Effect of fungicides commonly used for Fusarium head blight management on growth and fumonisin production by Fusarium proliferatum.

Fumonisin occurrence was reported in wheat grains and F. proliferatum has been suggested to be the main contributor to its presence in wheat. Thus, a survey was performed in order to study the impact of four commercial fungicides used in Argentina for controlling Fusarium head blight disease (epoxiconazole+metconazole, tebuconazole, pyraclostrobin+epoxiconazole, and prothioconazole) on growth and fumonisin production of two F. proliferatum strains in relation to water activity (aW; 0.99, 0.97, 0.95) and temperature (15°C and 25°C). Most fungicides reduced growth rates when compared to the control (reduction increased as fungicide concentration increased), and reduced fumonisin production when they were used at high doses; however, most fungicides enhanced fumonisin production at sublethal doses, with the exception of prothioconazole. Thus, fungicides used for FHB management could enhance fumonisin production by F. proliferatum strains present in wheat grains.

Toxigenic fungal species and natural occurrence of mycotoxins in crops harvested in Argentina.

Mycotoxins are secondary metabolites produced by fungal species that mainly belong to Aspergillus, Fusarium, Penicillium and Alternaria, which can grow in a variety of crops including cereals, oilseeds and fruits. Consequently, their prevalence in foods and by-products not only affects human and animal health but also causes important losses in both domestic and international markets. This review provides data about toxigenic fungal species and mycotoxin occurrence in different crops commonly grown in Argentina. This information will be relevant to establish adequate management strategies to reduce the impact of mycotoxins on human food and animal feed chains and to implement future legislation on the maximum permitted levels of these fungal metabolites.

Sinorhizobium meliloti low molecular mass phosphotyrosine phosphatase SMc02309 modifies activity of the UDP-glucose pyrophosphorylase ExoN involved in succinoglycan biosynthesis.

In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This study demonstrated that the chromosomal ORF SMc02309 from Sinorhizobium meliloti 2011 encodes a protein with significant sequence similarity to low molecular mass protein-tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighbouring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyses p-nitrophenyl phosphate (p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on Medicago sativa roots.

Interacting Abiotic Factors Affect Growth and Mycotoxin Production Profiles of Alternaria Section Alternaria Strains on Chickpea-Based Media.

Chickpea is susceptible to fungal infection and mycotoxin contamination. Argentina exports most of its chickpea production; thus, its quality is of concern. The Alternaria fungal genus was found to be prevalent in chickpea samples from Argentina. The species within this genus are able to produce mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA). In this context, we evaluated the effect of water activity (0.99, 0.98, 0.96, 0.95, 0.94, 0.92, and 0.90 aW), temperature (4, 15, 25, and 30 °C), incubation time (7, 14, 21, and 28 days), and their interactions on mycelial growth and AOH, AME, and TA production on chickpea-based medium by two A. alternata strains and one A. arborescens strain isolated from chickpea in Argentina. Maximum growth rates were obtained at the highest aW (0.99) and 25 °C, with growth decreasing as the aW of the medium and the temperature were reduced. A. arborescens grew significantly faster than A. alternata. Mycotoxin production was affected by both variables (aW and temperature), and the pattern obtained was dependent on the strains/species evaluated. In general, both A. alternata strains produced maximum amounts of AOH and AME at 30 °C and 0.99-0.98 aW, while for TA production, both strains behaved completely differently (maximum levels at 25 °C and 0.96 aW for one strain and 30 °C and 0.98 aW for the other). A. arborescens produced maximum amounts of the three toxins at 25 °C and 0.98 aW. Temperature and aW conditions for mycotoxin production were slightly narrower than those for growth. Temperature and aW conditions assayed are those found during chickpea grain development in the field, and also could be present during storage. This study provides useful data on the conditions representing a risk for contamination of chickpea by Alternaria toxins.

Influence of abiotic factors (water activity and temperature) on growth and aflatoxin production by Aspergillus flavus in a chickpea-based medium.

The effect of water activity (aW; 0.87, 0.90, 0.92, 0.94, 0.96, 0.98 and 0.99), temperature (15, 25, and 30 °C), incubation time (5, 10, 14, and 21 days), and their interactions on mycelial growth and aflatoxin production in a chickpea-based medium by three Aspergillus flavus strains isolated from chickpea grains in Argentina was evaluated. Maximum growth rates were obtained at the highest aW (0.99) and 30 °C, with growth decreasing as the aW of the medium was reduced. Maximum levels of aflatoxins were produced at 0.99 aW and 25 °C after 5 days of incubation for two strains, and at 25 °C and 0.96 aW after 21 days of incubation for the third strain. The aflatoxin concentrations varied considerably depending on the aW and temperature interactions assayed. Two-dimensional profiles of aW by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from aflatoxin accumulation on chickpea. This study provides useful baseline data on conditions representing a high and a low risk for contamination of chickpea by aflatoxins which is of greater concern because this pulse is destined mainly for human consumption.

