Population dynamics of potentially harmful algal blooms in Bizerte Lagoon,Tunisia

The population dynamics of potentially harmful phytoplankton in the semi-closed, coastal Bizerte Lagoon, Tunisia, in the south-western Mediterranean, were examined from November 2007 to February 2009 at six sampling stations, three situated in areas of mussel and oyster farming. The harmful species monitored included the potential producers of amnesic shellfish poisoning (Pseudo-nitzschia spp.), paralytic shellfish poisoning (Alexandrium spp.), diarrheic shellfish poisoning (Dinophysis spp. and Prorocentrum spp.), ichthyotoxins (Cochlodinium polykrikoides, Akashiwo sanguinea and Karenia mikimotoi), yessotoxins (Gonyaulax spinifera) and the discolouration of water (Neoceratium lineatum and Protoperidinium sp.). These were numerically dominated by potentially toxic species of the diatom genus Pseudo-nitzschia, which were present year-round at all stations. Prorocentrum spp., Dinophysis spp. and Neoceratium lineatum were the most abundant and recurrent harmful dinoflagellates, exhibiting their highest densities at the shellfish farming stations. Alexandrium spp. bloomed only on one occasion, reaching its highest densities at a shellfish farming station. Canonical correspondence analyses revealed significant relationships between the harmful phytoplankton species monitored and the environmental conditions. The widespread distribution of harmful phytoplankton in Bizerte Lagoon, with the permanent presence of Pseudo-nitzschia spp. and the high frequency of dinoflagellate blooms in the shellfish areas, suggests a potential risk of shellfish poisoning events in the region.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

海水来源的548株真菌的近海及远海分布模式及其抑菌活性分析

[目的]海洋真菌是新颖天然产物的理想来源.本研究旨在加深对可培养海洋真菌多样性的了解,以及挖掘具有应用潜力的海洋真菌.[方法]采用膜过滤法分离近海和远海海水样品中的真菌,通过菌株分离纯化、ITS基因序列测序,分析浅海带、半深海带和深海带海水样品中可培养真菌的多样性.在此基础上,采用固体平板法筛选具有抑菌活性的菌株.[结...     

Fungi in sediments of the sea of Japan and their biologically active metabolites

The most abundant marine fungi encountered in various regions of the Sea of Japan belong to the generaPenicillium, Aspergillus, Wardomyces, Trichoderma, Chrysosporium, andChaetomium. Facultative marine fungi of the generaScytalidium, Verticillium, andOidiodendron and obligate marine fungi of the genusDendryphiella are much less abundant. The composition of marine sediments and the anthropogenic load on them were found to influence the abundance and species diversity of fungi, as well as the occurrence of fungal strains producing hemolytically active substances. The biodiversity of mycobiota and the abundance of hemotoxin-producing fungi in marine sediments may be used to evaluate the anthropogenic load on marine biocenoses. Hemolytic compounds were produced by 57% of the fungi isolated from marine sediments. The hemolytic activity ofChaetomium spiculipilium was revealed in the fraction of the culture liquid containing extracellular fatty acids and pigments. The fatty acid composition of this marine fungus was determined.     

TOXIN PROFILE,PIGMENT COMPOSITION,AND LARGE SUBUNIT RDNA PHYLOGENETIC ANALYSIS OF AN ALEXANDRIUM MINUTUM (DINOPHYCEAE) STRAIN ISOLATED FROM THE FLEET LAGOON,UNITED KINGDOM1

Paralytic shellfish toxins, pigment composition, and large subunit (LSU) rDNA sequence were analyzed for a clonal culture of Alexandrium minutum Halim isolated in 2000 from the coastal Fleet Lagoon, Dorset, United Kingdom. The HPLC pigment analysis revealed the presence of chl a, peridinin, and diadinoxanthin as major pigments and chl c₁+c₂ and c₃, diatoxanthin, and β‐carotene as minor components. The toxins responsible for paralytic shellfish poisoning were analyzed by HPLC with postcolumn derivatization and fluorescence detection. The paralytic shellfish poisoning toxin profile of the Fleet Lagoon strain of A. minutum in exponential growth phase was dominated by gonyautoxin‐3 up to 54%, whereas gonyautoxin‐2 made up 10% and saxitoxin (STX) 36%. The average toxicity of the culture was 3.8 pg STX Eq·cell^?1, and total toxin content varied from 5.6 fmol·cell^?1 on day 1 to a maximum of 16.8 fmol·cell^?1 during the early stationary phase. Sequence analysis of the LSU rDNA revealed the strain to be closely related to several European strains of A. minutum and one isolated from Australian waters, although most of these do not produce STX. The shallow Fleet Lagoon may provide a favorable environment for A. minutum to bloom, and the presence of highly potent saxitoxins in this strain indicates potential for future shellfish contamination.     

