Ecology and taxonomy of chitinoclasticCytophaga and related chitin-degrading bacteria isolated from an estuary

A total of 103 strains of estuarine, Chitinoclastic bacteria isolated from water, and sediment samples collected from the upper Chesapeake Bay, including 17 freshwater and 11 seawater isolates, were subjected to numerical taxonomy analysis. The isolates included 44 yellow-orange pigmented strains classified asCytophaga-like bacteria (CLB) of theCytophagaceae. Salt requirement of the strains ranged from tolerance to 1% NaCl to an absolute requirement for NaCl, with 1% NaCl satisfying this requirement. The largest phenon consisted of facultatively anaerobic, oligo-nitrophilic, and flexirubin pigment-producing freshwater and estuarine isolates, and included reference strains of bothCytophaga johnsonae Stanier andCytophaga aquatilis Strohl and Tait. Other phena, containing a smaller number of strains, comprised marine and estuarine isolates which did not produce flexirubin pigments, and required organic nitrogen for growth and for production of chitinolytic enzymes. Salt-requiring, flexirubin pigment-producing, chitin-degrading strains were, on occasion, isolated from estuarine samples and represented phena found in estuaries. Most of theCytophaga isolates, as well as chitin-degrading species not of the genusCytophaga that were isolated from Chesapeake Bay, clustered in phena representing previously described species of aerobic, zymogenic, chitinoclastic bacteria. When the frequency of occurrence of features related to environmental parameters, viz., pH, salinity, temperature range of growth, and growth on media lacking organic nitrogen, was calculated, ecological groupings of strains in the 2 major phena of CLB could be distinguished among the estuarine, chitin-degrading bacteria.     

Blue-green algae (cyanobacteria): prospects and perspectives

Summary Photosynthetic, prokaryotic blue-green algae (cyanobacteria) occur in a wide range of natural habitats of diverse ionic composition and as such, represent an important source of biological material for biosolar energy conversion programs using saline water. The gasvacuolate, filamentous Spirulina is grown in seminatural culture in Lake Texcoco, Mexico, as a major source of single-cell protein for animal nutrition. Pilot-scale trials in other areas of the world have also demonstrated the suitability of blue-green algae, including Spirulina, for growth under brackish conditions. The carbohydrate accumulation profiles of blue-green algae differ in isolates from freshwater, marine and hypersaline habitats, with a trend towards sucrose or trehalose accumulation in stenohaline freshwater strains grown in media containing NaCl, while euryhaline and marine forms frequently accumulate glucosylglycerol. Many halotolerant isolates from hypersaline habitats accumulate glycinebetaine in response to osmotic stress. This knowledge may provide scope for future improvement in the N₂ fixation rates of blue-green algae in saline media, using betaine-accumulating N₂-fixing strains in preference to other, saltsensitive isolates.     

Isolation of ACC deaminase producing PGPR from rice rhizosphere and evaluating their plant growth promoting activity under salt stress

Aims

Bacteria possessing ACC deaminase activity reduce the level of stress ethylene conferring resistance and stimulating growth of plants under various biotic and abiotic stresses. The present study aims at isolating efficient ACC deaminase producing PGPR strains from the rhizosphere of rice plants grown in coastal saline soils and quantifying the effect of potent PGPR isolates on rice seed germination and seedling growth under salinity stress and ethylene production from rice seedlings inoculated with ACC deaminase containing PGPR.

Methods

Soils from root region of rice growing in coastal soils of varying salinity were used for isolating ACC deaminase producing bacteria and three bacterial isolates were identified following polyphasic taxonomy. Seed germination, root growth and stress ethylene production in rice seedlings following inoculation with selected PGPR under salt stress were quantified.

Results

Inoculation with selected PGPR isolates had considerable positive impacts on different growth parameters of rice including germination percentage, shoot and root growth and chlorophyll content as compared to uninoculated control. Inoculation with the ACC deaminase producing strains reduced ethylene production under salinity stress.

Conclusions

This study demonstrates the effectiveness of rhizobacteria containing ACC deaminase for enhancing salt tolerance and consequently improving the growth of rice plants under salt-stress conditions.     

