Aspergillus osmophilus sp. nov., and a new teleomorph for A. proliferans

A new species of Aspergillus and a new teleomorph for A. proliferans, both isolated from cereals in Iran, are described using morphological and molecular data. A combined sequence dataset of the ITS region, partial β-tubulin and partial calmodulin genes resolved the relationships of members of section Aspergillus largely in concordance with morphological traits of ascospores. Aspergillus osmophilus sp. nov. is differentiated from the closest species, A. xerophilus by possessing larger ascospores, conidia and associated fruiting bodies. Both species are strongly xerophilic and possess ascospores with lobate-reticulate convex surfaces. The newly discovered teleomorph for A. proliferans is characterized by delicately roughened ascospores with a shallow or distinct furrow and finely roughened to irregular equatorial crests.     

Phylogenetic relationship between different race representative populations of Fusarium oxysporum f. sp. ciceris in respect of translation elongation factor-1α, β-tubulin,and internal transcribed spacer region genes

Genetic diversity of 70 isolates of Fusarium oxysporum f. sp. ciceris originated from various states of India representing eight races causing wilt in chickpea (Cicer arietinum) was analyzed using translation elongation factor-1α (TEF-1α), β-tubulin, and internal transcribed spacer (ITS) gene regions. TEF-1α, β-tubulin, and ITS gene-specific markers produced ~720-, ~500-, and ~550-bp amplicons, respectively, in all the isolates of the pathogen. A phylogenetic tree constructed from the sequences generated in the present study along with the sequences of foreign isolates of Fusarium species available in NCBI database sharing more than 90 % nucleotide sequence similarity grouped the isolates into two major clusters. Most of the isolates of the present study showed more or less similar grouping pattern in case of the three gene sequences. Each group had the isolates representing different races as well as place of origin indicating low level of diversity among the isolates in respect of these gene sequences. Except TEF-1α, the groups generated by β-tubulin and ITS gene sequences did not correspond to the state of origin and races of the pathogen. However, the groups of TEF-1α partially corresponded to the place of origin as well as races of the pathogen. The isolates did not show any race-specific grouping patterns; however, most of the isolates representing race 1 clustered separately.     

Molecular identification and biodiversity of potential allergenic molds (Aspergillus and Penicilium) in the poultry house: first report

The production of poultry has proved to be a significant source of fungi. Penicillium and Aspergillus are among the most important species, because they can cause irritations, infections, allergies, spoilage of food and beverages and are able to produce dangerous mycotoxins. Detection and identification of the species in these genera is very significant, because it provides relevant information about the properties of the responsible strains. Therefore, their taxonomic was determined using a phenotypic and molecular (ITS and partial β-tubulin sequences) methods. Investigations were conducted in the poultry house located near Wroc?aw in Lower Silesia (Poland). Using morphological analyses, 215 fungal strains were identified. Among them, 56 belonged to the Penicilium and Aspergillus genera. The results obtained from sequence analysis corresponded well with the morphological identification. Classification at species level was possible in most cases using the sequence data. Most isolates belong to Penicilium genus, with the follow dominating species (P. chrysogenum, P. polonicum and P. olsonii).     

Aspergillus bertholletius sp. nov. from Brazil Nuts

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial β-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial β-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).     

