Karyology and hyphal characters as taxonomic criteria in Ascomycetous black yeasts and related fungi

Mycelial development of seventy-three strains of black yeasts and related fungi were studied, and numbers of nuclei per hyphal cell were counted. Two main patterns were apparent in expanding hyphae, viz. (1) uninucleate expanding hyphal cells, septum formation strictly following mitosis, and (2) multinucleate, branched, aseptate hyphal tips, septa being formed in a later stage, leading to oligo- or uninucleate mature cells. Characteristic genera in the two groups areExophiala andAureobasidium, respectively. InZasmidium and in someRamichloridium species all mycelial cells are oligonucleate. The character is indicative for relationships at the family level in black yeasts.     

Combined effects of endo- and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts

Effects of endo- and exogenous trehalose on viability of two antagonistic yeasts, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) Harrison, were investigated after being treated with rapid-freezing, slow-freezing and freeze-drying, respectively. The accumulation of intracellular trehalose in the two yeasts was induced by culturing the yeast cells in trehalose-containing medium, which significantly enhanced viabilities of both yeasts in the slow-freezing test. Trehalose, as an exogenous protectant, at the concentration of 5% or 10% could markedly increase survivals of the two yeasts when subjected to freeze-drying. When combined with exogenous trehalose as a protective substance, the yeasts containing high intracellular trehalose level showed higher viabilities as compared to those containing low levels under both freezing and freeze-drying stresses. The highest survival of C. laurentii and R. glutinis were 90% and 97% after freeze-drying, respectively, compared to 63% and 28% for the yeasts with lower intracellular trehalose levels. These results may be due to the fact that a combined effect occurred between endo- and exogenous trehalose of yeast cells. The combined effect on C. laurentii and R. glutinis also resulted in the highest level of biocontrol efficacy against blue mold in apple fruit caused by Penicillium expansum Link, and reduced the disease indexes to 45 and 56, respectively, compared to 94 and 81 in the untreated control. Meanwhile, the combination of endo- and exogenous trehalose significantly increased population of both yeasts in apple wounds, especially at the first 48 h after inoculation, which might explain the reason of the improvement in biocontrol effects of the two yeasts.     

Production of volatile organic compounds (VOCs) by yeasts isolated from the ascocarps of black (<Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt.) and white (<Emphasis Type="Italic">Tuber magnatum</Emphasis> Pico) truffles

Twenty-nine yeast strains were isolated from the ascocarps of black and white truffles (Tuber melanosporum Vitt. and Tuber magnatum Pico, respectively), and identified using a polyphasic approach. According to the conventional taxonomic methods, MSP-PCR fingerprinting and sequencing of the D1/D2 domain of 26S rDNA, the strains were identified as Candida saitoana, Debaryomyces hansenii, Cryptococcus sp., Rhodotorula mucilaginosa, and Trichosporon moniliiforme. All isolates assimilated l-methionine as a sole nitrogen source and produced the volatile organic compounds (VOCs), 2-methyl butanol, 3-methyl butanol, methanethiol, S-methyl thioacetate, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dihydro-2-methyl-3(2H)-thiophenone and 3-(methylthio)-1-propanol (MTP). ANOVA analysis of data showed significant (P<0.01) differences in VOCs produced by different yeasts, with MTP as the major component (produced at concentrations ranging from 19.8 to 225.6 mg/l). In addition, since some molecules produced by the isolates of this study are also characteristic of truffle complex aroma, it is possible to hypothesize a complementary role of yeasts associated with this ecosystem in contributing to final Tuber spp. aroma through the independent synthesis of yeast-specific volatile constituents.     

Capnobotryella renispora on roof tile

Capnobotryella renispora J. Sugiyama is described from a specimen on roof tile and in culture. Its relationship with the black yeasts is discussed.     

Yeast communities of the cactusPilosocereus arrabidae and associated insects in the Sandy Coastal Plains of Southeastern Brazil

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the mothSigelgaita sp. in the cactusPilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactusPilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent speciesPichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp.,Myxozyma mucilagina andSporopachydermia sp. A, representing about 80% of the total isolates.Pichia sp. A and aCandida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genusKloeckera, basidiomicetous anamorphs of the genusCryptococcus, the black yeastAureobasidium pullulans, Pichia sp. A, aCandida domercqii-like species, and some cactophilic yeasts, especiallyClavispora opuntiae. The feeding site ofSigelgaita sp. larvae hadClavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.     

