Antonio Bottalico

Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe

The Fusarium species predominantly found associated with Fusarium head blight (FHB) in wheat and other small-grain cereals all over Europe are F. graminearum, F. avenaceum and F. culmorum. Among the less frequently encountered species are several others which are less pathogenic or opportunistic, but also toxigenic. These include F. poae, F. cerealisF. equisetiF. sporotrichioidesF. tricinctum and, to a lesser extent, F. acuminatumF. subglutinansF. solaniF. oxysporumF. verticillioidesF. semitectum and F. proliferatum. The species profile of FHB is due to several factors, primarily climatic conditions, particularly rain and the temperature at flowering stage, but also agronomic factors, such as soil cultivation, nitrogen fertilization, fungicides, crop rotation, and host genotype. The most frequently encountered Fusarium mycotoxins in FHB in Europe has proved to be deoxynivalenol and zearalenone produced by F. graminearum and F. culmorum with the former more common in southern (warmer) and the latter in northern (colder) European areas. Nivalenol was usually found associated with deoxynivalenol and its derivatives (mono-acetyldeoxynivalenols), together with fusarenone-X, formed by F. graminearumF. cerealisF. culmorum and, in northern areas, by F. poae. Moreover, from central to northern European countries, moniliformin has been consistently reported, as a consequence of the widespread distribution of F. avenaceum whereas the occurrence of T-2 toxin derivatives, such as T-2 toxin and HT-2 toxin, and diacetoxyscirpenol have been recorded in conjunction with sporadic epidemics of F. sporotrichioides and F. poae. Finally, beauvericin and various enniatins have recently been found in Finnish wheat colonized by F.avenaceum and F. poae.

Toxigenic Fusarium species and mycotoxins associated with maize ear rot in Europe

Several Fusarium species occurring worldwide on maize as causal agents of ear rot, are capable of producing mycotoxins in infected kernels, some of which have a notable impact on human and animal health. The main groups of Fusarium toxins commonly found are: trichothecenes, zearalenones, fumonisins, and moniliformin. In addition, beauvericin and fusaproliferin have been found in Fusarium-infected maize ears. Zearalenone and deoxynivalenol are commonly found in maize red ear rot, which is essentially caused by species of the Discolour section, particularly F. graminearum. Moreover, nivalenol and fusarenone-X were often found associated with the occasional occurrence of F. cerealis, and diacetoxyscirpenol and T-2 toxin with the occurrence of F. poae and F. sporotrichioides, respectively. In addition, the occurrence of F. avenaceum and F. subglutinans usually led to the accumulation of moniliformin. In maize pink ear rot, which is mainly caused by F. verticillioides, there is increasing evidence of the wide occurrence of fumonisin B1. This carcinogenic toxin is usually found in association with moniliformin, beauvericin, and fusaproliferin, both in central Europe due to the co-occurrence of F. subglutinans, and in southern Europe where the spread of F. verticillioides is reinforced by the widespread presence of F. proliferatum capable of producing fumonisin B1, moniliformin, beauvericin, and fusaproliferin.

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.

Natural occurrence ofAlternaria mycotoxins in olives—their production and possible transfer into the oil

A limited survey of the natural occurrence of the major Alternaria mycotoxins, i.e. alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), and tenuazonic acid (TA) has been carried out on olives and related processing products (oil and husks). The toxigenicity of Alternaria strains isolated from olives and the possible mycotoxin transfer into the oil have also been investigated. Four out of 13 olive samples were contaminated by 2 to 4 Alternaria mycotoxins. The highest contamination was found in a badly damaged sample containing 2.9, 2.3, 1.4 and 0.3 mg/kg of AME, AOH, ALT and TA, respectively. No mycotoxins were detected in olive-oil destined for human consumption (6 samples) or olive-husks (3 samples) collected from oil-mills after the first pressing of olives. An oil sample produced in our laboratory by processing the most contaminated olive sample contained AME (0.79 mg/kg) and AOH (0.29 mg/kg). The estimated mycotoxin amount transferred into the oil was 4% for AME, 1.8% for AOH, and zero for ALT and TA (considering 15% the oil yield). Although Alternaria species, mostly A. alternata (Fr.) Keissler, were present at various extent on all the examined olive samples, mycotoxins were only detected in samples of physically damaged olives. The production of mycotoxins by A. alternata isolated from olives was much higher (up to 3 order of magnitude for TA) on rice cultures than on olive cultures.

