savastanoi in different olive cultivars. All the selected bacteria were shown to produce variable amounts of the plant growth hormone indole-3-acetic acid (IAA). Amplification of the intergenic transcribed spacers (ITS) between 16S and 23S rDNA genes, allowed the clustering of the isolates into seven distinct groups. All isolates from ITS group 1 were positive to the Pseudomonas savastanoi pv. savastanoi specific iaa L gene as shown by PCR. Partial sequencing of 16S rDNA gene confirmed the identity of these isolates to Pseudomonas savastanoi strains and allowed to tentatively assign the other isolates from the remaining ITS groups to Pantoea oleae/agglomerans, Burkholderia cepacia, Pseudomonas putida, Stenotrophomonas maltophilia and Hafnia alvei. Identification of endophytic knot-derived isolates revealed association of various saprophytic and putative human pathogenic bacteria with P. savastanoi pv. savastanoi in knot environment of olive infected trees.

Uptake of Pb2+ by a cyanobacterium belonging to the genus Synechocystis, isolated from East Kolkata Wetlands

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 mug ml(-1) Pb(2+ )and growth was completely inhibited at 400 mug ml(-1) Pb(2+). All experiments were carried out with 100 mug ml(-1) Pb(2+) in which growth was the maximum. 91.67% of the total Pb(2+) got adsorbed to the outer surface of the cell and 1% of the total Pb(2+) entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb(2+) were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR-SmtB like regulator gene was expressed in both the strains after Pb(2+) induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb(2+) induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb(2+).

Lactococcus lactis subspecies lactis also causes white muscle disease in farmed giant freshwater prawns Macrobrachium rosenbergii

From May to August 2001 in Taiwan, 27 farms for the giant freshwater prawn Macrobrachium rosenbergii experienced white tail disease outbreaks in animals approximately 3 to 5 mo old, with total lengths from 6 to 8 cm. Examination of the infected prawns revealed not only previously reported Lactococcus garvieae (16 farms) but also the novel L. lactis subsp. lactis (10 farms). One farm had shrimp infected with both bacteria. In the farms with L. lactis infections, the cumulative mortality was approximately 25 to 60%. Gross signs of disease were opaque and whitish muscles, while histopathology included marked edema and necrotic lesions, with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain/heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Eleven isolates from different farms were identified as L. lactis subsp. lactis using API 20 Strep and Rapid ID32 Strep tests and using PCR assays specific for the L. lactis subsp. lactis 16S rDNA gene (650 bp amplicon) and for the 16S to 23S rDNA interspacer region (380 bp amplicon). In addition, sequencing of the full 16S rDNA genes of 2 of the isolates (MR17 and MR26; GenBank Accession Numbers AF493058 and AF493057, respectively) revealed 99.9% identity between the isolates and 98.7% identity to several complete 16S rRNA sequences of L. lactis subsp. lactis at GenBank. Experimental infections with our isolates gave gross signs and histopathological changes similar to those seen in naturally infected prawns. The mean lethal dose of 4 isolates and the reference strain L. lactis subsp. lactis BCRC 10791 ranged from 4.2 x 10(6) to 2.5 x 10(7) colony-forming units prawn(-1), indicating virulence similar to that previously reported for L. garvieae. This is the first report confirming L. lactis subsp. lactis as a pathogen in juvenile and adult prawns from aquaculture.

Phylogeny of γ-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.

Suppression of Pythium ultimum root rot of sorghum by rhizobacterial isolates from Ethiopia and South Africa

