Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

DNA homologies of 14 strains of Chlorella protothecoides were determined. All strains are related by a high degree of DNA similarity (96–102% D) with the exception of strain 211-11 a which proved to belong to C. kessleri. There is, however, no detectable DNA homology with strains of the genus Prototheca which is supposed to have evolved from C. protothecoides by loss of photosynthetic pigments. Even within Prototheca the low degree of DNA similarity indicates a heterogeneity similar to that observed in the genus Chlorella.     

Physiological and biochemical contributions to the taxonomy of the genus Prototheca

16 strains of the genus Prototheca do not produce extracellular amylolytic enzymes. The base composition of their DNA shows rather continuous values from 62% to 78% GC (guanine + cytosine). Their assignment to four species and their possible relationship with Chlorella protothecoides are discussed.Abbreviations Used GC guanine + cytosine - SSC saline sodium citrate     

Simple new test for presumptive differentiation between genus Candida and genus Prototheca

A differential test was made between genus Candida and genus Prototheca using a new and very simple differential test.A total of 59 strains of Candida and 78 strains of Prototheca were used. The basis of the test was the differential use of a disc carrying 60 mcg of Rybostamicin to which all the Candida were resistant and the Prototheca inhibited.     

Identification of a unicellular,non-pigmented alga that mediates growth inhibition in anuran tadpoles: a new species of the genus Prototheca (Chlorophyceae: Chlorococcales)

A non-pigmented, unicellular alga isolated from the faeces of British anuran tadpoles and which is associated with growth inhibition in these tadpoles, was described and identified using cytological, ultrastructural, nutrient assimilation and immunological studies. The alga possessed all the distinctive morphological features of the genus Prototheca, it grew weakly on Prototheca Isolation Medium (PIM), it required thiamine for continued growth and replication, and it could assimilate the five major substrates used to speciate the protothecans. All of these characteristics, together with previous nucleic acid hybridisation studies, indicated that the microorganism belonged to the genus Prototheca. There are currently five species recognised as valid (Pore, 1985 & 1986): Prototheca zopfii Kruger, 1884, P. wickerhamii Tubaki & Soneda, 1959, P. moriformis Kruger, 1884, P. stagnora Cooke, 1968 and P. ulmea Pore, 1986.The immunology showed that the new species was related to two of the protothecans, but overall it showed that the alga was antigenically distinct from the other protothecans tested in the immunoassay. This, together with its inability to grow strongly on PIM, its ability to assimilate a wide rage of carbon substrates and its ability to mediate growth inhibition in anuran tadpoles, indicated a new species of Prototheca. We therefore propose the name Prototheca richardsi sp. n.     

MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Pun?ochá?ová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.     

Symbiotic effectiveness of strains of Rhizobium leguminosarum biovar phaseoli isolated from soils of Rwanda

We have isolated 48 strains of Rhizobium leguminosarum biovar phaseoli from nodules of Phaseolus vulgaris L. cultivated on 32 different soils at 22 various locations in Rwanda, Central Africa. The symbiotic effectiveness of the strains was appraised in the greenhouse by measuring shoots dry matter and total plant nitrogen content after six weeks of growth. Of the strains tested 19%, 58% and 23% were rated very effective, effective and ineffective, respectively. A very significant correlation (r=0.96, P<0.01) was observed between shoots dry matter and total N content. By using the total nitrogen balance method, it was estimated that in the presence of a very effective strain, up to 86% of the N present in the shoots comes from N₂ fixation. No significant correlations were observed between the symbiotic effectiveness of the strains and the pH of the soils from which they originated, the tolerance of the strains to acidity or their ability to produce organic acids. The nine very effective strains selected were highly competitive against two ineffective strains with the two P. vulgaris cultivars Rubona-5 and Kiryumukwe.Contribution no 367, Station de recherches, Agriculture Canada.Contribution no 367, Station de recherches, Agriculture Canada.     

