Mycological research最新文献

Basidiomycetes present specific problems with regard to their preservation, because most of them do not form resistant propagules in culture but exist only as mycelium. Usually these fungi can only be preserved by serial transfer on agar (labour-intensive procedures that can increase the danger of variation or loss of physiological or morphological features), or cryopreserved in liquid nitrogen (expensive). Cryopreservation at −80 °C and lyophilisation could be good alternatives.

In this work we set up and tested six protocols of cryopreservation at −80 °C, and 12 protocols of lyophilisation on 15 isolates of white-rot fungi (WRF) belonging to 10 species. The tested protocols were mainly characterized by the use of different growth media, protectants, time and number of perfusion with protectants and finally by the typology and origin of the samples to be cryopreserved (mycelium/agar plug, whole colony) or to lyophilise (mycelium/agar plug, mycelium fragment, whole colony). Cryopreservation and lyophilisation outcomes were checked, at morphological (macro- and microscopic features), physiological (growth rate and laccase, Mn-independent and Mn-dependent peroxidases activities) and genetic level (Amplified Fragment Length Polymorphisms analysis – AFLP). Vitality of all fungi was successfully preserved by all cryopreservation protocols at −80 °C, and by two lyophilisation methods. Our results showed that cryopreservation at −80 °C did not produce morphological changes in any isolate, while two isolates were affected by lyophilisation. None of the physiological features were lost, even though growth rate and enzyme activities were somehow influenced by all preservation methods. AFLP analysis showed that only the two isolates that varied in their morphology after lyophilisation produced a different DNA fingerprint pattern in comparison with that obtained before lyophilisation. These findings provide evidence that cryopreservation at −80 °C and lyophilisation are suitable alternatives to liquid nitrogen cryopreservation for preservation of some WRF strains.

The Agaricales is the largest and most diverse order of mushroom-forming Basidiomycota, with over 100 natural groups recognized in recent Fungal Tree of Life studies. Most agarics are either saprotrophic or ectomycorrhizal fungi, but the family Hygrophoraceae is in part characterized by a unique and remarkable diversity of lichenized forms. The most familiar of these is the chlorolichen genus Lichenomphalia, whose phylogenetic position in the Agaricales has been established. Recent limited evidence suggested that Hygrophoraceae also contains cyanolichens in the genus Dictyonema, which indicates a remarkable concentration and diversity of lichen-formers in a single family of agarics. To demonstrate the relationships of lichen-formers to other fungi in the family, we assembled ribosomal sequences from 52 species representing recognized groups within the Hygrophoraceae, among them new sequences representing Acantholichen and most species and forms of Dictyonema. The molecular data were evaluated using parsimony, likelihood, Bayesian, and distance analyses, including coding of ambiguous regions by means of INAASE and ARC, all of which indicate that Dictyonema and Acantholichen form a monophyletic clade derived from the primarily bryophilous genus Arrhenia and sister to the enigmatic Athelia pyriformis, a species unrelated to the Atheliales for which we are proposing a new genus name Eonema. The chlorolichen genus Lichenomphalia may be polyphyletic. Fungi in the Dictyonema–Acantholichen clade are typically tropical, entirely lichenized, and associate with cyanobacterial photobionts. Our data indicate a transition from agaricoid–omphalinoid basidiomes observed in Arrhenia to stereoid–corticioid forms in Dictyonema, and also support a previous suggestion of a connection between loss of clamp connections and lichenization. The diverse basidiome and thallus morphologies and nutritional ecologies of these fungi indicate a remarkable evolutionary flexibility that appears to have developed in part as a consequence of symbiosis.

Fusarium oxysporum is a ubiquitous species complex of soilborne plant pathogens that comprises many different formae speciales, each characterized by a high degree of host specificity. In this study, the evolutionary relationships between different isolates of the F. oxysporum species complex have been examined, with a special emphasis on the formae speciales lycopersici and radicis-lycopersici, sharing tomato as host while causing different symptoms. Phylogenetic analyses of partial sequences of a housekeeping gene, the elongation factor-1α (EF-1α) gene, and a gene encoding a pathogenicity trait, the exopolygalacturonase (pgx4) gene, were conducted on a worldwide collection of F. oxysporum strains representing the most frequently observed vegetative compatibility groups of these formae speciales. Based on the reconstructed phylogenies, multiple evolutionary lineages were found for both formae speciales. However, different tree topologies and statistical parameters were obtained for the cladograms as several strains switched from one cluster to another depending on the locus that was used to infer the phylogeny. In addition, mating type analysis showed a mixed distribution of the MAT1-1 and MAT1-2 alleles in the F. oxysporum species complex, irrespective of the geographic origin of the tested isolates. This observation, as well as the topological conflicts that were detected between EF-1α and pgx4, are discussed in relation to the evolutionary history of the F. oxysporum species complex.

