PHENOTYPIC PLASTICITY IN DRAPARNALDIA (CHLOROPHYTA: CHAETOPHORACEAE). I. EFFECTS OF THE CHEMICAL ENVIRONMENT1

Eight axenic isolates of Draparnaldia spp. were in defined media under controlled condidom of day length, light intensity and temperature to study effects of the nutrientions on genera; pheuotype and development of main text cells. Phosphate (0–6,8 mg/l P), magnesium (0-9.6 mg/l Mg), sulfate (1.2 –14.0 mg/l S) and chloride (0.2-48.2mg/l Cl) had no effect. Small, but significant, concentration-independent, isolate-specific differences due to sodium and patassium (as the phosphate salt) occured in main axis cell length. The presence of calcium (>1.7 mg/l) was required by all isolates for main axis differentiation. In the absence of calcium, only zoospores and stunted Stigeoclonium- like plants were produced. Nitrate (>5 mg/l N) depressed main axis cell size;at higher levels (> 15 mg/l N) hair prooduction was depressed and several isolates developed a very typical Cloniophora appearance, Several of the isolates did not produce typical Draparnaldia- like phenotypes under any of the chemical formulations tried; therefore, it is suggested that chemical factors alone do not account for either the phemotypec plasticity of Draparnaldia in the field or the failure of others to grow typical plants in culture.     

Molecular characterization and mating type analysis of oyster mushroom (<Emphasis Type="Italic">Pleurotus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>) using single basidiospores for strain improvement

Existence of variability in morphological traits and growth rate of mycelium of homokaryotic single basidiospores can be exploited for the development of inter-strainal hybrids. We isolated 182 single basidiospores from mushroom bodies of P. sajor-caju, P. florida, P. eous and one wild relative of Pleurotus called Hypsizygus ulmaris. The single spores were isolated using a new technique that is less prone to contamination and more efficient than the common techniques used by earlier workers. All the isolates showed a varied range of cultural morphology. Mating types of all the isolates within the species were identified on the basis of hyphal fusion via anastomosis with the tester strains. Four compatible pairs of isolates with well prominent tuft in the contact zone were selected for dikaryon isolation. Dikaryons were used for spawn preparation and mushroom cultivation. The dikaryotic isolates with their replicates were evaluated for spawn run period, yield and biological efficiency. 42 isolates (10 di- and 32 mono-karyotic isolates) were analyzed with RAPD genetic markers. Phenotypic characters and mating types of all the 42 isolates analyzed genetically were correlated with their genetic polymorphism data. The isolates showed very large diversity both at the phenotypic and the genotypic level. Available phenotypic and genotypic data can further help in the selection of monosporous isolates for developing inter-strainal hybrids which can lead to better prospects for genetic improvement in different species of Pleurotus.     

Phenotypic variation of Rhizoctonia oryzae in sheath disease of rice in Myanmar

Similarity in the cultural characteristics among closely related species of Rhizoctonia creates confusion and uncertainity in diagnosis. The present research was conducted to study the existence of phenotypic groups among isolates of R. oryzae in Myanmar. It was aimed to study the variation in phenotypic and molecular profiles among some isolates of R. oryzae and R. zeae. We found the occurrence of two distinct phenotypic groups of R. oryzae and a group of R. zeae. Ribosomal DNA-ITS sequencing was conducted and the resulting dendrogram agreed with those of the morphological grouping. A genetic distance of 0.064–0.072 wts was found between the R. oryzae and R. zeae groups. A pairwise distance of 0.014 and 0.022 was found between the RO1 and RO2 groups of R. oryzae. Our research revealed the existence of two distinct phenotypes in the isolates of R. oryzae collected from rice sheath in Myanmar and their differentiating features with R. zeae.     

