Different growth and physiological responses to experimental warming of two dominant plant species <Emphasis Type="Italic">Elymus nutans</Emphasis> and <Emphasis Type="Italic">Potentilla anserina</Emphasis> in an alpine meadow of the eastern Tibetan Plateau

The effects of experimental warming on the growth and physiology of grass Elymus nutans and forb Potentilla anserina were studied by using open-top chambers (OTCs) in an alpine meadow of the eastern Tibetan Plateau. The warming treatment increased mean air and soil surface temperatures by 1.53°C and 0.50°C, respectively, but it reduced soil relative water content in the surface layer. Experimental warming enhanced the growth and gas exchange of E. nutans, while it reduced those of P. anserina. Experimental warming resulted in an increased efficiency of photosystem II (PSII) in E. nutans, while decreasing it in P. anserina; significantly stimulated non-photochemical quenching, antioxidative enzymes and non-enzymes in both species; and significantly reduced malondialdehyde content in E. nutans, while promoting it in P. anserina. The results of this study indicated that the two species showed different growth responses to experimental warming and their different physiological performances further indicated that experimental warming alleviated the negative effect of low temperature on the growth and development of E. nutans, but limited the competitive ability of P. anserina in the study region.     

Métabolisme de l’Acide 2-Aminoéthylphosphonique (Ciliatine) chez le Poulet

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.     

Cloning, Sequencing, and Transgenic Expression ofPodospora curvicollaandSordaria macrosporaeEF1A Genes: Relationship between Cytosolic Translation and Longevity in Filamentous Fungi

We have cloned and sequenced the gene encoding the translation elongation factor eEF1A from two filamentous fungi,Podospora curvicollaandSordaria macrospora.These fungi are close relatives ofPodospora anserinaand also show senescence syndromes. Comparison of the sequences of the deduced proteins with that ofP. anserinareveals that the three proteins differ in several positions. Replacement of theP. anserinagene by either of the two exogenous genes does not entail any modification inP. anserinaphysiology; the longevity of the fungus is not affected. No alteration ofin vivotranslational accuracy was detected; however, the exogenous proteins nonetheless promoted a modification of the resistance to the aminoglycoside antibiotic paromomycin. These data suggest that optimization of life span between these closely related fungi has likely not been performed during evolution through modifications of eEF1A activity, despite the fact that mutations in this factor can drastically affect longevity.     

The crucial role of the Pls1 tetraspanin during ascospore germination in Podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a ΔPaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the ΔPaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.     

Dedikaryotization of higher fungi in submerged culture

Shaker experiments were done with submerged propagation of the oyster pleurotus (Pleurotus ostreatus/Jacq. ex. Fr./Kummer). The starting material was dikaryotic or monokaryotic mycelium obtained under stationary conditions. During submerged cultivation in a wort-containing medium on a cyclic shaker at 240 r.p.m. in flasks with articulated surface, dedikaryotization took place and the culture was predominantly monokaryotic after 10–14 days. Agitation of the medium favours the formation of monokaryotic forms. The typical mushroom flavour is associated with the dikaryotic form of mycelium so that submerged cultivation does not produce higher fungal mycelium in its aromatic form.     

Perstraction of intracellular pigments by submerged cultivation of <Emphasis Type="Italic">Monascus</Emphasis> in nonionic surfactant micelle aqueous solution

“Milking processing” describes the cultivation of microalgae in a water-organic solvent two-phase system that consists of simultaneous fermentation and secretion of intracellular product. It is usually limited by the conflict between the biocompatibility of the organic solvent to the microorganisms and the ability of the organic solvent to secret intracellular product into its extracellular broth. In the present work, submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle aqueous solution for pigment production is exploited, in which the fungus Monascus remains actively growing. Permeabilization of intracellular pigments across the cell membrane and extraction of the pigments to the nonionic surfactant micelles of its fermentation broth occur simultaneously. “Milking” the intracellular pigments in the submerged cultivation of Monascus is a perstraction process. The perstractive fermentation of intracellular pigments has the advantage of submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizing the pigments into nonionic surfactant micelles. It is shown as the marked increase of the extracellular pigment concentration by the submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle solution.     

Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina

In the filamentous fungus Podospora anserina, many pigmentation mutations map to the median region of the complex locus ‘14’, called segment ‘29’. The data presented in this paper show that segment 29 corresponds to a gene encoding a polyketide synthase, designated PaPKS1, and identifies two mutations that completely or partially abolish the activity of the PaPKS1 polypeptide. We present evidence that the P. anserina green pigment is a (DHN)-melanin. Using the powerful genetic system of PaPKS1 cloning, we demonstrate that in P. anserina trans-duplicated sequences are subject to the RIP process as previously demonstrated for the cis-duplicated regions.     