Effects of salinity on growth and on valve morphology of five estuarine diatoms

The effects of salinity on the growth and valve morphology of five benthic estuarine diatoms (Nitzschia pusilla, N. frustulum, N. palea, N. filiformis var. conferta and Eolimna subminuscula), isolated from both freshwater and brackish/marine habitats, were investigated. The four Nitzschia strains grew well over a broad salinity range, though some (N. pusilla, N. frustulum) showed a broader salinity range tolerance (from fully saline down to at least 9.5 ppt) than others (N. palea, N. filiformis var. conferta had reduced growth at salinities of 16 ppt and above). Salinity significantly affected the valve morphology of the five strains studied. However, there was no consistent pattern in either the morphological characters affected or the direction of the effects. Although significant, the effects of salinity on valve morphology were very small and therefore it seems that the taxonomic usefulness of some of the classical taxonomical characters is not undermined.     

Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.     

Influence of pH on copper and zinc sensitivity of ericoid mycobionts in vitro

The effect of pH on growth, metal uptake and toxicity in four isolates of ericoid mycobionts (two Hymenoscyphus ericae from unpolluted heathland sites and two H. ericae-type mycobionts from metal-contaminated mine spoil) was assessed in vitro. These isolates were incubated in liquid medium (10% Rorisons medium, glucose at 10 g l^–1) containing either 0.25 mM Cu or 2.0 mM Zn and adjusted to pH 2, 3, 4, 5 or 6. After 30 days incubation, dry mass and mycelial metal content were determined and growth was expressed as tolerance index, i.e. dry mass in the presence of metal as a percentage of dry mass in the absence of metal. Initial medium pH had a significant effect on both tolerance index and metal accumulation. Tolerance indices were highest at pH 2, with several isolates showing a stimulation of growth (i.e. tolerance index >100%) at this pH. Tolerance index decreased at higher initial pH values and growth of two mycobionts was completely inhibited (tolerance index=0) in the Cu-supplemented media at pH 6. Reduction in tolerance index coincided with an increase in mycelial accumulation of Cu and Zn. Practical and environmental implications of these results are discussed.     

Effect of extreme salinity conditions on the survival of Mytilopsis sallei Recluz (Pelecypoda)

Effects of salinity conditions, ranging from fresh water to 80, on the survival of marine molluscan fouling species, Mytilopsis sallei Recluz, have been studied in the laboratory. The results show that the species exhibits a wide tolerance to different salinity conditions including freshwater, showing normal activity up to 50 beyond which the higher salt concentration had a lethal effect. The effect of various salinity conditions on spawning has also been examined. The significance of wide range tolerance to salinity on the fouling in seawater cooling systems is discussed.     

The effect of gradual increase in salinity on the biomass productivity and biochemical composition of several marine,halotolerant<Emphasis Type="Bold">,</Emphasis> and halophilic microalgae

Open ponds are the preferred cultivation system for large-scale microalgal biomass production. To be more sustainable, commercial scale biomass production should rely on seawater, as freshwater is a limiting resource, especially in places with high irradiance. If seawater is used for both pond fill and evaporative volume makeup, salinity of the growth media will rise over time. It is not possible for any species to achieve optimum growth over the whole saline spectrum (from seawater salinity level up to salt saturation state). In this study, we investigated the effects of gradual salinity increase (between 35 and 233 ppt) on biomass productivity and biochemical composition (lipid and carbohydrate) of six marine, two halotolerant, and a halophilic microalgae. A gradual and slow stepped salinity increase was found to expand the salinity tolerance range of tested species. A gradual reduction in biomass productivity and maximum photochemical efficiency was observed as a consequence of increased salinity in all tested species. Among the marine microalgae, Tetraselmis showed highest biomass productivity (32 mg L^?1 day^?1) with widest salinity tolerance range (35 to 109 ppt). Halotolerant Amphora and Navicula were able to grow from 35 ppt to 129 ppt salinity. Halophilic Dunaliella was the only species capable of growing between 35 and 233 ppt and showed highest lipid content (56.2%) among all tested species. This study showed that it should be possible to maintain high biomass in open outdoor cultivation utilizing seawater by growing Tetraselmis, Amphora, and Dunaliella one after another as salinity increases in the cultivation system.     

