Polysaccharides and phenolic compounds as substrate for yeasts isolated from rotten wood and description of Cryptococcus fagi sp.nov.

Pieces of rotten wood collected in the forest were screened for the presence of yeasts. In spring time 3 tree species were sampled, followed by 9 species in summer. Yeast strains were identified by traditional methods. Identifications were confirmed by sequencing of ribosomal DNA in case of doubt. In total 14 yeast species of ascomycetous affiliation and 6 anamorphic basidiomycetous yeasts were isolated and identified. Most species were represented by only one strain, but Candida bertae by two and Trichosporon porosum by six strains, all from different wood samples. Three strains represented novel species, one of which is described as Cryptococcus fagi Middelhoven et Scorzetti. The type strain is CBS 9964 (JCM 13614). All strains were tested for growth on several polysaccharides as sole carbon source. Only some of these polymers supported growth of ascomycetous yeasts. Basidiomycetous yeasts assimilated soluble starch, pullulan, dextran, xylan, polygalacturonate, galactomannan and tannic acid or at least some of these. Cryptococcus podzolicus and T. porosum were the most active in this respect. None of the isolated strains grew on carboxymethyl cellulose, colloidal chitin, arabinogalactan and gum xanthan. Phenolic compounds were assimilated by several strains, belonging to the Trichosporonales and the Microbotryum and Stephanoascus/Blastobotrys clades, but not by members of the Tremellales (Cryptococcus musci excepted) and the Debaryomyces/Lodderomyces clade. Most of the ascomycetes assimilated n-hexadecane.     

Yeast species utilizing uric acid,adenine,n-alkylamines or diamines as sole source of carbon and energy

Yeast strains utilizing uric acid, adenine, monoamines or diamines as sole source of carbon and energy were isolated from several soil samples by the enrichment culture method. The most common species wasTrichosporon cutaneum. Strains ofCandida catenulata, C. famata, C. parapsilosis, C. rugosa, Cryptococcus laurentii, Stephanoascus ciferrii andTr. adeninovorans were also isolated. All strains utilizing uric acid as sole carbon source utilized some primaryn-alkyl-l-amines hydroxyamines or diamines as well. The ascomycetous yeast strains showing these characteristics all belonged to species known to assimilate hydrocarbons. Type strains of hydrocarbon-positive yeast species which were not found in the enrichment cultures generally assimilated putrescine, some type strains also butylamine or pentylamine, but none assimilated uric acid. Methanol-positive species were not isolated. Type strains of methanol-positive and of hydrocarbon-negative species did not assimilate uric acid, butylamine or putrescine. Assimilation of putrescine as sole source of carbon and energy may be a valuable diagnostic criterion in yeast taxonomy.     

Carbon assimilation and extracellular antigens of some yeast-like fungi

Many yeast-like fungi assimilated n-hexadecane, butylamine and putrescine as sole carbon sources. Methanol was not assimilated. This points to a physiological similarity to endomycetous, hydrocarbon-utilizing yeasts. Stephanoascus ciferrii assimilated uric acid, adenine and allantoin as sole source of carbon and nitrogen. All strains of Geotrichum candidum and many other yeast-like fungi assimilated acetoin and butan-2,3-diol. Assimilation tests for adenine, uric acid, allantoin, acetoin and butan-2,3-diol were found to be suitable for taxonomic purposes.Extracellular antigens immunologically related to those produced by Geotrichum candidum were detected in the cell-free culture liquids of several yeast-like fungi. The extracellular antigen excreted by Stephanoascus ciferrii was species-specific.     

Catabolism of benzene compounds by ascomycetous and basidiomycetous yeasts and yeastlike fungi

A literature review is given on growth of yeasts on benzene compounds and on the catabolic pathways involved. Additionally, a yeast collection was screened for assimilation of phenol and 3-hydroxybenzoic acid. Fifteen ascomycetous and thirteen basidiomycetous yeast species were selected and were tested for growth on 84 benzene compounds. It appeared that 63 of these compounds supported growth of one or more yeast species. The black yeastExophiala jeanselmei assimilated 54 of these compounds.The catechol branch of the 3-oxoadipate pathway and its hydroxyhydroquinone variant were involved in phenol and resorcinol catabolism of ascomycetes as well as of basidiomycetes. However, these two groups of yeasts showed characteristic differences in hydroxybenzoate catabolism. In the yeastlike fungusE. jeanselmei and in basidiomycetes of the generaCryptococcus, Leucosporidium andRhodotorula, the protocatechuate branch of the 3-oxoadipate pathway was induced by growth on 3- and 4-hydroxybenzoic acids. In threeTrichosporon species and in all ascomycetous yeasts tested, 4-hydroxybenzoic acid was catabolyzed via protocatechuate and hydroxyhydroquinone. These yeasts were unable to cleave protocatechuate. 3-Hydroxybenzoic and 3-hydroxycinnamic acids were catabolized in ascomycetous yeasts via the gentisate pathway, but in basidiomycetes via protocatechuate.Incomplete oxidation of phenol, some chlorophenols, cresols and xylenols was observed in cultures ofCandida parapsilosis growing on hydroquinone. Most compounds transformed by the growing culture were also converted by the phenol monooxygenase present in cell-free extracts of this yeast. They did not support growth.The relationship between the ability of ascomycetous yeasts to assimilate n-alkanes, amines and benzene compounds, and the presence of Coenzyme Q₉ is discussed.     