Fusarium species and their mycotoxins in infected corn in Italy.

Surveys of corn (infected plants and commercial kernels) forFusarium species and their mycotoxins were carried out on samples collected all over Italy and from some European and mediterranean countries.Investigations on samples of corn stalk and ear rot standing in the field, mainly collected in southern Italy, proved to be contaminated with zearalenone (ZON), zearalenols (ZOL), and deoxynivalenol (DON). TheFusarium species most frequently isolated, and their recorded toxigenic capability (in parentheses), were:F. moniliforme;F. culmorum (ZON, ZOL, DON, 3AcDON);F. equiseti (ZON, ZOL); andF. proliferatum (MF). Along with these species,F. graminearum group 2 (ZON, DON and/or 3AcDON or 15AcDON);F. chlamydosporum;F. acuminatum (type-A trichothecene derivatives); andF. semitectum were often found to be associated.F. heterosporum (ZON, ZOL);F. solani;F. crookwellense (ZON, ZOL, FUS, NIV);F. oxysporum (MF);F. avenaceum (MF);F. sporotrichioides (T-2 toxin and derivatives); andF. poae (DAS, MAS) were occasionally isolated.

Beauvericin and fumonisin B1 in preharvest Fusarium moniliforme maize ear rot in Sardinia

Six selected samples of preharvest maize ear rot, from different localities in Sardinia, Italy, were examined for causal Fusarium species and associated mycotoxins. All samples were almost exclusively found to be affected by Fusarium moniliforme, which was isolated from all infected ear sample kernels (100%). In two samples, in addition to F. moniliforme, F. proliferatum was also present but in a reduced percentage of kernels (up to 42%). All samples were found to be contaminated by fumonisin B1 (up to 250 mg/kg). Four samples were also found to be contaminated by beauvericin (up to 10 mg/kg), with higher concentration in samples also infected by F. proliferatum. When cultured on autoclaved maize kernels for 4 weeks at 25 degrees C, all 13 strains of F. moniliforme examined produced fumonisin B1 (up to 3750 mg/kg), whereas only three strains also produced beauvericin, but in very low amounts (5 mg/kg). In the same assay, four isolates of F. proliferatum also produced high amounts of fumonisin B1 (up to 2500 mg/kg) but this was associated with higher concentrations of beauvericin (up to 175 mg/kg). This is the first indication of the production of beauvericin by F. moniliforme, as well as of its co-occurrence with fumonisin B1 in preharvest F. moniliforme maize ear rot.

Deoxynivalenol and 3-acetyldeoxynivalenol - mycotoxins associated with wheat head fusariosis in Poland.

Samples of wheat naturally infected in the field byFusarium culmorum (W.G.Sm.) Sacc. andFusarium graminearum Schwabe were analyzed for deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone. No zearalenone was detected at levels higher than 0.5mg/kg. Deoxynivalenol was present in 100% and 3-acetyldeoxynivalenol in 80% of the examined samples at levels of 0.21 to 30.4 mg/kg and 0.54 to 29.54 mg/kg, respectively. The mycotoxin levels in the chaff were 5 to 50 times higher than in the kernels. This is the first report on natural occurrence of 3-acetyldeoxynivalenol in wheat. This toxin, in addition to deoxynivalenol, was highly correlated with wheat head fusariosis. These findings suggest that more attention should be given to the occurrence of 3-acetyldeoxynivalenol in cereal grains during the growth as well as during storage.

Moniliformin production by fusarium species.

