VEGETATIVE REPRODUCTION AND SEXUAL LIFE CYCLE OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM FROM TASMANIA,AUSTRALIA1

The toxic, chain-forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23-41 pm long, 27-36 pm wide). Vegetative growth (mean doubling time 3-4 days) was optimal at temperatures from 14.5-20° C, salinities of 23-34% and light irradiances of 50-300 μE·m^?2·s^?1. The sexual life cycle of G. catenatum was easily induced in a nutrient-deficient medium, provided compatible opposite mating types were combined (heterothallism). Gamete fusion produced a large (59-73 μm long, 50-59 μm wide) biconical, posteriorly biflagellate planozygote (double longitudinal flagellum) which after several days lost one longitudinal flagellum and gradually became subspherical in shape. This older planozygote stage persisted for up to two weeks before encysting into a round, brown resting cyst (42-52 μm diam; hypnozygote) with microreticulate surface ornamentation. Resting cysts germinated after a dormancy period as short as two weeks under our culture conditions, resulting in a single, posteriorly biflagellate germling cell (planomeiocyte). This divided to form a chain of two cells, which subsequently re-established a vegetative population. Implications for the bloom dynamics of this toxic dinoflagellate, a causative organism of paralytic shellfish poisoning, are discussed.     

Detection of Giardia in Environmental Waters by Immuno-PCR Amplification Methods

Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB). A 0.171-kbp segment of the giardin gene was PCR-amplified following “direct extraction” of Giardia DNA from seeded Cahaba river water concentrate with moderate turbidity (780 JTU's), but DNA purified from seeded Colorado river water concentrates with high turbidity (2 × 10⁵ JTUs) failed to amplify. However, if the cysts were first separated by the IMB approach from seeded Cahaba or Colorado river waters, and the DNA released by a freeze-boil Chelex?100 treatment, detection of G. muris by PCR amplification could be achieved at a sensitivity of 3 × 10⁰ or 3 × 10¹ cysts/ml, respectively. If, however, the G. muris cysts used to seed even moderately turbid river waters (780 JTUs) were formalin treated (which is conventionally used for microscopic examination), neither direct extraction nor IMB purification methods yielded amplifiable DNA. Use of immunomagnetic beads to separate Giardia cysts from complex matrices of environmental surface waters followed by DNA release and PCR amplification of the target giardin gene improved the reliability of detection of this pathogen with the required sensitivity. Received: 23 April 1997 / Accepted: 4 August 1997     

A NEW SPECIES OF ENSICULIFERA,E. IMARIENSE (DINOPHYCEAE), PRODUCING ORGANIC-WALLED CYSTS1

We describe a new organic-walled resting cyst from surface sediments of Imari Bay in western Japan. The cysts are spherical, 23–29 pm in diameter, and their surface is covered with spinous to membranous ornaments that are 5–7 μm long and 1.5–2.2 μm wide. The ornaments vary from slender and bifurcate to membranous and multifurcate distal extremities. No archeopyle was observed. The cyst shape is variable in both natural samples and clonal cultures. Vegetative cells are small and ovoid, 17–25 μm long and 14–21 μm wide, and are yellow-brown in color. The epitheca is conical with a conspicuous apical horn, and the hypotheca is hemispherical. The cingular transitional plate has a needle-like spine at its anterior right corner. The plate formula is Po, X, 4″3a, 7″, 5c, 5s 5″and 2″. Although vegetative cells of the present species correspond to Ensiculifera, it is distinct from other species in producing no calcareous cysts. No species of Ensiculifera has been reported to produce cysts composed of only an organic wall. The present species is provisionally placed in the genus Ensiculifera as E. imariense sp. nov.     

