DNA-based phylogenetic analyses have resolved the fungal genus Fusarium into multiple species complexes. TheF. incarnatum-equiseti species complex (FIESC) includes fusaria associated with several diseases of agriculturallyimportant 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 harmfullevels in cereals. High levels of cryptic speciation have been detected within the FIESC. As a result, it is often necessaryto 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 housekeepinggenes, 65 of the isolateswere resolvedwithin the Equiseti clade of the FIESC, and four isolateswere resolvedwithinthe Incarnatum clade. Seven isolates were resolved as a genealogically exclusive lineage, designated here asFIESC 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 withphylogeny 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 phylogeneticspecies investigated in this study.

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger 'aggregate' represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins ?-tubulin (benA), calmodulin (CaM), and the translation elongation factor-1 alpha (TEF-1?) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B2, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-?-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.

Fumonisins are a family of carcinogenic secondarymetabolites produced by members of the Fusariumfujikuroi species complex (FFSC) and rare strains ofFusarium oxysporum. In Fusarium, fumonisin biosyntheticgenes (FUM) are clustered, and the cluster isuniform in gene organization. Here, sequence analysesindicated that the cluster exists in five differentgenomic contexts, defining five cluster types. In FUMgene genealogies, evolutionary relationships betweenfusaria with different cluster types were largely incongruentwith species relationships inferred fromprimary-metabolism (PM) gene genealogies, and FUMcluster types are not trans-specific. In addition, synonymoussite divergence analyses indicated that threeFUM cluster types predate diversification of FFSC. Thedata are not consistent with balancing selection orinterspecific hybridization, but they are consistentwith two competing hypotheses: (i) multiple horizontaltransfers of the cluster from unknown donors to FFSCrecipients and (ii) cluster duplication and loss (birthand death). Furthermore, low levels of FUM gene divergencein F. bulbicola, an FFSC species, and F. oxysporumprovide evidence for horizontal transfer of thecluster from the former, or a closely related species, tothe latter. Thus, uniform gene organization within theFUM cluster belies a complex evolutionary history thathas not always paralleled the evolution of Fusarium.

Fumonisin contamination of maize is considered a serious problem in most maize-growing regions of the world,due to the widespread occurrence of these mycotoxins and their association with toxicosis in livestock andhumans. Fumonisins are produced primarily by species of Fusarium that are common in maize grain, but alsoby some species of Aspergillus sect. Nigri, which can also occur on maize kernels as opportunistic pathogens. Understandingthe origin of fumonisin contamination in maize is a key component in developing effectivemanagementstrategies. Although some fungi in Aspergillus sect. Nigri are known to produce fumonisins, little is knownabout the species which are common in maize and whether they make a measurable contribution to fumonisincontamination ofmaize grain. In thiswork,we evaluated populations of Aspergillus sect. Nigri isolated frommaizein USA and Italy, focusing on analysis of housekeeping genes, the fum8 gene and in vitro capability of producingfumonisins. DNA sequencing was used to identify Aspergillus strains belonging to sect. Nigri, in order to comparespecies composition between the two populations, which might influence specificmycotoxicological risks. Combinedbeta-tubulin/calmodulin sequences were used to genetically characterize 300 strains (199 from Italy and101 fromUSA)which grouped into 4 clades: Aspergilluswelwitschiae (syn. Aspergillus awamori, 14.7%), Aspergillustubingensis (37.0%) and Aspergillus niger group 1 (6.7%) and group 2 (41.3%). Only one strain was identified as Aspergilluscarbonarius. Species composition differed between the two populations; A. niger predominated amongthe USA isolates (69%), but comprised a smaller percentage (38%) of Italian isolates. Conversely, A. tubingensisand A. welwitschiae occurred at higher frequencies in the Italian population (42% and 20%, respectively) than inthe USA population (27% and 5%). The evaluation of FB2 production on CY20S agar revealed 118 FB2 producingand 84 non-producing strains distributed among the clades: A. welwitschiae, A. niger group 1 and A. niger group2, confirming the potential of Aspergillus sect. Nigri species to contribute to total fumonisin contamination ofmaize. A higher percentage of A. niger isolates (72.0%) produced FB2 compared to A. welwitschiae (36.6%). Thepercentage of FB2-producing A. niger strains was similar in the USA and Italian populations; however, the predominanceof A. niger in the USA population suggests a higher potential for fumonisin production. Some strainswith fum8 present in the genome did not produce FB2 in vitro, confirming the ineffectiveness of fum8 presence asa predictor of FB2 production.