Structural and functional features of gill epithelium and the brood pouch of pipefish <Emphasis Type="Italic">Sygnathus acusimilis</Emphasis> (Syngnathidae,Gasterosteiformes) during adaptation to dilute sea water

Study of the dilate sea-water tolerance of the pipefish Syngnathus acusimilis indicated that it successfully acclimatized to an abrupt change in water salinity from 5 to 32‰. Larvae in the brood pouch also successfully acclimatized to salinity change, and, after several days, juveniles left the brood pouch and continued to live. In freshwater, adult and fry perished during several minutes. In the gill epithelium of S. acusimilis, chloride cells of only a marine type were found. A comparison of the ultrastructure of chloride cells of gill epithelium and mitochondrion-rich cells of the brood pouch of S. acusimilis demonstrated their high similarity. The ultrastructure of cells of the brood pouch indisputably attests to their involvement in maintaining an osmotic and ionic homeostasis in the cavity of the brood pouch.     

Arbuscular mycorrhizal fungal propagules in a salt marsh

The tolerance of indigenous arbuscular mycorrhizal fungi (AMF) to stressful soil conditions and the relative contribution of spores of these fungi to plant colonization were examined in a Portuguese salt marsh. Glomus geosporum is dominant in this salt marsh. Using tetrazolium as a vital stain, a high proportion of field-collected spores were found to be metabolically active at all sampling dates. Spore germination tests showed that salt marsh spores were not affected by increasing levels of salinity, in contrast to two non-marsh spore isolates, and had a significantly higher ability to germinate under increased levels of salinity (20) than in the absence of or at low salinity (10). Germination of salt marsh spores was not affected by soil water levels above field capacity, in contrast to one of the two non-marsh spore isolates. For the evaluation of infectivity, a bioassay was established with undisturbed soil cores (containing all types of AM fungal propagules) and soil cores containing only spores as AM fungal propagules. Different types of propagules were able to initiate and to expand the root colonization of a native plant species, but spores were slower than mycelium and/or root fragments in colonizing host roots. The AM fungal adaptation shown by this study may explain the maintenance of AMF in salt marshes.     

Genetic relatedness of clinical and environmental isolates of lactose-positiveVibrio vulnificus

Environmental isolates of lactose-positiveVibrio vulnificus from different geographic areas were compared with clinical strains ofV. vulnificus on the basis of their phenotypic traits, virulence, DNA base composition, and DNA-DNA reasociation. EnvironmentalV. vulnificus strains were phenotypically indistinguishable from clinical isolates. These strains had a DNA base composition of 47–48 mol% guanine + cytosine and 85% reassociation at stringent temperature with DNA from clinicalV. vulnificus strains. These result indicateV. vulnificus strains from widely separated regions of the marine environment are indistinguishable from strains that have been agents of septicemia associated with shellfish consumption and of wound infections associated with seawater exposure.     

Salinity tolerant Trichoderma harzianum reinforces NaCl tolerance and reduces population dynamics of Fusarium oxysporum f.sp. ciceri in chickpea (Cicer arietinum L.) under salt stress conditions

The antagonistic potential of salinity tolerant (ST) Trichoderma (Th) isolates against Fusarium oxysporum f.sp. ciceri (foc) was tested, along with their capability to induce relative salt stress tolerance in chickpea with the aim to exploit their use as biological agents in reducing deleterious effects of salinity and controlling Fusarium wilt of chickpea under saline soil conditions. Under laboratory conditions, salt stress was created by supplementing nutrient medium with different concentrations of NaCl viz. 0, 70, 150 and 240?mM NaCl and a pot experiment was conducted using natural saline soil (EC – 6.6 dS?m^?1). Out of 45 Th isolates studied, only five isolates viz. Th-13, Th-14, Th-19, Th-33 and Th-50 were selected to be ST as these were able to grow and sporulate in growth medium containing up to 240?mM NaCl. In saline medium, ST isolates greatly surpassed salinity sensitive (SS) isolate with respect to growth rate, mycelial dry weight, sporulation and biological proficiency against foc. Out of five ST isolates that retained their tolerance to different salt stress levels, Th-14 and Th-19 showed maximum antagonism against foc. Under greenhouse conditions, chickpea plants obtained from seeds bioprimed with Th-14 and Th-19 performed well both at germination and seedling stage in comparison to control in saline soil. As compared to untreated plants, characterisation of Th treated plants confirmed that they had reinforced contents of proline along with relatively higher levels of total phenols, membrane stability index and superoxide dismutase activity while lower accumulation of hydrogen peroxide and malondealdehyde contents. ST isolates, Th-14 and Th-19 significantly reduced foc-induced wilt disease incidence in chickpea plants. The population density of both the Th isolates in rhizosphere far exceeded that of foc under both saline and non-saline soils. However, Th-14 was found more efficient in increasing relative salt stress tolerance in chickpea and reducing the foc growth in rhizosphere under present materials and conditions. These findings provide a novel paradigm for developing alternative, environmentally safe strategy to alleviate salt stress and manage fungal diseases such as foc that aggravates under saline soils.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

