PCR Assay for Identification of Aspergillus Carbonarius and Aspergillus Japonicus

Black Aspergilli, and in particular Aspergillus carbonarius, are the main causes of contamination of grapes and their by-products by ochratoxin A. A PCR-based method was developed to detect DNA of A. carbonarius and A. japonicus. Two pairs of primers (CARBO1/2 and JAPO1/2) designed from the calmodulin gene, produced PCR products of 371 and 583 bp for A. carbonarius and A. japonicus, respectively. Primer specificity was tested with DNA of 107 strains belonging to Aspergillus section Nigri isolated mostly from grapes in Europe. The sensitivity of primers CARBO1/2 and JAPO1/2 was 12.5 pg when using pure total genomic DNA of the two species. The developed primers provide a powerful tool for detection of the main ochratoxigenic producing Aspergillus species in grapes.     

Phylogeny and Molecular Diagnosis of Mycotoxigenic Fungi

Phylogenetic studies of the fungi that produce the five major groups of mycotoxins are reviewed, with a focus on studies employing ribosomal and/or β-tubulin (BenA) gene sequences. The toxins aflatoxin and ochratoxin A are produced by several Aspergillus and Penicillium species classified in the Trichocomaceae, Eurotiales. The toxins fumonisin, deoxynivalenol and zearalenone are produced by several Fusarium species classified in the Nectriaceae, Hypocreales. Studies of ribosomal genes have revealed that the present generic concepts for Aspergillus, Penicillium and Fusarium will require some adjustment in order to conform to phylogenetic principles. Phylogenetic studies have resulted in generally narrower species concepts in all three genera but there is good correlation between these species and mycotoxin production. The development of molecular diagnostics for the critical mycotoxigenic species is considered, with particular emphasis on the development of DNA hybridization probes that can be used to detect and identify multiple species using species and/or clade specific oligonucleotides designed from one or more genes. As an illustration, a virtual array for identifying Aspergillus species and groups of species producing aflatoxin is presented, based on oligonucleotides selected and optimized from a database of internal transcribed spacer and partial β-tubulin sequences assembled from GenBank. It was possible to design acceptable oligos for all species and groups in the complex using the β-tubulin gene, but only for one species and the larger group using the less variable internal transcribed spacer of the ribosomal DNA.     

Ochratoxin a in Grapes and Wine

The mycotoxin ochratoxin A is a potent nephrotoxin and a possible human carcinogen. It occurs in a variety of plant products, including wine, grape juice and dried vine fruits. Several surveys have shown that the range of ochratoxin A contents detected in wine produced in Europe varied between 0.01 and 3.4gl^–1. Both incidence and concentration of the toxin were higher in wines from southern regions and increased in the order white < rosè < red. In Italy, field trials were conducted in 1999 and 2000 to study fungi associated with grapes and their ability to produce ochratoxin. Aspergillus and/or Penicillium strains were present on grapes, starting from setting in a few vineyards. The highest level of grape colonisation was found at early veraison in 1999 and at ripening in 2000. In both years, 95% of strains belonged to the genus Aspergillus. Aspergillus niger aggregate was dominant, with about 50% of the ochratoxin-positive strains identified as A carbonarius. Other authors have confirmed the relevance of these fungi and underlined the contribution of A. carbonarius to the ochratoxin contamination of wine. This species is very invasive and colonises and penetrates berries, even without skin damage. It emerges that temperature, rain and relative humidity are the main factors that influence ochratoxin production in grapes.     

Epidemiology of Toxigenic Fungi and their Associated Mycotoxins for Some Mediterranean Crops