Diverse Deep-Sea Fungi from the South China Sea and Their Antimicrobial Activity

We investigated the diversity of fungal communities in nine different deep-sea sediment samples of the South China Sea by culture-dependent methods followed by analysis of fungal internal transcribed spacer (ITS) sequences. Although 14 out of 27 identified species were reported in a previous study, 13 species were isolated from sediments of deep-sea environments for the first report. Moreover, these ITS sequences of six isolates shared 84–92 % similarity with their closest matches in GenBank, which suggested that they might be novel phylotypes of genera Ajellomyces, Podosordaria, Torula, and Xylaria. The antimicrobial activities of these fungal isolates were explored using a double-layer technique. A relatively high proportion (56 %) of fungal isolates exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among four marine pathogenic microbes (Micrococcus luteus, Pseudoaltermonas piscida, Aspergerillus versicolor, and A. sydowii). Out of these antimicrobial fungi, the genera Arthrinium, Aspergillus, and Penicillium exhibited antibacterial and antifungal activities, while genus Aureobasidium displayed only antibacterial activity, and genera Acremonium, Cladosporium, Geomyces, and Phaeosphaeriopsis displayed only antifungal activity. To our knowledge, this is the first report to investigate the diversity and antimicrobial activity of culturable deep-sea-derived fungi in the South China Sea. These results suggest that diverse deep-sea fungi from the South China Sea are a potential source for antibiotics’ discovery and further increase the pool of fungi available for natural bioactive product screening.     

Penicillium daejeonium sp. nov., a new species isolated from a grape and schisandra fruit in Korea

Two isolates of monoverticillate Penicillium species were collected from a grape and schisandra fruit in Korea. Multigene phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and genes encoding β-tubulin (benA) and calmodulin (cmd), as well as morphological analyses revealed that the two isolates are members of the P. sclerotiorum complex in Penicillium subgenus Aspergilloides, but different from species of the P. sclerotiorum complex. The isolates are closely related to P. cainii, P. jacksonii, and P. viticola in terms of their multigene phylogeny, but their colony and conidiophore morphologies differ from those of closely related species. The name P. daejeonium is proposed for this unclassified new species belonging to the P. sclerotiorum complex in subgenus Aspergilloides.     

Aspergillus siamensis sp. nov. from soil in Thailand

A new species of Aspergillus section Fumigati, Aspergillus siamensis, isolated from coastal forest soil in Samaesarn island, Chonburi province, Eastern Thailand, is described and illustrated. This species is characterized by its broadly lenticular ascospores with two wide equatorial crests and finely spinulose and rugose convex surfaces and produced pale pinkish exudates after 14 d incubation on Czapek agar. The validation of this new species was supported further by analyses of the β-tubulin, calmodulin and actin gene sequences.     

Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity

The increasing availability of DNA sequence data enables exciting new opportunities for fungal ecology. However, it amplifies the challenge of how to objectively classify the diversity of fungal sequences into meaningful units, often in the absence of morphological characters. Here, we test the utility of modern multilocus Bayesian coalescent-based methods for delimiting cryptic fungal diversity in the orchid mycorrhiza morphospecies Serendipita vermifera. We obtained 147 fungal isolates from Caladenia, a speciose clade of Australian orchids known to associate with Serendipita fungi. DNA sequence data for 7 nuclear and mtDNA loci were used to erect competing species hypotheses by clustering isolates based on: (a) ITS sequence divergence, (b) Bayesian admixture analysis, and (c) mtDNA variation. We implemented two coalescent-based Bayesian methods to determine which species hypothesis best fitted our data. Both methods found strong support for eight species of Serendipita among our isolates, supporting species boundaries reflected in ITS divergence. Patterns of host plant association showed evidence for both generalist and specialist associations within the host genus Caladenia. Our findings demonstrate the utility of Bayesian species delimitation methods and suggest that wider application of these techniques will readily uncover new species in other cryptic fungal lineages.     

Preliminary Insights into the Phylogeography of Six Aquatic Hyphomycete Species

Aquatic hyphomycetes occur worldwide on a wide range of plant substrates decomposing in freshwaters, and are known to play a key role in organic matter turnover. The presumed worldwide distribution of many aquatic hyphomycete species has been based on morphology-based taxonomy and identification, which may overlook cryptic species, and mask global-scale biogeographical patterns. This might be circumvented by using DNA sequence data. The internal transcribed spacer (ITS) region from rDNA was recently designated as the most suitable barcode for fungal identification. In this study, we generated ITS barcodes of 130 isolates belonging to 6 aquatic hyphomycete species (Anguillospora filiformis, Flagellospora penicillioides, Geniculospora grandis, Lunulospora curvula, Tetrachaetum elegans and Tricladium chaetocladium), and collected from streams of Southwest Europe (86 isolates) and East Australia (44 isolates). European and Australian populations of 4 species (A. filiformis, F. penicillioides, G. grandis and T. elegans) grouped into different clades, and molecular diversity indices supported significant differentiation. Continents did not share haplotypes, except for T. chaetocladium. Overall results show substantial population diversity for all tested species and suggests that the biogeography of aquatic hyphomycetes may be species-specific.     