Aspergillus niger is a significant component of the fungal community on grapes. The mycotoxinfumonisin B2 (FB2) was recently detected in grape must and wine as well as in cultures of someA. niger strains isolated from grapes and raisins. This study examined 48 strains of Aspergillus sectionNigri for the presence of the fumonisin biosynthetic gene fum8 in relation to FB2 production. The fum8gene was detected in only 11 A. niger strains, 9 of which also produced FB2. Maximum parsimonyanalysis based on the calmodulin gene sequence indicated that the presence/absence of fum8 is notcorrelated with the phylogenetic relationship of the isolates. This is the first report correlating thepresence of a fumonisin biosynthetic gene with fumonisin production in A. niger from an important foodcrop. The results suggest that the absence of FB2 production in grape isolates of A. niger can resultfrom the absence of at least one gene essential for production.

Management of Calonectria spp. infections in nurseries requires scheduled fungicide applications, particularly with methyl benzimidazole carbamates (MBCs) and sterol demethylation inhibitors (DMIs). Due to rising concerns about the occurrence of MBC resistance in different Calonectria populations and variability in prochloraz efficacy in controlling these pathogens, a detailed study on prochloraz sensitivity distributions of Calonectria isolates belonging to the Calonectria scoparia complex was carried out. In total, 105 isolates collected in two distinct periods (1993 to 1996 and 2005 to 2009) were analyzed for prochloraz sensitivity. Based on DNA sequencing and phylogenetic analyses of ?-tubulin, histone H3, and translation elongation factor-1? gene sequences, 69 and 36 isolates were identified as C. pauciramosa and C. polizzii, respectively. The isolates collected more recently (group B) had a reduced prochloraz sensitivity, as indicated by greater values for the effective dose to reduce growth by 50% than those collected earlier (group A). The reduced sensitivity detected in vitro corresponded to partial loss of fungicide efficacy in controlling infections in red clover and feijoa under controlled and semi-field conditions, respectively. Frequent prochloraz application in nurseries for controlling Calonectria spp. infections is discouraged.

During the 2009 and the 2010 growing seasons, a root rot disease has been detected on young potted Persea americana plants in two nurseries located in the Catania and Messina provinces (eastern Sicily, Italy). A Cylindrocarpon sp. was consistently recovered from pieces of symptomatic tissues on Petri dishes containing potato dextrose agar. On the basis of morphological characteristics and molecular identification by DNA sequencing and phylogenetic analysis of internal transcribed spacer and ?-tubulin gene regions, the causal agent was identified as Ilyonectria (=Neonectria) macrodidyma. Koch's postulates were fulfilled by pathogenicity tests carried out on potted P. americana seedlings. To our knowledge, this is the first to report worldwide of the occurrence of a disease caused by I. macrodidyma on P. americana.