Molecular basis of epithelial Cl channels

Proximal straight tubules (PST) were dissected from rabbit kidneys, held by crimping pipettes in a chamber and bathed in a buffered isosmotic (295 mOsm/kg) solution containing 200 mm mannitol (MBS). Changes in tubule diameter were monitored on line with an inverted microscope, TV camera and image processor. The PST were then challenged for 20 sec with MBS made 35 mOsm/kg hyperosmotic by addition of either NaCl, KCl, mannitol (M), glycerol (G), ethylene glycol (E), glycine (g), urea (U), acetamide (A) or formamide (F). With NaCl, KCl, M, G, E, g, U, and A, tubules shrunk osmometrically within 0.5 sec and remained shrunk for as long as 20 sec without recovering their original volume (sometimes A showed some recovery). PST barely shrunk with F and quickly recovered their original volume. The permeability coefficients were 0 m/sec (NaCl, M, g, E and U), 1 m/sec (A), 84 m/sec (F) and 0.02 m/sec (G). The reflection coefficients = 1.0 (NaCl, KCl, M, G, E, g and U), 0.95 (A) and 0.62 (F). Similar values were obtained by substituting 200 mOsm/kg M in MBS by either NaCl, KCl, G, E, g, U, a or F. The olive oil/water partition coefficients are 5 (M), 15 (U), 85 (A) and 75 (F) (all x10^–5). Thus, part of F permeates the cell membrane through the lipid bilayer. The probing molecules van der Waals diameters are 7.4×8.2×12.0 (M), 3.6×5.2×5.4 (U), 3.8×5.2 ×5.4 (A) and (3.4×4.5×5.4 (F) Å. We conclude that only F clearly permeates the water channel (WCH). Water molecules must single file within the WCH. After subtraction of the bilayer permeability of the probes, we estimate for the WCH selectivity filter cross-section a diameter of 4.2–4.7 Å (if it is circular) and 3.6×4.2 Å (if it is rectangular). But if the oxygens facing the WCH lumen H bond with the molecules crossing the WCH, the WCH selectivity filter would be 3.3–3.8 Å (circular) and 3.6×4.0 Å (rectangular).This work was supported in part from grants from CONICIT, Consejo de Desarrollo Científico y Humanístico of UCV and Fundación Polar.     

Genetic characterisation of uninucleated cyst-producing Entamoeba spp. from ruminants

Six ssrRNA gene sequences were obtained by PCR amplification of DNA from uninucleated Entamoeba cysts isolated from fresh faeces of sheep, cows, a roe deer and a reindeer. Phylogenetic analysis using sequences of non-, uni-, quadri- and octonucleate cyst-producing Entamoeba spp. for comparison showed that all six isolates formed a separate clade nested within the clade of quadrinucleate cyst producers. The data indicate that Entamoeba bovis can be isolated from ruminant hosts other than cattle, and we suggest that organisms clustering with the sheep and cattle isolates analysed in the present study be named E. bovis.     

Isolation and characterization of Thermanaerothrix daxensis gen. nov., sp. nov., a thermophilic anaerobic bacterium pertaining to the phylum "Chloroflexi", isolated from a deep hot aquifer in the Aquitaine Basin

A new strictly anaerobic thermophilic multicellular filamentous bacterium (0.2–0.3 μm × >100 μm), designated GNS-1^T, was isolated from a deep hot aquifer in France. It was non-motile, and stained Gram-negative. Optimal growth was observed at 65 °C, pH 7.0, and 2 g L⁻¹ of NaCl. Strain GNS-1^T was chemoorganotrophic fermenting ribose, glucose, galactose, arabinose, fructose, mannose, maltose, sucrose, xylose, raffinose, pyruvate, and xylan. Yeast extract was required for growth. The end products of glucose fermentation were lactate, acetate, CO₂, and H₂. The G + C content of the DNA was 57.6 mol%. Its closest phylogenetic relative was Bellilinea caldifistulae with 92.5% similarity. Based on phylogenetic, genotypic and phenotypic characteristics, strain GNS-1^T (DSM 23592^T, JCM 16980^T) is proposed to be assigned to a novel species of a novel genus within the class Anaerolineae (subphylum I), phylum “Chloroflexi”, Thermanaerothrix daxensis gen. nov., sp. nov. The GenBank accession number is HM596746.     