Recent data on the epidemiology of the common mycotoxigenic species of Fusarium, Alternaria, Aspergillus and Penicillium in infected or colonized plants, and in stored or processed plant products from the Mediterranean area are reviewed. Emphasis is placed on the toxigenicity of the causal fungal species and the natural occurrence of well known mycotoxins (aflatoxins, ochratoxins, fumonisins, trichothecenes, zearalenone, patulin, Alternaria-toxins and moniliformin), as well as some more recently described compounds (fusaproliferin, beauvericin) whose toxigenic potential is not yet well understood. Several Fusarium species reported from throughout the Mediterranean area are responsible of the formation of mycotoxins in infected plants and in plant products, including: Fusarium graminearum, F. culmorum, F. cerealis, F. avenaceum, F. sporotrichioides and F. poae, which produce deoxynivalenol, nivalenol, fusarenone, zearalenone, moniliformin, and T-2 toxin derivatives in wheat and other small grains affected by head blight or scab, and in maize affected by red ear rot. Moreover, strains of F. verticillioides, F. proliferatum, and F. subglutinans, that form fumonisins, beauvericin, fusaproliferin, and moniliformin, are commonly associated with maize affected by ear rot. Fumonisins, were also associated with Fusarium crown and root rot of asparagus and Fusarium endosepsis of figs, caused primarily by F. proliferatum. Toxigenic A. alternata strains and associated tenuazonic acid and alternariols were commonly found in black mould of tomato, black rot of olive and citrus, black point of small cereals, and black mould of several vegetables. Toxigenic strains of A. carbonarius and ochratoxin A were often found associated with black rot of grapes, whereas toxigenic strains of A. flavus and/or P. verrucosum, forming aflatoxins and ochratoxin A, respectively, were found in moulded plant products from small cereals, peanuts, figs, pea, oilseed rape, sunflower seeds, sesame seeds, pistachios, and almonds. Finally, toxigenic strains of P. expansum and patulin were frequently found in apple, pear and other fresh fruits affected by blue mould rot, as well as in derived juices and jams.     

A survey of mold flora and of the mycotoxin deoxynivalenol in corn imported to ISRAEL

Shipments of corn imported to Israel from the U.S.A. during 1985 were sampled during unloading and examined for the presence of fungi and the trichothecene deoxynivalenol (DON). The most frequent species found on the corn wasAspergillus flavus followed byA. versicolor; three otherAspergilli — A. niger, A. ochraceus andA. fumigatus — were found at a much lower frequency. DON analysis was performed by electron capture detection gas chromatography after derivatization with heptafluorobutyryl imidazole. At fortification levels of 0.1, 1 and 2 μg/g, recoveries of 80—100% were obtained. Although DON was not detected in any of the samples analyzed, the observation (in the shipments) of fungi belonging to the generaFusarium andAspergillus suggests a potential danger of mycotoxin contamination. The relatively high moisture content of the samples increased the likelihood of such a hazard.     

Detection and Differentiation of Trichothecene and Enniatin-Producing Fusarium Species on Small-Grain Cereals

A large number of Fusarium species are associated with Fusarium head blight of wheat and other small-grain cereals as well as seedling blight and brown foot rot. Different Fusarium species tend to predominate under different environmental conditions and in different regions. In addition to causing disease, these fungi are of particular significance because they produce a number of mycotoxins including the trichothecenes and enniatins that contaminate infected grain. The nature and amount of the mycotoxins that accumulate will alter according to the species or even the particular isolates involved in the infection. It is highly desirable to be able to analyse such complex infections to determine which species and, preferably, which chemotypes are present, in order to understand the factors that affect the pathogenicity of each species and to evaluate the potential risk for contamination of grain with mycotoxins. This paper reports the development of molecular methods, based upon the polymerase chain reaction (PCR), for the detection of mycotoxigenic fungi. Several of the Fusarium species involved are closely related, making the development of specific assays problematic. We describe the development of primers specific to individual species and discuss how this work provides insight into fungal populations and relates to taxonomic studies. In some instances, it is desirable to detect the presence of potential mycotoxin producers rather than individual fungal species. Generic assays have been produced for several genes involved in trichothecene biosynthesis and for enniatin synthetase in order to permit the detection of species able to produce the associated mycotoxins. Additional work is under way to refine assays to enable detection related to the class of trichothecene and chemotype of isolate because of the potential risk posed to human and animal consumers by different trichothecenes.     