Evidence of concurrent local adaptation and high phenotypic plasticity in a polar microeukaryote

Here we investigated whether there is evidence of local adaptation in strains of an ancestrally marine dinoflagellate to the lacustrine environment they now inhabit (optimal genotypes) and/or if they have evolved phenotypic plasticity (a range of phenotypes). Eleven strains of Polarella glacialis were isolated and cultured from three different environments: the polar seas, a hyposaline and a hypersaline Antarctic lake. Local adaptation was tested by comparing growth rates of lacustrine and marine strains at their own and reciprocal site conditions. To determine phenotypic plasticity, we measured the reaction norm for salinity. We found evidence of both, limited local adaptation and higher phenotypic plasticity in lacustrine strains when compared with marine ancestors. At extreme high salinities, local lake strains outperformed other strains, and at extreme low salinities, strains from the hyposaline lake outperformed all other strains. The data suggest that lake populations may have evolved higher phenotypic plasticity in the lake habitats compared with the sea, presumably due to the high temporal variability in salinity in the lacustrine systems. Moreover, the interval of salinity tolerance differed between strains from the hyposaline and hypersaline lakes, indicating local adaptation promoted by different salinity.     

Isolation of aflatoxin from Acacia and the incidence of Aspergillus flavus in the Sudan

A series of environmental and clinical isolates of Sporothrix schenckii being studied in our laboratories under standard conditions of temperature, humidity and media, displayed, in some of the isolates, large dematiaceous hyphae. The large hyphae could be seen to produce the fine hyphae associated with S. schenckii on which typical microconidia developed. Typical microconidia also developed occasionally on the large hyphae, and strands of the unusually large hyphae also were seen to form arthroconidia. Some strains formed large, thin-walled cells on thin and/or thick hyphae resembling the balloon cells seen in mature colonies of Trichophyton tonsurans.All strains which have demonstrated these various and unusual structures histopathologically produced typical sporotrichosis in laboratory animals. When reisolated from these animals, the cultures again exhibited the unusual structures, as well as those typical of S. schenckii.     

Saprophytic competence of acid tolerant strains ofRhizobium trifolii in acid soil

Summary Laboratory prescreening ofRhizobium trifolii for acid tolerance, based upon the ability of rhizobia to grow in acid media (pH 4.2) containing Al (15 M), was successful for the selection of strains capable of survival in acid soil.Both sterile and non-sterile soils of varying acidity were inoculated with several strains ofR. trifolii.Acid tolerant strains generally had significantly higher populations at every sample period than an acid sensitive strain. Amelioration of soil acidity by liming improved persistence of all strains. Soil sterilization by autoclaving adversely affected survival of all strains at each soil acidity level.Paper Number 8766 of the Journal Series, North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Plasmids in tributyltin-resistant bacteria from fresh and estuarine waters

