Hypersaline waters in salterns - natural ecological niches for halophilic black yeasts

Hypersaline waters in salterns have so far been considered to be populated only with halophilic algae and bacteria and completely lacking halophilic fungi. In this paper we present population dynamics of polymorphic black yeasts, isolated from hypersaline waters (3-30% NaCl) of a saltern, in relation to different physicochemical parameters. Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, Aureobasidium pullulans and Cladosporium spp. were detected with the highest frequency just before the peak of halite (NaCl) concentration. Since H. werneckii, P. triangularis and T. salinum are not known outside saline environments, these results suggest that hypersaline water is their natural ecological niche.     

Phaeoannellomyces and the Phaeococcomycetaceae, new dematiaceous blastomycete taxa

Phaeoannellomyces McGinnis et Schell gen. nov., which is based upon P. elegans McGinnis et Schell sp. nov., is proposed for dematiaceous yeasts which produce percurrently proliferating conidiogenous cells. Cladosporium werneckii Horta is transferred to Phaeoannellomyces as P. werneckii (Horta) McGinnis et Schell comb. nov. because the most stable and distinctive synanamorph produced by this fungus consists of annellidic yeast cells. The Phaeococcomycetaceae McGinnis et Schell fam. nov. is proposed in the class Blastomycetes, division Fungi Imperfecti for the dematiaceous yeast genera Phaeoannellomyces and Phaeococcomyces de Hoog.     

Ergosterol biosynthesis in novel melanized fungi from hypersaline environments

Halotolerant and halophilic melanized fungi were recently described in hypersaline waters. A close study of the sterol composition of such fungi, namely Hortaea werneckii, Alternaria alternata, Cladosporium sphaerospermum, Cladosporium sp., and Aureobasidium pullulans revealed the dominance of ergosterol and the presence of 29 intermediates of its biosynthesis pathway. The presence or absence of intermediates from distinct synthesis routes gave insight into the operative synthetic pathways from 4,4,14-trimethylcholesta-8,24-dien-3 beta-ol (lanosterol) to ergosterol in melanized fungi and in Saccharomyces cerevisiae, a reference yeast cultured in parallel. In all studied melanized fungi, initial methylation at C-24 took place before C-14 and C-4 demethylation, involving a different reaction sequence from that observed in S. cerevisiae. Further transformation was observed to occur through various routes. In A. alternata, isomerization at C-7 takes place prior to desaturation at C-5 and C-22, and methylene reduction at C-24. In addition to these pathways in Cladosporium spp., H. werneckii, and A. pullulans, ergosterol may also be synthesized through reduction of the C-24 methylene group before desaturation at C-5 and C-22 or vice versa. Moreover, in all studied melanized fungi except A. alternata, ergosterol biosynthesis may also proceed through C-24 methylene reduction prior to C-4 demethylation. -- Méjanelle, L., J. F. Lòpez, N. Gunde-Cimerman, and J. O. Grimalt. Ergosterol biosynthesis in novel melanized fungi from hypersaline environments. J. Lipid Res. 2001. 42: 352--358.     

Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.     

Salt stress affects sterol biosynthesis in the halophilic black yeast Hortaea werneckii

Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia. Some of the adaptations of halophilic microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties. By modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes. We studied the regulation of sterol biosynthesis in H. werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols. We found some differences in the characteristics of HMG R and in its regulation by different environmental salinities in H. werneckii when compared to the mesophilic baker's yeast, Saccharomyces cerevisiae. Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts. From this perspective, H. werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on sterol biosynthesis in eukaryotes.     

Serological cross-reactivity between sporothrix schenckii and various unrelated fungi

The serological cross-reactivity ofSporothrix schenckii with various unrelated fungi was investigated by use of immunodiffusion tests. A rabbit antiS. schenckii serum was obtained, which reacted withCladosporium werneckii, C. carrionii, C. bantianum, Coccidioides immitis, Phialophora jeanselmei, P. gougerotii, P. dermatitidis, Fonsecaea pedrosoi, Aspergillus fumigatus, Histoplasma capsulatum andTrichophyton mentagrophytes, but not withSaccharomyces cerevisiae antigens. The serological determinants responsible for the cross-reactions were suggested to be D-galactosyl residues.     

