Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

The plant growth-promoting fungus Aspergillus ustus promotes growth and induces resistance against different lifestyle pathogens in Arabidopsis thaliana

To deal with pathogens, plants have evolved sophisticated mechanisms including constitutive and induced defense mechanisms. Phytohormones play important roles in plant growth and development, as well as in the systemic response induced by beneficial and pathogen microorganisms. In this work, we identified an Aspergillus ustus isolate that promotes growth and induces developmental changes in Solanum tuberosum and Arabidopsis thaliana. A. ustus inoculation on A. thaliana and S. tuberosum roots induced an increase in shoot and root growth, and lateral root and root hair numbers. Assays performed on Arabidopsis lines to measure reporter gene expression of auxin-induced/ repressed or cell cycle controlled genes (DR5 and CycB1, respectively) showed enhanced GUS activity, when compared with mock-inoculated seedlings. To determine the contribution of phytohormone signaling pathways in the effect elicited by A. ustus, we evaluated the response of a collection of hormone mutants of Arabidopsis defective in auxin, ethylene, cytokinin, or abscisic acid signaling to the inoculation with this fungus. All mutant lines inoculated with A. ustus showed increased biomass production, suggesting that these genes are not required to respond to this fungus. Moreover, we demonstrated that A. ustus synthesizes auxins and gibberellins in liquid cultures. In addition, A. ustus induced systemic resistance against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Pseudomonas syringae DC3000, probably through the induction of the expression of salicylic acid, jasmonic acid/ethylene, and camalexin defense-related genes in Arabidopsis.     

Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species

We examined nine Aspergillus japonicus isolates and 10 Aspergillus aculeatus isolates by using molecular and biochemical markers, including DNA sequences of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length polymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene could not be used to distinguish between A. japonicus and A. aculeatus but did show that these two taxa are more closely related to each other than to other species of black aspergilli. Aspergillus niger pyruvate kinase (pkiA) and pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which were used as probes in the RFLP analysis, revealed clear polymorphism between these two taxa. The A. niger pkiA and pelA probes placed six strains in an A. japonicus group and 12 isolates in an A. aculeatus group, which exhibited intraspecific variation when they were probed with the pelA gene. The secondary-metabolite profiles supported division of the isolates into the two species and differed from those of other black aspergilli. The strains classified as A. japonicus produced indole alkaloids and a polar metabolite, while the A. aculeatus isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid. A. aculeatus CBS 114.80 showed specific RFLP patterns for all loci examined. The secondary-metabolite profile of strain CBS 114.80 also differed from those of A. japonicus and A. aculeatus. Therefore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates of black aspergilli, which are important in relation to industrial use and legal protection of these organisms.     

Polyphasic taxonomy of Aspergillus fumigatus and related species

The variability within Aspergillus fumigatus Fresenius and related species was examined using macro-, micro-morphology, growth temperature regimes and extrolite patterns. In addition, DNA analyses including partial beta-tubulin, calmodulin and actin gene sequences were used. Detailed examination of strains, considered as A. fumigatus earlier, showed that they could be divided into four groups including A. fumigatus sensu stricto, A. lentulus and two new species. The intraspecific genetic variability within A. fumigatus sensu stricto was low, the sequence differences among 23 strains of the species was at most two bases in each partial beta-tubulin and calmodulin gene. However, intraspecific morphological diversity within the species was high and delineation of the species was equivocal. Therefore, beta-tubulin and calmodulin gene sequences could be critical determinants for the delineation of the A. fumigatus sensu stricto species. A. lentulus including isolates from clinical origin, Korean soil and from a dolphin clustered into an isolated group based on beta-tubulin, calmodulin and actin gene sequences, differing from A. fumigatus by morphological characters, growth temperature and extrolite profile. A. lentulus produces the extrolites auranthine, cyclopiazonic acid, a dimeric indole of unknown structure, neosartorin, some pyripyropens, terrein and some tryptoquivalins and tryptoquivalons. Two pair of isolates (CBS 117194, 117186 and 117520, 117519) clustered into separate groups from A. fumigatus and the other Aspergillus section Fumigati species, including the teleomorph Neosartorya, are proposed as two new species. A. fumigatiaffinis spec. nov. produces the extrolites auranthine, cycloechinulin, helvolic acid, neosartorin, palitantin, pyripyropens, tryptoquivalins and tryptoquivalons, and A. novofumigatus spec. nov. produces the extrolites cycloechinuline, helvolic acid, neosartorin, palitantin and terrein.     

Ribotoxin genes in isolates of Aspergillus section Clavati

Ribotoxins are ribosome inactivator proteins with high specificity against the sarcin/ricin domain of the 28S ribosomal RNA. We examined the presence of ribotoxin genes in isolates of species recently assigned to Aspergillus section Clavati using specific primer pairs. All species assigned to this section have been found to carry ribotoxin genes. Phylogenetic analysis of the sequences of the amplified gene fragments allowed us to classify the genes to different groups including the alpha-sarcin, gigantin, c-sarcin and mitogillin/restrictocin families. Two species, A. longivesica and N. acanthosporus produced ribotoxins which were only distantly related to gigantins and c-sarcins, respectively. Comparison of the protein sequences of the genes to known ribotoxin sequences revealed that all of them carry the presumed catalytic residues of ribotoxins, the cystein residues, and also the two Trp residues of alpha-sarcin conserved in all ribotoxins known so far. These data indicate that these genes probably encode active ribotoxins. Further studies are in progress to examine the secretion and activities of these new ribotoxins.     

