Giovanni Beccari

Brachypodium distachyon: a new pathosystem to study Fusarium head blight and other Fusarium diseases of wheat

BackgroundFusarium species cause Fusarium head blight (FHB) and other important diseases of cereals. The causal agents produce trichothecene mycotoxins such as deoxynivalenol (DON). The dicotyledonous model species Arabidopsis thaliana has been used to study Fusarium-host interactions but it is not ideal for model-to-crop translation. Brachypodium distachyon (Bd) has been proposed as a new monocotyledonous model species for functional genomic studies in grass species. This study aims to assess the interaction between the most prevalent FHB-causing Fusarium species and Bd in order to develop and exploit Bd as a genetic model for FHB and other Fusarium diseases of wheat.ResultsThe ability of Fusarium graminearum and Fusarium culmorum to infect a range of Bd tissues was examined in various bioassays which showed that both species can infect all Bd tissues examined, including intact foliar tissues. DON accumulated in infected spike tissues at levels similar to those of infected wheat spikes. Histological studies revealed details of infection, colonisation and host response and indicate that hair cells are important sites of infection. Susceptibility to Fusarium and DON was assessed in two Bd ecotypes and revealed variation in resistance between ecotypes.ConclusionsBd exhibits characteristics of susceptibility highly similar to those of wheat, including susceptibility to spread of disease in the spikelets. Bd is the first reported plant species to allow successful infection on intact foliar tissues by FHB-causing Fusarium species. DON appears to function as a virulence factor in Bd as it does in wheat. Bd is proposed as a valuable model for undertaking studies of Fusarium head blight and other Fusarium diseases of wheat.

Fusarium species, chemotype characterisation and trichothecene contamination of durum and soft wheat in an area of central Italy

BACKGROUND Fusarium head blight (FHB) of wheat is an important disease causing yield losses and mycotoxin contamination. The aim of the work was to detect and characterise trichothecene producing Fusarium species in durum and soft wheat cultivated in an area of central Italy in 2009 and 2010 and to determine trichothecene contamination by LC-MS/MS in the grain. RESULTS F. graminearum s. str. was the most frequent species. In 2009, the occurrence of F. avenaceum and F. poae was higher than in 2010. Among F. graminearum strains, the 15-acetyl deoxynivalenol (15-ADON) chemotype could be found more frequently, followed by nivalenol (NIV) and 3-ADON chemotypes, while all F. culmorum isolates belonged to the 3-ADON chemotype. All F. poae strains were NIV chemotypes. In vitro trichothecene production confirmed molecular characterisation. Durum wheat was characterised by a higher average DON contamination with respect to soft wheat, NIV was always detected at appreciable levels while type-A trichothecenes were mostly found in durum wheat samples in 2009 with 6% of samples exceeding the contamination level recently recommended by the European Union. CONCLUSION Climatic conditions were confirmed to be predominant factors influencing mycotoxigenic species composition and mycotoxin contaminations. However, NIV contamination was found to occur irrespective of climatic conditions, suggesting that it may often represent an under-estimated risk to be further investigated.

Infection by mycotoxigenic fungal species and mycotoxin contamination of maize grain in Umbria, central Italy

Surveys were carried out in 2006 and 2007 in Umbria (central Italy) to evaluate the presence of mycotoxigenic fungi and mycotoxins in maize grain sampled at harvest. Fusarium spp., were the most abundant species detected in maize kernels, followed by Aspergillus species of sections Flavi and Nigri and by Penicillium spp. Among Fusarium species, F. verticillioides was the most prevalent species, as detected by PCR directly on the kernels and on the fungi isolated from the kernels, followed by F. proliferatum and F. subglutinans. Fumonisins were the predominant mycotoxins with values, on average, of 4.3 and 5.7 mgkg(-1), in 2006 and 2007, respectively, with a maximum of 76.3 mgkg(-1) in the second year. Deoxynivalenol ranged from 0.2 to 3.98 mgkg(-1) in 2006 (average 1.04 mgkg(-1)) and from undetectable levels to 14 mgkg(-1) in 2007 (average 0.86 mgkg(-1)). Aflatoxins, analyzed only in 2007, averaged 26.3 μgkg(-1), with a maximum of 820 μgkg(-1). Zearalenone content was always very low. Results indicate that EU legal limits for these mycotoxins were rarely exceeded with low levels across most of the examined area, suggesting that this region could be considered suitable for the production of healthy maize.

