Phylogeny and a new species of Sparassis (Polyporales, Basidiomycota): evidence from mitochondrial atp6, nuclear rDNA and rpb2 genes

Three nuclear genes, lsu-rDNA (encoding nuclear large subunit rDNA), ITS (encoding the rDNA internal transcribed spacers and 5.8 S rDNA) and rpb2 (encoding the second largest subunit of RNA polymerase II), and the mitochondrial gene atp6 (encoding the sixth subunit of ATP synthase), were sequenced from all recognized Sparassis lineages. Sparassis latifolia sp. nov. from boreal coniferous forests in China is described based on morphological, ecological, geographical and molecular data. The nuclear gene phylogeny strongly supported groups corresponding to morphological differences, geographic distribution and host shifts among species that produce clamp connections, such as S. crispa from Europe, S. radicata from western North America and S. latifolia from Asia. The atp6 phylogeny however showed no divergence among these three species. For clampless Sparassis species, such as S. spathulata from eastern North America, S. brevipes and a new species from Europe, the atp6 phylogeny was congruent with the nuclear gene phylogeny. Sparassis cystidiosa is basal in the nuclear tree but sister to S. brevipes-S. spathulata clade in the ATP6 tree. The differences between the phylogenetic inferences from the atp6 gene and those from nuclear genes within Sparassis species are discussed.     

Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.     

Evidence for a new lineage of primary ambrosia fungi in Geosmithia Pitt (Ascomycota: Hypocreales)

Geosmithia is a genus of mitosporic filamentous fungi typically associated with phloeophagous bark beetles world-wide. During this study, the fungal associates of ambrosia beetles Cnesinus lecontei, Eupagiocerus dentipes, and Microcorthylus sp. from Costa Rica, were studied using morphology and DNA sequences. Fungal associates belonged to four undescribed Geosmithia species. Geosmithia eupagioceri sp. nov. and G. microcorthyli sp. nov. are evidently primary ambrosia fungi of their respective vectors E. dentipes and Microcorthylus species. They both have convergently evolved distinct morphological adaptations including the production of large, solitary and globose conidia, and yeast-like cells. Tunnels of C. lecontei contained an undescribed Geosmithia species, but its nutritional importance for its vector is unclear. An auxiliary ambrosia fungus, Geosmithia rufescens sp. nov., was found associated with both G. eupagioceri and the Geosmithia species associated with C. lecontei. G. microcorthyli is genetically quite similar to the phloem-associated Geosmithia sp. 8 from Europe. Large differences in morphology between these two species suggest the rapid co-evolution resulting from the close symbiosis of the former with its beetle host. The ITS rDNA sequences of G. microcorthyli and Geosmithia sp. 8 were not diagnostic, suggesting that alternative markers such as EF-1α, IGS rDNA or β-tubulin should be used, together with morphological and ecological data, for species delimitation in this genus. The primary ambrosia fungi described here are derived from phloem-associated ancestors, and represent two independent lineages of ambrosia fungi in the Hypocreales and a new ecological strategy within Geosmithia.     

Remarkable nutritional diversity of basidiomycetes in the Corticiales, including a new foliicolous species of Marchandiomyces (anamorphic Basidiomycota, Corticiaceae) from Australia

Fungi in the basidiomycete order Corticiales are remarkably diverse nutritionally, including a variety of saprotrophs, plant and fungal pathogens, and lichen-forming fungi. Tracing the origin of this diversity depends on a clearer understanding of the phylogenetic relationships of fungi in the order. One of its core members is the genus Marchandiomyces, originally established for lichen pathogens that form orange or coral bulbils. We describe here a new species in the genus, M. marsonii sp. nov., which is unusual in its appearance, habit, and geographic provenance. It is foliicolous on leaves of Pandanus (screw pines, Pandanaceae) and produces flattened, coral bulbils resembling apothecia of the ascomycete genus Orbilia. It is also the first member of the genus to be collected from Australia. An isolate of the new fungus and several additional cultures of related plant pathogenic fungi were obtained and investigated phylogenetically using parsimony, likelihood, and Bayesian analyses of nuclear small and large subunit ribosomal sequences. Our phylogeny makes clear that Marchandiomyces species and their close relatives contribute significantly to the ecological diversity of the Corticiales and that this diversity is derived mainly from lignicolous ancestors.     