Multiple gene genealogies and species recognition in the ectomycorrhizal fungus Paxillus involutus

Paxillus involutus (basidiomycetes, Boletales) is a common ectomycorrhizal fungus in the Northern Hemisphere. The fungus displays significant variation in phenotypic characters related to morphology, physiology, and ecology. Previous studies have shown that P. involutus contains several intersterility groups and morphological species. In this study, we have used concordance of multiple gene genealogies to identify genetically isolated species of P. involutus. Fragments from five protein coding genes in 50 isolates of P. involutus collected from different hosts and environments in Europe and one location in Canada were analysed using phylogenetic methods. Concordance of the five gene genealogies showed that P. involutus comprises at least four distinct phylogenetic lineages: phylogenetic species I (with nine isolates), II (33 isolates), III (three isolates), and IV (five isolates). The branches separating the four species were long and well supported compared with the species internodes. A low level of shared polymorphisms was observed among the four lineages indicating a long time since the genetic isolation began. Three of the phylospecies corresponded to earlier identified morphological species: I to P. obscurosporus, II to P. involutus s. str., and III to P. validus. The phylogenetic species had an overlapping geographical distribution. Species I and II differed partly in habitat and host preferences.     

Morphology,Physiology, and Virulence of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.     

Pulsed-field gel electrophoresis fingerprinting for identification of Azospirillum species

Pulsed-field gel electrophoresis (PFGE) was used to obtain macrorestriction fingerprints of restriction enzyme-cut DNA of natural isolates of Azospirillum spp. Metabolic profiles, along with other phenotypic characteristics, were compared with these fingerprints to differentiate among the azospirilla isolates. A wide diversity of phenotypes (e.g., colony color, motility, and accumulation of poly--hydroxybutyrate granules) was observed among the natural isolates of azospirilla. PFGE revealed that TCTAGA, the sequence recognized by Xba1, is rare in the genome of azospirilla. The PFGE fingerprint revealed that azospirilla associated with different crops have a very similar genetic background. PFGE fingerprints were more consistent in the identification of azospirilla isolates from specific hosts than the metabolic fingerprints. For further differentiation at strain level, metabolic, physiological, and morphological profiles provide additional information.Journal No. J-2920 of the Montana Agricultural Experiment Station.     

Actinoplanes capillaceus sp. nov., a new species of the genus Actinoplanes

Two motile actinomycete strains, K95–5561^T and K95–5562, were isolated from a soil sample collected at Sayama City, Saitama Prefecture, Japan. They produced bell shaped spore vesicles (sporangia) with hairy surfaces on substrate hyphae. When released into water, the sporangiospores became motile by a tuft of polar flagella. The chemotaxonomic and morphological characteristics together with 16S rRNA gene sequence data indicated that the two isolates belonged to the genus Actinoplanes. The two strains were assigned to a single species on the basis of phenotypic, notably cultural, morphological and physiological characteristics, and DNA-DNA pairing data. The two strains were distinguished from representatives of all validly described species of Actinoplanes using a combination of genotypic and phenotypic properties. It is, therefore, proposed that strains K95–5561 and K95–5562 be recognized as a new species of the genus Actinoplanes with the name Actinoplanes capillaceus sp. nov. The type strain of the species is strain K95–5561^T (=JCM 10268^T =IFO 16408^T). The invalidly proposed species `Ampullariella cylindrica', `Ampullariella pekinensis' and `Ampullariella pilifera' were assigned to Actinoplanes capillaceus on the basis of genotypic and phenotypic data.     

Amycolatopsis bartoniae sp. nov. and Amycolatopsis bullii sp. nov., mesophilic actinomycetes isolated from arid Australian soils