Bioactivity in free-living and symbiotic cyanobacteria of the genus Nostoc

Fifty cyanobacterial strains from different habitats(symbioses, soil, fresh and marine waters) belongingto the genus Nostoc were cultured and tested forbioactivity. Thirty-seven strains were isolated in ourlaboratory, the remaining were supplied by officialculture collections. All the organisms were grownunder controlled laboratory conditions. The biomasseswere lyophilised and extracted with ethanol:water toobtain a hydrophilic extract and then withdichloromethane:isopropanol to obtain a lipophilicextract. Both crude extracts were tested forantifungal (against Penicillium expansum andRhizoctonia solani) and antibacterial activity(against Agrobacterium vitis, Escherichiacoli and Staphylococcus epidermidis), and fortoxicity against Artemia salina nauplii.Twenty-four strains showed activity against at leastone of the target organisms. Bioactivity was equallydistributed between lipophilic and hydrophilicextracts, and was mostly directed against fungi (15strains) and Artemia nauplii (12 strains);antibacterial activity was less frequent (8 strains).The presence of bioactivity was independent of thestrain origin.     

In vitro antagonism of bioluminescent fungi by Trichoderma harzianum

Two species of bioluminescent fungi, Panellus stypticus and Omphalotus olearius were placed in contact with three different strains of interfungal pathogenic Trichoderma harzianum. Subsequent light emission by the luminous fungi and advance of the interfungal pathogens were compared. Relative differences among the pathogens were reflected in their rate of mycelial advance, the total area over which they produced spores upon the host fungi, and decreases in host bioluminescence. After ten days differences in the total surface areas of spore production varied from 1 to 53 per cent. Differences in the reduction of bioluminescence of the same material ranged over 2 orders of magnitude. Final reduction in luminescence ranged over 6 orders of magnitude. A marked reduction in bioluminescence was observed to precede the advance of spore production. The greatest reduction in luminescence was correlated with the presence of T. harzianum hyphae. Two strains of T. harzianum, NRRL 1698 and ATCC 58674, were effective against both bioluminescent fungi within the study period while a third strain, NRRL 13019, was only effective against Omphalotus olearius.     

Isolation and Analysis of Bacteria with Antimicrobial Activities from the Marine Sponge Haliclona simulans Collected from Irish Waters

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.     

Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters

Cylindrospermopsis raciborskii is a toxic bloom forming cyanobacteria that is a common component of the phytoplankton assemblage in temperate freshwaters, as well as in temperate climates. This species is of major concern in public health, due to its known ability to produce toxins, including cylindrospermopsin and paralytic shellfish poisoning toxin (PSP).In this study, M13 PCR fingerprinting, ERIC PCR fingerprinting and amplification of the internal transcribed spacer (ITS) region were used to characterize nine cultured strains of C. raciborskii, sourced from several freshwater lakes and rivers in Portugal, and two other Australian. Strains belonging to other taxa including Microcystis aeruginosa, Aphanizomenon spp., Planktothrix agardhii and Oscillatoria neglecta were also analysed to evaluate the taxonomical potential of the fingerprinting methods.Data obtained from genomic fingerprinting were used to perform hierarchical cluster analysis and demonstrated ability to differentiate strains at intra-specific level. However, the high level of variability prevents their use as an identification tool. ITS amplification displayed intra-specific polymorphism both in number and length of the obtained amplicons, but revealed itself as a good method for strain clustering. The unsuccessful amplification of peptide synthetase (PS) and polyketide synthase (PKS) genes pointed to the inability of Portuguese C. raciborskii strains to produce cylindrospermopsin. HPLC analysis further confirmed this lack of toxicity, since negative results were obtained for cylindrospermopsin and PSP toxins.     

Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group

The geographic range and bloom frequency of the toxic dinoflagellate Alexandrium minutum and other members of the A. minutum group have been increasing over the past few decades. Some of these species are responsible for paralytic shellfish poisoning (PSP) outbreaks throughout the world. The origins of new toxic populations found in previously unaffected areas are typically not known due to a lack of reliable plankton records with sound species identifications and to the lack of a global genetic database. This paper provides the first comprehensive study of minutum-group morphology and phylogeny on a global scale, including 45 isolates from northern Europe, the Mediterranean, Asia, Australia and New Zealand.Neither the morphospecies Alexandrium lusitanicum nor A. angustitabulatum was recoverable morphologically, due to large variation within and among all minutum-group clonal strains in characters previously used to distinguish these species: the length:width of the anterior sulcal plate, shape of the 1′ plate, connection between the 1′ plate and the apical pore complex, and the presence of a ventral pore. DNA sequence data from the D1 to D2 region of the LSU rDNA also fail to recognize these species. Therefore, we recommend that all isolates previously designated as A. lusitanicum or A. angustitabulatum be redesignated as A. minutum. A. tamutum, A. insuetum, and A. andersonii are clearly different from A. minutum on the basis of both genetic and morphological data.A. minutum strains from Europe and Australia are closely related to one another, which may indicate an introduction from Europe to Australia given the long history of PSP in Europe and its recent occurrence in Australia. A minutum from New Zealand and Taiwan form a separate phylogenetic group. Most strains of A. minutum fit into one of these two groups, although there are a few outlying strains that merit further study and may represent new species. The results of this paper have greatly improved our ability to track the spread of A. minutum species and to understand the evolutionary relationships within the A. minutum group by correcting inaccurate taxonomy and providing a global genetic database.     

Genetic variability of the invasive cyanobacteria <Emphasis Type="Italic">Cylindrospermopsis raciborskii </Emphasis>from Bir M’cherga reservoir (Tunisia)

Cylindrospermopsis raciborskii is an invasive freshwater cyanobacteria of tropical origin, also found in temperate regions. Due to its known ability to produce potent toxins, such as cylindrospermopsin and the paralytic shellfish poisoning, this species is of major concern from a water quality perspective. This study presents a genetic characterization of four C. raciborskii strains isolated from the Bir M’cherga Tunisian reservoir. The toxicity assessment was investigated via molecular biology tools, which suggested that all the isolated strains were not producing cylindrospermopsin, saxitoxin, or microcystin. This result was further confirmed by HPLC and MALDI-TOF analyses. However, we report for the first time in C. raciborskii the presence of mcyA and mcyE, two segments of the microcystin synthetase mcy cluster. All the strains were identified taxonomically based on the 16S rRNA sequences, and their phylogenetic relationships were assessed using the rpoC1 region. Tunisian strains formed a distinct clade separated from the other African strains.     

Indicator bacteria in freshwater and marine molluscs

The freshwater mussel Anodonta cygnea and four marine shellfish (mussels, Mytilus edulis; cockles, Cerastoderma edule; clams, Mya arenaria; Scrobicularia plana) from a total of six sites were surveyed for Escherichia coli, Clostridium perfringens, faecal streptococci, 25 and 37 °C coliforms, 25 °C and 37 °C total viable numbers and fluorescent pseudomonads. The A. cygnea from an urban lake contained greater numbers of the faecal indicator bacteria than animals from a rural lake. There were also differences in the other bacterial counts and these were discussed with respect to bacterial parameter and animal characteristics. When freshwater mussels were transferred from the city site to the rural site for 24 h the load of faecal indicator bacteria was eliminated or significantly reduced. Other bacterial types took longer to become stabilised. Loss of indicator bacteria from Anodonta was also demonstrated using cleansing in the laboratory. Very high bacterial numbers were found in some marine molluscs notably Scrobicularia plana and most shellfish contained significant numbers of the three faecal indicator bacteria at every sampling occasion. The relationship between bacterial types was discussed and it was concluded that in both freshwater and marine animals the bacterial numbers were determined more by sampling site than by species of shellfish.     