Role of oxidative stress in the extremely salt-tolerant yeast Hortaea werneckii

Eukaryotic halotolerant microorganisms are important as model organisms to understand the general mechanisms of resistance to environmental salinity. The ability of the extremely halotolerant black yeast Hortaea werneckii to combat oxidative stress was addressed, using hydrogen peroxide to generate the reactive oxygen species. Increasing environmental salinity was found to have no effect on its high ability to degrade hydrogen peroxide but resulted in a decrease in viability in response to externally added hydrogen peroxide, suggesting that the latter property determines the upper limit of the salt tolerance of H. werneckii. A refinement of the model of adaptation of H. werneckii to high-salinity environments is proposed.     

The halophilic fungus Hortaea werneckii and the halotolerant fungus Aureobasidium pullulans maintain low intracellular cation concentrations in hypersaline environments

Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na+ even when grown at high NaCl concentrations and can be thus considered Na+ excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K+ or Na+ for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H. werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment.     

Aspects of the progesterone response in Hortaea werneckii: Steroid detoxification, protein induction and remodelling of the cell wall

Progesterone in sublethal concentrations temporarily inhibits growth of Hortaea werneckii. This study investigates some of the compensatory mechanisms which are activated in the presence of progesterone and are most probably contributing to escape from growth inhibition. These mechanisms lead on the one hand to progesterone biotransformation/detoxification but, on the other, are suggested to increase the resistance of H. werneckii to the steroid. Biotransformation can detoxify progesterone efficiently in the early logarithmic phase, with mostly inducible steroid transforming enzymes, while progesterone biotransformation/detoxification in the late logarithmic and stationary phases of growth is not very efficient. The relative contribution of constitutive steroid transforming enzymes to progesterone biotransformation is increased in these latter phases of growth. In the presence of progesterone, activation of the cell wall integrity pathway is suggested by the overexpression of Pck2 which was detected in the stationary as well as the logarithmic phase of growth of the yeast. Progesterone treated H. werneckii cells were found to be more resistant to cell lysis than mock treated cells, indicating for the first time changes in the yeast cell wall as a result of treatment with progesterone.     

Salt stress and plasma-membrane fluidity in selected extremophilic yeasts and yeast-like fungi

We have investigated changes in plasma-membrane fluidity in relation to NaCl concentrations in yeasts and yeast-like fungi that were isolated from either subglacial ice or hypersaline waters. In both of these natural environments, these organisms are exposed to low water activity, due to either high NaCl concentrations or low temperatures. Our data indicate that the fluidity of the plasma membrane can be used as an indicator of fitness for survival in extreme environments. Fungi that can survive in such extreme environments, such as Hortaea werneckii in the hypersaline waters of salterns, and Cryptococcus liquefaciens in subglacial environments, showed similar profiles of plasma-membrane fluidity in response to raised salinity. The same was seen for ubiquitous fungi, which are generally adapted for different types of stress, such as Aureobasidium pullulans and Rhodotorula mucilaginosa. Representatives of both of these groups modulated their plasma-membrane fluidity differently. When salinity exceeded their optimal range, the ubiquitous stress-tolerant species (A. pullulans, Rh. mucilaginosa) showed increased plasma-membrane fluidity, whereas in the dominant extremophiles (H. werneckii, Cr. liquefaciens), it decreased. On the other hand, the plasma membranes of the fungi with a narrow ecological amplitude (Arctic A. pullulans and Rhodosporium diobovatum) showed different responses.     

Cladosporium fulvum circumvents the second functional resistance gene homologue at the Cf-4 locus (Hcr9-4E ) by secretion of a stable avr4E isoform

Introgression of resistance trait Cf-4 from wild tomato species into tomato cultivar MoneyMaker (MM-Cf0) has resulted in the near-isogenic line MM-Cf4 that confers resistance to the fungal tomato pathogen Cladosporium fulvum. At the Cf-4 locus, five homologues of Cladosporium resistance gene Cf-9 (Hcr9s) are present. While Hcr9-4D represents the functional Cf-4 resistance gene matching Avr4, Hcr9-4E confers resistance towards C. fulvum by mediating recognition of the novel avirulence determinant Avr4E. Here, we report the isolation of the Avr4E gene, which encodes a cysteine-rich protein of 101 amino acids that is secreted by C. fulvum during colonization of the apoplastic space of tomato leaves. By complementation we show that Avr4E confers avirulence to strains of C. fulvum that are normally virulent on Hcr9-4E-transgenic plants, indicating that Avr4E is a genuine, race-specific avirulence determinant. Strains of C. fulvum evade Hcr9-4E-mediated resistance either by a deletion of the Avr4E gene or by production of a stable Avr4E mutant protein that carries two amino acid substitutions, Phe(82)Leu and Met(93)Thr. Moreover, we demonstrate by site-directed mutagenesis that the single amino acid substitution Phe(82)Leu in Avr4E is sufficient to evade Hcr9-4E-mediated resistance.     