Fusarium proliferatum is a member of the Fusarium fujikuroi species complex (FFSC) involved in the maize ear rot together with Fusarium verticillioides, which is a very closely related species. Recently, different studies have detected natural fumonisin contamination in wheat kernels and most of them have shown that the main species isolated was F. proliferatum. Fusarium strains obtained from freshly harvested durum wheat samples (2008 to 2011 harvest seasons) from Argentina were characterized through a phylogenetic analysis based on translation elongation factor-1 alpha (EF-1?) and calmodulin (CaM) genes, determination of mating type alleles, and evaluation of fumonisin production capability. The strains were identified as F. proliferatum (72%), F. verticillioides (24%) and other Fusarium species. The ratio of mating type alleles (MAT-1 and MAT-2) obtained for both main populations suggests possible occurrence of sexual reproduction in the wheat fields, although this seems more frequent in F. proliferatum. Phylogenetic analysis revealed greater nucleotide variability in F. proliferatum strains than in F. verticillioides, however this was not related to origin, host or harvest year. The fumonisin-producing ability was detected in 92% of the strains isolated from durum wheat grains. These results indicate that F. proliferatum and F. verticillioides, among the fumonisin producing species, frequently contaminate durum wheat grains in Argentina, presenting a high risk for human and animal health.

An Aspergillus population (67 strains), isolated from maize in 2003, during the first outbreak of aflatoxin contamination documented in Northern Italy, was characterised according to gene sequencing data. All strains were identified as A. flavus by sequencing of ?-tubulin and calmodulin gene fragments. Furthermore, the strains were analysed for the presence of seven aflatoxin biosynthesis genes in relation to their capability to produce aflatoxin B1, targeting the regulatory genes aflR and aflS, and the structural genes aflD, aflM, aflO, aflP, and aflQ. The strains were placed into four groups based on their patterns of amplification products: group I (40 strains) characterised by presence of all seven amplicons; groups II (two strains) and III (nine strains), showing four (AflM, aflP, aflO, and aflQ) and three (aflO, aflP, aflQ) amplicons, respectively; and group IV (16 strains) characterised by total absence of PCR products. Only group I contained strains able to produce aflatoxin B1 (37 out of 40), whereas the strains belonging to the other groups and lacking three, four or all seven PCR products were non-producers. The results obtained in this study pointed out that A. flavus was the only species responsible for aflatoxin contamination in Northern Italy in 2003, and that the aflatoxin gene cluster variability existing in populations can be useful for understanding the toxicological risk as well as the selection of biocontrol agents.

The Pleurotus eryngii species complex is an economically important group which includes several closely related varieties, whose genetic discrimination is still not clear. One hundred and ten Italian strains of Pleurotus eryngii belonging to the varieties elaeoselini, eryngii, ferulae and thapsiae and P. nebrodensis were analysed by sequencing two housekeeping genes (ef1-a and rpb2), in order to find molecular markers for the identification of different varieties. Sequence analysis of partial ef1-a and rpb2 genes, allowed identification of some conserved nucleotide positions within each variety but variable among var. elaeoselini, var. eryngii, var. ferulae var. thapsiae and P. nebrodensis, allowing their discrimination. Phylogenetic analysis from the data of the two genes data set showed that var. elaeoselini, var. thapsiae, var. ferulae and var. eryngii are closely related to each other, and confirm P. nebrodensis as a separate clade.

Dried vine fruits may be heavily colonized by Aspergillus species. The molecular biodiversity of an Aspergillus population (234 strains) isolated from dried vine fruit samples of worldwide origin were analyzed by investigating four housekeeping gene loci (calmodulin, beta-tubulin, elongation factor 1-alpha, RPB2). Aspergillus Sect. Nigri was dominant and the strains were identified as A. tubingensis (138), A. awamori (38), A. carbonarius (27),A. uvarum (16) and A. niger (11). Four Aspergillus flavus strains were also identified from Chilean raisins. Two clusters closely related to the A. tubingensis species with a significant bootstrap (60% and 99%) were identified as distinct populations. Among the four loci, RPB2 showed the highest genetic variability. This is the first complete study on the worldwide distribution of black Aspergilli occurring on dried vine fruits identified by a molecular approach.