In this paper we have attempted to clarify the taxonomy and nomenclature of thirteen taxa of the genus Cortinarius subgenus Telamonia (sections Hydrocybe, Fraternii) well represented in the southwestern Mediterranean area of Europe (C. atrocoeruleus, C. bombycinus, C casimiri, C. contrarius, C. decipiens, C. fraternus, C. gallurae, C. hoffmannii, C. petroselineus, C. sertipes, C. subturibulosus, C. urdaibaiensis and C. vernus). To this end we have performed a combined study of morphological and molecular data (rDNA ITS sequences). The morphological analysis was carried out on 114 collections and the molecular analysis involved 31 of the 114 collections, including 11 type collections (types for C. casimiri and C. fraternus were not available). In addition, a study of spores under field emission scanning electron microscopy (FESEM) was conducted. The results of the combined analysis allowed us to asign the studied material to five species (C. casimiri s.l., C. decipiens s.l., C. gallurae, C. subturibulosus s.l. and C. vernus s.l.). Thus, all collections from more continental areas, which were originally identified as six different taxa (C. atrocoeruleus, C. contrarius, C. decipiens, C. fraternus, C. sertipes, C. flexipes fo. sertipes) corresponded to C. decipiens sensu lato, a widely distributed, genetically and morphologically variable species. Cortinarius casimiri is also found in such habitats, but it is confirmed as distinct taxon. Collections from Mediterranean sclerophyllous communities correspond to C. gallurae, C. vernus sensu lato and C. subturibulosus sensu lato. Due to close phylogenetic relationships we propose the new combinations C. casimiri var. hoffmannii (=C. decipiens var. hoffmannii non C. hoffmannii) and C. subturibulosus var. bombycinus (=C. bombycinus), and the new variety C. vernus var. nevadavernus (=C. vernus H. Lindstr. & Melot sensu auct.).

Previously, we identified three gene loci, Dic1, Dic2, and Dic3, that confer high-osmolarity adaptation and dicarboximide/phenylpyrrole fungicide sensitivity in Cochliobolus heterostrophus. Dic1 encoded a group III histidine kinase, but the other genes were not characterized. In the present study, we revealed that both Dic2 and Dic3 are involved in the Skn7 pathway. Dic2 encoded an Skn7-type response regulator, ChSkn7. Strain N4502 contained D359N in the response regulator domain of ChSkn7. Strain E4503 contained a deletion of 50 amino acids in the DNA-binding domain. Strain N4507 was a null mutant of the ChSkn7 gene. All of the dic2 mutant strains showed similar levels of sensitivity to high osmolarity and similar levels of resistance to fungicides. These results strongly suggested that both the DNA-binding domain and response regulator domain are essential for Skn7 function in osmotic adaptation and fungicide sensitivity. A western blot analysis revealed that Dic3 is not involved in the regulation of Hog1-type MAPKs. The Chssk1/dic3 double mutant strains clearly showed greater resistance to fungicides than the single mutant strains. An additive effect was also observed in the high-osmolarity experiments. On the other hand, the dic3/dic2 double mutant strains did not show higher levels of resistance to fungicides and greater sensitivity to KCl than the single mutant strains. These results strongly suggested that the dic3 locus confer high-osmolarity adaptation and fungicide sensitivity independently from Ssk1-Hog1 pathway, but not the Skn7 pathway. Moreover, the dic3 strain and all dic2 strains showed similar levels of sensitivity to high-osmolarity stress and similar levels of resistance to fungicides, suggesting Dic3 to have an essential role in the Skn7 pathway. Our results provide new insight into the functions of the Skn7 pathway in filamentous fungi.

