病原性丝状真菌的菌种保藏方法

目的探讨常见病原性丝状真菌的菌种保藏方法。方法将73株病原性丝状真菌经过纯化后,接种于马铃薯葡萄糖琼脂斜面分别在4℃和-80℃冷冻管保存,均用10%(v/v)的丙三醇作为保护剂。结果经过1 a左右的保存,将菌株复活,发现4℃斜面保藏法菌株的存活率为100%,-80℃冷冻管保藏法菌株的存活率为98.6%,有些毛癣菌属的菌株不耐低温而不适合-80℃保藏。结论病原真菌可以合理地结合上述2种方法保藏。若实验室配有低温冰箱,可以-80℃冷冻管保藏法为主,对于某些不耐低温的丝状真菌需要同时采用4℃斜面保藏法,则可以保证菌种的存活率。

Phylogeny and taxonomy of meristematic rock-inhabiting black fungi in the Dothideomycetes based on multi-locus phylogenies

The last decade has revealed an unexpected fungal diversity associated with natural rocks,often collected in environments influenced by harsh climatic conditions.Yet the phylogenetic affiliations and the taxonomy of many of these extreme fungi,mainly within Dothideomycetes,the largest class of Ascomycota,have only partially been described.In the present study we confirm that most rock inhabiting-fungi(RIF)are highly polyphyletic among Dothideomycetidae,mainly within the order Capnodiales,an order otherwise incorporating several families of major plant pathological importance.Novel taxa were identified within the two major and distinct clades of Teratosphaeriaceae,both comprising meristematic black fungi.Thirty one novel species and 13 new genera are proposed,based on ITS and partial nucLSU,RPB2 and BT2 sequences.

Ecology and phylogeny of black yeast-like fungi: diversity in unexplored habitats

The known biodiversity of black yeasts and their allies has exploded over the last decades.This even applies to medically significant genera such as Exophiala and Cladophialophora,where the number of accepted species has grown since the 1990s of the previous century from 9 to 44 and from 5 to 34,respectively.A first source of change no doubt is dissection of many supposed ubiquitous generalists into series of narrowly circumscribed molecular siblings,which often appear to be specialists with ecological preferences differing significantly between species.An early example of subdivision of classical species in black yeasts,using DNA homology techniques,concerned Exophiala jeanselmei.One of its siblings today is known to be a biofilm former in drinking water networks,while E.jeanselmei sensu stricto is thus far only known from subcutaneous infections in humans.

European Species of Hebeloma Section Theobromina

This paper addresses section Theobromina within the genus Hebeloma(Agaricales).We recognise seven European species within this section,three of which are described as new:Hebeloma alboerumpens,H.griseopruinatum and H.parvicystidiatum.The first two of these species appear to be ectomycorrhizal with Cistaceae:Cistus and Helianthemum.Hebeloma parvicystidiatum is more likely to be in mycorrhizal association with Quercus spp.We also provide a key to the European species within sect.Theobromina and an updated key of known Hebeloma associates of Cistus.Molecular analyses based on multiple loci further illustrate the distinctness of the newly described taxa and provide molecular evidence,supporting the morphological evidence,for the relationship that exists among species of this section.The ITS is the only one from the sequenced loci that,alongside with morphology,distinguishes among all of the species of sect.Theobromina.The section gains most of its molecular support from the MCM7 locus,followed by RPB2.