Antimicrobial, antitumor and in vivo cytotoxicity of actinomycetes inhabiting marine shellfish

Sixty-three actinomycete strains isolated from the marine shellfish Donax trunculus anatinus were phenotypically identified as ten genera, in addition to two unidentified strains. Their metabolic extracts exhibited wide antimicrobial activities towards 11 reference and clinical cultures; and 17.5% showed antitumor activities with solid tumor selectivity of four Nocardioides, Kitasatosporia and Streptomyces strains. Streptomyces 23-2B was particularly noted for its high antitumor activity against Ehrlich’s ascites carcinoma with plateau inhibitory effect at 500, 250 and 50 μg/ml concentrations, promising solid tumor selectivity and high cytotoxicity to human carcinoma of liver (HEPG2), cervix (HELA) and breast (MCF7) (IC₅₀: 3.89, 9.4 and 10 μg/ml, respectively). In vivo cytotoxicity of S.23-2B metabolites showed common sign of unimpaired kidney and liver functions, as indicated from non-significant elevation in serum enzymatic activities, urea, creatinine, total protein and albumin levels in response to 0.5 and 5 μg/g doses after alternate-day injection for 2 weeks. Microorganisms associated with the marine shellfish are suggested to be potential source of bioactive metabolites.     

青檀叶片内生和附生真菌组成及生态分布

对我国古老特有植物青檀叶片进行内生和附生真菌的研究,以了解青檀叶片内生和附生真菌的组成特点和探讨内生和附生真菌菌群之间的可能联系,为研究真菌资源多样性、植物附生和内生真菌的相互演化关系及真菌与宿主植物协同进化等提供有益参考资料。研究结果表明,从健康的青檀叶片获得可培养内生真菌839株,附生真菌1857株,共计2696株,鉴定其分属于4目,5科,43属。在目的分类水平上,内生和附生真菌均以丛梗孢目Moniliales为优势菌群,分别占90.23%和92.51%;在科的水平上,内生真菌以暗梗孢科Dematiaceae和丛梗孢科Moniliaceae为优势菌群,分别占47.56%和42.67%,附生真菌以丛梗孢科Moniliaceae和暗梗孢科Dematiaceae为优势菌群,分别占67.04%和25.47%;在属的水平上,内生真菌以黑团孢属Periconia和青霉属Penicillium为优势菌群,分别占31.47%和10.73%,附生真菌以小球霉属Glomerularia、膝葡孢属Gonatobotrys和青霉属Penicillium为优势菌群,分别占20.03%、13.95%和12.22%。青檀叶片内生真菌和附生真菌均存在的菌群数量达到23个属,占53.49%。内生真菌特有的属有6个,共分离19株,占0.70%,附生真菌特有的属有14个,共分离120株,占4.45%。内生真菌的Shannon-Wiener index(H')多样性指数(2.44)和Margalef index(R)丰富度指数(2.88)分别小于附生真菌ShannonWiener index(H')多样性指数(2.57)和Margalef index(R)丰富度指数(3.32),但两者的Evenness index(E)均匀度指数几乎相等。青檀叶片内生和附生真菌菌群组成具有较高的相似性,相似性系数达0.70。通过Fisher's exact test分析表明青檀叶片内生和附生真菌菌群组成无明显差异(P=0.072)。     

Effects of the estuarine dinoflagellate Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species

A series of experiments was conducted to examine effects of four strains of the estuarine dinoflagellate, Pfiesteria shumwayae, on the behavior and survival of larval and adult shellfish (bay scallop, Argopecten irradians; eastern oyster, Crassostrea virginica; northern quahogs, Mercenaria mercenaria; green mussels, Perna viridis [adults only]). In separate trials with larvae of A. irradians, C. virginica, and M. mercenaria, an aggressive predatory response of three strains of algal- and fish-fed P. shumwayae was observed (exception, algal-fed strain 1024C). Larval mortality resulted primarily from damage inflicted by physical attack of the flagellated cells, and secondarily from Pfiesteria toxin, as demonstrated in larval C. virginica exposed to P. shumwayae with versus without direct physical contact. Survival of adult shellfish and grazing activity depended upon the species and the cell density, strain, and nutritional history of P. shumwayae. No mortality of the four shellfish species was noted after 24 h of exposure to algal- or fish-fed P. shumwayae (strains 1024C, 1048C, and CCMP2089) in separate trials at ≤5 × 10³ cells ml⁻¹, whereas higher densities of fish-fed, but not algal-fed, populations (>7–8 × 10³ cells ml⁻¹) induced low (≤15%) but significant mortality. Adults of all four shellfish species sustained >90% mortality when exposed to fish-fed strain 270A1 (8 × 10³ cells ml⁻¹). In contrast, adult M. mercenaria and P. viridis exposed to a similar density of fish-fed strain 2172C sustained <15% mortality, and there was no mortality of A. irradians and C. virginica exposed to that strain. In mouse bioassays with tissue homogenates (adductor muscle, mantle, and whole animals) of A. irradians and M. mercenaria that had been exposed to P. shumwayae (three strains, separate trials), mice experienced several minutes of disorientation followed by recovery. Mice injected with tissue extracts from control animals fed cryptomonads showed no response. Grazing rates of adult shellfish on P. shumwayae (mean cell length ±1 standard error [S.E.], 9 ± 1 μm) generally were significantly lower when fed fish-fed (toxic) populations than when fed populations that previously had been maintained on algal prey, and grazing rates were highest with the nontoxic cryptomonad, Storeatula major (cell length 7 ± 1 μm). Abundant cysts of P. shumwayae were found in fecal strands of all shellfish species tested, and ≤45% of the feces produced viable flagellated cells when placed into favorable culture conditions. These findings were supported by a field study wherein fecal strands collected from field-collected adult shellfish (C. virginica, M. mercenaria, and ribbed mussels, Geukensia demissa) were confirmed to contain cysts of P. shumwayae, and these cysts produced fish-killing flagellated populations in standardized fish bioassays. Thus, predatory feeding by flagellated cells of P. shumwayae can adversely affect survival of larval bivalve molluscs, and grazing can be depressed when adult shellfish are fed P. shumwayae. The data suggest that P. shumwayae could affect recruitment of larval shellfish in estuaries and aquaculture facilities; shellfish can be adversely affected via reduced filtration rates; and adult shellfish may be vectors of toxic P. shumwayae when shellfish are transported from one geographic location to another.     