The obligately lichenicolous genus Lichenoconium represents a novel lineage in the Dothideomycetes

Lichenicolous fungi are obligately lichen-associated organisms that have evolved many times throughout the Ascomycota and Basidiomycota. Approximately 20% of lichenicolous ascomycetes are recognized only from asexual (anamorphic) characteristics, so the phylogenetic position of many groups has never been resolved. Here we present the first molecular phylogeny of Lichenoconium, a genus of strictly asexual, obligately lichenicolous species with broad geographic distributions and diverse host ecologies. We obtained nuclear and mitochondrial rDNA sequences from fungal cultures isolated from four species in the genus, including a new species, Lichenoconium aeruginosum sp. nov., collected in France, Luxembourg and Netherlands. Our multilocus phylogeny supports the monophyly of fungi in the genus Lichenoconium, and places the genus in the Dothideomycetes, an ascomycete class made up mainly of saprobes and plant-associated endophytes and pathogens. There are only a few recognized groups of lichen-formers in the Dothideomycetes, but Lichenoconium is not supported as being closely related to any of these, nor to any other recognized order within the Dothideomycetes. Given that Lichenoconium is but one of over 100 genera of anamorphic lichenicolous fungi, most of which have never been studied phylogenetically, we suggest that asexual lichenicolous fungi may represent novel and evolutionarily significant phylogenetic groups in the Kingdom Fungi.     

Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120.

A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli. The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATP synthase. This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E. coli. A unique feature of the Anabaena atp1 cluster is overlap between the coding regions for atpF and atpD. The atp1 cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1. The deduced translation products for the Anabaena sp. strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E. coli.     

Seven new species of Cis Latreille (Coleoptera: Ciidae) from southern Africa

Here we describe seven new species of Cis Latreille from southern Africa: Cis aster sp. nov., Cis makebae sp. nov., Cis mandelai sp. nov., Cis masekelai sp. nov., Cis neserorum sp. nov., Cis stalsi sp. nov. and Cis urbanae sp. nov. We also provide information on their host fungi and geographic distributions.     

Ascomycetes on Myrica gale in Sweden

The Ascomycete flora on Myrica gale has been investigated, mainly on the basis of Swedish material. It is found to be largely host specific. Four Discomycetes and nine Pyrenomycetes s. lat. are recorded as true, ± exclusive Myrica fungi; ten plurivorous Pyrenomycetes have occasionally been met with. The mycoflora on Myrica gale seems to be of a rather recent origin, composed of fairly modern species. Six new names are published: Gibbera gemmarum sp. nov., G. minutissima sp. nov., Melanomma myricae sp. nov., Semisphaeria sigmundii gen. nov. et sp. nov., Stomiopeltis myricae sp. nov., and Trichopezizella myricae comb. nov.     

Endophytic and epiphytic phyllosphere fungi of Camellia japonica: seasonal and leaf age-dependent variations

Seasonal and leaf age-dependent variations in the endophytic and epiphytic phyllosphere fungal assemblages of Camellia japonica were examined and compared. Live leaves of C. japonica were collected in four seasons (May, Aug, Nov, Feb), and fungi were isolated from healthy-looking leaves of 0, 1, 2 and 3 y old. The infection rate and total number of endophytic fungi increased May-Feb, and species richness of endophytes increased as leaves aged. In contrast the infection rate of epiphytic fungi was 100% for all leaf ages at every sampling date. The total number of epiphytic fungi isolated was greatest in May and lowest in Aug. The species richness of epiphytes did not differ significantly by season or leaf age. Eight fungal species were recorded as major phyllosphere fungi of C. japonica. Seasonal variations were detected for the frequencies of Colletotrichum gloeosporioides, C. acutatum, and epiphytes Pestalotiopsis sp.1, Aureobasidium pullulans, Phoma sp.1 and Ramichloridium sp., whereas the frequency of the endophyte Geniculosporium sp.1 varied with leaf age. The frequency of the epiphyte Cladosporium cladosporioides varied with both season and leaf age.     

Diversity of acaropathogenic fungi in Poland and other European countries

The occurrence, species diversity and some aspects of taxonomical affinity and host selectivity of acaropathogenic fungi associated with phytophagous, saprotrophic and predacious mites in Poland and other European countries were investigated on wild and cultivated plants, in insect feeding sites under the bark and in decayed wood. From among 33 species of fungi affecting mites only five species of Entomophthorales were separated and the most numerous were Neozygites floridana mostly on Tetranychus urticae, N. abacaridis on a few eriophyid species, and Conidiobolus coronatus attacking gamasid mites most frequently of the genus Dendrolaelaps. The most frequent mite pathogens occurring in mite communities on plants and in wood infested by insects were of the genus Hirsutella. Until now 13 of their form-species have been recognized in these habitats, but only H. kirchneri, H. necatrix and H. thompsonii (including its variety synnematosa) can be treated as exclusive oligophagous pathogens of phytophagous mites, though their potential host range seems to embrace only selected eriophyid or tarsonemid mites. Taxonomical differentiation of fungal strains was based on close morphological observations and molecular analysis of ITS region sequences. Two new species of acaropathogenic fungi were described in these studies. Hirsutella danubiensis sp. nov. was found in the tetranychid T. urticae, whereas H. vandergeesti sp. nov. affected phytoseiid mites of the genera Amblyseius, Neoseiulus, Seiulus and Typhlodromus, and the tarsonemid Tarsonemus lacustris.     

