Structure Analysis of the MatA Locus of Sexual Compatibility in the Edible Mushroom Pleurotus ostreatus.

The edible oyster mushroom Pleurotus ostreatus is one of the most cultivated species worldwide. Morphogenesis associated with the maturation of fruit bodies is controlled by two unlinked loci of sexual compatibility matA and matB with multiple alleles (tetrapolar system of sexual compatibility). Quantitative analysis of the alleles of mating compatibility loci in 17 natural isolates collected in the Moscow region was performed in mon-mon (monokaryons-monokaryon) and di-mon (dikaryon-monokaryon) crossings. Four monokaryotic testers strains which were heteroallelic at both mating type loci were obtained for each of the five natural mushroom isolates by using original technique of sterile spore prints on Petri dishes and mon-mon crossing. Twelve natural isolates were crossed via di-mon mating with the four monokaryotic testers M-38. Genetic analysis of the alleles of sexual compatibility loci in 17 natural isolates revealed multiple alleles at both loci: at least ten alleles at matA locus and eight alleles at matB locus. Structural organization analysis of the matA locus was performed in silico for homokaryotic strains PC9 and PC15 based on the whole-genome sequencing data available at DOE Joint Genome Institute. The matA locus has an extremely divergent structure: there are one copy of the homeodomain gene hd1 and one copy of the hd2 gene in the PC9 strain, whereas the matA locus of the PC15 strain is composed by two copies of hd1.1 and hd1.2 genes (class HD1 homeodomain proteins) and one copy of hd2 gene (class HD2 proteins). Comprehensive analysis of amino acid sequences of HD1 and HD2 homeodomain proteins demonstrated that the proteins have a globular structure with the nuclear localization and contain a variable N-terminus and a more conserved DNA-binding domain with a specific conserved motif  WFXNXR in the third ɑ-helix. The results suggest that multiple alleles of the matA locus of sexual compatibility in basidiomycete fungi is achieved due to both different copy number of the coding hd genes within the locus and the variability of the coding gene sequences.

[Molecular Polymorphism of ß-Galactosidase LAC4 Genes in Dairy and Natural Strains of Kluyveromyces Yeasts].

The ability to ferment lactose is a characteristic peculiarity of dairy Kluyveromyces lactis yeasts; the vast majority of other yeast species are not able to assimilate this disaccharide. Molecular polymorphism of LAC4 genes encoding ß-galactosidase controlling lactose fermentation is not well studied, and the published data concern only a single strain (K. lactis var. lactis NRRL Y-1140) isolated from cream in the United States. We studied ß-galactosidase genes in lactose-fermenting К lactis strains isolated from dairy products and natural sources in different regions of the world using molecular karyotyping, Southern hybridization, and sequencing. It was established that the ability to ferment lactose in К. lactis var. lactis dairy yeasts is controlled by at least three polymeric LAC loci with different chromosomal localization: LAC1 (chromosome III), LAC2 (II), and LAC3 (IV). Most of the strains we studied had the LAC2 locus. A comparative analysis of ß-galactosidases of the Kluyveromyces genus yeasts and these enzymes from other yeasts was conducted for the first time. Phylogenetic analysis detected significant differences between the LAC4 proteins of yeasts of the Kluyveromyces genus (K. lactis, К. marxianus, К. aestuarii, К. nonfermentans, К. wickerhamii), Scheffersomyces stipitis, Sugiyamaella lignohabitans, and Debaryomyces hansenii. A correlation between ß-galactosidase sequences and ecological origin (dairy products and natural sources) of Kluyveromyces strains was found. The group of dairy strains is heterogeneous and includes К. lactis var. lactis and К. marxianus yeasts (99.80-100% similarity), which indicates a common origin of their LAC4 genes. Phylogenetic analysis of ß-galactosidases indicates a close genetic relationship of dairy and hospital strains of К. lactis var. lactis and К. marxianus. Clinical isolates are able to ferment lactose and appear to originate from the dairy yeasts.

[New strains of basidiomycetes that produce bioethanol from lignocellulose biomass].

Sixty six isolates were screened for ability of bioethanol production; dynamics of product accumulation and substrate utilization were investigated for two selected strains Trametes hirsuta MT-24.24 and Trametes versicolor IT-1. The strains' efficiency was evaluated as bioethanol production by 1 g biomass. Strain T. versicolor IT-1 producing over 33 g/L of the ethanol for 9 d was selected. Direct conversion of Na-carboxymethyl cellulose, microcrystalline cellulose and straw was shown with ethanol yields of 2.1, 1.6 and 1.7 g/L, respectively, for 9 d fermentation time.

[Phylogenetic analysis of Pleurotus species].

