Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences

The taxonomy of Aphanizomenon flos-aquae strain NH-5, a producer of cyanotoxins, was re-evaluated by comparison with six other Aphanizomenon strains using morphological characteristics and 16S rRNA gene sequences. Strain NH-5 was concluded to be improperly identified as Aph. flos-aquae based upon (1) lack of bundle formation in the trichomes, (2) location of akinetes next to heterocytes, (3) lower similarities (less than 97.5%) in the 16S rRNA gene sequences relative to Aph. flos-aquae strains, and (4) comparison within a phylogenetic tree constructed from 16S rRNA gene sequences. The Aphanizomenon strains investigated in this study are classified to four morphological groups as described by the classical taxonomy of Komárek & Kovácik (1989). This classification was supported from the phylogenetic results of 16S rRNA gene sequences. This study also discusses the generic boundaries between Aphanizomenon and Anabaena.     

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)1

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena–like and Aphanizomenon–like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.     

Trichome structure of fourAphanizomenon taxa (Cyanophyceae) from Czechoslovakia,with notes on the taxonomy and delimitation of the genus

The dimension and variation range of terminal and intercalary cells, heterocysts, and akinetes of fourAphanizomenon taxa occurring in Czechoslovakia were studied. Statistical and graphical procedures were used for evaluation. With regard toA. flos-aquae, the results support the distinction of two varieties (var.flos-aquae and var.klebahnii). In the samples determined asA. gracile two clearly distinguishable morphological types could be found; one of them is recognized as a new species:A. flexuosum. Its diacritical features are established and problems of the intrageneric taxonomy ofAphanizomenon and its demarcation from the genusAnabaena are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Taxonomic studies of planktic species of Anabaena based on morphological characteristics in cultured strains

Fifty (50) strains of planktic species of Anabaena (cyanobacteria), including collections from Japan and China and from different culture collections, were induced to form akinetes at low temperature (15 °C). Their morphologies were then observed and described. Fourty seven strains successfully formed akinetes and these were classified into 20 species comprising seven with straight trichomes and 13 with coiled trichomes. Three strains, which did not form akinetes, were separated into two taxonomic groups, but could not be identified to any described species. In addition, a key to the planktic species of Anabaena described in the study is presented.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

A hypothesis on the role of Aphanizomenon in translocating phosphorus

A mechanism for the upward translocation of phosphorus by flake-forming Aphanizomenon is hypothesized. Aphanizomenon germinates on oxic sediments and rises into the water column, then the flakes diurnally migrate. Observations from Spring Lake, Minnesota, U.S.A., indicate that Aphanizomenon translocates phosphorus to the summertime epilimnion. However, the observations cannot discriminate between which of two mechanisms related to Aphanizomenon flakes, upon germination or with subsequent diurnal migrations, is more important.     

Cyanobacterial blooms and water quality in Greek waterbodies

The cyanobacterial species composition of nine Greek waterbodies of different type and trophic status was examined during the warm period of the year (May–October). Cyanobacterial water blooms were observed in all waterbodies. Forty-six cyanobacterial taxa were identified, 11 of which are known to be toxic. Eighteen species are reported for the first time in these waterbodies, 8 of which are known to produce toxins. Toxin producing species were found in all of the waterbodies and were primarily dominant in bloom formations (e.g., Microcystis aeruginosa, Anabaena flos-aquae, Aphanizomenon flos-aquae and Cylindrospermopsis raciborskii). Cosmopolitan species (e.g., M. aeruginosa), pantropic (e.g., Anabaenopsis tanganyikae) and holarctic species (e.g., Anabaena flos-aquae) were encountered. Shallow, eutrophic waterbodies had blooms dominated by Microcystis species and were characterized by phytoplankton association M. Anabaena and Aphanizomenon species of association H were dominant in waterbodies with low dissolved inorganic nitrogen and thermal stratification in the summer. Total cyanobacterial biovolumes (CBV) ranged from 7 to 9,507 cm³ m⁻³ and were higher than Alert Level 2 and Guidance Level 2 (10 cm³ m⁻³; World Health Organization; WHO) in seven of the waterbodies. Chlorophyll a concentrations ranged from 6 to 90,000 mg m⁻³ and were higher than Alert Level 2 and Guidance Level 2 (50 mg m⁻³; WHO) in eight of the waterbodies. There is also an elevated risk of acute toxicosis (Guidance Level 3; WHO) in five waterbodies. Water of an undesirable quality, hazardous to humans and animals occurs in several Greek waterbodies.     

Trichome morphology provides phylogenetically informative characters for Tremandra, Platytheca and Tetratheca (former Tremandraceae)

Summary Trichomes of Tremandra R.Br. ex DC., Platytheca Steetz and Tetratheca Sm. (Elaeocarpaceae, former Tremandraceae), together with two outgroup species of Elaeocarpus L., are illustrated using scanning electron microscopy, and their distribution on various plant organs is documented. Various trichomes types were identified that relate taxa: simple hairs, stellate hairs, short glandular trichomes, long glandular trichomes, and three forms of tubercules. Both outgroup and ingroup taxa have simple hairs. Stellate hairs are confirmed as unique to Tremandra. Prominent and sculptured multicelled tubercules, some bearing a stout hair, are characteristic of Platytheca. Smaller multicelled tubercules occur in both Platytheca and Tetratheca, except for the Western Australian taxon Te. filiformis Benth. (possibly plesiomorphic). Unicellular tubercules (papilla) characterise two species of Tetratheca. Short glandular trichomes, usually found on the ovary, also occur in both of these genera but not in all species (possibly secondary losses), while long glandular trichomes, usually on stems and leaves, occur only in some groups of Tetratheca. Within Tetratheca, Western Australian taxa that have five-merous flowers fall into three ‘groups’: seven species (together with one from South Australia) that have short glandular trichomes but no long glandular trichomes; six species that have long glandular trichomes but no short glandular trichomes; and four species that have both trichome types. All other species of Tetratheca have four-merous flowers and form two ‘groups’: 12 eastern species (including one from South Australia) that have both short glandular trichomes and long glandular trichomes; 4 western species and six eastern species that lack short glandular trichomes. On the basis of these characters, a phylogenetic hypothesis for the three genera is presented.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Phytoplankton vertical distributions and composition in Baltic Sea cyanobacterial blooms

We studied the vertical structure of the phytoplankton community in two toxic cyanobacterial blooms in the offshore Baltic Sea. In 1994, vertically separated potentially toxic, diazotrophic and mixotrophic species (belonging to Cyanophyceae, Dinophyceae and Prymnesiophyceae) dominated. In 1997, picocyanobacteria, mainly in colonies, made up 40–50% of the total phytoplankton carbon biomass in the top 20 m both day and night. Colony-forming species of picocyanobacteria seem to be occasionally important and hitherto underestimated in the Baltic Sea.We found species-specific depth distribution patterns. Nodularia spumigena and Anabaena spp. were observed mainly above 10 m depth, while Aphanizomenon sp. was mostly found deeper, especially at night. Dinophysis norvegica was only abundant near the seasonal pycnocline and showed very limited diurnal migration. Other flagellates, including small Cryptophyceae and 10 identified Chrysochromulina species, occurred down to 40 m depth. Their vertical migration may help to retrieve nutrients from below the summer pycnocline.We conclude that considerable differences in dominating functional groups may occur between years/bloom stages, and that the vertical distribution pattern of many species is recurring at similar environmental conditions, suggesting species-specific niche-separation.