The status of two mesophilic filamentous actinomycetes isolated from an arid Australian soil sample was determined using a polyphasic taxonomic approach. The isolates had chemical and morphological properties consistent with their classification in the genus Amycolatopsis, assignments that were supported by analysis of 16S rRNA gene sequence data. Isolate SF26^T formed a distinct phyletic line and hence was sharply separated from its nearest phylogenetic neighbour, Amycolatopsis sacchari DSM 44468^T. In contrast, isolate SF27^T formed a subclade in the Amycolatopsis tree with Amycolatopsis vancoresmycina DSM 44592^T but was separated readily from the latter by DNA:DNA pairing data. The two isolates were distinguished from one another and from their respective nearest phylogenetic neighbours using a range of phenotypic properties. These data indicate that the two isolates should be recognized as new species in the genus Amycolatopsis. The names proposed for these new taxa are Amycolatopsis bartoniae sp. nov. and Amycolatopsis bullii sp. nov. with isolates SF26^T (=NCIMB 14706^T = NRRL B-2846^T) and SF27^T (=NCIMB 14707^T = NRRL B-24847^T) as the respective type strains.     

Distribution and identification of red yeasts in deep-sea environments around the northwest Pacific Ocean

We isolated 99 yeast strains, including 40 red yeasts, from benthic animals and sediments collected from the deep-sea floor in various areas in the northwest Pacific Ocean. Comparing the yeast isolates from animals and sediments collected from shallow locations, the proportion of red yeasts differed considerably, comprising 81.5% and 10.6% of the isolates from animals and sediments, respectively. All of the red yeast isolates belonged to the genera Rhodotorula and Sporobolomyces. On the basis of morphological and physiological characteristics, the isolates were identified as R. aurantiaca, R. glutinis, R. minuta and R. mucilaginosa of the genus Rhodotorula, and S. salmonicolor and S. shibatanus of the genus Sporobolomyces. Only R. glutinis and R. mucilaginosa were isolated from sediments. All of the others were isolated from animal sources. Phylogenetic analyses based on internal transcribed spacer (ITS) regions and 5.8S rRNA gene sequences allowed us to establish the precise taxonomic placement of each of the isolates and thereby investigate the intraspecific relationships among the isolates. Twenty-two strains identified as members of R. glutinis, which showed a wide distribution in the deep-sea, and five isolates identified as R. minuta, which were isolated only from benthic animals, showed substantial heterogeneity within the species. The isolates phenotypically identified as Sporobolomyces species and R. mucilaginosa phylogenetically occupied the placements corresponding to these species. Some strains assigned to known species on the basis of phenotypic features should be regarded as new species as suggested by the results of molecular analysis.     

Studies on the diversity of the distinct phylogenetic lineage encompassing Glomus claroideum and Glomus etunicatum

Morphological and molecular characters were analysed to investigate diversity within isolates of the Glomus claroideum/Glomus etunicatum species group in the genus Glomus. The inter- and intra-isolate sequence diversity of the large subunit (LSU) rRNA gene D2 region of eight isolates of G. claroideum and G. etunicatum was studied using PCR-single strand conformational polymorphism (SSCP)-sequencing. In addition, two isolates recently obtained from Southern China were included in the analysis to allow for a wider geographic screening. Single spore DNA isolation confirmed the magnitude of gene diversity found in multispore DNA extractions. An apparent overlap of spore morphological characters was found between G. claroideum and G. etunicatum in some isolates. Analysis of the sequence frequencies in all G. etunicatum and G. claroideum isolates (ten) showed that four LSU D2 sequences, representing 32.1% of the clones analysed for multispore extraction (564) were found to be common to both species, and those sequences were the most abundant in four of the ten isolates analysed. The frequency of these sequences ranged between 23.2% and 87.5% of the clones analysed in each isolate. The implications for the use of phenotypic characters to define species in arbuscular mycorrhizal fungi are discussed. The current position of G. claroideum/G.etunicatum in the taxonomy of the Glomeromycota is also discussed.     