Endophytic fungi associated with leaves of Betulaceae in Japan

Diversity and species composition of endophytic fungi on leaves of 11 tree species in Betulaceae were studied, with reference to climatic, tree species, and seasonal variations. A total of 186 fungal isolates were obtained from 190 leaves collected in a subalpine forest, a cool temperate forest, and a subtropical forest in Japan, and were divided into 46 operational taxonomic units (OTUs) according to the base sequences of D1-D2 region of large subunit rDNA. The 2 most frequent OTUs were Muscodor sp. and Nemania sp. in Xylariaceae, followed by Gnomonia sp., Glomerella acutata , Apiosporopsis sp., Asteroma sp., and Cladosporium cladosporioides . The similarities of OTU composition in endophytic fungal assemblages on leaves of Betulaceae were generally low among the forests of different climatic regions. Fungal OTU compositions were relatively similar between 2 Betula species in the subalpine forest, whereas 7 tree species in the cool temperate forest were divided into 3 groups according to the similarity of endophytic fungal assemblages on the leaves, with 4 Carpinus species assigned into 2 of the 3 groups. The similarity of endophytic fungal assemblages between August and October was relatively high in the subalpine forest, whereas the seasonal changes were generally greater (i.e., the similarities among sampling dates were lower) in the cool temperate forest.     

New species and distribution records of Clavulina (Cantharellales, Basidiomycota) from the Guiana Shield

Two new species of Clavulina Schroet. (Clavulinaceae, Cantharellales, Basidiomycota) and new distribution records for Clavulina amazonensis Corner and Clavulina sprucei (Berk.) Corner are described from the Pakaraima Mountains of Guyana, in the central Guiana Shield region. These fungi occur in rainforests dominated by ectomycorrhizal (ECM) trees of the leguminous genus Dicymbe (Fabaceae subfam. Caesalpinioideae). Macromorphological, micromorphological and habitat data are provided for each species. Nuclear ribosomal DNA sequences of the internal transcribed spacer region and 28S subunit were obtained for new species and from representative collections of C. amazonensis and C. sprucei. The two new species, Clavulina kunmudlutsa sp. nov. and Clavulina tepurumenga sp. nov., constitute important edible fungi for the Patamona Amerindians. Our specimens of C. sprucei represent the first reports of the species since 1853 as well as a range extension of nearly 1500 km, while sequence data from basidiomata as well as ECM roots suggest that this taxon consists of a cryptic species complex.     

Eleven new species of Sugiyamaella and Candida from forest habitats

Eleven new yeasts from forest habitats are described from nucleotide sequence analysis of the nearly entire large subunit rRNA gene, ITS1-5.8S-ITS2 rRNA gene, mitochondrial small subunit rRNA gene, and the cytochrome oxidase II gene. All are members of the Sugiyamaella clade. Three are heterothallic species of Sugiyamaella and eight species are assigned to Candida. The Sugiyamaella species are: Su. americana sp. nov., type strain NRRL YB-2067 (CBS 10352), mating type a, and isotype NRRL YB-4197 (CBS 10353), mating type alpha; Su. chiloensis sp. nov., type strain NRRL Y-17646 (CBS 8168), mating type a, and isotype NRRL Y-27101 (CBS 5927), mating type alpha, anamorph Ca. bertae; Su. japonica sp. nov., type strain NRRL YB-2788 (CBS 10354), diploid, NRRL YB-2764 (CBS 10355), mating type a, and NRRL YB-2799 (CBS 10356), mating type alpha. The Candida species with type strains are: Ca. boreocaroliniensis sp. nov. NRRL YB-1835 (CBS 10344), Ca. floridensis sp. nov. NRRL YB-3827 (CBS 10350), Ca. grinbergsii sp. nov. NRRL Y-27117 (CBS 5924), Ca. lignohabitans sp. nov. NRRL YB-1473 (CBS 10342), Ca. marilandica sp. nov. NRRL YB-1847 (CBS 10346), Ca. marionensis sp. nov. NRRL YB-1336 (CBS 10341), Ca. neomexicana sp. nov. NRRL YB-2450 (CBS 10349), and Ca. pinicola sp. nov. NRRL YB-2263 (CBS 10348).     