两种栽培型广藿香内生真菌群落组成变化

为探索内生真菌与广藿香互作间对宿主活性成分形成机制的影响,该研究以成分差异较大的牌香和湛香为对象,采用传统形态学方法对所获菌株归类,通过真菌通用引物ITS1/ITS4扩增菌株rDNA-ITS序列,鉴定其分类地位并研究其多样性。结果表明：(1)用PDA和LBA培养基对苗期、分枝期和成株期广藿香茎叶组织块进行内生真菌分离,共获得3 070株菌株,其中牌香(PX)分离出1 624株,鉴定出1 319株,分属于36属;湛香(ZX)分离出1 446株,鉴定出994株,分属于33属。牌香分离出7种特有内生真菌,分别为香柱菌(Epichloe typhina)、盘长孢状刺盘孢菌(Colletotrichum gloeosporioides)、座腔孢菌(Botryosphaeria sp.)、丝核菌(Rhizoctonia sp.)及截盘多毛孢菌(Truncatella sp.),并首次分离到疫霉菌(Phytophthora sp.)和指疫霉菌(Sclerophthora sp.),这2种菌属于卵菌门内生菌。湛香分离出拟青霉菌(Paecilomyces sp.)和尾孢菌(Cercospora sp.)...     

块菌(松露)宿主可培养内生菌群落结构与多样性研究

探究四川凉山彝族自治州块菌主产区华山松内生菌群的结构及多样性。在会东县选取4个点(新田乡、新云乡、淌塘镇、雪山乡)的块菌宿主华山松的根、茎、叶为实验材料,通过不同培养基分离样品根、茎、叶的内生细菌、真菌、放线菌,DNA分子鉴定分离菌株的种属,最终分离得到细菌46株,其中欧文氏菌属(Erwinia)5株,沙门氏菌属(Salmonell)1株,杆菌属(Bacillus)22株,葡萄球菌属(Staphylococcus)2株,假单胞菌属(Pseudomonas)2株,类芽胞杆菌属(Paenibacillus)2株,布丘氏菌属(Buttiauxella)2株,肠杆菌属(Enterobacte)3株,爱文氏菌属(Ewingella)1株,泛菌属(Rahnella)1株,拉恩氏菌属(Rahnella Izard)2株,其他属3株;真菌19株,均为子囊菌门(Ascomycota),其中青霉属(Penicillium)4株,疱霉属(Phoma)1株,子囊菌门未知菌1株,篮状菌属(Cladosporium)1株,分枝孢子菌属(Sydowia)1株,曲霉属(Aspergillus)1株,其他属10株;放线菌33株,为链霉菌属(Streptomyces)22株,短小杆菌属(Curtobacterium)2株,短杆菌属(Brevibacterium)1株,其他属8株。研究结果表明,来自不同地点、不同植株部位、不同培养基分离得到的块菌宿主华山松内生菌有差异,证实微生物在土壤、块菌、宿主植物之间有着复杂的相互作用,为块菌的人工栽培提供参考。