Cyphellophora and its relatives in Phialophora: biodiversity and possible role in human infection

Cyphellophora is a genus of black yeast-like fungi characterised by having simple phialides with multiseptate,curved conidia.Judging from SSU and LSU data,Cyphellophora was found to be located in a well-supported clade within the Chaetothyriales comprising a number of species occurring on human skin and nail.Cyphellophora is phylogenetically close to Phialophora europaea,P.reptans and P.oxyspora,though morphologically these species produce singlecelled phialoconidia rather than multiseptate ones.Pseudomicrodochium suttonii and P.fusarioides have dark colonies and phylogenetically fit in with Cyphellophora;the type species of Pseudomicrodochium,P.aciculare,has similar,septate conidia but has a hyaline thallus.In the present study,multilocus phylogenetic analyses were combined with morphology and physiology.Sequences of the internal transcribed spacer region,the DNA dependent RNA polymerase II largest subunit and the partial beta tubulin gene were analysed for a set of 30 strains.Two novel species,Cyphellophora pauciseptata and Phialophora ambigua were discovered.Cyphellophora eucalypti was reduced to synonymy of C.guyanensis.The role of the studied fungi between colonization and infection of human skin was discussed.Putative virulence factors for these black yeast-like fungi were hypothesized to be the ability to assimilate monoaromatic hydrocarbons,to produce melanin pigments,and to tolerate the temperature of epidermal human skin.

Global ITS diversity in the Sporothrix schenckii complex

Molecular phylogeny has revealed that sporotrichosis is caused by several Sporothrix species which differ in clinical behavior.The complex is embedded within Ophiostoma,a genus mainly comprising fungi that live in association with bark beetles,but differs by a high virulence towards humans and other mammals.The different ecology is corroborated by phylogenetic separation.The aim of the present study was to determine the validity of the rDNA internal transcribed spacer(ITS)region as a marker for diagnostics of species in the clinical group,using betatubulin sequences to calibrate species delimitations.The topology of the two gene trees was concordant,and all clinically relevant Sporothrix species could easily be recognized by means of the ITS region.An increased geographic sampling did not affected delimitation success in the clinical clade of the S.schenckii complex.

Multilocus analysis of the Exophiala jeanselmei clade containing black yeasts involved in opportunistic disease in humans

To confirm species delimitations in the‘jeanselmeiclade’in the Chaetothyriales,four independent markers were analysed,and phylogenetic trees were reconstructed using different algorithms.Reproductive isolation within the complex and reproductive modes in the species involved were determined,in order to explore specific borderlines using Genealogical Concordance Phylogenetic Species Recognition(GCPSR).Sequences of the Internal Transcribed Spacer(ITS),partial Translation Elongation Factor 1-α(TEF1),β-Tubulin(BT2),and Actin(ACT1)genes were analysed for a set of 81 strains.Phylogenetic reconstruction was performed using neighbour-joining(NJ),maximum parsimony(MP),maximum likelihood(ML)and Bayesian analysis(BA)to evaluate the concordance of topologies obtained under different optimization criteria.Incongruence among lineages was detected by Partition Homogeneity Test(PHT).Most Exophiala species analysed in this study seemed to be preponderantly clonal.A group of species around Exophiala spinifera showed a tendency toward human pathogenicity,whereas species around E.bergeri were often associated with environments rich in hydrocarbons.Though the tree topologies were not completely identical when different algorithms were used,the four gene lineages were nearly congruent and the trees of the combined multilocus data set did not provide more phylogenetic information than those of separate data sets.ITS data are sufficient for identification of species in the‘jeanselmei-clade’.

Novel Phialophora species from leaf-cutting ants (tribe Attini)

Ants in the tribe Attini(Hymenoptera,Formicidae)maintain a 50 million-year-old lifestyle of co-evolution with symbiotic basidiomycetous fungi which they cultivate as essential source of nutrition.However,other microorganisms have been reported from ant habitats indicating a higher diversity of consistently associated species than established to date.Recently,black yeast-like fungi have been reported as a possible symbiont,like a competitor of the actinomycete bacteria that grow in the integument of the insects.During the mating season,gynes of Atta capiguara and A.laevigata were collected from nests located in Botucatu,SP,Brazil and sampled using flotation technique.Phylogenetic analysis based on ITS,partial 28S(LSU)andβ-tubulin sequences revealed the occurrence of two novel species of Phialophora among the melanized fungi isolated.