A total of 132 Fusarium isolates belonging to 19 species sensu Nelson et al (1983) originating from Poland, Italy, and international cultures collections were examined for their ability to produce mycotoxin moniliformin.Moniliformin was produced by the following isolates:—F acuminatum Ell & Ev: 2 out of 2,130 – 2670mg/kg—F avenaceum (Fr) Sacc 18 out of 18,70 – 2670mg/kg—F anthophilum (A Braun) Wollenw. 1 out of 3, 200mg/kg—F dlamini Marasas et al: 2 out of 3,130 – 470mg/kg—F oxysporum Schlecht emend Snyd Hans: 4 out of 9,130 – 270 mg/kg—F proliferatum (Matsushima) Nirenberg: 3 out of 7,130 – 400 mg/kg—F solani (Mart) Appel & Wollenw: 1 out of 14,670 mg/kg—F subglutinans (Wollenw & Reinking) Nelson et al: 8 out of 20,70 – 1660 mg/kg—F tricinctum (Corda) Sacc: 2 out of 9,130 – 1330 mg/kgIn cultures ofF beomiforme Nelson, Toussoun & Burgess,F chlamydosporum Wollenw & Reinking,F compactum I Wollenw/ Gordon, F equiseti /Corda/Sacc,F poae I Peck / Wollenw,F moniliforme Sheldon,F napiforme Marasas, Nelson & Rabie,F nygamai Burgess & Timbold,F poly phialidicum Marasas et al,F sporotrichioides Sherb moniliformin was not detected.The highest amounts of moniliformin byF avenaceum using solid substrate were formed on rice and lower on oats kernels.

Trichothecene mycotoxins produced byFusarium sporotrichioides strain P-11

A highly toxic strain ofFusarium sporotrichioides Sherb. (P-11) isolated from wheat in Poland produced on rice culture up to 11 trichothecenes, which are: T-2 toxin (750 ppm), neosolaniol (300 ppm), HT-2 toxin (75 ppm), acetyl T-2 toxin (35ppm), 3′-hydroxy-T-2 (20ppm), T-2 triol (12.5ppm), 3′-hydroxy-HT-2 (1.2ppm), 4-acetoxy-T-2 tetraol (1.1 ppm), 15-acetoxy-T-2 tetraol (0.65 ppm), 8-acetoxy-T-2 tetraol (0.45 ppm), and T-2 tetraol (0.2 ppm). The presence of most of these trichothecenes, including the 3′-hydroxy-derivatives, in the excreta of animals treated with T-2 toxin indicates the existence of some correlation between T-2 toxin metabolism in animals and microorganisms, respectively.

Production of type A trichothecenes and enniatin B byFusarium sambucinum Fuckel sensu lato

Twenty-nineFusarium isolates, representing three new taxa originated by Nirenberg fromF. sambucinum Fuckel sensu lato, namely:F. sambucinum Fuckel sensu stricto,F. venenotum Nirenb., andF. torulosum (Berk. & Curt.) Nirenb., were tested for in vitro production of toxic secondary metabolites on autoclaved corn kernels.F. sambucinum sensu stricto was able to produce type A trichothecenes and enniatin B (EB). In particular, amongst the 14 isolates tested, 5 produced only diacetoxyscirpenol (DAS) (up to 700 µg/g); 1 produced only neosolaniol (NEOS) (250 µg/g); 2 produced T-2 toxin (T-2) + NEOS (up to 175 and 150 µg/g, respectively); 1 produced NEOS + DAS (300 and 100 µg/g, respectively); and 5 produced DAS + EB (up to 500 and 140 µg/g, respectively). All six isolates ofF. venenotum were able to produce only DAS (up to 100 µg/g).F. torulosum produced no trichothecenes, but four out of nine tested isolates were able to produce EB (up to 140 µg/g). Zearalenones and type B trichothecenes were not found. The toxicity of the culture extracts towardsArtemia salina L. was correlated in general with the occurrence of the above toxins, except for someF. torulosum strains. However, the lack of correlation between the amounts of toxins recovered and toxic activity observed in theGeotrichum candidum Link ex Pers. andA. salina assays suggested the presence of unknown toxic compounds.