Natural mechanisms for cereal resistance to the accumulation of <Emphasis Type="Italic">Fusarium</Emphasis> trichothecenes

This review describes the naturally occurring mechanisms in cereals that lead to a reduction of Fusarium trichothecene mycotoxin accumulation in grains. A reduction in mycotoxin contamination in grains could also limit fungal infection, as trichothecenes have been reported to act as virulence factors. The mechanisms explaining the low toxin accumulation trait, generally referred to as type V resistance to Fusarium, can be subdivided into two classes. Class 1 includes mechanisms by which the plants chemically transform the trichothecenes, leading to their degradation or detoxification. Among the detoxification strategies, glycosylation of trichothecenes is a natural process already reported in wheat. According to the structure and the toxicity of trichothecenes, two other detoxification processes, acetylation and de-epoxidation, can be expressed, at least in transgenic plants. Class 2 comprises mechanisms that lead to reduced mycotoxin accumulation by inhibition of their biosynthesis through the action of plant endogenous compounds. These include both grain constitutive compounds and compounds induced in response to pathogen infection. There are already many compounds with antioxidant properties, like phenolic compounds, peptides or carotenoids, and with prooxidant properties, like hydrogen peroxide or linoleic acid-derived hydroperoxides, that have been described as ‘modulators’ of mycotoxin biosynthesis. This review addresses for the first time different studies reporting specific in vitro effects of such compounds on the biosynthesis of Fusarium mycotoxins. A better understanding of the natural processes limiting accumulation of trichothecenes in the plant will open the way to the development of novel breeding varieties with reduced ‘mycotoxin risk’.     

Control of infection of tomato fruits by <Emphasis Type="Italic">Alternaria</Emphasis> and mycotoxin production using plant extracts

Tomato fruits are susceptible to infection by Alternaria species. In addition, Alternaria species may contaminate the fruits with mycotoxins. There is thus interest in control systems to minimise pathogenicity and control toxin production. The objectives of this study were to examine the effect of plant extracts of Eucalyptus globulus and Calendula officinalis on the growth of strains of Alternaria alternata and A. arborescens, on pathogenicity of tomato fruits and mycotoxin production. The growth bioassays showed that the ethanolic and chloroformic fractions of E. globulus were the most effective in reducing growth of A. alternata (66–74 %) and A. arborescens (86–88 %), respectively at 2500 μg/g. The effects of plant extracts on mycotoxin biosynthesis were variable and strain dependent. The most effective fractions in decreasing mycotoxin accumulation were the ethanolic and chloroformic extracts of E. globulus, which reduced tenuazonic acid by 89 %, alternariol by 75–94 % and almost complete inhibition of alternariol monomethyl ether. All the tested fractions reduced percentage of infected tomato fruits when compared to the controls. The ethanolic and chloroformic fractions of E. globulus completely inhibited growth of A. alternata and A. arborescens on unwounded fruits and reduced the aggressiveness on wounded fruits of strains of both species significantly.     

Fusarium – Problematik bei Körnermais (Zea Mays L.)

In the last few years in maize stalk and ear rots caused by fusarium spp. was increasing, often causing contamination with mycotoxins. Most prevalent was fusarium graminearum, mainly producing Deoxynivalenol (DON) in kernels and stalk. DON is suspected to cause several diseases in humans and livestock. In our study, injection of conidial suspension in the silk channel was the most successful method for artificial inoculation. Due to significant differences between several hybrids, it could be concluded that resistance against the fungus is genetically controlled. Intensity of resistance is varying extremely, depending on environment. The mycotoxin concentration of the kernels was positively correlated with percentage of visible infected kernels, obtaining levels up to 95?ppm. Susceptible hybrids even showed in the rest plant extremely high levels of DON (up to 60?ppm). Naturally infected plants showed high mycotoxin concentrations above and below the main ear, exceeding the recommended level of forage for cattle. High mycotoxin levels in kernels and rest plants show the importance of resistance breeding against fusarium.     

Molecular biology and biotechnology for reduction of Fusarium mycotoxin contamination

Fusarium head blight (FHB) is a devastating disease of important cereal crops resulting in significant yield loss and mycotoxin contamination. Persistent outbreaks of FHB in Europe and North America have led to various efforts to understand the mechanisms of resistance to this disease and mycotoxin biosynthesis. In this minireview, we summarize basic and applied studies conducted in our laboratories into reducing mycotoxin contamination in FHB.     