Improvement by soil yeasts of arbuscular mycorrhizal symbiosis of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) colonized by<Emphasis Type="Italic"> Glomus mosseae</Emphasis>

The effects of the soil yeasts Rhodotorula mucilaginosa, Cryptococcus laurentii and Saccharomyces kunashirensis on the arbuscular mycorrhizal (AM) fungus Glomus mosseae (BEG 12) was studied in vitro and in greenhouse trials. The presence of yeasts or their soluble and volatile exudates stimulated the percentage spore germination and hyphal growth of G. mosseae. Percentage root length colonized by G. mosseae and plant dry matter of soybean (Glycine max L. Merill) were increased only when the soil yeasts were inoculated prior to the AM fungus. Higher beneficial effects on AM colonization and plant dry matter were found when the soil yeasts were inoculated as an aqueous solution rather than as a thin agar slice. Although soluble and volatile exudates of yeasts benefited the AM symbiosis, their modes of action were different.This revised version was published online in May 2004 with corrections to the section of the article.     

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (¹³C, ¹⁵N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of ¹³C and ¹⁵N sources, and this medium can be reused several times with supplementation of the ¹³C source only.     

刺梨自然发酵过程中非酿酒酵母多样性分析

【目的】分析刺梨果实自然发酵过程中非酿酒酵母菌群特征,为筛选优质刺梨非酿酒酵母提供参考。【方法】基于Illumina MiSeq高通量测序技术和WL营养琼脂鉴定培养基纯种分离技术,分析刺梨果实自然发酵1 d (F1)、3 d (F3)、5 d (F5)和15 d (F15) 4个阶段及YPD培养基富集培养样本中非酿酒酵母种群组成和多样性。【结果】高通量测序分析结果共获得182个OTUs (operational taxonomic units,OTUs),归属于81个属107个种;物种多样性分析结果表明,刺梨果实自然发酵前期,优势非酿酒酵母为汉逊酵母(Hanseniasporasp.)和伯顿丝孢毕赤酵母(Hyphopichiaburtonii),二者在样本F1中分别占42.59%和26.85%;随着自然发酵的不断进行,二者的比例逐渐降低,在第15天(F15),Hanseniaspora sp.和H. burtonii比例降低至7.73%和0.52%。相反,Pichia sporocuriosa和未培养的酵母,随着自然发酵不断进行所占比例逐渐增大,分别由F1中的0.23%和0.33%增至F15中的37.26%和32.62%。此外,采用WL营养琼脂鉴定培养基纯种分离和鉴定技术,从刺梨上分离到Hanseniasporasp.、H.burtonii、克鲁维毕赤酵母(Pichia kluyveri)、P. sporocuriosa和异常威克汉姆酵母(Wickerhamomyces anomalus) 5种类型的可培养非酿酒酵母。【结论】刺梨果实上存在着丰富的非酿酒酵母菌资源,研究刺梨自然发酵过程中非酿酒酵母多样性,为酵母资源开发和利用奠定基础。     

Growth and macromolecular content of the dimorphic fungus Aureobasidium pullulans and the effect of hydroxyurea and other inhibitors

The growth kinetics and the macromolecular content of the yeast and ethanol-induced hyphal forms of Aureobasidium pullulans were studied. During the morphological transition from yeasts to hyphae, both the protein and RNA content decreased significantly, the mycelial form containing only 76% of the amount of protein in the yeasts, and 38% of the RNA. The DNA was the only component tested whose level increased during the transition. Among several compounds inhibiting macromolecular synthesis, only hydroxyurea showed a remarkable effect on the morphology of A. pullulans, inducing the mycelial morphology. The macromolecular composition of hydroxyurea-treated cultures changed with time in a way similar to that of the ethanol-Tween 80-ammonia medium, and to that of carbon-starved cultures, without ethanol or glucose.     

Coenzyme Q systems in ascomycetous black yeasts

72 Strains belonging to 44 species of ascomycetous black yeasts were analyzed for their coenzyme Q systems. Prevalent were Q-10 and dihydrogenated Q-10 systems. Members of the Dothidealean suborder Dothideineae have Q-10 (H₂), while those belonging to the suborder Pseudosphaeriineae mostly have Q-10. The anamorph genus Exophiala Carmichael and the teleomorph genus Capronia Sacc. seem to be heterogenous.     