Isolation,Antimicrobial Activity,and Metabolites of Fungus <Emphasis Type="Italic">Cladosporium</Emphasis> sp. Associated with Red Alga <Emphasis Type="Italic">Porphyra yezoensis</Emphasis>

Cladosporium sp. isolate N5 was isolated as a dominant fungus from the healthy conchocelis of Porphyra yezoensis. In the re-infection test, it did not cause any pathogenic symptoms in the alga. Twenty-one cultural conditions were chosen to test its antimicrobial activity in order to obtain the best condition for large-scale fermentation. Phenylacetic acid, p-hydroxyphenylethyl alcohol, and L-beta-phenyllactic acid were isolated from the crude extract as strong antimicrobial compounds and they are the first reported secondary metabolites for the genus Cladosporium. In addition, the Cladosporium sp. produced the reported Porphyra yezoensis growth regulators phenylacetic acid and p-hydroxyphenylacetic acid. No cytotoxicity was found in the brine shrimp lethality test, which indicated that the environmental-friendly Cladosporium sp. could be used as a potential biocontrol agent to protect the alga from pathogens.     

Osmoadaptation-dependent activity of microsomal HMG-CoA reductase in the extremely halotolerant black yeast Hortaea werneckii is regulated by ubiquitination

We have investigated regulation of HMG-CoA reductase (HMGR) in one of the most salt-tolerant fungi, Hortaea werneckii, under different salinities and at the level of protein degradation. Two different HwHMGR isoenzymes were identified, specific to mitochondria and endoplasmic reticulum: HwHmg1 and HwHmg2, respectively. The activity of microsomal HwHmg2 is highest under hypo-saline and extremely hyper-saline conditions, and down-regulated under optimal growth conditions. We show that this is due to intense ubiquitination and proteasomal degradation of HwHmg2. The activity of the truncated mitochondrial HwHmg1 is constant under different growth conditions, suggesting an osmoadaptation-directed fate for mevalonate utilization in H. werneckii.     

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.     

Heat resistance of Cladosporium isolated from laboratory animal facilities

Heat resistance tests for the saprophyte, Cladosporium, isolated from laboratory animal facilities were carried out. In testing the effects of moderate and high temperature conditions, C. sphaerospermum (C. s) and C. cladosporioides (C. c) were found to grow on media in temperatures less than 32 degrees C, but did not in temperature of 35 degrees C and over. The colony diameter of Cladosporium became smaller as temperature increased. The death time of C. s treated with moist heat was within 12 min at 48 degrees C and that of C. c was within 26 min at 43 degrees C. Both Cladosporium species could not survive for more than 1 min at 55 degrees C. On the other hand, Cladosporium treated with dry heat could not survive more than 69-12 min (C. s) and 39-9.5 min (C. c) at 70-100 degrees C. From these results, it can be seen that Cladosporium was definitely sensitive to heat treatment, and the authors assume that heat is a means of prevention in laboratory animal facilities.     

木霉(Trichoderma spp.)对三种引起大棚蔬菜病害病原菌的影响

通过木霉属(Trichoderma) 3菌株与双鸭山蔬菜大棚中的黄瓜枯萎病菌(FusariumoxysporumSchlecht.f.cucumerinum)、黄瓜果腐病菌(PhytophthoracapsiciLeonian)、菜豆叶枯病菌(Cladosporiumsp .)的对峙培养试验,结果表明:绿色木霉1(TrichodermaviridePers.exGray 1)可作为双鸭山蔬菜大棚中的黄瓜枯萎病、黄瓜果腐病、菜豆叶枯病3种病害的生物防治拮抗菌加以利用,该拮抗菌对菜豆叶枯病菌抑制效果最好;绿色木霉2 (Tricho dermaviride 2 )对黄瓜果腐病菌抑制效果最好;而哈茨木霉(TrichodermaharzianumRifai)对以上3种病原菌都有抑制效果,对菜豆叶枯病菌抑制效果最好。从试验结果还可看出,绿色木霉2对黄瓜枯萎病菌和菜豆叶枯病菌的生长有促进作用。