Fusarium temperatum, a mycotoxin-producing pathogen of maize

In a recent study, a population of Fusarium strains isolated from maize in Belgium was described as a new species, F. temperatum, that is morphologically similar and phylogenetically closely related to F. subglutinans, a species in the American clade of the Gibberella fujikuroi species complex. In fields, the F. temperatum:F. subglutinans ratio was very high, suggesting that F. temperatum outcompetes its sister species F. subglutinans. This raised the question whether this novel species contributes to the final rot symptoms observed on maize plants at harvest, as well as to the potential mycotoxin contamination. Results of the pathogenicity tests by soil and toothpick inoculation demonstrate the ability of F. temperatum to cause seedling malformation and stalk rot under greenhouse conditions. Screening of 15 Fusarium mycotoxins showed the ability of F. temperatum to produce moniliformin, beauvericin, enniatins and fumonisin B₁. The results indicate that F. temperatum can produce mycotoxins and cause maize diseases and, therefore, poses a potential risk to maize production and to the safety of human food and animal feed.     

Mycotoxin Genetics and Gene Clusters

Fungi produce low molecular weight secondary metabolites such as antibiotics and mycotoxins. Antibiotics cure diseases whereas mycotoxins cause diseases in plants, animals and human beings. Species such as Aspergillus, Fusarium, Penicillium and Stachybotrys are known to produce mycotoxins that accumulate in processed foods and feeds, although the incidence of infection occurs before processing, during the active growth of the organism. Among the mycotoxins, aflatoxins produced by Aspergillus flavus and A. parasiticus have been extensively studied at the molecular level. A complex biosynthetic pathway involving sixteen steps is mediated by individual major genes. These fungi have eight linkage groups, but the aflatoxin/sterigmatocystin (AF/ST) metabolic pathway genes have been mapped to only three linkage groups; ten of them belong to linkage group VII, and one of each to linkage group II and VIII. These genes are involved in both the regulatory and biosynthetic pathways and are clustered on the respective chromosomes. Clustering of genes in fungi indicates an evolutionary trend among genes that orchestrate gene function. Being linked together they segregate as a unit, thereby conferring a selective advantage to the organism. The evolution of gene clusters takes place through vertical or horizontal gene transfer. In fungi, horizontal gene transfer is most effective. Functionally, the mechanism of evolution of mycotoxin gene clusters in fungi seems to be similar to the evolution of a super-gene. The possible implications of evolutionary parallelism of gene clusters and super-genes is briefly explored.     

Multiple facets of response to fungicides – the influence of azole treatment on expression of key mycotoxin biosynthetic genes and candidate resistance factors in the control of resistant <Emphasis Type="Italic">Fusarium</Emphasis> strains

The spread of fungicide resistant and/or tolerant phytopathogenic fungi is an important factor affecting crop protection. However, the mechanisms of fungal response to fungicide application are not entirely characterised. In particular, the contribution of previously known resistance factors and the final influence of fungicide treatments on metabolism of surviving mycelia (e.g. mycotoxin increased release and biosynthesis potentially causing contamination of the crops) merit investigation, in order to improve future molecular diagnostics of fungicide resistant strains. The performed experiments have shown distinct expression changes for homologs of a known fungicide resistance factor Flr1 (yeast; DHA1 family of major facilitator superfamily transporters) after azole application in cultured fusaria. Two distantly related homologs of that gene were selected, based on the unsupervised clustering and phylogenetic analysis of transporter sequences. One of these (FGSG_02865), was found to occur across the Fusarium sambucinum complex (F. graminearum, F. culmorum, F. cerealis) and was upregulated starting 24 h after fungicide treatments. This delayed response may point to possible involvement of DHA1 antiporters in a generalised response to stress resulting from fungicide treatment. Additional expression profiling was conducted for the mycotoxin biosynthetic genes (trichothecene and zearalenone gene clusters) in strains of Fusarium sambucinum complex cereal pathogens. The expression changes, when subjected to treatment with the fungicides (flusilazole, carbendazim), show that even an effective treatment (in this study, the benzimidazole fungicide carbendazim) applied to the grown mycelium, can result in enhanced activation of mycotoxin biosynthetic genes in fungal cells which survive the treatment. Our results suggest that increased mycotoxin contamination can be strongly influenced not only by the amount or the type of antifungal compound, but also the timing of fungicide exposition (stage of infection).     