New species of Aspergillus producing sterigmatocystin.

A number of species belonging to the genus Aspergillus were evaluated for their toxicity to ducklings and the ability to produce sterigmatocystin. Three new species capable of producing sterigmatocystin were found, namely, Aspergillus aurantio-brunneus, Aspergillus quadrilineatus, and Aspergillus ustus. All three were toxic to ducklings. The production of sterigmatocystin by Aspergillus rugulosus was confirmed, and the toxicity of Aspergillus stellatus and Aspergillus multicolor is described.     

New species of Aspergillus producing sterigmatocystin.

A number of species belonging to the genus Aspergillus were evaluated for their toxicity to ducklings and the ability to produce sterigmatocystin. Three new species capable of producing sterigmatocystin were found, namely, Aspergillus aurantio-brunneus, Aspergillus quadrilineatus, and Aspergillus ustus. All three were toxic to ducklings. The production of sterigmatocystin by Aspergillus rugulosus was confirmed, and the toxicity of Aspergillus stellatus and Aspergillus multicolor is described.     

Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the internal transcribed spacer regions and the 5.8 S rRNA gene

Phenotypic features and sequences of the internal transcribed spacer (ITS) regions and the 5.8 S rRNA gene of type or neotype strains and other isolates of the 17 species currently assigned to Aspergillus section Circumdati and some potentially related species were analyzed. Parsimony analysis of sequence data indicated that Aspergillus section Circumdati is paraphyletic. Aspergillus campestris, A. lanosus, and A. dimorphicus with A. sepultus were found to be more closely related to Aspergillus sections Candidi, Flavi, and Cremei, respectively. These results were also supported by phenotypic data. A. robustus and A. ochraceoroseus were found not to be related to any of the species examined. Species of the proposed revised Aspergillus section Circumdati formed two main clades, which could also be distinguished based on phenotypic methods. Phylogenetic analysis of sequence data of other isolates assigned to species of the revised section indicates that either some of these isolates were misidentified or species concepts of A. ochraceus, A. melleus, and A. petrakii should be reconsidered.     

Delineation of the European Armillaria species based on the sequences of the internal transcribed spacer (ITS) of ribosomal DNA

Variation within the internal transcribed spacer (ITS) of the ribosomal RNA gene of 15 isolates representing seven European Armillaria species, was examined by sequencing of the PCR-amplified products. The analysis of an 744-bp region showed that the 5.8S gene appeared to be highly conserved in the 15 isolates and in other Basidiomycetes and Ascomycetes, whereas ITS1 and especially ITS2 spacers exhibited polymorphisms due to base substitutions, insertions or deletions of up to eight nucleotides. An initial dendrogram for the full sequence was drawn using cluster analysis (UPGMA), and a tree was constructed using the maximum parsimony method. Both methods indicated that the isolates could be divided into four major groups. One group, consisting of A. ectypa , was distinct from all the other species. Examination of the other groups indicated that A. tabescens and A. mellea were in a separated cluster, with a significant variation between the two isolates of the latter species. A. gallica and A. cepistipes constituted another closely related group distinguishable from A. ostoyae and A. borealis , these latter two species exhibiting the highest similarity. The results are consistent with, and discussed in regard to, the relationships estimated previously by pairing tests, morphological and physiological comparisons, as well as by restriction fragment length polymorphism of the rDNA.     

Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi

Most aspergilli that produce aflatoxin are members of Aspergillus section Flavi, however isolates of several Aspergillus species not closely related to section Flavi also have been found to produce aflatoxin. Two of the species, Aspergillus ochraceoroseus and an undescribed Aspergillus species SRRC 1468, are morphologically similar to members of Aspergillus section Circumdati. The other species have Emericella teleomorphs (Em. astellata and an undescribed Emericella species SRRC 2520) and are morphologically distinctive in having ascospores with large flanges. All these aflatoxin-producing isolates were from tropical zones near oceans, and none of them grew on artificial media at 37 C. Aflatoxins and sterigmatocystin production were quantified by high-pressure liquid chromatography (HPLC) and confirmed by HPLC-mass spectrometry (LC-MS) detection. Phylogenetic analyses were conducted on these four species using A. parasiticus and Em. nidulans, (which produce aflatoxin and the aflatoxin precursor sterigmatocystin, respectively) for comparison. Two aflatoxin/sterigmatocystin biosynthesis genes and the beta tubulin gene were used in the analyses. Results showed that of the new aflatoxin-producers, Aspergillus SRRC 1468 forms a strongly supported clade with A. ochraceoroseus as does Emericella SRRC 2520 with Em. astellata SRRC 503 and 512.     

Occurrence of Indole Alkaloids among Secondary Metabolites of Soil Aspergillus Spp.