Grazing impact of heterotrophic dinoflagellates and ciliates on common red-tide euglenophyte Eutreptiella gymnastica in Masan Bay, Korea

The euglenophyte Eutreptiella gymnastica is a common red tide causative species. However, there have been no studies on the grazing impact of heterotrophic protists on this species. To investigate the grazing impact of heterotrophic protists on E. gymnastica, we measured daily the abundances of E. gymnastica and co-occurring potential heterotrophic protistan grazers in Masan Bay, Korea, in August 2004 when an E. gymnastica red tide occurred. In addition, we tested whether the common heterotrophic dinoflagellates Gyrodinium dominans, Oxyrrhis marina, Pfiesteria piscicida, Polykrikos kofoidii, Protoperidinium bipes, and Stoeckeria algicida and the naked ciliates Strobilidium sp. (30–40 μm in cell length) and Strombidinopsis sp. (70–100 μm in cell length) were able to feed on E. gymnastica. We also measured their growth and ingestion rates on E. gymnastica as a function of prey concentration. Finally, we calculated the grazing coefficients by combining field data on the abundance of the heterotrophic dinoflagellate and ciliate grazers and co-occurring E. gymnastica with laboratory data on ingestion rates obtained in this study. The maximum abundance of E. gymnastica in Masan Bay in August, 2004 was 7575 cells ml⁻¹, while those of Gyrodinium spp., P. kofoidii, P. bipes, the naked ciliates (≤50 μm in cell length), and naked ciliates (>50 μm in cell length) were 50, 9, 58, 32, and 3 cells ml⁻¹, respectively. The maximum growth rate of G. dominans on E. gymnastica (1.13 d⁻¹) was higher than that of O. marina (0.81 d⁻¹) or P. bipes (0.77 d⁻¹). However, E. gymnastica did not support positive growth of P. kofoidii, Strobilidium sp., and Strombidinopsis sp. (−0.04 ∼ −2.8 d⁻¹). The maximum ingestion rates of G. dominans, P. kofoidii, P. bipes, O. marina, and Strobilidium sp. on E. gymnastica (2.1–2.7 ng C predator⁻¹ d⁻¹) were similar, but they were much lower than that of Strombidinopsis sp. (156 ng C predator⁻¹ d⁻¹). The calculated grazing coefficients for P. bipes, small heterotrophic Gyrodinium spp. (25–35 μm in cell length), naked ciliates (≤50 μm in cell length), P. kofoidii, and naked ciliates (>50 μm in cell length) on E. gymnastica were up to 0.77, 0.61, 0.22, 0.07 and 0.03 d⁻¹, respectively (i.e., up to 54%, 46%, 20%, 7%, and 3% of E. gymnastica populations were removed by the population of each of these heterotrophic protistan grazers in 1 d, respectively). The results of the present study suggest that P. bipes, small heterotrophic Gyrodinium spp., and naked ciliates (≤50 μm in cell length) sometimes have considerable potential grazing impact on the populations of E. gymnastica.     

Factors Affecting High-Oxygen Survival of Heterotrophic Microorganisms from an Antarctic Lake

