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。

Environmental siblings of black agents of human chromoblastomycosis

Agents of human chromoblastomycosis,a skin disease almost exclusively caused by members of the order Chaetothyriales,are assumed to be traumatically inoculated into the skin with sharp environmental materials such as plant thorns or wooden splinters carrying the respective opportunist.In the supposition that such fungi should have their main habitat in the environment,we investigated the occurrence of black fungi in living areas of patients with chromoblastomycosis.In South America Fonsecaea agents are prevalent as agents of the disease,while also related Cladophialophora species,known from other types of skin infections,are known from the continent.Ninety environmental isolates were preliminarily selected as possible agents of chromoblastomycosis,based on morphology.Judging from ITS sequence data isolates were attributed to the genera Cladophialophora,Cyphellophora,Exophiala,Fonsecaea,Phialophora,and Veronaea.A total of 45 fungi morphologically identified as Fonsecaea or Cladophialophora isolated from debris and thorns of living prickly plants in Brazil were processed for taxonomic studies.Phylogenetic analysis revealed that the isolates indeed belonged to the Chaetothyriales,but only rarely an agent of chromoblastomycosis was concerned;only two strains of F.pedrosoi and one F.monophora were isolated from debris plants.The remaining isolates belonged to hitherto unknown molecular siblings of Fonsecaea.Two novel taxa are introduced.

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.

Extremotolerant rock inhabiting black fungi from Italian monumental sites

A wide sampling for isolating highly destructive and extremotolerant black fungi was performed from Italian monuments in selected historical sites which include the "Cortile della Pigna",Vatican Museum and the St Peter colonnade(Vatican City State),the monumental cemetery of Bonaria(Cagliari),and other monuments in the city of Cagliari.Thirty out of seventy-four strains isolated were identified basing on ITS Blastn comparison.Based on multilocus phylogeny and morphological data,one new genus and species Lithophila guttulata,five new species Knufia marmoricola,K.vaticanii,K.karalitana,K.mediterranea and Exophiala bonariae,order Chaetothyriales and one new genus and species,Saxophila tyrrhenica,and two new species Vermiconia calcicola and Devriesia sardiniae,order Capnodiales,were proposed.Ecological considerations are put forward.

Diversity and delimitation of Rhizopus microsporus

Rhizopus microsporus has been used for centuries in the production of oriental fermented foods,but the species is also known as a toxin producer and from severe human infections.To study the diversity and species delimitation of Rhizopus microsporus,48 isolates from the reference collection of the CBS-KNAW Fungal Biodiversity Centre,comprising nine environmental,ten clinical,and 23 foodborne strains,in addition to six strains from unknown sources and representing all existing varieties of the species,were examined.Sequence diversity was based on the internal transcribed spacer(ITS),and on a part of the actin(ACT)and translation elongation factor 1-α(TEF)genes.Differences in physiological properties were assessed including temperature relationships.Spore morphology was studied,mating type tests were performed,and MALDI-ToF profiles were generated.Clinical and food-associated strains as well as members of different varieties mated successfully and consequently they belong to a single biological species.Molecular differences did not match with any other parameter investigated.Based on these results the varieties of Rhizopus microsporus are reduced to synonyms.

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.

Taxonomy of Ochroconis, genus including opportunistic pathogens on humans and animals

The genus Ochroconis(Sympoventuriaceae,Venturiales)is revised and currently contains 13 species for which the phylogenetic position has been determined using multilocus sequencing.The older generic name Scolecobasidium is considered to be of doubtful identity because the type specimen is ambiguous.Within the Ochroconis lineage,phylogenetic distances of all markers analyzed are exceptionally large,both between and within species.A new genus Verruconis is proposed for the neurotropic opportunist Ochroconis gallopava.Species accepted within the lineages are keyed out on the basis of phenotypic characters.Main ecological traits within each species are discussed.Verruconis species are thermophilic and one of them is an important agent of infection in the brain,while Ochroconis is mesophilic,several species causing infections in cold-blooded animals.

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.

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.

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’.

A multigene phylogeny reveals that Ochroconis belongs to the family Sympoventuriaceae (Venturiales,Dothideomycetes)

Ochroconis is a genus of ascomycete fungi that includes oligotrophic saprobes and some opportunistic species causing infections in vertebrates.The most important of these opportunists is the neurotropic species Ochroconis gallopava,which occurs in birds and occasionally in immunocompromised humans.Other Ochroconis species have been isolated from superficial infections of cats,dogs and fish.In their natural environment,these species are found in litter,soil,and on moist surfaces.Some thermophilic species have been isolated from hot springs,industrial effluents,and self-heated plant material.Although their ecology and epidemiology has been investigated,their classification within the ascomycetes is still unknown.Here,the phylogenetic placement of Ochroconis is investigated using a four-gene phylogeny(nuLSU,nuSSU,mtSSU and RPB2).The results show that Ochroconis and its recently described sister genus Verruconis belong to the order Venturiales(Dothideomycetes)and are nested within the Sympoventuriaceae,a family recently erected for a group of plant-saprobes or-pathogens sister to the Venturiaceae.They form a well-supported monophyletic group together with five species of the anamorphic genus Fusicladium isolated from leaf and needle litters.

Mountain tips as reservoirs for new rock-fungal entities: Saxomyces gen. nov. and four new species from the Alps

As part of a worldwide sampling nine black fungi were isolated from rocks collected in four distinct sites of the Alps at high altitudes.Based on a nucSSU,nucLSU and mtSSU multi-locus phylogeny,seven of them were found to cluster into a distinct and wellsupported clade in a basal position within the Class Dothideomycetes.As in other rock fungi these new groups of isolates were characterized by a meristematic growth and a scarcely differentiated morphology with highly melanized and thick-walled toruloid hyphae.Nonetheless,few peculiar characters were also observed as convoluted hyphal tips and the production of spherical propagules.The new genus and species Saxomyces alpinus and S.penninicus,are here described based on morphological and molecular data,in a yet to be defined order of the Dothideomycetes.The remaining two black fungi clustered in Cryomyces,a genus previously exclusively found in rocks from the McMurdo Dry Valleys in Antarctica.These two isolates were genetically distant from other Antarctic Cryomyces species based on ITS sequences,and they showed a peculiar morphology;they are here described as the new species C.montanus and C.funiculosus.Implications of our results on the evolution,adaptation and dispersal of rock-inhabiting fungi under extreme conditions are discussed.

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.