Occurrence and Population Size of <Emphasis Type="Italic">Malassezia</Emphasis> spp. in the External Ear Canal of Dogs and Cats Both Healthy and with Otitis

Malassezia yeasts are commensal organisms of human and animal skin that occasionally act as pathogens; lipid-dependent species are associated with human skin disorders. The aim of this work was to evaluate the occurrence, risk factors and population size of different species of Malassezia in the external ear canal of dogs and cats with and without otitis externa. Between 2001 and 2003, 107 healthy animals (25 cats and 82 dogs) and 123 animals with chronic otitis exteirna (48 cats and 75 dogs) were clinically examined. Sterile cotton swabs were used to collect specimens from the external ear canal and culture tests and cytological examinations were performed to detect the presence and population size of Malassezia yeasts. Malassezia yeasts were isolated from 72.9% and 40% of the cats and from 57.3% and 28.0% of the dogs, with and without otitis externa, respectively. Malassezia was frequently isolated from animals under 5 years of age. The highest prevalence of positive samples was reported in winter for cats and in autumn for dogs. Dogs with pendulous ears showed a higher incidence of infection than dogs with erect ears. More Malassezia yeasts were recovered from animals with otitis (i.e. 59.6 colony forming units – CFUs – for cats and 66.0 CFUs for dogs) than healthy animals. Out of the 413 isolates obtained from animals with and without otitis, 403 (97.6%) were identified as M. pachydermatis and 10 (2.4%) as M. globosa. A statistical evaluation of the occurrence of Malassezia yeasts in dogs and cats revealed that predisposing factors for Malassezia infections are sampling period for cats, and type of ear for dogs. The largest population of Malassezia yeasts was detected in animals with otitis, suggesting a role in the occurrence of lesions.     

短梗霉产liamocins研究进展

短梗霉真菌(Aureobasidium spp.)是一种世界性的酵母样真菌,因其产生黑色素而被称为黑酵母.短梗霉的许多菌株都能分泌细胞外脂质liamocins.Liamocins具有表面活性、良好的抗癌和抗菌活性.本文综述了分泌liamocins的短梗霉的多样性及影响其产生liamocins的因素,总结了liamoci...     

Methanol utilization by a strain of Aspergillus niger: influence on the synthesis and activity of pectinases

During the production of pectinases by a strain of Aspergillus niger isolated from rotten lemons, methanol was liberated into the medium due to the cleavage of the pectin molecule used as the carbon source. The methanol was subsequently consumed by the microorganism but neither the synthesis nor the activity of pectinesterase and polygalacturonase was affected. Although not studied in detail, the mechanism involved in the utilization of methanol is similar to that described for methylotrophic yeasts.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

The shelf life and effectiveness of granular formulations of Metschnikowia pulcherrima and Pichia guilliermondii yeast isolates that control postharvest decay of citrus fruit

Our overall objectives were to prepare commercially acceptable formulations of the postharvest biological control yeasts, Metschnikowia pulcherrima and Pichia guilliermondii, which have a long storage life and to determine the effectiveness of these formulations to control postharvest green and blue moulds on citrus fruit. Yeasts, grown on a cane molasses-based medium, were combined with talc or kaolin carriers and various adjuvants and the viability of yeast in 12 formulations was determined over a 6 month period. Formulation no. 11, containing talc, sodium alginate, sucrose, and yeast extract, for both yeasts had a significantly higher viable yeast cell content over a 6 month storage period. Among the formulations, three formulations (formulations no. 5, 6, and 11) were selected for additional in vivo testing because they had higher levels of viability amongst yeast cell populations during storage and were easier to resuspend remained in suspension more easily. These formulations were tested on Satsuma mandarin and grapefruit to control green and blue moulds. Formulations no. 5, 6, and 11 for both yeasts effectively controlled green mould, while only formulation no. 11 with either yeast isolate M. pulcherrima (isolate M1/1) or P. guilliermondii (isolate P1/3) effectively controlled both blue and green moulds.     