We sought to determine factors relating to the survival of heterotrophic microorganisms from the high-dissolved-oxygen (HDO) waters of Lake Hoare, Antarctica. This lake contains perpetual HDO about three times that of normal saturation (40 to 50 mg liter⁻¹). Five isolates, one yeast and four bacteria, were selected from Lake Hoare waters by growth with the membrane filter technique with oxygen added to yield dissolved concentrations 14 times that in situ, 175 mg liter⁻¹. One bacterial isolate was obtained from the microbial mat beneath the HDO waters. This organism was isolated at normal atmospheric oxygen saturation. The bacteria were gram-negative rods, motile, oxidase positive, catalase positive, and superoxide dismutase positive; they contained carotenoids. The planktonic isolates grew in media containing 10 mg of Trypticase soy (BBL Microbiology Systems)-peptone (2:1) liter⁻¹ but not at 10 g liter⁻¹. Under low-nutrient levels simulating Lake Hoare waters (10 mg liter⁻¹), two of the planktonic isolates tested were not inhibited by HDO. Growth inhibition by HDO increased as nutrient concentration was increased. A carotenoid-negative mutant of one isolate demonstrated a decreased growth rate, maximal cell density, and increased cell lysis in the death phase under HDO compared with the parent strain. The specific activity of superoxide dismutase was increased by HDO in four of the five bacterial isolates. The superoxide dismutase was of the manganese type on the basis of inhibition and electrophoretic studies. The bacterial isolates from Lake Hoare possess several adaptations which may aid their survival in the HDO waters, as well as protection due to the oligotrophic nature of the lake.     

Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258–1.407 μg/mL, the medium inhibition concentration (IC₅₀) were 0.727–0.892 μg/mL and detection limit (IC₁₀) were 0.029–0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21–5.94 × 10⁷ M⁻¹. The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%–100.8% with a CV at 2.5%–9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.     

Studies on protozoan parasites of Australian fishes. Notes on coccidian parasites with description of three new species

In 1990 several species of the generaEimeria andGoussia were found during the examination of some marine and freshwater fishes in New South Wales for protozoan parasites. In freshwater hosts,Goussia carpelli (Léger & Stankovitch, 1921) was found inCarassius auratus from a fish farm andEimeria anguillae Léger & Hollande, 1922 inAnguilla australis from feral eels. A new species,G. piekarskii n. sp., is described from the small intestine of feralGambusia holbrooki. Spherical or subspherical oöcysts average 9.2 μm in diameter, the average size of sporocysts being 7.6 × 4.2 μm. From coastal marine fishes,Goussia thelohani labbé, 1896 was identified in the liver parenchyma ofAcanthopagrus australis andRhabdosargus sarda. G. cruciata (Thélohan, 1892) Labbé, 1896 was found in the liver parenchyma ofPseudocaranx dentex. Both species were found for the first time outside the European area and in completely different hosts.Eimeria sillaginis Molnár & Rohde, 1988 is redescribed from the intestine ofSillago ciliata, and includes a new structure on its sporocyst wall.E. nesowai n. sp. is described from the pyloric caeca and intestine ofGerres ovatus. Subspherical oöcysts average 12.9 μm in diameter, the average size of sporocysts being 7 × 4.2 μm.E. rohdei n. sp. was found to infect the pyloric caeca ofMonacanthus chinensis. The average sizes of oöcysts and sporocysts are 7.5 μm diameter and 5.1 × 3 μm, respectively.     

Collection and evaluation of epididymal sperm in captive agoutis (Dasyprocta aguti)

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1-2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 μL, pH 6.9, sperm concentration 748 x 10⁶ sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 μm long and 3.13 ± 0.35 μm wide, with an area of 13.01 ± 2.01 μm². Midpieces were 5.33 ±0.44 μm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 μm, and overall sperm length was 40.12 ± 2.44 μm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species.     

Production of ciguatoxin and maitotoxin by strains of Gambierdiscus australes,G. pacificus and G. polynesiensis (Dinophyceae) isolated from Rarotonga,Cook Islands