Fusarium chaquense, sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina.

The Chaco wetland is among the most biologically diverse regions in Argentina. In collections of fungi from asymptomatic native grasses (Poaceae) from the wetlands, we identified isolates of Fusarium that were morphologically similar to F. armeniacum, but distinct from it by their production of abundant microconidia. All the isolates had identical, or nearly identical, partial sequences of TEF1 and RPB2. But they were distinct from reference sequences from F. armeniacum and Fusarium species closely related to it. Phylogenetic analysis of 34 full-length housekeeping gene sequences retrieved from whole genome sequences of three Chaco wetland isolates, 29 genes resolved the isolates as an exclusive clade within the F. sambucinum species complex. Based on results of the morphological and phylogenetic analysis, we concluded that the Chaco wetland isolates are a distinct and novel species, herein described as Fusarium chaquense, sp. nov., which is closely related to F. armeniacum. F. chaquense in culture can produce the trichothecenes T-2 and HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol, as well as beauvericin and the pigment aurofusarin. Genome sequence analysis also revealed the presence of three previously described loci required for trichothecene biosynthesis. This research represents the first study of Fusarium in a natural ecosystem in Argentina.

Ecophysiology of Fusarium chaquense a Novel Type A Trichothecene Producer Species Isolated from Natural Grasses.

Fusarium chaquense, a recently formally described novel species, has been identified as an T-2 toxin (T-2), HT-2 toxin (HT-2) and other toxins producer in natural grasses (Poaceae) from Argentina. The major objective of this study was to describe the effect of water activity (aW, 0.995, 0.98, 0.95, 0.93 and 0.91), temperature (15, 25 and 30 °C) and incubation time (5, 15 and 25 days) on growth and to evaluate the production of T-2, HT-2 toxins and beauvericin (BEA) by two F. chaquense strains in a grass-based media. The results showed a wide range of conditions for F. chaquense growth and mycotoxin production. Both strains had a maximum growth rate at the highest aW (0.995) and 25 °C. Regarding mycotoxin production, more T-2 than the other analysed mycotoxins were produced by the two strains. T-2 production was favoured at 0.995 aW and 30 °C, while HT-2 production at 0.98-0.95 aW and 15 °C. The maximum levels of BEA were produced at 0.995 aW and 25-30 °C. Two-dimensional profiles of aW by temperature interactions were obtained from these data in order to identify areas where conditions indicate a significant risk of mycotoxins accumulation on grass. For its versatility on growth and mycotoxin production in a wide range of aW and temperatures, F. chaquense would have an adaptive advantage over other Fusarium species, and this would explain its high frequency of isolation in natural grasses grown up in the Chaco wetlands.

A loop-mediated isothermal amplification (LAMP) based assay for the rapid and sensitive group-specific detection of fumonisin producing Fusarium spp.

Fumonisins are mycotoxins that contaminate maize and maize-based food products, and feed. They have been associated with nerve system disorders in horses, pulmonary edema in swine as well as neural tube defects and esophageal cancer in humans. The fum1 gene codes for a polyketide synthase involved in the biosynthesis of fumonisins. It is present in the genomes of all fumonisin producing Fusarium spp. Reliable detection of fum1 can provide an estimate of the toxicological potential of cultures and food sources. Therefore, a fum1 specific LAMP assay was developed and tested with purified DNA of 48 different species from the Fusarium fujikuroi species complex (FFSC). The fum1 gene was detected in 22 species among which F. fujikuroi, F. globosum, F. nygamai, F. proliferatum, F. subglutinans and F. verticillioides were the most prominent fumonisin producers. None out of 92 tested non-Fusarium species showed cross reactions with the new assay. The lowest limit of detection (LOD) was 5 pg of genomic DNA per reaction for F. fujikuroi, F. nygamai and F. verticillioides. Higher LODs were found for other LAMP positive species. Apart from pure genomic DNA, the LAMP assay detected fumonisin-producers when 103 conidia/reaction were used as template after mechanical lysis. LAMP-results were well correlated with FB1 production. This is the first report on fumonisin production in strains of F. annanatum, F. coicis, F. mundagurra, F. newnesense, F. pininemorale, F. sororula, F. tjataeba, F. udum and F. werrikimbe. Usefulness of the LAMP assay was demonstrated by analyzing fumonisin contaminated maize grains. The new LAMP assay is rapid, sensitive and reliable for the diagnosis of typical fumonisin producers and can be a versatile tool in HACCP concepts that target the reduction of fumonisins in the food and feed chain.