Les levures des Drosophiles de savane d'Afrique intertropicale (Savanes De Lamto,Côte d'ivoire)

Yeasts from crops of Drosophilidae (154 flies belonging to 22 species) of Lamto savannas (Ivory Coast) were studied. 50 yeast strains (belonging to 19 species) were isolated and studied. The most frequently isolated yeasts wereHanseniaspora andPichia membranaefaciens s.l.; these yeasts ferment and assimilate few of the carbon compounds. Some crops contained until 100 000 yeast cells. In 25% of the flies, these yeasts belonged to several different species.     

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.     

Utilization of low molecular weight aromatic compounds by heterobasidiomycetous yeasts: taxonomic implications.

The utilization of low molecular weight aromatic compounds implies the operation of complex metabolic pathways. In order to investigate the taxonomic relevance of this property among heterobasidiomycetous yeasts, both at the species level and at higher taxonomic ranks, the capacity to assimilate twenty such compounds was tested in a total of 332 strains representing approximately 200 species. The substrates most frequently utilized were protocatechuic, caffeic, and p-hydroxybenzoic acids, whereas cinnamic, sinapic, and syringic acids and guaiacol were never assimilated. The assimilation of the majority of the aromatic compounds investigated correlated with the utilization of protocatechuic acid. Among the Urediniomycetes, the members of the Sporidiales and those of the Naohidea-Rhodotorula minuta clade showed a good ability to utilize aromatic compounds, whereas the members of the Agaricostilbum-Kondoa group were more heterogeneous, in agreement with the four subclades known. Among the Tremellomycetidae, the members of the Cystofilobasidium and Tremella clades showed a reduced or null ability to utilize aromatic compounds. In contrast, the members of the Trichosporon clade were able to utilize phenol and similar substrates, and the representatives of the Filobasidium clade assimilated various aromatic compounds, including those requiring more complex catabolic routes. Assimilation tests using, as sole carbon and energy sources, low molecular weight aromatic compounds appear to be potentially useful in taxonomic studies of basidiomycetous yeasts. In those species in which a considerable number of strains was investigated, variable assimilation patterns were frequently observed. The possibility that such discrepant results indicate an incorrect species delimitation is discussed.     

Utilization of phenol by hydrocarbon assimilating yeasts

77 Ascomycetous, basidiomycetous as well as imperfect yeast strains of 46 different species and 20 genera were tested for growth with the substrates n-octane, n-hexadecane, and phenol. Of 59 yeast strains with ascomycetous cell wall structure 33 grew on hydrocarbons and 32 on phenol. No yeast strain out of 26 which are unable to use n-alkanes as a source of carbon and energy grew on phenol. In comparison with the latter 32 out of 33 n-hexadecane assimilating yeasts were also capable of using phenol. All n-octane utilizing yeasts of this group also assimilate phenol as a carbon source for growth.The correlation of the hydrocarbon assimilation with the phenol assimilation seems to be not so strong in the basidiomycetous yeasts. 7 out of 18 strains from this group grew on n-hexadecane and 13 on phenol.Furthermore, it could be shown that the use of hydrocarbons and phenol (as well as methanol) is strongly correlated with the coenzyme Q structure of the respective yeast strain.The results are discussed with respect to the particular chemical properties of the substrates used and the fact that coenzyme Q structure is considered to be an important marker of evolutionary relationships among yeasts.     

The yeast flora of some decaying mushrooms on trunks of living trees

Several ascomycetous and basidiomycetous yeasts were isolated from rotten mushrooms on the trunks of beech and tamarisk trees. One strain, identified as the novel species Cryptococcus allantoinivorans, assimilated allantoin as the sole carbon source. Phylogenetically it belongs to the C. laurentii complex, Papiliotrema bandonii being the closest relative. Some ascomycetous strains could not be distinguished from Pichia guillermondii, but deviated considerably in rDNA sequences. In addition to these species, both decaying mushrooms were inhabited by more common species, viz. Candida albicans, C. saitoana, Rhodotorula mucilaginosa, Trichosporon asahii, T. multisporum and T. porosum. The basidiomycetous yeasts, except R. mucilaginosa, assimilated some polysaccharides of plant origin.     