We performed phylogenetic analysis for ten Pleurotus species, based on internal transcribed spacer (ITS) sequences of rDNA. A phylogenetic tree was constructed on the basis of 31 oyster fungi strains of different origin and 10 reference sequences from GenBank. Our analysis demonstrates that the tested Pleurotus species are of monophyletic origin. We evaluated the evolutionary distances between these species. Classic genetic analysis of sexual compatibility based on monocaryon (mon)-mon crosses showed no reproductive barriers within the P. cornucopiae-P. euosmus species complex. Thus, despite the divergence (subclustering) between commercial strains and natural isolates of P. ostreatus revealed by phylogenetic analysis, there is no reproductive isolation between these groups. A common allele of the matB locus was identified for the commercial strains Sommer and L/4, supporting the common origin of these strains.

[Cultural Properties and Taxonomic Position of Helminthosporium-Like Fungal Isolates from the White Sea].

Morphological, cultural and physiological characteristics of 19 Helminthosporium-like hyphomycetes isolated from the White Sea were studied. Taxonomic status of the isolates was verified using molecular genetics techniques. One of the isolates was identified as Alternatia sp., while the rest of marine isolates belonged to the species Paradendriphiella salina (G.K. Sutherl.) Woudenb. & Crous. The specific features of the isolates studied were characterized as adaptive. Optimum salinity for their growth was 1-2% NaCl, which is lower than the value for the known open ocean isolates. This is probably due to relatively low salinity of White Sea (22-24 per thousand) as compared with the ocean water (35 per thousand). While the temperature optimum for growth was 22 per thousand, growth and sporulation occurred at 6 degrees C, which has not been reported for marine fungi isolated from warmer seawater. All isolates studied grew and sporulated efficiently on the medium supplied with the Fucus algae extract and in the sea water layer. Conidia of the isolates submerged in the sea water were propagated efficiently, unlike the soil-born fungi. Holoblastic conidiogenesis was demonstrated by light and scanning electron microscopy, confirming the separation of P. salina from the genus Scolecobasidium.

[DNA marking of some quantitative trait loci in the cultivated edible mushroom Pleurotus ostratus (Fr.) Kumm].

Fungi of the genus Pleurotus, in particular, species Pleurotus ostreatus (common oyster mushroom) are among most cultivated fungi in the world. Due to intense rates of development of studies in this field, efficient breeding programs are highly required in the search for new P. ostreatus strains. The principal traits used worldwide for selecting strains are intensity of fruitbearing, fruit body cap color (for some consumptive markets), and mycelium growth rate. In this connection, the objective of this work was to study these quantitative traits and to find molecular markers, which could be employed to accomplish breeding programs. In general, we found 12 genomic loci (quantitative trait loci, QTLs) controlling mycelium growth rate of oyster and six QTLs responsible for the fruit body cap color. The genetic map of P. ostreatus was constructed, and all markers of quantitative traits found by us were located on this genetic map. The obtained linkage map can be a useful tool for the accomplishment of breeding programs to improve economically important traits of oyster mushroom.

[The commercially cultivated edible oyster mushrooms Pleurotus sajor-caju and P. pulmonarius are two separate species, similar in morphology but reproductively isolated].

Two closely related commercially cultivated oyster mushroom species, Pleurotus pulmonarius and P. sajor-caju have been differentiated by traditional mating experiments as well as analysis of the variable ITS and IGS sequences of the ribosomal gene cluster. Molecular analysis of the variable ITS and IGS regions has allowed neither reliable differentiation between the morphologically similar species P. pulmonarius and P. sajor-caju nor confirmation of species identity of the P. sajor-caju strains CS-32, H-1, and H-2. Analysis of the sexual (mating) compatibility between haploid tester strains of these two species in monokaryon-monokaryon mating experiments has demonstrated complete reproductive isolation between P. pulmonarius and P. sajor-caju, thereby confirming that these are separate species.

[Differentiation of closely related oyster fungi Pleurotus pulmonarius and P. ostreatus by mating and molecular markers].

Classical matings and RAPD-PCR analysis were used to differentiate two closely related basidial fungi, Pleurotus pulmonarius and P. ostreatus, which are widespread in Russian forest biocenoses with moderate climate. Monokaryon-monokaryon (mon-mon) and dikaryon--monokaryon (di-mon) matings demonstrated complete reproductive isolation of the two species, which have partly overlapping morphological traits. The prevalence of a particular species in nature was shown to depend to a great extent on the natural conditions, namely, the day and night temperature ranges. The clustering of natural Pleurotus strains displayed two trends: one was associated with natural reproductive isolation of the two species (D = 0.61) and the other, with the geographical factor (D = 0.39). A relatively recent origin is suggested for the divergence of the two species and the reproductive barrier between them. Adaptation to natural conditions was considered to be the main factor causing the divergence of natural Pleurotus populations and, eventually, allopatric speciation.

[Comparative analysis of Pleurotus ostreatus natural isolates].