The isolation and culturing of toxic Gambierdiscus species is desirable for obtaining reliable sources of toxins responsible for Ciguatera Fish Poisoning (CFP) in order to provide reference materials and purified standards for chemical analyses and bioassays. Gambierdiscus polynesiensis CAWD212, G. pacificus CAWD213 and G. australes CAWD149 and 216 have been isolated from samples of sea water from the Cook Islands. G. polynesiensis produced ciguatoxins (CTX; 18.2 pg per cell) and G. australes CAWD149 produced maitotoxin (MTX; 8.3 pg per cell) and in both extracts putative MTX-3 was detected. G. pacificus also produced putative MTX-3. The toxicities of extracts of G. polynesiensis and G. pacificus were similar by i.p. injection (LD_50s of 1.0 mg/kg and 0.8 mg/kg respectively), but when administered by gavage, the MTX-3 analogue producing G. pacificus (LD₅₀ 251 mg/kg) was 30-times less toxic than the CTX-producing G. polynesiensis (LD₅₀ 7.9 mg/kg). Consumption of fish contaminated by CFP has caused illness and even death in the Cook Islands and throughout the Pacific region and is now considered a globally neglected disease. Large-scale cultures of these organisms are now available, and isolation of the toxins they produce is in progress.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.     

Isolations of α-glucosidase-producing thermophilic bacilli from hot springs of Turkey

From 42 different hot springs in six provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the α-glucosidase production capacities by using pNPG as a substrate. α-Glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced α-glucosidase at significant levels comparable with that of the reference strains tested; thus, five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g), were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes in α-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to α-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a, and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular α-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in α-glucosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable α-1,4-glucosidase-producing bacilli that have biotechnological potential with α-glucosidases located at different cell positions were obtained. The text was submitted by the authors in English.     

Molecular phylogeny of the spiny-surfaced species of the dinoflagellate Prorocentrum with the description of P. thermophilum sp. nov. and P. criophilum sp. nov. (Prorocentrales,Dinophyceae)

Spiny-surfaced species of Prorocentrum form harmful algal blooms, and its taxonomic identity is obscure due to the size and shape variability. Molecular phylogenies reveal two major clades: one for P. cordatum with sequences mainly retrieved as P. minimum, and the other for P. shikokuense with sequences also retrieved as P. dentatum and P. donghaiense. Several closely related clades still need to be characterized. Here, we provide nuclear SSU and LSU rRNA genes, and nuclear ITS region (ITS1-5.8S gene-ITS2) sequences of the strain CCMP3122 isolated from Florida (initially named P. donghaiense) and strains Prorocentrum sp. RCC6871–2 from the Ross Sea, Antarctica. We describe Prorocentrum thermophilum sp. nov. based on the strain CCMP3122, a species also distributed in the open waters of the Gulf of Mexico, New Zealand, and the Arabian Gulf; and Prorocentrum criophilum sp. nov. based on the strain RCC6872, which is distributed in the Antarctic Ocean and Arctic Sea. Prorocentrum thermophilum is roundish (~14 μm long, ~12 μm wide), with an inconspicuous anterior spine-like prolongation under light microscopy, valves with tiny, short knobs (5–7 per μm²), and several (<7) large trichocyst pores (~0.3 μm) in the right valve, as well as smaller pores (~0.15 μm). Prorocentrum criophilum is round in valve view (~11 μm long, 10 μm wide) and asymmetrically roundish in lateral view, the periflagellar area was not discernible under light microscopy, valves with very tiny, short knobs (6–10 per μm²), and at least 12 large pores in the right valve. Other potentially undescribed species of spiny-surfaced Prorocentrum are discussed.     