Yeasts in an industrial malting ecosystem

The malting ecosystem consists of two components: the germinating cereal grains and the complex microbial community. Yeasts and yeast-like fungi are an important part of this ecosystem, but the composition and the effects of this microbial group have been largely unknown. In this study we surveyed the development of yeasts and yeast-like fungi in four industrial scale malting processes. A total of 136 malting process samples were collected and examined for the presence of yeasts growing at 15, 25 and 37°C. More than 700 colonies were isolated and characterized. The isolates were discriminated by PCR-fingerprinting with microsatellite primer (M13). Yeasts representing different fingerprint types were identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene. Furthermore, identified yeasts were screened for the production of α-amylase, β-glucanase, cellulase and xylanase. A numerous and diverse yeast community consisting of both ascomycetous (25) and basidiomycetous (18) species was detected in the various stages of the malting process. The most frequently isolated ascomycetous yeasts belonged to the genera Candida, Clavispora, Galactomyces, Hanseniaspora, Issatchenkia, Pichia, Saccharomyces and Williopsis and the basidiomycetous yeasts to Bulleromyces, Filobasidium, Cryptococcus, Rhodotorula, Sporobolomyces and Trichosporon. In addition, two ascomycetous yeast-like fungi (black yeasts) belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Knowledge of the microbial diversity provides a basis for microflora management and understanding of the role of microbes in the cereal germination process.     

Yeasts in malting,with special emphasis on <Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> (synonym <Emphasis Type="Italic">Pichia anomala</Emphasis>)

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. The production of malt (malting) exploits the biochemical reactions of a natural process, grain germination. In addition to germinating grain, the malting process includes another metabolically active component: a diverse microbial community that includes various types of bacteria and fungi. Therefore, malting can be considered as a complex ecosystem involving two metabolically active groups. Yeasts and yeast-like fungi are an important part of this ecosystem, but previously the significance of yeasts in malting has been largely underestimated. Characterization and identification of yeasts in industrial processes revealed 25 ascomycetous yeasts belonging to 10 genera, and 18 basidiomycetous yeasts belonging to 7 genera. In addition, two ascomycetous yeast-like fungi belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Several ascomycetous yeast strains showed strong antagonistic activity against field and storage moulds, Wickerhamomyces anomalus (synonym Pichia anomala) being the most effective species. Malting studies revealed that W. anomalus VTT C-04565 effectively restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. In order to broaden the antimicrobial spectrum and to improve malt brewhouse performance, W. anomalus could be combined with other starter cultures such as Lactobacillus plantarum. Well-characterized microbial mixtures consisting of barley and malt-derived microbes open up several possibilities to improve malt properties and to ensure the safety of the malting process.     

Molecular phylogenetics and generic assessment in the tribe Morindeae (Rubiaceae–Rubioideae): How to circumscribe Morinda L. to be monophyletic?

Most of the species of the family Rubiaceae with flowers arranged in head inflorescences are currently classified in three distantly related tribes, Naucleeae (subfamily Cinchonoideae) and Morindeae and Schradereae (subfamily Rubioideae). Within Morindeae the type genus Morinda is traditionally and currently circumscribed based on its head inflorescences and syncarpous fruits (syncarps). These characters are also present in some members of its allied genera, raising doubts about the monophyly of Morinda. We perform Bayesian phylogenetic analyses using combined nrETS/nrITS/trnT-F data for 67 Morindeae taxa and five outgroups from the closely related tribes Mitchelleae and Gaertnereae to rigorously test the monophyly of Morinda as currently delimited and assess the phylogenetic value of head inflorescences and syncarps in Morinda and Morindeae and to evaluate generic relationships and limits in Morindeae. Our analyses demonstrate that head inflorescences and syncarps in Morinda and Morindeae are evolutionarily labile. Morinda is highly paraphyletic, unless the genera Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included. Morindeae comprises four well-supported and morphologically distinct major lineages: Appunia clade, Morinda clade (including Sarcopygme and the lectotype M. royoc), Coelospermum clade (containing Pogonolobus and Morinda reticulata), and Gynochthodes–Morinda clade. Four possible alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four major lineages of Morindeae as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in the tribe and the phylogenetic and taxonomic distinctness of its newly delimited genera.     