First large-scale isolation of Prototheca zopfii from milk produced by dairy herds in Italy

A total of 1045 milk samples collected from infected and non infected quarters of 269 cows were investigated. This study showed that 4.7% of samples possessed cells of Prototheca spp. (10⁶cells/ml). The presence of other pathogenic microorganisms was also monitored. Prototheca spp. isolates were classified on the basis of current taxonomic guidelines and identified as P. zopfii. Susceptibility tests carried out in vitro by using 25 antibiotic compounds revealed that the strains of P. zopfii. were susceptible only to nystatin and amphotericin B (58 and 33% of total strains, respectively). The present study represents the first large-scale investigation carried out in Italy on the isolation of this achlorophyllous yeast-like microalga in milk samples produced by dairy herds.     

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk. This revised version was published online in June 2006 with corrections to the Cover Date.     

Low oxygen is optimal for luciferase synthesis in some bacteria

The synthesis of the bioluminescent systems in many strains of two species of the genus Photobacterium which were isolated as symbionts is greater at low oxygen concentrations, where aerobic growth is blocked. In strains of two other species, one Photobacterium of symbiotic orgin, and one (genus Beneckea) whose luminous members are not known to be involved in symbiotic associations, a different response is observed. At low oxygen concentrations, where there is an inhibition of growth, there is also a similar decrease in the synthesis, of the luminescent system. These species-specific differences may indicate important ecological differences along with distinctive differences in the molecular control mechanisms involved in the synthesis of luciferase.     

Field and culture studies of species of Gelidium (Gelidiales,Rhodophyta) from their northern limit of distribution in Europe

Two species of Gelidium, provisionally referred to as G. pusillum and G. latifolium, are included in the Scandinavian flora and reach their northern limit of distribution on the Norwegian west coast. Small frond sizes due to adverse growth conditions, extreme phenotypic variability and lack of sexual reproduction make identification of specimens very difficult. Both species were isolated into unialgal culture and were compared with cultured strains referable to G. pusillum from Ireland and France. Temperature and salinity requirements and tolerance ranges were determined and discussed in view of distribution along the Norwegian coast. Little or no growth occurs below 9 °C, corresponding to a northern summer growth limit. On the Norwegian Skagerrak coast, low winter temperature rather than reduced salinity is the limiting factor.     

DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

Grazer-induced changes in the desmid Staurastrum

In aquatic environments, predator kairomones have been shown to affect morphology of prey species. Past work on the interaction between zooplankton and phytoplankton was based mainly on the Daphnia–Scenedesmus model. Algae of the genus Staurastrum can produce mucilage, causing cell clumping and settling out of the water column. These clumps are too large to be eaten by daphniids. Thus, we hypothesised that this may be a grazer defence. We investigated whether Daphnia magna induces the formation of mucus globules in Staurastrum, how this occurs, and if the formation of clumps inhibits growth in juvenile Daphnia. Eight strains of Staurastrum were used to check whether mucus extrusion is induced by the presence of Daphniaor possibly by a chemical excreted by Daphnia magna. None of the strains reacted to the presence of Daphnia water alone, animals had to be present to induce clumping. Mechanical action (gentle stirring) caused the same strains to clump. The ecological relevance of clumping was then investigated. The different Staurastrum strains were used as food in a growth experiment with ecologically relevant densities of neonates of Daphnia hyalina. These small daphniids did not cause the same clumping observed for Daphnia magna when present in experiments at high densities. We observed that juvenile daphniids grew less well on strains with larger cell size.     

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.     

Occurrence and antagonistic potential of Stenotrophomonas strains isolated from deep-sea invertebrates

Stenotrophomonas maltophilia is known to be of significance as opportunistic pathogen as well as a source of biocontrol and bioremediation activities. S. maltophilia strains have been isolated from rhizospheres, soil, clinical material, aquatic habitats, but little is known about Stenotrophomonas strains recovered from marine environments. During a survey of the biodiversity of Pseudomonas-like bacteria associated with deep-sea invertebrates six Stenotrophomonas strains were isolated from sponge, sea urchin, and ophiura specimens collected from differing Pacific areas, including the Philippine Sea, the Fiji Sea and the Bering Sea. 16S rRNA gene sequence analysis confirmed an assignment of marine isolates to the genus Stenotrophomonas as it placed four strains into the S. maltophilia CIP 60.77^T cluster and two related to the S. rhizophila DSM 14405^T. Together with a number of common characteristics typical of S. maltophilia and S. rhizophila marine isolates exhibited differences in pigmentation, a NaCl tolerance, a range of temperatures, which supported their growth, substrate utilization pattern, and antibiotics resistance. Strains displayed hemolytic and remarkable inhibitory activity against a number of fungal cultures and Gram-positive microorganisms, but very weak or none against Candida albicans. This is the first report on isolation, taxonomic characterization and antimicrobial activity of Stenotrophomonas strains isolated from deep-sea invertebrates.     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055–1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.     