Fumonisin occurrence in wheat-based products from Argentina.

In Argentina, wheat is the most consumed cereal by the human population. Since fumonisins occurence in wheat grains and wheat-based products have been reported worldwide, a survey was conducted in order to determine fumonisin contamination in 91 wheat-based products (white wheat flour samples, wheat flour used at bakery products and whole-wheat flour samples) collected from different retail stores of Rio Cuarto city in Argentina using HPLC-MS/MS. Sixty-seven samples (74%) showed contamination by fumonisins. From these samples, 16 showed fumonisin levels between LOD and LOQ (between 0.01 to 0.05 ng/g), while fumonisins (FB1 + FB2) in quantifiable samples ranged from 0.05 ng/g to 18.9 ng/g. Although FB1 was more prevalent, FB2 was foun3d in higher levels than FB1. Overall, fumonisin prevalence was high, but concentrations were far below EU or USA limits set for maize and maize-based products.

Preliminary Study on the Use of Chitosan as an Eco-Friendly Alternative to Control Fusarium Growth and Mycotoxin Production on Maize and Wheat.

The objectives of the present study were to determine the combined effects of chitosan and water activity (aW) on growth and mycotoxin production in situ on the two most important Fusarium species (F. proliferatum and F. verticillioides) present on maize, and on F. graminearum, the main pathogen causing Fusarium head blight on wheat. Results showed that low-molecular-weight chitosan with more than 70% deacetylation at the lowest dose used (0.5 mg/g) was able to reduce deoxynivalenol (DON) and fumonisin (FBs) production on irradiated maize and wheat grains. Growth rates of F. graminearum also decreased at the lowest chitosan dose used (0.5 mg/g), while F. verticillioides and F. proliferatum growth rates were reduced at 0.98 aW at the highest chitosan dose used (2 mg/g). Since mycotoxins are unavoidable contaminants in food and feed chains, their presence needs to be reduced in order to minimize their effects on human and animal health and to diminish the annual market loss through rejected maize and wheat; in this scenario, pre- and post-harvest use of chitosan could be an important alternative.

Influence of water activity and temperature on growth and fumonisin production by Fusarium proliferatum strains on irradiated wheat grains.

Wheat is the most important cereal consumed by the Argentine population. In previous studies performed in durum and common wheat grains in this country it has been observed fumonisin contamination as well as high incidence of Fusarium proliferatum. Fumonisins are toxic fungal metabolites, and consumption of fumonisin-contaminated maize has been epidemiologically associated with oesophageal cancer and neural tube defects in some human populations. Using irradiated wheat-grains, the effects of abiotic factors, temperature (15, 25, and 30°C) and water activity (aW; 0.995, 0.98, 0.96, 0.94, 0.92, and 0.88), on mycelial growth and fumonisin biosynthesis were compared for three F. proliferatum strains isolated from wheat grains in Argentina. Although all isolates showed similar profiles of growth, the fumonisin production profiles were slightly different. Maximum growth rates were obtained at the highest aW (0.995) and 25°C, with growth decreasing as the aW of the medium was reduced. Maximum amounts of total fumonisins (FB1, FB2 and FB3) were produced at 0.995 aW and 15°C for 2 strains, and at 25°C and 0.995 aW for the third one. Fumonisins concentrations varied considerably depending on the aW and temperature interactions assayed. Studied strains showed different fumonisin production profiles. Two-dimensional profiles of aW by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk of fumonisins accumulation on wheat. As a result, temperature and aW conditions that resulted in fumonisins production are those found during wheat grain development (especially milk and dough stages) in the field. This is the first study made using irradiated wheat grains and provides useful baseline data on conditions representing a low or a high risk for fumonisins contamination of wheat grains which is of concern because this cereal is destined mainly for human consumption.

Abiotic conditions leading to FUM gene expression and fumonisin accumulation by Fusarium proliferatum strains grown on a wheat-based substrate.

Fusarium proliferatum produces fumonisins B not only on maize but also on diverse crops including wheat. Using a wheat-based medium, the effects of abiotic factors, temperature and water activity (aW), on growth, fumonisin biosynthesis, and expression of FUM genes were compared for three F. proliferatum strains isolated from durum wheat in Argentina. Although all isolates showed similar profiles of growth, the fumonisin production profiles were slightly different. Regarding FUM gene transcriptional control, both FUM8 and FUM19 expression showed similar behavior in all tested conditions. For both genes, expression at 25°C correlated with fumonisin production, regardless of the aw conditions. However, at 15°C, these two genes were as highly expressed as at 25°C although the amounts of toxin were very weak, suggesting that the kinetics of fumonisin production was slowed at 15°C. This study provides useful baseline data on conditions representing a low or a high risk for contamination of wheat kernels with fumonisins.