Bacteria isolated from the rhizosphere of sorghum in Ethiopia and from different grass species within the Nylsvlei Nature Reserve in South Africa were evaluated for their in-vitro and in-vivo antagonistic activity against Pythium ultimum that causes root rot in sorghum. Root rot caused by this pathogen in sorghum is characterized by blackened discolorations with white mycelial growth on the surface of the roots. Statistically significant disease suppression was achieved by 15 isolates from sorghum rhizosphere in Ethiopia (⩾50% disease suppression), whereas eight isolates from the rhizosphere of grasses at Nylsvlei Nature Reserve in South Africa resulted in ⩾80% disease suppression. The isolates maintained themselves at a level ⩾105cfu/g indicating that they are also highly competent in sorghum rhizosphere. Isolates which showed the best overall performance in-vitro and in-vivo were identified by means of the API system and sequencing of the bacterial 16S rDNA genes. The majority of these isolates were identified as members of the genus Bacillus, 56% of which were B. cereus. Three other isolates were identified as Brevibacterium laterosporus, Pseudomonas fluorescens and Serratia marcescens. Selected modes of action studies indicated that production of antibiotic substances and siderophores as well as induction of systemic resistance in sorghum were exhibited by some of the most effective isolates. The results of this study offer a significant impetus to the application of plant growth promoting rhizobacteria for use as biological control agents against P. ultimum in sorghum.

A mitochondrial phylogeography of Brachidontes variabilis (Bivalvia: Mytilidae) reveals three cryptic species

This study examined genetic variation across the range of Brachidontes variabilis to produce a molecular phylogeography. Neighbour joining (NJ), minimum evolution (ME) and maximum parsimony (MP) trees based on partial mitochondrial DNA sequences of 16S-rDNA and cytochrome oxidase (COI) genes revealed three monophyletic clades: (1) Brachidontes pharaonis s.l. from the Mediterranean Sea and the Red Sea; (2) B. variabilis from the Indian Ocean; (3) B. variabilis from the western Pacific Ocean. Although the three clades have never been differentiated by malacologists employing conventional morphological keys, they should be ascribed to the taxonomic rank of species. The nucleotide divergences between Brachidontes lineages (between 10.3% and 23.2%) were substantially higher than the divergence between congeneric Mytilus species (2.3–6.7%) and corresponded to interspecific divergences found in other bivalvia, indicating that they should be considered three different species. Analysis of the 16S-rDNA sequences revealed heteroplasmy, indicating dual uniparental inheritance (DUI) of mtDNA in the species of Brachidontes collected in the Indian Ocean, but not in the species in the Pacific nor in the species in the Red Sea and the Mediterranean Sea. When we employed the conventional estimate of the rate of mitochondrial sequence divergence (2% per million years), the divergence times for the three monophyletic lineages were 6–11 Myr for the Indian Ocean and Pacific Ocean Brachidontes sp. and 6.5–9 Myr for the Red Sea and Indian Ocean Brachidontes sp. Thus, these species diverged from one another during the Miocene (23.8–5.3 Myr). We infer that a common ancestor of the three Brachidontes species probably had an Indo-Pacific distribution and that vicariance events, linked to Pleistocene glaciations first and then to the opening of the Red Sea, produced three monophyletic lineages.

Use of metabolic and molecular methods for the identification of a Bacillus strain isolated from paper affected by foxing

Foxing of paper is a deterioration phenomenon occurring in the form of brown-yellowish spots, the abiotic and/or biotic causes of which are not yet completely understood. Nevertheless, microbiological infection has been recognized that may contribute to paper damage and therefore it becomes important to know the taxonomic position and the degradative activity of the potential infectious biological agents which mostly are fungi, but also bacteria and yeasts. A cellulolytic bacterial strain isolated from a foxed paper sample exhibited morphological and physiological characteristics of the Bacillus genus. To study its taxonomic position, different identification methods were used: the Biolog system, the direct amplified polymorphic DNA-polymerase chain reaction analysis (DAPD-PCR) and the partial sequencing of the 16S rDNA gene. Biolog system and partial sequencing of 16S rDNA gene assigned the strain to the Paenibacillus polymyxa species. DAPD-PCR analysis indicated a high similarity with Bacillus circulans, by comparing the isolated strain with some closely related Bacillus species.