Cibacron blue 3G-A inhibits cell separation of gram-positive bacteria

A triazine dye, Cibacron blue 3G-A (CB), is an inhibitor of cell separation of staphylococcal spp. therefore, we examined the effect of CB on growth of grampositive bacteria other than Staphylococcus. CB added to the medium of growing cultures of strains of genus Micrococcus, Streptococcus, Lactobacillus and Bacillus caused inhibition of cell separation. Moreover, in case of Bacillus and Lactobacillus, individual cells were elongated as filament. Strains of the genus Micrococcus were as sensitive to CB as genus Staphylococcus in which the minimum concentrations of CB needed for inhibition of cell separation ranged from 15 to 100 M. Other strains belong to genus Streptococcus, Bacillus and Lactobacillus were less sensitive; the minimum concentrations were 100 M–25 mM.     

Chemotaxonomic discrimination among the fungal genera Tolypocladium, beauveria and Paecilomyces

Fungi of the genera Beauveria and Paecilomyces were found to produce cyclotetradepsipeptides, beauverolides. Production of beauverolides was not detected at the genus Tolypocladium. Analysis of beauverolides therefore provides a very simple chemotaxonomic test which seems to be suitable for fast discrimination between the genera Beauveria vs Tolypocladium and complementing morphological examination. A GC-MS study of β-hydroxy acid distribution in the beauverolide hydrolyzates revealed that all strains prdouce γ-methyl-β-hydroxy acids only. Their occurrence thus cannot be used as a taxonomic marker of different species within the genera Beauveria and Paecilomyces.     

田菁根际促生菌的筛选及其促生耐盐效果

【目的】为探究盐生植物田菁及其根际功能微生物改良盐碱地的效果,本研究从黄河三角洲盐碱区田菁根际土壤中分离促生菌,并明确其耐盐促生效果。【方法】采用选择培养方法从田菁根际土壤中分离固氮菌、解磷菌以及解钾菌,并进行16S rRNA分子生物学鉴定。之后对菌株的耐盐及促生特性进行测定,筛选性状优良菌株进行玉米促生作用研究。【结果】共分离得到105株根际促生菌,其中N102兼具多种促生特性且耐盐性达15%。田菁种子发芽试验表明,N102可显著提高田菁发芽率(47%,P<0.05)、芽长(48.5%,P<0.05)和根长(60%,P<0.05);玉米盆栽试验结果表明,N102对盐胁迫下玉米的株高、根长、叶绿素含量、地上部干重以及根干重具有显著的促进作用。经系统发育分析,N102与Enterobacter soli ATCC BAA-2102 (NR117547)序列相似度为99.30%,鉴定属于Enterobacter属。【结论】菌株N102具有多种植物促生耐盐特性,具有开发成有效促进盐碱地作物生长的微生物肥的良好前景。     

The growth of Epidermophyton floccosum and E. stockdaleae at different temperatures

The ability of 17 strains of genus Epidermophyton (15 strains belonging to Epidermophyton floccosum, one to E. floccosum var. nigricans and one to E. stockdaleae) to grow at different temperatures (4 °C, 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C) was stated.The strains were inoculated on Sabouraud Dextrose Agar and regularly controled over a period of 14 days when the plates were incubated at 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C, and over a period of 70 days when the temperature was 4 °C. The optimal growth of E. floccosum was observed at 28 °C and 31 °C, and no signs of growth were recorded neither at 4 °C nor at 40 °C. The optimal development of E. stockdaleae was observed at 25 °C and 28 °C. This species grew from 4 °C to 31 °C.