Natural occurrence of fusarium head blight,mycotoxins and mycotoxin‐producing isolates of Fusarium in commercial fields of wheat in Hubei

Combined analyses of the natural occurrence of fusarium head blight (FHB), mycotoxins and mycotoxin‐producing isolates of Fusarium spp. in fields of wheat revealed FHB epidemics in 12 of 14 regions in Hubei in 2009. Mycotoxin contamination ranged from 0·59 to 15·28 μg g^?1 in grains. Of the causal agents associated with symptoms of FHB, 84% were Fusarium asiaticum and 9·5% were Fusarium graminearum, while the remaining 6·5% were other Fusarium species. Genetic chemotyping demonstrated that F. asiaticum comprised deoxynivalenol (DON), 3‐acetyldeoxynivalenol (3‐AcDON), 15‐acetyldeoxynivalenol (15‐AcDON) and nivalenol (NIV) producers, whereas F. graminearum only included DON and 15‐AcDON producers. Compared with the chemotype patterns in 1999, there appeared to be a modest shift towards 3‐AcDON chemotypes in field populations during the following decade. However, isolates genetically chemotyped as 3‐AcDON were present in all regions, whereas the chemical 3‐AcDON was only detected in three of the 14 regions where 3‐AcDON accounted for 15–20% of the DON and acetylated forms. NIV mycotoxins were detected in seven regions, six of which also yielded NIV chemotypes. The number of genetic 3‐AcDON producers was positively correlated with amounts of total mycotoxins (DON, NIV and acetylated forms) or DON in wheat grains. Chemical analyses of wheat grains and rice cultures inoculated with different isolates from the fields confirmed their genetic chemotypes and revealed a preferential biosynthesis of 3‐AcDON and 4‐AcNIV in rice. These findings suggest the importance of chemotyping coupled with species identification for improved prediction of mycotoxin contamination in wheat.     

United States Department of Agriculture-Agricultural Research Service research on pre-harvest prevention of mycotoxins and mycotoxigenic fungi in US crops

Mycotoxins (ie toxins produced by molds) are fungal metabolites that can contaminate foods and feeds and cause toxic effects in higher organisms that consume the contaminated commodities. Therefore, mycotoxin contamination of foods and feeds results is a serious food safety issue and affects the competitiveness of US agriculture in both domestic and export markets. This article highlights research accomplished by Agricultural Research Service (ARS) laboratories on control of pre-harvest toxin contamination by using biocontrol, host-plant resistance enhancement and integrated management systems. Emphasis is placed on the most economically relevant mycotoxins, namely aflatoxins produced by Aspergillus flavus, Link, trichothecenes produced by various Fusarium spp and fumonisins produced by F verticillioides. Significant inroads have been made in establishing various control strategies such as development of atoxigenic biocontrol fungi that can outcompete their closely related, toxigenic cousins in field environments, thus reducing levels of mycotoxins in the crops. Potential biochemical and genetic resistance markers have been identified in crops, particularly in corn, which are being utilized as selectable markers in breeding for resistance to aflatoxin contamination. Prototypes of genetically engineered crops have been developed which: (1) contain genes for resistance to the phytotoxic effects of certain trichothecenes, thereby helping reduce fungal virulence, or (2) contain genes encoding fungal growth inhibitors for reducing fungal infection. Gene clusters housing the genes governing formation of trichothecenes, fumonisins and aflatoxins have been elucidated and are being targeted in strategies to interrupt the biosynthesis of these mycotoxins. Ultimately, a combination of strategies using biocompetitive fungi and enhancement of host-plant resistance may be needed to adequately prevent mycotoxin contamination in the field. To achieve this, plants may be developed that resist fungal infection and/or reduce the toxic effects of the mycotoxins themselves, or interrupt mycotoxin biosynthesis. This research effort could potentially save affected agricultural industries hundreds of millions of dollars during years of serious mycotoxin outbreaks.     

Systematics of key phytopathogenic Fusarium species: current status and future challenges