Aspergillus felis sp. nov., an Emerging Agent of Invasive Aspergillosis in Humans,Cats, and Dogs

We describe a novel heterothallic species in Aspergillus section Fumigati, namely A. felis (neosartorya-morph) isolated from three host species with invasive aspergillosis including a human patient with chronic invasive pulmonary aspergillosis, domestic cats with invasive fungal rhinosinusitis and a dog with disseminated invasive aspergillosis. Disease in all host species was often refractory to aggressive antifungal therapeutic regimens. Four other human isolates previously reported as A. viridinutans were identified as A. felis on comparative sequence analysis of the partial β-tubulin and/or calmodulin genes. A. felis is a heterothallic mold with a fully functioning reproductive cycle, as confirmed by mating-type analysis, induction of teleomorphs within 7 to 10 days in vitro and ascospore germination. Phenotypic analyses show that A. felis can be distinguished from the related species A. viridinutans by its ability to grow at 45°C and from A. fumigatus by its inability to grow at 50°C. Itraconazole and voriconazole cross-resistance was common in vitro.     

Aspergillus huiyaniae sp. nov., a teleomorphic species in sect. Fumigati isolated from desert soil in China

Aspergillus huiyaniae, a new teleomorphic species isolated from desert soil in Xinjiang, China, was described and illustrated. Aspergillus huiyaniae is characterized by its yellowish white to pale yellow cleistothecia, broadly lenticular ascospores with two equatorial crests and irregularly ribbed to slightly reticulate convex surfaces, and subglobose to ovate or broadly ellipsoidal conidia with smooth walls. This species was supported further by the analyses of the β-tubulin, calmodulin and actin gene sequences.     

Colletotrichum fructicola and C. siamense are involved in chilli anthracnose in India

Chilli anthracnose is a major problem in India and worldwide. In this study, we investigated the phylogenetic relationships of 52 fungal isolates associated with chilli anthracnose in southern India. All the 52 isolates were sequenced for partial ITS/5.8S rRNA and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes and showed affinities with Colletotrichum siamense and C. fructicola within Colletotrichum. gloeosporioides species complex. Further, a reduced subset of 17 selected isolates was made and in a maximum parsimony analysis of a multigene data-set including partial ITS/5.8S rRNA, actin (act), calmodulin (cal), chitin synthase (chs1), gapdh and β-tubulin (tub2) gene sequence data, these fungal isolates clustered with the type strain of C. fructicola, except for strain MTCC 3439 that showed phylogenetic affinities with C. siamense. The pathogenicity tests involving two representative isolates: UASB-Cg-14 and MTCC 3439, confirmed the involvement of C. fructicola and C. siamense in the development of disease symptoms on fresh chilli fruits. This is the first report of the association of C. fructicola and C. siamense in causing chilli anthracnose in India.     

Proven Invasive Pulmonary Aspergillosis in Stem Cell Transplant Recipient Due to <Emphasis Type="Italic">Aspergillus sublatus</Emphasis>, a Cryptic Species of <Emphasis Type="Italic">A. nidulans</Emphasis>

Invasive fungal disease represents one of the severe complications in haematopoietic stem cell transplant recipients. We describe a case of a patient treated for relapse of chronic lymphoblastic leukaemia 6 years after HSCT. The patient was treated for invasive pulmonary aspergillosis but died 3 months later from multiple organ failures consisting of haemorrhagic necrotizing fungal pneumonia, refractory chronic hepatic graft versus host disease and cytomegalovirus hepatitis. Autopsy samples revealed histopathological evidence of fungal hyphae and an unusual Aspergillus nidulans-like species was isolated in pure culture. More precise identification was achieved by using scanning electron microscopy of ascospores and sequencing of calmodulin gene, and the isolate was subsequently re-identified as A. sublatus (section Nidulantes) and showed good in vitro susceptibility against all classes of antifungals. Commonly used ITS rDNA region and β-tubulin gene fail to discriminate A. sublatus from related pathogenic species, especially A. quadrilineatus and A. nidulans. Although this is the first case of proven IPA attributed to A. sublatus, we demonstrated that at least some previously reported infections due to A. quadrilineatus were probably caused by this cryptic species.     