Degradation of benzene compounds by yeasts in acidic soils

Attemps were made to demonstrate the role of yeasts in the degradation of benzene compounds under natural soil conditions. Yeasts were isolated from acidic sandy soil supplied with benzene compounds. For this purpose the slant culture method was used. Growth on the benzene compounds took place on solid growth media at 10°C. Several yeast species were isolated: Leucosporidium scottii, Rhodotorula aurantiaca, Rhodotorula mucilaginosa, Trichosporon dulcitum, Trichosporon moniliiforme and Schizoblastosporion starkeyi-henricii. Cryptococcus humicolus and Cryptococcus laurentii were isolated from liquid enrichment cultures. All these strains assimilated several benzene compounds in pure culture.Cresol removal from contaminated soil was speeded up by inoculation with Rhodotorula aurantiaca G36. It was demonstrated that this yeast utilized this compound in competition with the soil microflora.     

Trichosporon insectorum sp. nov., a new anamorphic basidiomycetous killer yeast

Three killer yeasts, isolated from the gut of insects in Panama and artisanal cheese in Brazil, were shown to be related to the Ovoides clade of the genus Trichosporon. Sequencing of the D1/D2 region of the LSU rDNA and physiological characterization revealed a distinct taxonomic position in relation to known species of the genus. Conspecificity of the three killer isolates was reinforced by similar M13 fingerprinting and killer profiles. We propose a new species in this genus: Trichosporon insectorum. The type strain is CBS 10422^T (syn. NRRL Y-48120). This anamorphic species produces arthroconidia but not appressoria, and its killer character seems to be associated with dsRNA.     

Three new species of Candida from apple cider: C. anglica, C. cidri and C. pomicola

Three new anamorphic ascomycetous yeasts are described: Candida anglica (type strain NRRL Y-27079, CBS 4262), Candida cidri (type strain NRRL Y-27078, CBS 4241), and Candida pomicola (type strain NRRL Y-27083, CBS 4242). These three species were isolated from cider produced in the United Kingdom, and their identification was determined from unique nucleotide sequences in the species-specific D1/D2 domain of large subunit (26S) ribosomal DNA. Phylogenetic analysis of D1/D2 sequences placed C. anglica near Candida fragi, C. cidri near Pichia capsulata, and C. pomicola in the Pichia holstii clade.     

Nitrate assimilation in Candida nitratophila and other yeasts

Nitrate assimilation has been studied in four species of yeasts; Candida nitratophila, Candida utilis, Hansenula anomala and Rhodotorula glutinis. Ammonium-grown cultures of these organisms did not assimilate nitrate but acquired the capacity to do so after a 3 h period of nitrogenstarvation. Ammonium inhibited nitrate assimilation completely in nitrate-grown cultures of R. glutinis. With Candida spp. ammonium and nitrate were assimilated simultaneously but each was assimilated at a lower rate than when either was supplied alone. Nitrogen-starved cultures of C. nitratophila contained enough nitrate reductase activity to sustain high rates of nitrate assimilation. Results indicate that the high levels of nitrate reductase in nitrate-grown cultures of C. nitratophila do not limit nitrate assimilation. Nitrate assimilation appears to be limited by nitrate uptake and/or the supply of reducing equivalents for nitrate reduction in these cultures.     

Identity and biodegradative abilities of yeasts isolated from plants growing in an arid climate

Plants harvested in the Canary Islands Lanzarote and Fuerteventura were analyzed for the yeasts inhabiting their surface. Half of the isolates (22 out of 44) were identified as Debaryomyces hansenii. Black ascomycetes, viz. Hortaea werneckii and two Hormonema species were represented by 7 strains. Basidiomycetous yeasts, viz. Cryptococcus sp. (8 strains), Rhodotorula sp. (5 strains), Cerinosterus cyanescens (1 strain) and Pseudozyma sp. (1 strain) constituted a minority of 33%. Thirty strains were screened for their ability to assimilate various plant constituents including lipids of the cuticle and the cell membrane, hemicelluloses, nitrogenous compounds (protein, nucleic acids, amino acids) and benzene compounds. All strains were able to assimilate or to hydrolyze lipids, lecithin included. Many strains of D. hansenii, H. dematioides, H. werneckii, C. cyanescens, Cr. laurentii, Pseudozyma sp. and Rh. glutinis were proteolytic. Hemicelluloses like xylan and pectin were assimilated by black ascomycetous yeasts, Cryptococcus sp., Pseudozyma sp. and Rh. glutinis. Ferulic and hydroxycinnamic acids, gallic and tannic acids were assimilated by some strains of H. dematioides, C. cyanescens, Pseudozyma sp. and Rhodotorula sp.     

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twentyone of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.In memoriam of Professor R. Y. Stanier