Two-dimensional environmental profiles of growth and fumonisin production by Fusarium proliferatum on a wheat-based substrate.

The effect of water activity (aW; 0.995, 0.99, 0.98, 0.96, 0.94, 0.92, and 0.90), temperature (15, 25, and 30°C), incubation time (7, 14, 21 and 28days), and their interactions on mycelial growth and fumonisin production on wheat-based medium by three Fusarium proliferatum strains isolated from wheat in Argentina was evaluated. Maximum growth rates were obtained at the highest aW (0.995) and 30°C, with growth decreasing as the aW of the medium was reduced. Maximum amounts of total fumonisins (FB1, FB2 and FB3) were produced at 0.99 aW and 25°C after 21 and 28days of incubation for 2 strains, and at 15°C and 0.98 aW after 28days of incubation for the third strain. The fumonisin concentrations varied considerably depending on the aW and temperature interactions assayed. The studied strains had different fumonisin production profiles. F. proliferatum ITEM 15661 and ITEM 15664 produced FB1 and FB2 whereas F. proliferatum ITEM 15654 was able to produce FB1, FB2 and FB3. Interestingly, fumonisin production profiles for each particular strain were related to incubation temperatures. Fumonisins were produced from 15 to 30°C and at aW values of 0.92 to 0.995 after 21 to 28days of incubation. However at 7 and 14days of incubation small amounts of fumonisin were produced at aW lower than 0.94. Two-dimensional profiles of aW by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from fumonisin accumulation on wheat. Temperature and aW conditions that resulted in fumonisin production are those found during wheat grain development (especially milk and dough stages) in the field. This study provides useful base line data on conditions representing a high and a low risk for contamination of wheat by fumonisins which is becoming of greater concern because this cereal is destined mainly for human consumption.

Combined effect of chitosan and water activity on growth and fumonisin production by Fusarium verticillioides and Fusarium proliferatum on maize-based media.

The objectives of the present study were to determine the in vitro efficacy of chitosan (0.5, 1.0, 2.0 and 3.0mg/mL) under different water availabilities (0.995, 0.99, 0.98, 0.96 and 0.93) at 25°C on lag phase, growth rate and fumonisin production by isolates of Fusarium verticillioides and Fusarium proliferatum. The presence of chitosan affected growth and fumonisin production, and this effect was dependent on the dose and aW treatment used. The presence of chitosan increased the lag phase, and reduced the growth rate of both Fusarium species significantly at all concentrations used, especially at 0.93 aW. Also, significant reduction of fumonisin production was observed in both Fusarium species at all conditions assayed. The present study has shown the combined effects of chitosan and aW on growth and fumonisin production by the two most important Fusarium species present on maize. Low molecular weight (Mw) chitosan with more than 70% of degree of deacetylation (DD) at 0.5mg/mL was able to significantly reduce growth rate and fumonisin production on maize-based media, with maximum levels of reduction in both parameters obtained at the highest doses used. As fumonisins are unavoidable contaminants in food and feed chains, their presence needs to be reduced to minimize their effects on human and animal health and to diminish the annual market loss through rejected maize. In this scenario post-harvest use of chitosan could be an important alternative treatment.

Evaluation of ability of ferulic acid to control growth and fumonisin production of Fusarium verticillioides and Fusarium proliferatum on maize based media.

The aim of this study was to evaluate the efficacy of ferulic acid (1, 10, 20 and 25 mM) at different water activity (aw) values (0.99, 0.98, 0.96 and 0.93) at 25 °C on growth and fumonisin production by Fusarium verticillioides and Fusarium proliferatum on maize based media. For both Fusarium species, the lag phase significantly decreased (p ≤ 0.001), and the growth rates increased (p ≤ 0.001) at the lowest ferulic acid concentration used (1mM), regardless of the aw. However, high doses of ferulic acid (10 to 25 mM) significantly reduced (p ≤ 0.001) the growth rate of both Fusarium species, regardless of the a(w). In general, growth rate inhibition increased as ferulic acid doses increased and as media aw decreased. Fumonisin production profiles of both Fusarium species showed that low ferulic acid concentrations (1-10mM) significantly increased (p ≤ 0.001) toxin production, regardless of the aw. High doses of ferulic acid (20-25 mM) reduced fumonisin production, in comparison with the controls, by both Fusarium species but they were not statistically significant in most cases. The results show that the use of ferulic acid as a post-harvest strategy to reduce mycotoxin accumulation on maize needs to be discussed.