OBSERVATION OF SAND-DWELLING TOXIC DINOFLAGELLATES (DINOPHYCEAE) FROM WIDELY DIFFERING SITES, INCLUDING TWO NEW SPECIES

Dinoflagellate associations, including toxic and potentially toxic benthic species, were examined in sand from South Water Cay and Carrie Bow Cay, Belize. The inshore sand habitat in localized areas of warm shallow lagoonal waters supported blooms of toxic assemblages of dinoflagellates. In the sand, the dominant microalgae were dinoflagellates; cyanobacteria were a minor component and diatoms were absent. Ciliates and nematodes were present. Assemblages of microorganisms in colored sand were examined for 4 consecutive days after which a storm washed away the patch. The sand-dwelling dinoflagellate assemblage included 16 species where densities ranged from as low as 1.3% to 15% of total cell densities. The dominant species was Scrippsiella subsalsa, having 1.8 × 10⁵ to 2.6 × 10⁵ cells g^-1 sand. Toxic dinoflagellates identified in the sand were Gambierdiscus toxicus, Ostreopsis lenticularis, Prorocentrum lima, Prorocentrum mexicanum, and Amphidinium carteri. The potentially toxic Ostreopsis labens, Gambierdiscus belizeanussp. nov., and Coolia tropicalis sp. nov. were also identified. Toxic and potentially toxic species represented 36% to 60% of total microalgal cell assemblage. The morphology of a new sand-dwelling species, Gambierdiscus belizeanus sp. nov., was examined with the scanning electron microscope. The plate formula was P_o, 3′, 7″, 6c, s?, 5?, 1p, and 2″″.Dimensions of G. belizeanus cells were 53–67 pm long, 54–63 μm wide, and 92–98 μm in dorsoventral depth. Cells were deeply areolated, ellipsoid in apical view, and compressed anteroposteriorly. The cells of G. belizeanus were identified by the cell's long, narrow, pentagonal, posterior intercalary plate (1p) wedged between the wide postcingular plates 2″’and 4″; 1p occupied 20% of the width of the hypotheca. The plate formula for Coolia tropicalis sp. nov. was P_o, 3′, 7″, 7c, 8s?, 5″″, and 2″″, Cell size ranges were 23–40 μm long, 25–39 μm wide, and 35–65 μm in dorsoventral diameter. Cells were spherical, smooth, and covered with scattered round pores. The epitheca was smaller than the hypotheca. Precingular plates 1″ and 7″ were small and narrow, and the first apical plate 1″ and precingular plate 6″ were the largest plates on the epitheca. The apical pore was straight and 7 μm long, and was situated in the apical plate complex. Cells of C. tropicalis were distinguished from C. monotis by the wedge-shaped plate 1′, a four-sided 3’plate, and a short apical pore.     

Nivalenol and 15‐acetyldeoxynivalenol Chemotypes of Fusarium graminearum Clade Species are Prevalent on Maize throughout China

Fusarium graminearum clade species are among the main causative agents of Gibberella ear rot (GER) in maize and responsible for the various trichothecene mycotoxins accumulated in contaminated maize grains. In this study, a total of 620 isolates from diseased maize ears collected from 59 districts in 19 provinces throughout China, previously identified morphologically as Fusarium graminearum clade, was genetically characterized at the species level based on SCAR (Sequence Characterized Amplified Region) and for their potential capability of mycotoxin production using the genetic chemotyping assay. The results showed that 359 isolates were F. asiaticum (SCAR 5), which consisted of 97% nivalenol (NIV)‐chemotypes, 0.8% 3‐acetyldeoxynivalenol (3‐ADON)‐producing isolates and 2.2% 15‐acetyldeoxynivalenol (15‐ADON) producers, whereas the remaining 261 isolates were identified as F. graminearum sensu stricto (SCAR 1), all of which produced 15‐ADON mycotoxins. This high proportion of NIV producers present in F. asiaticum is different from the chemotype patterns in F. asiaticum populations isolated from wheat and barley, where DON and its acetylated chemotypes were the predominant mycotoxins. Moreover, the majority of NIV producers (59.1%) and all the 3‐ADON‐producing strains were derived from the warmer regions in southern China, whereas most of the 15‐ADON‐producing strains (78.4%) were isolated from the colder regions in northern China. Our study is the first report of NIV chemotypes of F. asiaticum and 15‐ADON chemotypes of F. graminearum sensu stricto that were associated with the GER of maize in China.     