Summary Twenty-six tributyltin (TBT)-resistant bacterial strains isolated from sediments were examined for the presence of plasmids. Plasmids of the size reported to carry metal resistance genes were not found in 15 of the strains, indicating that resistance does not have to be plasmid-mediated. Attempts to cure plasmid-containing strains using acridine organge, ethidium bromide, novobiocin or sodium dodecylsulfate, or by growth at elevated temperature were not successful, nor were plasmids transferred from TBT-resistant strains into TBT-sensitive organisms by electroporation. In a broth mating experiment however, plasmid pUM505, a conjugative plasmid known to encode chromium resistance inPseudomonas aeruginosa PAO1, was introduced into TBT-sensitiveBeijerinckia sp. MC-27 isolated from freshwater sediment. The TBT tolerance of theBeijerinckia sp. increased 100-fold, from 8.4 M TBT inBeijerinckia sp. MC-27 to 840 M TBT inBeijerinckia sp. MC-27 (pUM505) on solid medium. The plasmid was transferred at a frequency of approximately 6×10^–4. TBT-resistant transconjugants grew faster in media containing TBT and lost their enhanced TBT tolerance and the plasmid upon serial transfer in medium without TBT. Spontaneous mutants of the donorP. aeruginosa lost both TBT resistance and the plasmid. Therefore, TBT resistance in bacteria can be plasmid-mediated. To our knowledge, this is the first report that resistance to a tin compound can be plasmid-mediated.     

Distribution of ultramicrobacteria in a gulf coast estuary and induction of ultramicrobacteria

The abundance of ultramicrobacteria (i.e., bacteria that pass through a 0.2m filter) in a subtropical Alabama estuary was determined during a 1-year period. Although phenotypic and molecular characterization indicated that the population of ultramicrobacteria was dominated byVibrio species, species ofListonella andPseudomonas were also abundant. Vibrios occurred with the greatest frequency in waters whose salinities were less than 14, and were the most abundant species of the total ultramicrobacterial population year-round, whilePseudomonas species were absent or considerably reduced during the winter months. The total number of ultramicrobacteria showed an inverse relationship to total heterotrophic bacteria as measured by colony-forming units (CFU)/ml and to water quality as measured by several parameters. Analysis by generic composition indicated that both salinity and temperature significantly affected the distribution of these organisms. Laboratory studies revealed that strains of vibrios under starvation in both static and continuous-flow microcosms could be induced to form cells that passed through 0.2 and/or 0.4m filters. Cells exposed to low nutrients became very small; some grew on both oligotrophic (5.5 mg carbon/liter) and eutrophic (5.5 g carbon/liter) media; and some few cells grew only on oligotrophic media. By passing selected vibrio strains on progressively diluted nutrient media, cells were also obtained that were small, that passed through 0.4m filters, and that could grow in oligotrophic media. These results suggest that ultramicrobacteria in estuaries (at least some portion of the population) may be nutrientstarved or low nutrient-induced forms of certain heterotrophic, eutrophic, autochthonous, estuarine bacteria.     

The Genetic Control of Cell Growth and Development in Yeast Saccharomyces cerevisiae: Disturbed Sporulation in Diploids with a Decreased Activity of the Ras/cAMP Signal Transduction Pathway

Seven haploid strains (four with the MAT mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast. Previous phenotypic analysis assigned six of these strains to a physiological group of strains with changed activity of the Ras/cAMP signal transduction pathway. The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media. Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency). The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway. All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34°C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose). Sporulation proved to depend on temperature and medium composition. The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.     

Effect of inbreeding and salinity on quantitative characters and asymmetry of Tisbe holothuriae (Humes)

Effects of differing salinities on morphological characters have been found in marine and brackish populations of Tisbe holothuriae. The results suggested an active role of salinity as a factor promoting divergence. In order to test if the observed differentiation could be due to the level of heterozygosity, a study was performed in strains with low and high homozygosity. Six quantitative traits, and their respective fluctuating asymmetry, have been measured in outbred (coefficient of consanguineity, F = 0) and inbred (F = 0.5) strains, raised at three salinities; 20, 25 and 35. The analysis of variance was applied to test if the morphological measures were the same, and if the variations induced by decreased salinity had the same trend, in the two strains. Wilcoxon test was applied to the asymmetry, when it was not possible to render the variances homogeneous. The results showed that the measures and the effects of salinity on morphometry differed in the strains with low and high homozygosity. No effect of salinity on asymmetry was found. Length measures showed higher asymmetry in the most homozygous strain.