A new species, Sphaerodes mycoparasitica (Ascomycetes, Melanosporales), was isolated from isolates of Fusarium avenaceum and Fusarium graminearum originating from wheat fields in Saskatchewan, and from Fusarium oxysporum originating from asparagus fields in Quebec, Canada. The species is characterized by a unique combination of ascospore size, shape (fusiform and triangular) and wall ornamentation (reticulate and smooth). Also, conidia are produced from simple phialides on the surface of ascoma peridial wall, on ascoma surrounding hyphae, and on irregularly branched conidiophores arising from hyphae. The closest relation of S. mycoparasitica is Sphaerodes quadrangularis, which has no detected anamorphic stage. The description of S. mycoparasitica, its phylogenetic position—based on DNA sequences of large subunit ribosomal RNA gene (LSU)—as well as a key for all known Sphaerodes species are provided.

Common ragweed (Ambrosia artemisiifolia) is an invasive and highly allergenic plant species, on which two species, Plasmopara halstedii and Plasmopara angustiterminalis, have been recognized to cause downy mildew disease. In this study, morphological and molecular patterns of seven Plasmopara specimens collected from A. artemisiifolia in Canada, Hungary, and USA were compared with those of P. halstedii and P. angustiterminalis from Helianthus and Xanthium, respectively. Analyses of partial sequences of three genes, namely those for the large subunit (28S) of rDNA, cytochrome c oxidase subunit II (COX2), and NADH dehydrogenase subunit I (ND1) of mtDNA, were carried out to examine the phylogenetic relationships among these specimens using both Bayesian and maximum parsimony methods. All the phylogenetic analyses revealed that the downy mildew pathogens infecting A. artemisiifolia in Hungary and North America clearly represent a lineage distinct from other Plasmopara taxa investigated. The shape of sporangia and the width of trunks and branches also allowed the separation of the specimens parasitic to A. artemisiifolia from P. halstedii on Helianthus annuus and P. angustiterminalis on Xanthium strumarium. Surprisingly, the Hungarian and the Canadian specimens were more closely related to each other than to those from the USA based on COX2 and ND1 mtDNA data, although the D1/D2/D3 sequences of 28S rDNA were identical in all these Plasmopara specimens. The regional distribution of the mtDNA haplotypes seen in this study suggests a transatlantic migration has occurred and would be interesting to follow up with a more detailed sampling. To investigate the diversity within P. halstedii sensu lato, infecting different host plant species, specimens from six asteraceous genera, Ambrosia, Flaveria, Helianthus, Siegesbeckia, Solidago, and Xanthium, were also included in molecular analyses. These represented six distinct lineages according to the host plant genera. These findings might serve as a basis for a taxonomical reassessment of the P. halstedii complex and also for the delimitation of several well-defined species within this complex.

NADP-Glutamate dehydrogenase (NADP-GDH) located at the interface of carbon and nitrogen metabolism has the potential to dictate fungal carbon flux. NADP-GDH from Aspergillus terreus, itaconate producer and an opportunistic pathogen, was purified to homogeneity using novel reactive dye-affinity resins. The pure enzyme was extensively characterized for its biochemical and kinetic properties and compared with its well studied Aspergillus niger counterpart. The A. terreus NADP-GDH was more stable and showed non-competitive ammonium inhibition with respect to glutamate. It exhibited hyperbolic 2-oxoglutarate saturation albeit with a weak substrate inhibition. This is in contrast to the allosteric nature of the enzyme from other Aspergilli. Differential susceptibility to chymotrypsin is also consistent with the absence of substrate cooperativity and conformational changes associated with A. terreus NADP-GDH. The non-allosteric nature of A. terreus NADP-GDH provides a unique opportunity to assess the contribution of allostery in metabolic regulation.

The maize pathogens Fusarium verticillioides (Fv) and Fusarium proliferatum (Fp) are morphologically very similar to one another, so Fp isolates have been often mistaken as Fusarium moniliforme (the former name of Fv). The only presently accepted morphological discriminator between these species is the presence/absence of polyphialides. Here, a collection of 100 Fusarium strains, isolated from infected maize kernels on plants grown in north-western Italy, were assigned as Fv or Fp on the basis of the presence/absence of polyphialides. This classification was tested on a subset of isolates by sexual crosses, ITS and calmodulin sequencing and AFLP profiling. An ITS-RFLP assay was extended to the full collection and to a number of Fv and Fp isolates of different geographical origin and hosts. The ITS region is proposed as taxonomically informative for distinguishing between Fp and Fv.