Molecular and morphological analyses of solitary forms of brackish Thalassiosiroid diatoms (Coscinodiscophyceae), with emphasis on their phenotypic plasticity

Blooms of centric diatoms are a common feature in the Bilbao estuary during summer when river flow is at its lowest and water temperature is above 20ºC. To gain insight into the specific composition of these diatom blooms, net samples and cultures of estuarine isolates were analysed under the scanning electron microscope (SEM) and by molecular analyses of the Internal Transcribed Spacers 1 and 2 plus the coding region 5.8S (ITS region) and the 28S rRNA gene. Seven species of solitary centric diatoms belonging to four genera were found in the estuary including: Conticribra weissflogii, Cyclotella atomus var. atomus, Cyclotella cryptica, Cyclotella marina, Cyclotella meneghiniana, Discostella pseudostelligera and Thalassiosira pseudonana. Dominant species during blooms were C. meneghiniana and Co. weissflogii in the upper estuary and D. pseudostelligera and T. pseudonana in the middle estuary. The morphological traits used to differentiate between species pairs of similar morphology (C. meneghiniana/C. cryptica or D. pseudostelligera/D. woltereckii) were observed to vary with environmental conditions, denoting a great deal of phenotypic plasticity which would hinder accurate identification of the species when using morphological approaches alone.     

Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley

A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%–7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the γ-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA–DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA–DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA–DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595). Received: July 20, 1999 / Accepted: October 29, 1999     

Utilization of a polyphasic approach in the taxonomic reassessment of antibiotic- and enzyme-producing <Emphasis Type="Italic">Bacillus</Emphasis> spp. isolated from the Philippines

Summary The taxonomy of 58 locally isolated antibiotic- and enzyme-producing Bacillus isolates deposited at the Philippine National Collection of Microorganisms (PNCM)-BIOTECH was reassessed in this study using a polyphasic approach since they had been only partially identified prior to deposition in the culture collection. The isolates had 41.1–69% G + C, and possessed the characteristic diaminopimelic acid (DAP) and fatty acid methyl ester (FAMEs) properties of Bacillus species. Molecular analysis using specific PCR primers differentiated the isolates into two major groups, the Bacillus cereus group and the Bacillus subtilis group. To further differentiate these isolates, they were subjected to 39 phenotypic tests. Using the dichotomous key constructed for Bacillus, 45 isolates maintained their original identities, five were named at the species level, and 12 were re-identified and renamed. These results showed that the classical phenotypic tests allowed the reclassification of the isolates, while modern techniques of chemotaxonomy and the molecular approach led only to genus and cluster classification and confirmation.     

Biosystematic studies on novel streptomycetes from soil

Members of three putatively novel Streptomyces species, designated Streptomyces groups A, B and C, were repeatedly isolated from environmental samples taken from four hay meadow plots at Cockle Park Experimental Farm, Northumberland (UK). Representative isolates were found to have properties consistent with their classification in the genus Streptomyces and were recovered in three taxa using different phenotypic criteria, namely morphological and pigmentation properties, rapid enzyme tests, and whole-organism fatty acid, protein electrophoretic and pyrolysis mass-spectrometric data. The isolates were rapidly characterised as three taxonomic groups using pyrolysis mass spectrometry. The three taxa were also distinguished from one another and from validly described species of Streptomyces using rapid enzyme tests based on the fluorophores 7-amino-methylcoumarin and 4-methylumbelliferone, and computer-assisted identification procedures. The results indicate that selective isolation and rapid characterisation of streptomycetes using pyrolysis mass spectrometry provide a practical way of determining the phenotypic species diversity of streptomycetes in natural habitats. The experimental data also indicate that representative sampling of cultivable streptomycetes from soil can best be achieved using a multi-step extraction procedure coupled with the use of selective isolation procedures.     