Effect of polycyclic aromatic hydrocarbons on laccase production by white rot fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

The effect of polycyclic aromatic hydrocarbons (PAHs) on the time course of laccase production by the fungus Pleurotus ostreatus D1 under conditions of submerged cultivation on Kirk’s medium has been studied. It has been shown that phenanthrene, fluoranthene, pyrene, and chrysene actively induce this enzyme, whereas fluorene and anthracene had a smaller effect. Addition of Mn²⁺ ions to cultivation medium elevates the laccase activity twofold and more in the presence of all the studied PAHs. Electrophoresis under nondenaturing conditions demonstrates induction of additional laccase forms by xenobiotics. Ligninolytic peroxidase activities are undetectable under the conditions used.     

A mitochondrial respiratory mutant of Podospora anserina obtained by short-term submerged cultivation of senescent mycelium

A spontaneous long-lived isolate of Podospora anserina obtained by relatively short-term submerged cultivation of the wild-type senescent culture and conventionally termed ??immortal?? was shown to be a cox1 mutant. As a respiratory mutant, the isolate in question is characterized by dysfunction of the cytochrome respiratory chain, activation of alternative respiration leading to a low level of reactive oxygen species production, and the lack of accumulation of ??-senDNA, the specific factor of P. anserina senescence. Absence of visible vegetative incompatibility was shown in the fungal mutants carrying respiratory defects. It was discovered that the P. anserina female sex organs could be fertilized not only by microconidia but also by the fragments of vegetative mycelium. Partial nonobservance of monoparental female inheritance of mitochondria associated with fertilization by vegetative mycelium was also revealed.     

Effect of a mitochondria-targeted antioxidant on ageing of <Emphasis Type="Italic">Podospora anserina</Emphasis>: Ultrastructural study

A beneficial effect of mitochondria targeted antioxidant (MTA) SkQ1 added to the culture medium on life span of Podospora anserina was revealed. As was shown earlier, optimal concentration was 400 nM. SkQ1 was shown to increase P. anserina life span 2.4 times, at the same time maintaining native cell ultrastructure: degradative alterations were not revealed. Significant reorganization of P. anserina cell ultrastructure was detected. These changes were not described earlier. The greatest structural changes proceeded in mitochondria. Specific character of these alterations suggests an essential role of oxidative stress in senescence of P. anserina.     

WD-repeat instability and diversification of the <Emphasis Type="Italic">Podospora anserina hnwd</Emphasis> non-self recognition gene family

Background  

Genes involved in non-self recognition and host defence are typically capable of rapid diversification and exploit specialized genetic mechanism to that end. Fungi display a non-self recognition phenomenon termed heterokaryon incompatibility that operates when cells of unlike genotype fuse and leads to the cell death of the fusion cell. In the fungus Podospora anserina, three genes controlling this allorecognition process het-d, het-e and het-r are paralogs belonging to the same hnwd gene family. HNWD proteins are STAND proteins (signal transduction NTPase with multiple domains) that display a WD-repeat domain controlling recognition specificity. Based on genomic sequence analysis of different P. anserina isolates, it was established that repeat regions of all members of the gene family are extremely polymorphic and undergoing concerted evolution arguing for frequent recombination within and between family members.     

Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development

Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here we investigated the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation, and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperaturerelated role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures as they characteristically occur in nature.     

Polysaccharides from mycelium of the fungus <Emphasis Type="Italic">Cunninghamella japonica</Emphasis>

Preliminary data on the polysaccharide composition of mycelium of the fungus Cunninghamella japonica (synonymous with C. echinulata) grown by the method of submerged cultivation were obtained. Mild acidic hydrolysis of mycelium resulted in the formation of glucose, mannose, and galactose; while the treatment with acid under drastic conditions afforded glucosamine as a product of hydrolysis of chitin and chitosan, their total content was about 35%. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water, 2% aqueous NaOH, and 10% AcOH; their monosaccharide composition was characterized. The yield of chitosan extracted with AcOH was insignificant. Additional purification of the fraction obtained after extraction with alkali afforded polysaccharide which was a linear (1 → 3)-α-D-glucopyranan according to the data of NMR spectroscopy and the chemical methods of structural analysis. The presence of this polysaccharide, as well as a low content of chitosan and polyuronides, distinguishes the studied strain C. japonica from most of the known Mucorales.     