This review is intended to provide plant pathologists and other scientists with a current overview of the most important Fusarium phytopathogens and mycotoxin producers. Knowledge of Fusarium species diversity and their evolutionary relationships has increased dramatically due to the application of multilocus molecular phylogenetics and genealogical concordance phylogenetic species recognition over the past 15 years. Currently Fusarium is estimated to comprise at least 300 genealogically exclusive phylogenetic species; however, fewer than half have been formally described. The most important plant pathogens reside in the following four groups: the F. fujikuroi species complex noted for Bakanae of rice, ear rot of maize, pitch canker of pine and several species that contaminate corn and other cereals with fumonisin mycotoxins; the F. graminearum species complex including the primary agents causing Fusarium head blight of wheat and barley that contaminate grain with trichothecene mycotoxins; the F. oxysporum species complex including vascular wilt agents of over 100 agronomically important crops; and the F. solani species complex, which includes many economically destructive foot and root rot pathogens of diverse hosts. Several other Fusarium phytopathogens reported from Japan and nested within other species complexes are reviewed briefly. With the abandonment of dual nomenclature, a broad consensus within the global community of Fusarium researchers has strongly supported the unitary use of the name Fusarium instead of several teleomorph names linked to it. Plant pathologists and other scientists needing accurate identifications of Fusarium isolates are encouraged to use Fusarium-ID and Fusarium MLST, Internet accessible websites dedicated to the molecular identification of Fusarium species.     

Incidence and diversity of the fungal genera Aspergillus and Penicillium in Portuguese almonds and chestnuts

Almonds (Prunus dulcis (Miller) D.A. Webb) and European (sweet) chestnuts (Castanea sativa Miller) are of great economic and social impact in Mediterranean countries, and in some areas they constitute the main income of rural populations. Despite all efforts to control fungal contamination, toxigenic fungi are ubiquitous in nature and occur regularly in worldwide food supplies, and these nuts are no exception. This work aimed to provide knowledge on the general mycobiota of Portuguese almonds and chestnuts, and its evolution from field to the end of storage. For this matter, 45 field chestnut samples and 36 almond samples (30 field samples and six storage samples) were collected in Trás-os-Montes, Portugal. All fungi belonging to genus Aspergillus were isolated and identified to the section level. Fungi representative of other genera were identified to the genus level. In the field, chestnuts were mainly contaminated with the genera Fusarium, Cladosporium, Alternaria and Penicillium, and the genus Aspergillus was only rarely found, whereas almonds were more contaminated with Aspergillus. In almonds, Aspergillus incidence increased significantly from field to the end of storage, but diversity decreased, with potentially toxigenic isolates belonging to sections Flavi and Nigri becoming more significant and widespread throughout storage. These fungi were determined to be moderately associated, which can be indicative of mycotoxin co-contamination problems if adequate storage conditions are not secured.     

Fungal invasion and mycotoxin contamination of stored sorghum grain as influenced by threshing methods

Sanitation from harvest to storage is a key factor in eliminating sources of infection and reducing levels of mycotoxigenic fungal invasion, and thereby mycotoxin contamination. This study was conducted at Haramaya University, Eastern Ethiopia to investigate the effects of threshing methods on fungal invasion and mycotoxin contamination of sorghum grain. The methods included threshing on bare ground, cow-dung-painted ground, concrete asphalt and canvas. The threshed grain was kept separately in bags in three replications in a completely randomized design and stored for five months. The results revealed that all the sorghum grain samples taken from different threshing methods were contaminated with both Aspergillus and Fusarium species. There were variations in fungal invasion between samples taken immediately after threshing and stored samples. The level of Aspergillus spp. invasion was much higher in the latter in sorghum grain threshed on bare ground. The concentration of aflatoxin B₁ was gradually increased, whereas the total fumonisin concentration was decreased with the storage duration both in 2013 and 2014. On stored sorghum grain, the highest (1.97 µg kg^?1) and the lowest (0.70 µg kg^?1) mean aflatoxin B₁ concentrations were recorded from sorghum grains threshed on bare ground and on canvas, respectively. Sorghum grain obtained from canvas threshing method had significantly lower mean total fumonisin content (142.5, 54.9 µg kg^?1) than grain from the other methods at threshing. The current work clearly demonstrated the effect that threshing might have on fungal invasion and mycotoxin contamination of sorghum grains. .     

Mykotoxinbildung während der Infektion der Weizenähre durch den pathogenen Pilz Fusarium graminearum

Fusarium graminearum is one of the most destructive pathogens of cereals and a threat to food and feed production worldwide. Infection especially of wheat leads to yield losses and mycotoxin contamination. Deoxynivalenol is a hazardous mycotoxin inhibiting the biosynthesis of proteins. Its production by the fungus is a prerequisite for the colonization of the wheat spike. Combining molecular genetics with bioimaging techniques allows a fascinating insight into the pathobiology of this cereal pathogen.