A comparative analysis is performed of the polymorphism of the Pleurotus ostreatus (Fr.) Kumm naturally occurring strains isolated from the natural substrates found in two geographically remote Russian natural preserves, the Central Arboreal Biosphere Tver State Preserve (CABTSP) and the Moscow State University Zvenigorod Biological Station (ZBS, Moscow oblast), and within the city of Moscow. The results of the frequency analysis for the isozyme loci alleles and for the sexual and vegetative incompatibility groups are presented; the genetic structure and the interpopulation relations among 58 P. ostreatus dikaryotic strains are estimated. The natural samples from the Moscow and Tver oblasts are shown to have a high degree of polymorphism with a genetic differentiation of 0.743; in spite of their territorial remoteness, they are, however, actively exchanging genetic material. The natural fungal isolates form two reproductively isolated groups.

[Interspecific genetic variability of the oyster mushroom Pleurotus ostreatus as revealed by allozyme gene analysis]. / Vnutrividovoe gennoe raznoobrazie veshenki ustrichnoi, Pleurotus ostreatus, izuchennoe po sovokupnosti allozimnykh genov.

Allozyme variation in natural populations of basidiomycetes fungus Pleurotus ostreatus (88 individuals) from three regions of central Russia was studied. The species was shown to have 92.86% of polymorphic allozyme loci and expected heterozygosity He = 0.49. The mean number of alleles per locus was 3.5. The genetic differences among populations were supported by F-statistics (FST = 0.750). The low level of inbreeding (FIS = 0.018) suggests that the P. ostreatus populations are panmictic, and the main reproduction mode involves basidiospores dispersing at long distances. Using cluster analysis, geographically isolated populations and intersterile groups were differentiated within the complex P. ostreatus species.

[Transposable elements are the factors involved in various rearrangements and modifications of fungal genomes]. / Transpozony kak faktory razlichnykh perestroek i modifikatsii v genomakh gribov.

Data on transposable elements in fungal genomes are reviewed. Possible role of transposons in the pathogenetic processes and regulation of mating compatibility are discussed. The transposition-inducing factors and mechanisms responsible for transposition within the genome are considered.

[Use of molecular markers for differentiation of cultivated strains of oyster and button mushrooms]. / Ispol'zovanie molekuliarnykh markerov dlia differentsiatsii kul'tiviruemykh shtammov veshenki i shampin'ona.

Analysis of commercial strains of two edible mushrooms, Pleurotus ostreatus and Agaricus bisporus, using PCR and isozyme electrophoresis techniques allowed us to differentiate groups of genetically similar and distant strains. Among the commercial strains of P. ostreatus, the level of genetic variation was higher suggesting a broader genetic basis employed in breeding of this mushroom. The cultivars and hybrids of, A. bisporus, showed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.

[The biosynthesis of low-molecular-weight nitrogen-containing secondary metabolite-alkaloids by the resident strains of Penicillium chrysogenum and Penicillium expansum isolated on the board of the Mir space station ]. / Biosintez nizkomolekuliarnykh azotsoderzhashchikh vtorichnykh metabolitov-alkaloidov shtammami-rezidentami gribov vidov Penicillium chrysogenum i Penicillium expansum, vydelennykh na orbital'noi stantsii "Mir".

The analysis of the absorption spectra of the low-molecular-weight nitrogen-containing secondary metabolites--alkaloids--of 4 Penicillium chrysogenum strains and 6 Penicillium expansum strains isolated on board the Mir space station showed that all these strains synthesize metabolites of alkaloid origin (roquefortine, 3,12-dihydroroquefortine, meleagrin, viridicatin, viridicatol, isorugulosuvin, rugulosuvin B, N-acetyl-tryptamine, and a "yellow metabolite" containing the benzoquinone chromophore).

[Residential colonization of orbital complex "Mir" environment by penicillium chrysogenum and problem of ecological safety in long-term space flight]. / Rezidentnoe zaselenie sredy na orbital'nom komplekse "Mir" penicillium chrysogenum i problema ékologicheskoi bezopasnosti v dlitel'nom kosmicheskom polete.

Results of many years of the survey of highly specific evolution of quantitative and species composition of microflora of the MIR environment are reviewed. Analysis of the data enabled listing of microorganisms-declinous fungi with the ability of residential colonization of structural materials of the interior and equipment of habitable modules of the space station. Results of the studies of variability and level of similarity/affinity on the basis of DNA, polymorphism of strains isolated in space flight, convincingly confirmed this characteristic in the Penicillium chrysogenum cultures. In view of the common origin determined from the signs of genetic alliance, the P. chrysogenum strains isolated on MIR in 1995 can be considered descendants of the cultures found at the beginning of the MIR operation. This ecological expansion of P. chrysogenum in the space station environment gains in prominence due to the fact that representative of this particular species known for its active biodestructive nature were, as a rule, detected in the areas where structural materials of the SALYUT and MIR space stations incurred biological degradation.