Novel Cryphonectriaceae from La Réunion and South Africa,and their pathogenicity on <Emphasis Type="Italic">Eucalyptus</Emphasis>

Fungi in the Cryphonectriaceae are important canker pathogens of plants in the Melastomataceae and Myrtaceae (Myrtales). These fungi are known to undergo host jumps or shifts. In this study, fruiting structures resembling those of Cryphonectriaceae were collected and isolated from dying branches of Syzygium cordatum and root collars of Heteropyxis natalensis in South Africa, and from cankers on the bark of Tibouchina grandifolia in La Réunion. A phylogenetic species concept was used to identify the fungi using partial sequences of the large subunit and internal transcribed spacer regions of the nuclear ribosomal DNA, and two regions of the β-tubulin gene. The results revealed a new genus and species in the Cryphonectriaceae from South Africa that is provided with the name Myrtonectria myrtacearum gen. et sp. nov. Two new species of Celoporthe (Cel.) were recognised from La Réunion and these are described as Cel. borbonica sp. nov. and Cel. tibouchinae sp. nov. The new taxa were mildly pathogenic in pathogenicity tests on a clone of Eucalyptus grandis. Similar to other related taxa in the Cryphonectriaceae, they appear to be endophytes and latent pathogens that could threaten Eucalyptus forestry in the future.     

Nature and bioactivities of endolichenic fungi in Pseudocyphellaria sp., Parmotrema sp. and Usnea sp. at Hakgala montane forest in Sri Lanka

Aim:  The aim of this study was to investigate the nature and bioactivities of endolichenic fungi in three abundant lichens, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp. in the lower elevation of Hakgala montane forest in Sri Lanka.
Methods and Results:  Endolichenic fungal strains, fungi that live asymptomatically in the lichen thallus, much the same way as endophytic fungi live within healthy plant tissues, were isolated from three abundant lichen species, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp., at Hakgala montane forest in Sri Lanka, using the surface sterilization method. Nine endolichenic fungal strains were isolated from Parmotrema sp. and Usnea sp. separately, while 11 endolichenic fungi were recovered from the lichen Pseudocyphellaria sp. Isolation of endolichenic fungus Chrysosporium sp. 2 was common to all three lichen species. Substrate utilization patterns and antifungal activities of eight endolichenic fungal species were evaluated and the results revealed that all the test fungi were able to produce at least one enzyme to utilize the test substrates. Nigrospora sp., Chrysosporium sp. 1 and 2 and Cladosporium sp. showed antifungal activities on growth of some selected plant pathogenic fungi.
Conclusions:  Endolichenic fungal strains (29) were isolated from the lichens Parmotrema sp., Usnea sp. and Pseudocyphellaria sp. in Sri Lanka. Chrysosporium sp. 2 was common in all three lichens. Some of these endolichenic fungal strains showed antifungal activities against common plant pathogenic fungi and they are capable of utilizing the substrates by producing specific enzymes.
Significance and Impact of the Study:  The diversity and prevalence of the endolichenic fungi have not been studied extensively and this is the first report of isolation and identification of endolichenic fungi in lichens available in Sri Lanka.     

Quantitative analysis of phytopathogenic ascomycota on leaves of pedunculate oaks (Quercus robur L.) by real-time PCR

Leaves of oak trees are often infected by various pathogenic fungi. As it is difficult to remove such organisms quantitatively from the leaf surface and as it is often impossible to grow these organisms independently from their host, there are almost no molecular data available from these oak leaf specific pathogens. For the quantitative removal of the microorganisms a procedure was developed combining a wax and microorganism freezing method with a DNA extraction technique. For the development of a species specific detection, DNA of pathogenic filamentous fungi was isolated from hyphae of the upper leaf surface of Quercus robur. Three different species could be identified as (i) Cladosporium sp., (ii) Ramularia sp. and (iii) Microsphaera alphitoides by amplifying and sequencing an 18S-28S segment of their rDNA. For the final quantification a real-time PCR protocol was established allowing the species specific quantification of the three pathogenic filamentous fungi. The whole procedure was successfully applied to quantify the amount of the three species on oak leaves collected in autumn.     

Three new anascosporic genera of the Saccharomycotina: Danielozyma gen. nov., Deakozyma gen. nov. and Middelhovenomyces gen. nov

Three new non-ascosporic, ascomycetous yeast genera are proposed based on their isolation from currently described species and genera. Phylogenetic placement of the genera was determined from analysis of nuclear gene sequences for D1/D2 large subunit rRNA, small subunit rRNA, translation elongation factor-1α and RNA polymerase II, subunits B1 and B2. The new taxa are: Deakozyma gen. nov., type species Deakozyma indianensis sp. nov. (type strain NRRL YB-1937, CBS 12903); Danielozyma gen. nov., type species Danielozyma ontarioensis comb. nov. (type strain NRRL YB-1246, CBS 8502); D. litseae comb. nov. (type strain NRRL YB-3246, CBS 8799); Middelhovenomyces gen. nov., type species Middelhovenomyces tepae comb. nov. (type strain NRRL Y-17670, CBS 5115) and M. petrohuensis comb. nov. (type strain NRRL Y-17663, CBS 8173).