Isolation and characterization of heat resistant enterotoxigenic Staphylococcus aureus from a food poisoning outbreak in Indian subcontinent

Outbreaks of staphylococcal food poisoning (SFP) are very common across the world, however, there is hardly any report of SFP from the Indian subcontinent. An outbreak occurred in the state of Madhya Pradesh (India) after the consumption of a snack called “Bhalla” made up of potato balls fried in vegetable oil. More than 100 children and adults who ate the snack suffered from the typical symptoms of SFP and required hospitalization. Food and clinical samples were found to contain a large number of enterotoxigenic Staphylococcus aureus. All enterotoxigenic isolates produced a combination of SEB and SED enterotoxins and were sensitive to oxacillin and vancomycin. Isolates were characterized by molecular biology tools, viz., SDS-PAGE, amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD) and nucleotide sequencing of seb, sed, and 16S rDNA genes. Results of these studies suggested that the isolates, irrespective of their isolation from food or clinical samples, were clonal in origin. Further, seb gene sequence of isolates showed nucleotide variations at multiple sites when compared with other sequences available in the database. Representative isolates, one each from food and clinical samples, were found to be highly heat resistant ( D 60 ∼ 15–16 min). Isolates obtained in the current outbreak need to be further studied to find out the impact on food safety guidelines with respect to thermal processing.

Molecular and biochemical characterizations of human oral lactobacilli as putative probiotic candidates

The objective of this study was to characterize the lactobacilli from the human oral cavity as a potential source of probiotic strains. Samples were collected from four different locations within the oral cavity: surface of healthy tooth, oral mucous membrane, surface of tooth decay and deep tooth decay. On the basis of morphological and biochemical properties eight categories were formed and 26 isolates were selected for further characterization. The isolates were determined as Lactobacillus sp. using primers specific for 16S rDNA. Sequencing of 16S rDNA genes and repetitive sequence-based polymerase chain reactions were used for determination to species and subspecies levels. Predominant species were Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus salivarius and Lactobacillus paracasei subsp. paracasei, while Lactobacillus acidophilus, Lactobacillus cellobiosus, Lactobacillus delbrueckii subsp. lactis and Lactobacillus gasseri were also present. The isolates Lactobacillus salivarius BGHO1, Lactobacillus fermentum BGHO36 and BGHO64, Lactobacillus gasseri BGHO89 and Lactobacillus delbrueckii subsp. lactis BGHO99 exhibited antagonistic action on the growth of Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, Salmonella enteritidis, Streptococcus pneumoniae and Streptococcus mutans, but not on growth of Candida albicans. Moreover, the isolates L. salivarius BGHO1 and L. gasseri BGHO89 were tolerant to low pH and high concentration of bile salts. Taken together, these findings imply that L. salivarius BGHO1 and L. gasseri BGHO89 might be subjects for additional investigation as potential probiotic strains.

Phenotypic and genetic diversity of rhizobia isolated from nodules of the legume genera Astragalus, Lespedeza and Hedysarum in northwestern China

Twenty-nine rhizobial isolates from root nodules of the wild Legumes Astragalus, Lespedeza and Hedysarum growing in the northwestern region of China, were characterized by numerical taxonomy, RFLP and sequencing of PCR-amplified 16S rDNA genes, and cross-nodulation with selected Legume species. Based on the results from numerical taxonomy, the isolates could be divided into two main groups (Clusters 1 and 2) and some single isolates at 82% similarity. CLuster 1 contained six isolates from Astragalus, Lespedeza and Hedysarum spp. Cluster 2 consisted of nine isolates from Astragalus and Hedysarum species. The phytogenetic analysis based on 16S rRNA gene sequences showed that SH199, representing cluster 1, belonged to the Rhizobium-Agrobacterium group, and SH290B, representing cluster 2, was closely related to R. galegae and R. huautlense.

A NEW SPECIES OF RHABDIAS FROM LUNGS OF THE WOOD FROG, RANA SYLVATICA, IN NORTH AMERICA: THE LAST SIBLING OF RHABDIAS RANAE ?

Rhabdias bakeri n. sp. is described from specimens found in lungs of the wood frog, Rana sylvatica, from North Dakota. The new species has previously been mistakenly identified as Rhabdias ranae Walton, 1929, a common parasite of the leopard frog, Rana pipiens. The new species differs from R. ranae and Rhabdias joaquinensis Ingles, 1935 by the shape and size of pseudolabia, shape and size of buccal capsule, and wider esophageal bulb. Molecular analysis based on the partial sequences of nuclear 18S rDNA gene, complete sequences of internal transcribed spacer region, and partial sequences of 28S gene demonstrates clear differences between Rhabdias from Ra. sylvatica and Ra. pipiens, and supports the status of R. bakeri as a new species.