Molecular analysis of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within β-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.     

A PCR-based method to characterise and identify benzimidazole resistance in Helminthosporium solani

Control of Helminthosporium solani, the cause of silver scurf in potato tubers, has been impaired by selection of benzimidazole-resistant strains as a result of repeated use of the fungicide thiabendazole. Identification of thiabendazole-resistant strains of H. solani by conventional techniques takes several weeks. Primers designed from conserved regions of the fungal β-tubulin gene were used to PCR amplify and sequence a portion of the gene. A point mutation was detected at codon 198 in thiabendazole-resistant isolates causing a change in the amino acid sequence from glutamic acid to alanine or glutamine. Species-specific PCR primers designed to amplify this region were used in conjunction with a restriction endonuclease to cause cleavage in sensitive isolates only and thus provide a rapid diagnostic test to differentiate field isolates.     

A beauvericin hot spot in the genus Isaria

Beauvericin is a naturally occurring cyclohexadepsipeptide originally described from Beauveria bassiana but also reported from several Fusarium species as well as members of the genus Isaria. Twenty-six isolates of Isaria species and its Cordyceps teleomorph, and ten taxonomically close strains including Beauveria, Nomuraea and Paecilomyces species were sequenced and tested for beauvericin production. Trees using ITS rDNA and β-tubulin sequence data were constructed and used to infer the phylogenetic distribution of beauvericin production. A group comprising Isaria tenuipes and its known teleomorph Cordyceps takaomontana, Isaria cicadae and its Cordyceps teleomorph, Isaria japonica and Isaria fumosorosea, showed positive beauvericin production which correlated well with combined ITS rDNA and β-tubulin phylogenies. The results suggested that beauvericin can serve as a chemotaxonomic marker for these limited species of the I. tenuipes complex.     

Analysis of Claviceps africana and C. sorghi from India using AFLPs,EF-1α gene intron 4, and β-tubulin gene intron 3

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1α gene intron 4 and β-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1α gene intron 4, the β-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.     

Intraspecific Differentiation of Colletotrichum gloeosporioides sensu lato Based on In Silico Multilocus PCR-RFLP Fingerprinting

Colletotrichum gloeosporioides sensu lato is one of the most common and widely distributed plant pathogens in the world. Understanding fungal biodiversity is hinged on accurate identification and delimitation at the inter- and intraspecific levels. Sequences of the ITS1-5.8S-ITS2 region (ITS), β-tubulin (TUB), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of 30 C. gloeosporioides sensu lato isolates, collected from anthracnose infected papaya fruits grown in the main production areas in Trinidad, were analyzed by in silico PCR-RFLP analysis with the aim of identifying which gene region(s) had the highest level of intraspecific polymorphism. Restriction site polymorphisms generated from 13 restriction enzymes enabled the identification of specific enzymes that were successful at intraspecific discrimination of the C. gloeosporioides isolates. Genetic distance values were reflective of the level of polymorphisms obtained for the four different gene regions. In both cases (calculated genetic distance and percentage of polymorphic loci from RFLP profiles), ACT and ITS gene regions had the highest level of restriction site polymorphisms and genetic diversity, GPDH and TUB had the lowest. Cluster analysis based on PCR-RFLP genetic distance data revealed sub-specific placement of the isolates which appeared to be gene-dependent. The implications of these findings are discussed relative to biodiversity monitoring and the need for multilocus, polyphasic investigations which must take into account the possibility of exaggerated estimates of genetic diversity.