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SummaryFumonisins are a family of carcinogenic secondary metabolites produced by members of the Fusarium fujikuroi species complex (FFSC) and rare strains of Fusarium oxysporum. In Fusarium, fumonisin biosynthetic genes (FUM) are clustered, and the cluster is uniform in gene organization. Here, sequence analyses indicated that the cluster exists in five different genomic contexts, defining five cluster types. In FUM gene genealogies, evolutionary relationships between fusaria with different cluster types were largely incongruent with species relationships inferred from primary-metabolism (PM) gene genealogies, and FUM cluster types are not trans-specific. In addition, synonymous site divergence analyses indicated that three FUM cluster types predate diversification of FFSC. The data are not consistent with balancing selection or interspecific hybridization, but they are consistent with two competing hypotheses: (i) multiple horizontal transfers of the cluster from unknown donors to FFSC recipients and (ii) cluster duplication and loss (birth and death). Furthermore, low levels of FUM gene divergence in F. bulbicola, an FFSC species, and F. oxysporum provide evidence for horizontal transfer of the cluster from the former, or a closely related species, to the latter. Thus, uniform gene organization within the FUM cluster belies a complex evolutionary history that has not always paralleled the evolution of Fusarium.

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The still-high mortality for lung cancer urgently requires the availability of new, noninvasive diagnostic tools for use in early diagnosis and screening programs. Recently, exhaled breath condensate (EBC) has been proposed as a useful tool to obtain biological information on lung cancer disease. This study provides, for the first time, evidence that DNA alterations already described in lung cancer are detectable in EBC from patients with non-small cell lung cancer (NSCLC) and in healthy subjects. Thirty patients with histologic evidence of NSCLC and 20 healthy subjects were enrolled in the present study. All subjects had allelotyping analysis of DNA from EBC (EBC-DNA) and from whole blood (WB-DNA) of a selected panel of five microsatellites (D3S2338, D3S1266, D3S1300, D3S1304, D3S1289) located in chromosomal region 3p. Results from healthy subjects and subjects with cancer, and from EBC and WB, were compared. In addition, the relationships with smoking habit and clinicopathologic tumor features were considered. Microsatellite alterations (MAs) were found in 53% of EBC-DNA and in 10% of WB-DNA loci investigated in patients with NSCLC (p < 10(-6)); conversely, MAs were present only in 13% of EBC-DNA and in 2% of WB-DNA informative loci in healthy subjects. In patients with NSCLC, a direct association between number of MAs detected in EBC-DNA and tobacco consumption was observed. We conclude that EBC-DNA is highly sensitive in detecting MA information unique to patients with lung cancer. Furthermore, MA information seems to be directly related with tobacco consumption, and is potentially applicable to screening and early diagnostic programs for patients with NSCLC.

The ability to produce fumonisin mycotoxins varies among members of the black aspergilli. Previously, analyses of selected genes in the fumonisin biosynthetic gene (fum) cluster in black aspergilli from California grapes indicated that fumonisin-nonproducing isolates of Aspergillus welwitschiae lack six fum genes, but nonproducing isolates of Aspergillus niger do not. In the current study, analyses of black aspergilli from grapes from the Mediterranean Basin indicate that the genomic context of the fum cluster is the same in isolates of A. niger and A. welwitschiae regardless of fumonisin-production ability and that full-length clusters occur in producing isolates of both species and nonproducing isolates of A. niger. In contrast, the cluster has undergone an eight-gene deletion in fumonisin-nonproducing isolates of A. welwitschiae. Phylogenetic analyses suggest each species consists of a mixed population of fumonisin-producing and nonproducing individuals, and that existence of both production phenotypes may provide a selective advantage to these species. Differences in gene content of fum cluster homologues and phylogenetic relationships of fum genes suggest that the mutation(s) responsible for the nonproduction phenotype differs, and therefore arose independently, in the two species. Partial fum cluster homologues were also identified in genome sequences of four other black Aspergillus species. Gene content of these partial clusters and phylogenetic relationships of fum sequences indicate that non-random partial deletion of the cluster has occurred multiple times among the species. This in turn suggests that an intact cluster and fumonisin production were once more widespread among black aspergilli.

A novel species, Aspergillus uvarum sp. nov., is described within Aspergillus section Nigri. This species can be distinguished from other black aspergilli based on internal transcribed spacers (ITS), b-tubulin and calmodulin gene sequences, by AFLP analysis and by extrolite profiles. Aspergillus uvarum sp. nov. isolates produced secalonic acid, common to other Aspergillus japonicus-related taxa, and geodin, erdin and dihydrogeodin, which are not produced by any other black aspergilli. None of the isolates were found to produce ochratoxin A. The novel species is most closely related to two atypical strains of Aspergillus aculeatus, CBS 114.80 and CBS 620.78, and was isolated from grape berries in Portugal, Italy, France, Israel, Greece and Spain.

DNA-based phylogenetic analyses have resolved the fungal genus Fusarium into multiple species complexes. The F. incarnatum-equiseti species complex (FIESC) includes fusaria associated with several diseases of agriculturally important crops, including cereals. Although members of FIESC are considered to be only moderately aggressive, they are able to produce a diversity of mycotoxins, including trichothecenes, which can accumulate to harmful levels in cereals. High levels of cryptic speciation have been detected within the FIESC. As a result, it is often necessary to use approaches other than morphological characterization to distinguish species. In the current study, we used a polyphasic approach to characterize a collection of 69 FIESC isolates recovered from cereals in Europe, Turkey, and North America. In a species phylogeny inferred from nucleotide sequences from four housekeeping genes, 65 of the isolates were resolved within the Equiseti clade of the FIESC, and four isolates were resolved within the Incarnatum clade. Seven isolates were resolved as a genealogically exclusive lineage, designated here as FIESC 31. Phylogenies based on nucleotide sequences of trichothecene biosynthetic genes and MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) were largely concordant with phylogeny inferred from the housekeeping gene. Finally, Liquid Chromatography (Time-Of-Flight) Mass Spectrometry [LC-(TOF-)MS(/MS)] revealed variability in mycotoxin production profiles among the different phylogenetic species investigated in this study.

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