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine B, which was formed by A. fumigatus. -Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Properties of Aspergillus tamarii,A. caelatus and related species from acidic tea field soils in Japan

Fungi in Aspergillus section Flavi include both aflatoxin producers and non-producers. Aspergillus caelatus is a recently described non-aflatoxigenic species in this section, which has some common characteristics with A. tamarii, such as yellowish brown color and double walled spores. In contrast to the morphological similarities, all of the A. caelatus isolates tested produced no cyclopiazonic acid whereas most isolates of A. tamarii produce this compound. There are six nucleotide differences that distinguish the DNA sequences of these two species in the regions of ITS1, ITS2, 5.8S rDNA and 28S rDNA and this is a consistent difference. Both species were isolated from acidified field soils, but A. tamarii isolates were more common than A. caelatus in highly acidic soils. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions,and description of Thauera phenylacetica sp. nov., Thauera aminoaromaticasp. nov., and Azoarcus buckelii sp. nov

Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5-1 and B5-2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromaticasp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcus anaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.     

An amendment of Aspergillus section Candidi based on chemotaxonomical evidence

A novel 2,2'-epoxy-terphenyllin, candidusin C, in addition to the well known secondary metabolites terphenyllin, 3-hydroxyterpenyllin and chlorflavonin, has been isolated from the chemically unexplored fungus Aspergillus campestris. The latter three are known secondary metabolites from Aspergillus candidus and therefore a large number of Aspergilli were screened for production of these compounds to see whether they could be regarded as chemotaxonomical indicators of section membership in the monotypic Aspergillus section Candidi. The results indicated that A. campestris and A. taichungensis should be placed in Candidi and this was further confirmed by morphological and physiological similarities. Three species outside the section Candidi produced candidusin related secondary metabolites: Aspergillus arenarius, A. ellipticus and Penicillium raistrickii. Chlorflavonin, however, was only found in section Candidi.     

Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal

Three new aflatoxin-producing species belonging to Aspergillus section Flavi are described. They are Aspergillus mottae, A. sergii and A. transmontanensis. These species were isolated from Portuguese almonds and maize. An investigation examined morphology, extrolite production and DNA sequence data to characterize these isolates and describe the new species. Phylogenetic analysis showed that A. transmontanensis and A. sergii form a clade with A. parasiticus whereas A. mottae shares a most recent common ancestor with the combined A. flavus and A. parasiticus clade.     

Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus

In a prior study, we identified seven clinical isolates of an Aspergillus sp. that were slow to sporulate in multiple media and demonstrated decreased in vitro susceptibilities to multiple antifungals, including amphotericin B, itraconazole, voriconazole, and caspofungin. These isolates were initially considered to be variants of Aspergillus fumigatus because of differences in mitochondrial cytochrome b sequences and unique randomly amplified polymorphic DNA PCR patterns (S. A. Balajee, M. Weaver, A. Imhof, J. Gribskov, and K. A. Marr, Antimicrob. Agents Chemother. 48: 1197-1203, 2004). The present study was performed to clarify the taxonomic status of these organisms by phylogenetic analyses based on multilocus sequence typing of five genes (the beta-tubulin gene, the rodlet A gene, the salt-responsive gene, the mitochondrial cytochrome b gene, and the internal transcribed spacer regions). Results revealed that four of the seven variant isolates clustered together in a clade very distant from A. fumigatus and distinct from other members of the A. fumigatus group. This new clade, consisting of four members, was monophyletic with strong bootstrap support when the protein-encoding regions were analyzed, indicating a new species status under the phylogenetic species concept. Phenotype studies revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48 degrees C. Our findings suggest the presence of a previously unrecognized, potentially drug-resistant Aspergillus species that we designate A. lentulus.     

Presence of a single genotype of the newly described species Mycobacterium immunogenum in industrial metalworking fluids associated with hypersensitivity pneumonitis

Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.     

Taxonomy and phylogeny of new wood- and soil-inhabiting Sporothrix species in the Ophiostoma stenoceras-Sporothrix schenckii complex

Sporothrix, one of the anamorph genera of Ophiostoma, includes the important human pathogen S. schenckii and various fungi associated with insects and sap stain of wood. A survey of fungi from wood utility poles in South Africa yielded two distinct groups of Sporothrix isolates from different geographical areas. DNA sequence and morphological data derived in this study showed that isolates in these groups represent two novel species in the S. schenckii-O. stenoceras species complex. A new species isolated from pine poles and rosebush wood and phylogenetically closely related to S. pallida is described here as Sporothrix stylites. Phylogenetic analyses also confirmed the synonymy of S. albicans and S. nivea with S. pallida. Sporothrix stylites and S. pallida also are related closely to the isolates from soil, previously treated as "environmental" isolates of S. schenckii. Soil isolates are clearly distinct from human isolates of S. schenckii. We describe the former here as Sporothrix humicola. The isolates from eucalypt poles group peripheral to most other species in the S. schenckii-O. stenoceras complex and are newly described as Sporothrix lignivora. Phylogenetic analyses of sequences of isolates from soil and wood together with those of clinical isolates showed that the human-pathogenic strains form an aggregate of several cryptic species.