Bacillus locisalis sp. nov., a new haloalkaliphilic species from hypersaline and alkaline lakes of China, Kenya and Tanzania

A polyphasic taxonomic study was performed on seven Bacillus-like bacteria isolated from three hypersaline and alkaline lakes located in China, Kenya and Tanzania. All strains were moderately halophilic and alkaliphilic, Gram positive, motile rods. The DNA G+C content from the seven isolates ranged from 42.2 to 43.4 mol% and their major fatty acid was anteiso-C_15:0. Strain CG1^T, selected as representative strain of the isolates, possesses meso-diaminopimelic acid in the cell wall peptidoglycan, MK-7 as the predominant menaquinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids. Comparative 16S rRNA gene sequence analysis indicated that the isolates belonged to the genus Bacillus. The seven isolates shared 97.7–99.9% 16S rRNA gene sequence similarity, and formed a branch that was distinct from the type strains of the recognized species of the genus Bacillus. They were most closely related to Bacillus agaradhaerens DSM 8721^T (92.6–93.8% 16S rRNA sequence similarity). DNA–DNA hybridization values between the seven isolates were 85–100%. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus locisalis sp. nov. is proposed. The type strain is CG1^T (CCM 7370^T = CECT 7152^T = CGMCC 1.6286^T = DSM 18085^T).     

Longidorus carniolensis sp. n. (Nematoda, Longidoridae) from vineyard soil in Slovenia

A new needle nematode, Longidorus carniolensissp. n., recovered from the soil around the roots of grapevine Vitis vinifera L. from Slovenia, is described and illustrated. Longidorus carniolensisis an amphimictic species, characterised by females with a moderately long (L=5.6-8.2 mm) and plump (a=51-72.4, ave. 66.3) body, assuming a spiral to C-shape when heat relaxed. Head region continuous, anteriorly almost flat, lip region 23-25 μm wide; guiding ring situated posteriorly (42-47 μm, 43-50 μm in males), odontostyle long (ave. 146.6 (136-157) μm); pharyngeal glands with normal location, their nuclei of approximately equal size; tail bluntly conoidal to almost hemispherical. Males abundant, spicules slender and long (122-145 μm), ventromedian supplements 13-17, irregularly spaced, preceded by an adanal pair. Four juvenile stages present, the first stage juvenile with bluntly conoidal tail. Codes for identifying the new species when using the key by Chen et al. (1997) are: A 56, B 4, C 4, D 1, E 4, F 35, G 1, H 1, I 2. The new species is morphologically the most similar to Longidorus poessneckensis Altherr, 1974, Longidorus macrosoma Hooper, 1961, Longidorus caespiticola Hooper, 1961, Longidorus helveticus Lamberti et al., 2001, Longidorus macroteromucronatus Altherr, 1974, Longidorus pius Barsi & Lamberti, 2001, Longidorus raskii Lamberti & Agostinelli, 1993, Longidorus kheirii Pedram et al. 2008, Longidorus silvae Roca, 1993, Longidorus iuglandis Roca et al., 1985, Longidorus vinearum Bravo & Roca, 1995 and Longidorus major Roca & d'Erico, 1987, but differs from these species either by the body and odontostyle length, position of guide ring, head region and tail shape or the shape of the first stage juvenile tail. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other speciesof the genus Longidorus with known sequences. Relationships of Longidorus carniolensissp. n. with other Longidorus species based on analysis of this DNA fragment and morphology are discussed.     

Trichothecenes produced by Stachybotrys atra from Eastern Europe.

A total of 17 isolates of Stachybotrys atra isolated from various parts of Hungary and Czechoslovakia were grown on rice, and the toxin production of each isolate was analyzed by high-performance liquid chromatography. Of the 17 isolates, 14 produced macrocyclic trichothecenes (satratoxins F, G, and H, roridin E, and verrucarin J) as well as trichoverrols A and B. Most isolates produced satratoxins G and H in higher quantities than the other trichothecenes. The yield (in milligrams) of trichothecenes produced by one isolate grown on 800 g of rice was as follows: roridin E, 12; satratoxin F, 10; satratoxin G, 75; satratoxin H, 100; trichoverrol A, 15; and trichoverrol B, 30.