Phenotypic and Molecular Identification of <Emphasis Type="Italic">Sporothrix</Emphasis> Isolates from an Epidemic Area of Sporotrichosis in Brazil

Sporotrichosis has significantly increased in Brazil in the last decade, particularly in the state of Rio de Janeiro, with the occurrence of an epidemic related to zoonotic transmission from cats to humans. Recently, four new phylogenetic species were incorporated into the Sporothrix species complex based on the phenotypic and molecular characteristics, and a new species name (Sporothrix brasiliensis) was proposed for some of the Sporothrix isolates from this epidemic. This study describes the characterization of 246 isolates obtained from patients attending the Laboratory of Infectious Dermatology, IPEC-FIOCRUZ, between 1998 and 2008, together with one environmental sample. Two hundred and six of the isolates (83.4%) were characterized as S. brasiliensis, 15 (6.0%) as S. schenckii, and one (0.5%) as S. mexicana. Twenty-five isolates (10.1%) could not be identified according to their phenotype and were classified as Sporothrix spp. The calmodulin gene was sequenced to confirm the identity of these isolates. The molecular analysis demonstrated that 24 of the isolates were S. brasiliensis, with the remainder being a S. globosa isolate. The isolate characterized phenotypically as S. mexicana was clustered on the S. schenckii clade. The correlation between molecular data and phenotypic characteristics described in this study is fundamental to the identification of the Sporothrix complex.     

Identification of Two Multidrug-Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Clonal Lineages with a Countrywide Distribution in Hungary

The aim of this study was to identify class 1 integrons from extended-spectrum and metallo-β-lactamase-negative, multidrug-resistant Pseudomonas aeruginosa clinical isolates from Hungary and to characterize the isolates by phenotypic and molecular methods. Fourteen selected P. aeruginosa isolates resistant to ceftazidime, gentamicin, and ciprofloxacin were subjected to serotyping, random amplification of polymorphic DNA (RAPD), integron content analysis, and a phenotypic test to detect high-level production of AmpC. Four representative isolates were further analyzed by multilocus sequence typing. Two P. aeruginosa multidrug-resistant clonal lineages were identified with a countrywide distribution. The first lineage is characterized by serotype O4, RAPD genotype A, sequence type ST175, and the presence of a class 1 integron harbouring aadB and aadA13 gene cassettes in its variable region. The second lineage is characterized by serotype O6, RAPD genotype B, sequence type ST395, and a class 1 integron carrying a single aadB cassette. The corresponding isolates were recovered from altogether 11 towns in Hungary. ST175 and ST395 are the presently calculated founders of two distinct P. aeruginosa clonal complexes that appear to have a wide geographical distribution also outside Hungary. The multidrug-resistant phenotype associated with these two clonal lineages might have contributed to an increase in their frequency and to their subsequent diversification. Both P. aeruginosa lineages displayed ≥8-fold synergy with boronic acid/ceftazidime combinations, suggesting an AmpC-mediated resistance to ceftazidime. Our observations underscore the role of class 1 integrons in the spread of aminoglycoside resistance by clonal dissemination among P. aeruginosa clinical isolates in Hungary.     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Long‐term stasis and short‐term divergence in the phenotypes of microsnails on oceanic islands

Phenotypic divergence is often unrelated to genotypic divergence. An extreme example is rapid phenotypic differentiation despite genetic similarity. Another extreme is morphological stasis despite substantial genetic divergence. These opposite patterns have been viewed as reflecting opposite properties of the lineages. In this study, phenotypic radiation accompanied by both rapid divergence and long‐term conservatism is documented in the inferred molecular phylogeny of the micro land snails Cavernacmella (Assimineidae) on the Ogasawara Islands. The populations of Cavernacmella on the Sekimon limestone outcrop of Hahajima Island showed marked divergence in shell morphology. Within this area, one lineage diversified into types with elongated turret shells, conical shells and flat disc‐like shells without substantial genetic differentiation. Additionally, a co‐occurring species with these types developed a much larger shell size. Moreover, a lineage adapted to live inside caves in this area. In contrast, populations in the other areas exhibited no morphological differences despite high genetic divergence among populations. Accordingly, the phenotypic evolution of Cavernacmella in Ogasawara is characterized by a pattern of long‐term stasis and periodic bursts of change. This pattern suggests that even lineages with phenotypic conservatism could shift to an alternative state allowing rapid phenotypic divergence.