An extensive use of Weissella (W.) confusa is currently being made for the production of a variety of fermented foods and beverages although some strains of this species have emerged as opportunistic pathogens for humans and animals. Nevertheless, no rapid methods are available for the reliable identification of W. confusa.We developed a novel PCR using AFLP (Amplified Fragment Length Polymorphism)-derived primers for the rapid and unequivocal identification of W. confusa. Fluorescent AFLP of 30 strains of W. confusa, Leuconostoc citreum, Lactobacillus (Lb.) brevis, Lb. rossiae, Lb. plantarum and Lb. buchneri allowed us to detect, purify and sequence several W. confusa specific AFLP fragments. The homology search in BLAST of a 303 bp nucleotide sequence revealed a d77% identity of the purified fragment with the lepA gene of several lactic acid bacteria. A PCR assay targeting 225 bp of this fragment was developed and tested against the DNA of 109 strains, including 34 foodborne and clinical W. confusa and 75 strains of 47 phylogenetically closely and distantly related species, resulting in 100% specificity with a detection limit of 16 pg. Being the first species-specific PCR to date developed for the rapid and unambiguous identification of W. confusa, this novel assay could be a reliable and efficient tool for detecting W. confusa not only in food and beverages, but also in clinical specimens, thus contributing to clarify its real significance in human and animal infections.

Aspergillus section Nigri populations isolated from seven growing regions from Argentina were characterizedby sequencing in order to identify species responsible for production of ochratoxin A (OTA) and fumonisins(FBs). Sequences of genes encoding calmodulin, ?-tubulin, the second largest subunit of RNA polymerase IIand translation elongation factor 1 alpha were analysed. The phylogenetic analysis showed the presence of sixlineages: A. carbonarius, A. tubingensis, A. niger, A. japonicus, A. homomorphus and A. foetidus grouped in fourmajor clusters. The molecular tools used allowed the identification for the first time of A. homomorphus fromvineyards. OTA production confirmed the importance of A. carbonarius as the main ochratoxigenic speciesisolated and, to a variable degree, of A. niger and A. tubingensis, which were by far the most commonlyoccurring species on grapes in Argentina. The only strains able to produce OTA and fumonisins (B2-B4) belongto the A. niger cluster.

Aflatoxins are highly toxic carcinogens that contaminate crops worldwide. Previous studies conducted in Nigeria and Ghana found high concentrations of aflatoxins in pre- and post-harvest maize. However, little information is available on the population structure of Aspergillus Sect. Flavi in West Africa. We determined the incidence of Aspergillus Sect. Flavi and the level of aflatoxin contamination in 91 maize samples from farms and markets in Nigeria and Ghana. Aspergillus spp. were recovered from 61/91 maize samples and aflatoxins B-1 and/or B-2 occurred in 36/91 samples. Three samples from the farms also contained aflatoxin G(1) and/or G(2). Farm samples were more highly contaminated than were samples from the market, in terms of both the percentage of the samples contaminated and the level of mycotoxin contamination. One-hundred-and-thirty-five strains representative of the 1163 strains collected were identified by using a multilocus sequence analysis of portions of the genes encoding calmodulin, beta-tubulin and actin, and evaluated for aflatoxin production. Of the 135 strains, there were 110 - Aspergillus flavus, 20 - Aspergillus tamarii, 2 - Aspergillus wentii, 2 - Aspergillus flavofurcatus, and 1 - Aspergillus parvisclerotigenus. Twenty-five of the A. flavus strains and the A. parvisclerotigenus strain were the only strains that produced aflatoxins. The higher contamination of the farm than the market samples suggests that the aflatoxin exposure of rural farmers is even higher than previously estimated based on reported contamination of market samples. The relative infrequency of the A. flavus S-BG strains, producing small sclerotia and high levels of both aflatoxins (B and G), suggests that long-term chronic exposure to this mycotoxin are a much higher health risk in West Africa than is the acute toxicity due to very highly contaminated maize in east Africa. (C) 2014 Elsevier Ltd. All rights reserved.

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.