Experimental pulmonary candidiasis

The initial interaction of Candida albicans with pulmonary tissue of B6D2/F₁ mice was investigated. The LD₅₀ for mice challenged intravenously (IV) was approximately 3 × 10⁵ yeasts, whereas the LD₅₀ by the intratracheal (IT) route was in excess of 10⁸ yeasts. Mice challenged IV died of progressive yeast growth in the kidneys. In contrast, mice challenged IT rapidly eliminated the entire inoculum by the first day after challenge. Resident pulmonary alveolar macrophages (PAM) killed upwards of 70% of C. albicans in an in vitro killing assay. At effector: target ratios favoring the effector cell population resident PAM were able to restrict the formation of yeast germ tubes to only 30% of the yeasts, whereas at equivalent ratios virtually all of the intracellular yeasts produced germ tubes. Evaluation of the ability of PAM, harvested from genetically different strains of inbred mice, to kill C. albicans in vitro showed that killing ability was a property of resident PAM from mice with the black 6 background. It was discovered that during the initial stages of infection in vivo, the expression of the F4/80 surface molecule was down regulated, and the expression of the Mac 1 surface molecule upregulated. There were no quantitative changes in expression of either Mac 2, Mac 3, Ly 5 or the 5C6 surface epitopes. Taken together, the data show that pulmonary tissue is quantitatively very resistant to C. albicans infection, because of the ability of resident PAM to rapidly phagocytize and kill yeasts. Killing of C. albicans by resident PAM may be a property of a subset of this mononuclear phagocyte population and was accompanied by alterations in the expression of surface molecules.Presented as part of the Everett S. Beneke Symposium in Mycology, May 27, 1988.     

Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Three new species of anamorphic yeasts phenotypically and phylogenetically related toCandida mesenterica: The description ofCandida fungicola sp. nov.,Candida sagamina sp. nov., andCandida fukazawae sp. nov. isolated from fruit bodies of mushrooms

Four strains of anamorphic yeasts isolated from fruit bodies of mushrooms collected in Japan were found to represent three new species of the genusCandida. These species resembleCandida mesenterica in characteristics commonly employed in the classification of yeasts. On the basis of DNA-DNA reassociation, however, they were clearly distinguished fromC. mesenterica and from one another. Three new species,Candida fungicola, Candida sagamina, andCandida fukazawae, are proposed for these yeasts. The analysis of SSU rDNA sequences suggested that these three species were closely related to each other and toC. mesenterica andC. suecica.     

L-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation

l-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603^T and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to d-glucose and d-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from d-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated—arabitol from l-arabinose and xylitol from d-xylose. Different l-arabinose concentrations and oxygen conditions were tested to better understand l-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from l-arabinose (and also xylitol from d-xylose) than C. arabinofermentans PYCC 5603^T. In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex l-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in l-arabinose-assimilating yeasts.     

Biodiversity of yeasts isolated from the indigenous forest of Argan <Emphasis Type="Italic">(Argania spinosa</Emphasis> (L.) Skeels) in Morocco

In this study we have isolated and characterized yeasts from the soil, leaves and fruits of the indigenous Moroccan Argan tree (Argania spinosa) in two locations: the coastal city of Essaouira and a drier, more stressed environment in Taroudant city. Factorial and classification analyses of the metabolic profiles showed that the yeasts from the soil and those from the fruit seemed to form distinctive groups while those from the leaves were common to the two groups. Associating the profiles with yeast species, the soil isolates seemed to be dominated by profiles associated with basidiomycetous yeasts (Bullera variabilis, association to Filobasidium capsuligenum, and Rhodotorula glutinis) while those of the fruits were associated with ascomycetous yeasts (Pichia angusta and Zygoascus hellenicus). Most profile groups were shared between the leaves and one of the other biotopes owing to the semi-deciduous character of the Argan leaves that dominate in the rhizospheric soil and to the fibrous and low flesh fruits of Argan. Although most metabolic profile groups were represented in both sampling locations, certain groups were encountered only in Taroudant samples among which a group of four yeasts that grew at 44 °C. The Taroudant samples also presented the two most osmo-tolerant yeasts capable of growing at 15% NaCl and 125% sucrose. Some of the yeast strains showed very promising activities of polygalacturonase (0.40 units/g protein) without any pectinesterase activity while others strongly inhibited the gray rot mould Botrytis cinerea, and could be good candidates for the post-harvest control of this mould on fruits.