Identification of toxigenic fungal species associated with maize ear rot: Calmodulin as single informative gene

Accurate identification of fungi occurring on agrofood products is the key aspect of any prevention and pest management program, offering valuable information in leading crop health and food safety. Fungal species misidentification can dramatically impact biodiversity assessment, ecological studies, management decisions, and, concerning toxigenic fungi, health risk assessment, since they can produce a wide range of toxic secondary metabolites, referred to as mycotoxins. Since each toxigenic fungal species can have its own mycotoxin profile, a correct species identification, hereby attempted with universal DNA barcoding approach, could have a key role in mycotoxins prevention strategies. Currently, identification of single marker for species resolution in fungi has not been achieved and the analysis of multiple genes is used, with the advantage of an accurate species identification and disadvantage of difficult setting up of PCR-based diagnostic assays. In the present paper, we describe our strategy to set up a DNA-based species identification of fungal species associated with maize ear rot, combining DNA barcoding approach and species-specific primers design for PCR based assays. We have (i) investigated the appropriate molecular marker for species identification, limited to mycobiota possibly occurring on maize, identifying calmodulin gene as single taxonomically informative entity; (ii) designed 17 sets of primers for rapid identification of 14 Fusarium, 10 Aspergillus, 2 Penicillium, and 2 Talaromyces species or species groups, and finally (iii) tested specificity of the 17 set of primers, in combination with 3 additional sets previously developed

A polyphasic approach for characterization of a collection of cereal isolates of the Fusarium incarnatum-equiseti species complex

"Available online 22 June 2016"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.This work was supported by the EU project EC KBBE-2007-222690-2 MYCORED

Molecular characterization of an Aspergillus flavus population isolated from maize during the first outbreak of aflatoxin contamination in Italy

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 \u3b2-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

Variation in the fumonisin biosynthetic gene cluster in fumonisin-producing and nonproducing black aspergilli

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 fumonisinproducing 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

Genetic Diversity in Fusarium graminearum from a Major Wheat-Producing Region of Argentina

The Fusarium graminearum species complex (FGSC) is a group of mycotoxigenic fungi that are the primary cause of Fusarium head blight (FHB) of wheat worldwide. The distribution, frequency of occurrence, and genetic diversity of FGSC species in cereal crops in South America is not well understood compared to some regions of Asia, Europe and North America. Therefore, we examined the frequency and genetic diversity of a collection of 183 FGSC isolates recovered from wheat grown during multiple growing seasons and across a large area of eastern Argentina, a major wheat producing region in South America. Sequence analysis of the translation elongation factor 1−α and β-tubulin genes as well as Amplified Fragment Length Polymorphism (AFLP) analyses indicated that all isolates were the FGSC species F. graminearum sensu stricto. AFLP analysis resolved at least 11 subgroups, and all the isolates represented different AFLP haplotypes. AFLP profile and geographic origin were not correlated. Previously obtained trichothecene production profiles of the isolates revealed that the 15-acetyldeoxynivalenol chemotype was slightly more frequent than the 3-acetyldeoxynivalenol chemotype among the isolates. These data extend the current understanding of FGSC diversity and provide further evidence that F. graminearum sensu stricto is the predominant cause of FHB in the temperate main wheat-growing area of Argentina. Moreover, two isolates of F. crookwellense and four of F. pseudograminearum were also recovered from wheat samples and sequenced. The results also suggest that, although F. graminearum sensu stricto was the only FGSC species recovered in this study, the high level of genetic diversity within this species should be considered in plant breeding efforts and development of other disease management strategies aimed at reducing FHB