Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins

The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (rum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin

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

Fumonisin B2 by Aspergillus niger in the grape–wine chain: an additional potential mycotoxicological risk

Fumonisins are mycotoxins with cancer-promoting activity and are associated with a number of animal and human diseases. The potential risk of contamination by fumonisin B2 (FB2), although at low levels, has been demonstrated in must and wine. Black aspergilli in general and Aspergillus niger in particular are considered to be the major responsible agents of FB2 contamination in grape and its by-products. Contamination by FB2 therefore is yet another safety concern of grape and wine producers, as ochratoxin A, produced mainly by A. carbonarius, may prove to be a major mycotoxicological problem in the grape–wine chain

Fumonisin and beauvericin chemotypes and genotypes of the sister species Fusarium subglutinans and Fusarium temperatum

Fusarium subglutinans and Fusarium temperatum are common maize pathogens that produce mycotoxins and cause plant disease. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant. Our objective was to clarify this situation by determining both the chemotypes and genotypes for strains from both species. We analyzed 25 strains from Argentina, 13 F. subglutinans and 12 F. temperatum strains, for toxin production by ultraperformance liquid chromatography mass spectrometry (UPLC-MS). We used new genome sequences from two strains of F. subglutinans and one strain of F. temperatum, plus genomes of other Fusarium species, to determine the presence of functional gene clusters for the synthesis of these toxins. None of the strains examined from either species produced fumonisins. These strains also lack Fum biosynthetic genes but retain homologs of some genes that flank the Fum cluster in Fusarium verticillioides. None of the F. subglutinans strains we examined produced beauvericin although 9 of 12 F. temperatum strains did. A complete beauvericin (Bea) gene cluster was present in all three new genome sequences. The Bea1 gene was presumably functional in F. temperatum but was not functional in F. subglutinans due to a large insertion and multiple mutations that resulted in premature stop codons. The accumulation of only a few mutations expected to disrupt Bea1 suggests that the process of its inactivation is relatively recent. Thus, none of the strains of F. subglutinans or F. temperatum we examined produce fumonisins, and the strains of F. subglutinans examined also cannot produce beauvericin. Variation in the ability of strains of F. temperatum to produce beauvericin requires further study and could reflect the recent shared ancestry of these two species.Fil: Fumero, María Verónica. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Villani, Alessandra. Institute of Sciences of Food Production; ItaliaFil: Susca, Antonia. Institute of Sciences of Food Production; ItaliaFil: Haidukowski, Miriam. Institute of Sciences of Food Production; ItaliaFil: Cimmarusti, María. Institute of Sciences of Food Production; ItaliaFil: Toomajian, Christopher. Kansas State University; Estados UnidosFil: Leslie, John Franklin. Kansas State University; Estados UnidosFil: Chulze, Sofia Noemi. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Moretti, Antonio. Institute of Sciences of Food Production; Itali

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

Variation in fumonisin and ochratoxin production associated with differences in biosynthetic gene content in Aspergillus niger and A. welwitschiae isolates from multiple crop and geographic origins

The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that i) isolates of both species differed in ability to produce the mycotoxins; ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin

Pathogenicity of Fumonisin-producing and Nonproducing Strains of Aspergillus Species in Section Nigri to Maize Ears and Seedlings.

Species of Aspergillus section Nigri are commonly associated with maize kernels, and some strains can produce fumonisin mycotoxins. However, there is little information about the extent to which these fungi contribute to fumonisin contamination in grain, the damage they cause to maize ears, or their effects on maize seed germination and seedling health. We compared fumonisin-producing and nonproducing strains of A. niger, A. welwitschiae, A. phoenicis, A. tubingensis, and A. carbonarius from the United States and Italy in laboratory and field studies to assess their ability to contribute to fumonisin contamination, to cause maize ear rot, and to affect seed germination and seedling growth. In laboratory experiments, some strains of each Aspergillus species reduced germination or seedling growth, but there was high variability among strains within species. There were no consistent differences between fumonisin-producing and nonproducing strains. In field studies in Iowa and Illinois, strains were variable in their ability to cause ear rot symptoms, but this was independent of the ability of the Aspergillus strains to produce fumonisins. Contamination of grain with fumonisins was not consistently increased by inoculation with Aspergillus strains compared with the control, and was much greater in F. verticillioides-inoculated treatments than in Aspergillus-inoculated treatments. However, the ratio of the FB analogs FB2 and FB1 was altered by inoculation with some Aspergillus strains, indicating that FB2 production by Aspergillus strains occurred in the field. These results demonstrate the pathogenic capabilities of strains of Aspergillus in section Nigri, but suggest that their effects on maize ears and seedlings are not related to their ability to produce fumonisins, and that fumonisin contamination of grain caused by Aspergillus spp. is not as significant as that caused by Fusarium spp

Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger Strains Isolated from Grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and β-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 μg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 μg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 μg/liter), and none of the 19 strains of Aspergillus “uniseriate” produced ochratoxin A above the level of detection (4 μg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy

Embryo Culture, In Vitro Propagation, and Molecular Identification for Advanced Olive Breeding Programs

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (Xf) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long time required for seed germination caused by the inhibition effect of the woody endocarp, the seed integument, and the endosperm. Starting from F1 seeds by cross-breeding, the embryo culture was compared with traditional seed germination, evaluating the effectiveness of in vitro multiplication of the plantlets for large-scale production. The isolated embryos were established on a new medium based on Rugini ‘84 macroelements, Murashige & Skoog ‘62 microelements, with Nitsch J. P. & Nitsch C. ‘69 vitamine and subcultured on Leva MSM modified. The results obtained confirmed that in vitro culture of olive embryos is a valid tool for increasing the percentage and speed of germination, helping to reduce the time of the olive breeding programs, offering the possibility to effectively propagate plantlets for further experiments

In vitro study of fum gene expression and fumonisins production in Fusarium verticillioides under different ecological conditions.

F. verticillioides is a fungus associated to maize ears all over the world. It can cause Fusarium pink ear rot and produces fumonisins (FBs), mycotoxins found in both maize kernels and its derivatives. They are toxic to humans, in particular FB1 is classified as possibly carcinogenic. Fumonisins biosynthetic pathway is regulated by several genes belonging to FUM cluster whose behaviour in different ecological conditions was poorly studied. The aim of this work was to investigate the behaviour of 2 F. verticillioides strains grown in vitro both on a FB-inducing (Malt extract agar-MEA) and FB-inhibiting (Czapeck yeast agar-CYA) media. Fungal growth, FBs production and gene expression were studied in different temperature (T; 20-30) and water activity (aw; 0.90-0.99) regimes, in liquid cultures incubated for 7-21 days. The expression of two genes: FUM21 and FUM2 was considered; relative quantitation of gene expression was performed by RealTime (RT)-PCR. The results showed that, with aw fixed at 0.99, maximum FUM21 and FUM2 expression was observed at 30\ub0C after 14 days of incubation, while FBs production increased from 7 to 21 days. At fixed T (25\ub0C), fungal growth at 0.90 aw was not observed after 21 days of incubation. Gene expression increased with incubation time both at 0.95 and 0.99 aw, FB production increased till to 21 and 14 days respectively at 0.95 and 0.99 aw. In all the conditions studied the expression of the 2 genes considered followed a very similar trend, but FUM2 was much more expressed than FUM21 and more influenced by aw conditions