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.

First Report of Pear Decline Phytoplasmas on Pear in Serbia.

During August of 2004, pear (Pyrus communis L.) plants with typical symptoms of pear decline (PD) were observed in orchards in central Serbia. The affected plants showed premature reddening and upward rolling of leaves that often showed down-turned petioles. In some cases, premature defoliation was observed. Although a similar decline of pear was observed earlier, until now, the causal agent had not been identified. DNA was extracted with a chloroform/phenol procedure from fresh leaf midribs and branch phloem scrapes of four symptomatic and one asymptomatic pear plants separately. A nested polymerase chain reaction assay (PCR) was used for phytoplasma detection (first PCR round with P1/P7 (4) phytoplasma universal primer pair, followed by nested PCR with group 16SrX specific primers f01/r01) (3). With these primers, the expected products from phloem scrapes and midrib extracts of symptomatic plant samples were obtained. Restriction fragment length polymorphism (RFLP) analyses of the f01/r01 amplicon, with RsaI and SspI restriction enzymes, discriminating among 16SrX subgroup phytoplasmas, showed profiles corresponding to those of the apple proliferation phytoplasma group, 16SrX-C subgroup, "Candidatus Phytoplasma pyri" (2). A 1,155-bp sequence of 16S rDNA gene for one of the PA2f/r (1) amplicons obtained in nested PCR on P1/P7 products from one of the leaf midrib samples was deposited in GenBank (Accession No. AY949984); both strands of the fragment were sequenced with the Big Dye Terminator reaction kit (Applied Biosystems, Foster City, CA). The sequences were analyzed with the Chromas 1.55 DNA sequencing software (Technelysium, Queensland, Australia) and aligned with BLAST software ( http://www.ncbi.nlm.nih.gov ). The blast search showed 100% homology of this sequence with that of PD strain Y16392, confirming the identity with PD of the phytoplasma detected. To our knowledge, this is the first report of pear decline phytoplasmas in Serbia.

Differentiation between Bovine and Ovine Strains of Histophilus somni Based on the Sequences of 16S rDNA and rpoB Gene

Nucleotide sequences of 16S rDNA and rpoB gene of 25 bovine and 6 ovine Histophilus somni strains were determined to detect subtle differences between the host animal species. The 1465 nucleotide residues of the 16S rDNA exhibited levels of sequence similarities of 99.4% or more. The high sequence similarity of the 16S rDNA of recently described species H. somni was confirmed in the 31 strains from cattle and sheep. These results suggested that the intra-specific diversity of 16S rDNA was limited in bovine and ovine strains of H. somni. The specific association of strains was also observed in the 311 bp region of rpoB gene which sequence similarities were 98.6% or more. However, the phylogenetic tree analysis of the rpoB gene showed that the ovine strains appeared to form a subgroup recovered in 70% of the bootstrap trees. In the 311 bp region of the ovine strains, a HincII restriction endonuclease site was detected. The PCR-amplified rpoB DNA of 46 bovine and 20 ovine H. somni strains were examined for the digestion with HincII. As the results, 17 strains of ovine strains were cleaved by the enzyme but none of the bovine strains appeared to possess the restriction site. The restriction enzyme analysis of rpoB gene may be useful to differentiate ovine strains from bovine strains of H. somni.

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing.

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil

To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.

Endophytic Pseudomonas spp. populations of pathogen-infected potato plants analysed by 16S rDNA- and 16S rRNA-based denaturating gradient gel electrophoresis

The diversity of abundant and metabolically active pseudomonads in potato plants was analysed using a culture-independent approach. The effect of two plant varieties, Agria and Bionta, as well as the presence of a plant pathogen Erwinia carotovora ssp. atroseptica on this bacterial group was tested. A combination of Pseudomonas-specific PCR, DGGE analysis, cloning and sequencing of partial 16S rDNA genes was performed using DNA and RNA extracted from potato stem tissue. Sequence analysis revealed a high species diversity, with the most prominent ones being Pseudomonas stutzeri and Pseudomonas gingeri. Some species showed high rRNA contents indicating high metabolic activity. Both, highly abundant and metabolically active Pseudomonaspopulations were more affected by the plant genotype than by the presence of E. carotovora.