<Emphasis Type="Italic">Pseudocercospora griseola</Emphasis> Causing Angular Leaf Spot on <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Produces 1,8-Dihydroxynaphthalene-Melanin

Pseudocercospora griseola is the causal agent of angular leaf spot of common bean (ALS). It has undergone parallel coevolution with its host and two major groups have been defined, “Andean” (P. griseola f. griseola) and “Mesoamerican” (P. griseola f. mesoamericana). The aim of this study was to analyze the nature and the level of the dark pigment synthesized by the representatives of each group. After 21 days of incubation on potato dextrose agar medium, P. griseola f. griseola isolate S3b developed colonies with diameters of 17.5 ± 1.3 mm and concentric rings of pigmentation. Isolate T4 of P. griseola f. mesoamericana presented smaller colonies (9.9 ± 0.3 mm) with a uniform dark-gray color. Both isolates, S3b and T4, produced the same pigment, a 1,8-dihydroxynaphthalene-melanin, although different in quantity and structural features as suggested by the IR spectrum. The P. griseola f. griseola isolate S3b had a higher growth rate and melanin content as well as smaller sensitivity to melanin synthesis inhibitors compared to the isolate T4 of P. griseola f. mesoamericana. These results suggest a possible link between melanin and growth in P. griseola.     

Mitochondrial morphogenesis and ultrastructure of basidiomycetes from genera Agaricus and Pleurotus

Supravital study of rhodamine-stained mitochondria in cells of aerial and submerged micelium of 31 strains from 9 species from genus Agaricus (A. arvensis Schaeff., A. bisporus (Lange) Imbach, A. bitorquis (Quel.) Sacc., A. campestris L., A. excellens, (F.H. Müller) F.H. Müller, A. macrocarpus (F.H. Müller) F.H. Müller, A. silvaticus Schaeff., A. silvicola (Vittad.) Peck, A. xanthodermus Genev) and of 2 strains from 2 species from genus Pleurotus (P. ostreatus (Jacg.) P. Kumm., P. pulmonarius (Fr.) Quel.) was carried out. Mitochondrial morphogenesis in micelial cells of species from Agaricus and Pleurotus genera has many common features in distribution of mitochondria in micelial cells both under the favorable growth conditions and during chondriom reorganization (fission and fragmentation of mitochondria to small subunits) under unfarovable growth conditions and during aging. During the balanced growth of mycelium of heterokaryotic strains from genera Agaricus and Pleurotus for 7–14 days on agar media as well as during cultivation of mycelium of oyster mushroom and several strains of field mushroom in submerged culture, distribution of mitochondria by the type 1 (small granular mitochondria in the apical zone—1, long rod-like mitochondria in the subapical zone—2 and short rod-like and granular mitochondria formed as a result of fragmentation of rod-like mitochondria in mature mycelium cells—3) was observed. Under unfarovable growth conditions (starvation), in mycelium cells of homokaryotic strains of champignon, during long cultivation of all strains and species from the studied genera and during cultivation on liquid wort, for majority of champignon, distribution of mitochondria by the type 2 was observed (sphere-like mitochondria in all cells of the studied mycelium zones). Mitochondrial profiles at ultrastructural level had specific features, such as associations of the outer mitochondrial membrane with cytoplasmic ribosomes and changes in cristae structure. The preliminary test for apoptosis-like phenotype of the submerged mycelium cells of Bs94 champignon that hardly grows in the submerged culture gave positive result. Thus, mitochondrial morphogenesis in mycelium cells of Agaricus and Pleurotus species under different conditions and terms of cultivation occurs similarly and is determined by a complex of physiological and biochemical processes reflecting the state of mycelium cells.     

Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development

Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system, and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here, we investigate the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperature-related role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures, as they characteristically occur in nature.Key words: Podospora anserina, aging, mitochondria, protein quality control, temperature, i-AAA protease     

Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation

The noncoagulative type of pellet formation can be induced in submerged cultivation of the filamentous fungus Rhizopus nigricans. The size and constitution of the hyphal agglomerates obtained varied with changes in inoculum size and agitation speed for given media composition and cultivation conditions. The physiological state of mycelium, used for a further process of biotransformation, was estimated by following the growth kinetics, pH value and substrate utilization during submerged cultivation. Namely, differences in pellet morphology and physiology affect the ability of R. nigricans to hydroxylate progesterone at the 11α position. A repeated batch procedure revealed the best maintenance of biotransformation capacity for pellets, obtained from the growth phase of cultivation at high agitation speed and with low inoculum size.