Phylogenetic analysis of intestinal microflora indicates a novel Mycoplasma phylotype in farmed and wild salmon.

All studies of the microbial community of the gastrointestinal tract of salmon to date have employed culture-based approaches, typically on pond- or tank-raised, freshwater animals. We present a phylogenetic survey of the bacterial populations present in the distal intestine of salmon from three different marine locations in Europe. This was accomplished through PCR amplification, cloning, and sequencing of partial 16S rDNA genes from microbial community DNA isolated from the contents of the GI tract distal to the pyloric ceca. Using this approach, the intestinal microbial communities of wild salmon from Scotland and pen-raised salmon from Scotland and Norway were compared. The predominating bacterial populations detected were Acinetobacter junii and a novel Mycoplasma phylotype. This Mycoplasma phylotype apparently comprised approximately 96% of the total microbes in the distal intestine of wild salmon. Substantial differences in intestinal microbial community composition and diversity were observed between the two groups of pen-raised salmon, which, in addition to geographical separation, were raised on different feeds. The microbial profiles found in this study were substantially different from those indicated in earlier culture-based studies for several species of fish, presumably because of the culture-independent techniques employed. Further, analysis of short-chain fatty acids in the digestive tract indicated that the decreasing redox gradient from proximal to distal reaches common to homeothermic animals was absent in salmon, and that the bacterial fermentation levels were much lower than are reported in homeothermic animals.

Application of Cecidomyiid Galls to the Systematics of the Genus Machilus (Lauraceae) in Taiwan

The species of the genus Machilus (Lauraceae) in Taiwan sustain diverse cecidomyiid galls induced by the insects of the genus Daphnephila (Cecidomyiidae). This work examines the feasibility of applying cecidomyiid galls to the systematics of the genus Machilus. Amplified fragment length polymorphism (AFLP) was used to analyze the 38 gall-bearing trees of four Machilus taxa including Machilus kusanoi, M. thunbergii, M. zuihoensis var. zuihoensis, and M. zuihoensis var. mushaensis. The UPGMA cluster analysis of the AFLP data revealed three distinct clusters, including M. kusanoi, M. thunbergii, and M. zuihoensis variety complex. Machilus zuihoensis var. zuihoensis and M. zuihoensis var. mushaensis were indistinguishable from the three primer combinations. These two varieties could be considered the same taxon. PCR and DNA sequencing methods were used to analyze the nucleotide sequences of the mitochondrial 16SrDNA gene of the twenty gall midges from three types of galls from four Machilus taxa. The phylogenetic tree from the partial 16SrDNA sequence by UPGMA method of proportion distance revealed that the gall midges can be divided into three groups according to gall types. The phylogenetic tree cannot separate the. twovarieties of M. zuihoensis within each group. Machilus zuihoensis var. zuihoensis and M. zuihoensis var. mushaensis cannot be distinguished according to the AFLP or DNA sequencing methods, and they are more closely related to M. thunbergii than to M. kusanoi. The systematic relationships among the Machilus from the data of host plants are congruent with the data from the gall inducers. Results in this study imply that the gall inducers of genus Daphnephila provide information for resolving the plant systematic relationships based on molecular techniques.

Evidence for two genetically and chemically defined host races of Tyria jacobaeae (Arctiidae, Lepidoptera)

A widely distributed host race of Tyria jacobaeae lives on Senecio jacobaea and related species and accumulates pyrrolizidine alkaloids (“PA race”), another race, which is restricted to the Alps and found on Petasites paradoxus, sequesters sesquiterpenes, such as petasol and isopetasol. Nucleotide sequences of the mitochondrial 16S rDNA gene show 1% sequence divergence, indicating that genetical differences exist between the PA exploiting and the terpene-sequestering host races of T. jacobaeae. This finding suggests that both host races of T. jacobaeae must have been separated for some time already, possibly since the Pleistocene.