Characterization of Fusarium verticillioides strains isolated from maize in Italy: Fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B₁, B₂, and B₃ were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg⁻¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.

Presence of Fusarium Species and Other Toxigenic Fungi in Malting Barley and Multi-Mycotoxin Analysis by Liquid Chromatography–High-Resolution Mass Spectrometry

A study was carried out on 43 malting barley samples collected in 2013 across the Umbria region (central Italy) to determine the incidence of the principal mycotoxigenic fungal genera, to identify the Fusarium species isolated from the grains, and to detect the presence of 34 fungal secondary metabolites by liquid chromatography-high-resolution mass spectrometry. The multimycotoxin-method development involved the evaluation of both a two-step solvent and QuEChERS protocol for metabolite extraction. The former protocol was selected because of better accuracy, which was evaluated on the basis of spike-recovery experiments. The most frequently isolated fungal species belonged to the genera Alternaria and Fusarium. The predominant Fusarium species was F. avenaceum, followed by F. graminearum. HT-2 toxin was the most frequently detected mycotoxin, followed by enniatin B, enniatin B1, T-2 toxin, and nivalenol. As a consequence of the observed mixed fungal infections, mycotoxin co-occurrence was also detected. A combination of mycological and mycotoxin analyses allowed the ability to obtain comprehensive information about the presence of mycotoxigenic fungi and their contaminants in malting barley cultivated in a specific geographic area.

First report of Miscanthus × giganteus rhizome rot caused by Fusarium avenaceum, Fusarium oxysporum and Mucor hiemalis

In 2007, after the failure of establishment of a miscanthus experimental field, rhizome rot was observed and several fungi were isolated from diseased tissue, namely Fusarium avenaceum, F. oxysporum and Mucor hiemalis. All three fungi proved to be causal agents of the disease. This is the first report of a miscanthus rhizome rot caused by F. avenaceum, F. oxysporum and M. hiemalis.

Changes in the Fusarium Head Blight Complex of Malting Barley in a Three-Year Field Experiment in Italy

In this study, conducted for three years on eleven malting barley varieties cultivated in central Italy, the incidence of different mycotoxigenic fungal genera, the identification of the Fusarium species associated with the Fusarium Head Blight (FHB) complex, and kernels contamination with deoxynivalenol (DON) and T-2 mycotoxins were determined. The influence of climatic conditions on Fusarium infections and FHB complex composition was also investigated. Fusarium species were always present in the three years and the high average and maximum temperatures during anthesis mainly favored their occurrence. The FHB complex was subject to changes during the three years and the main causal agents were F. poae, F. avenaceum, F. tricinctum and F. graminearum, which, even if constantly present, never represented the principal FHB agent. The relative incidence of Fusarium species changed because of climatic conditions occurring during the seasons. The FHB complex was composed of many different Fusarium species and some of them were associated with a specific variety and/or with specific weather parameters, indicating that the interaction between a certain plant genotype and climatic conditions may influence the presence of Fusarium spp. causing infections. With regard to mycotoxin contamination, T-2 toxin, in some cases, was found in kernels at levels that exceeded EU recommended values.

The cereal pathogen Fusarium pseudograminearum produces a new class of active cytokinins during infection

The fungal pathogen Fusarium pseudograminearum causes important diseases of wheat and barley. During a survey of secondary metabolites produced by this fungus, a novel class of cytokinins, herein termed Fusarium cytokinins, was discovered. Cytokinins are known for their growth-promoting and anti-senescence activities, and the production of a cytokinin mimic by what was once considered as a necrotrophic pathogen that promotes cell death and senescence challenges the simple view that this pathogen invades its hosts by employing a barrage of lytic enzymes and toxins. Through genome mining, a gene cluster in the F. pseudograminearum genome for the production of Fusarium cytokinins was identified and the biosynthetic pathway was established using gene knockouts. The Fusarium cytokinins could activate plant cytokinin signalling, demonstrating their genuine hormone mimicry. In planta analysis of the transcriptional response to one Fusarium cytokinin suggests extensive reprogramming of the host environment by these molecules, possibly through crosstalk with defence hormone signalling pathways.

Risks Related to the Presence of Fungal Species and Mycotoxins in Grapes, Wines and Other Derived Products in the Mediterranean Area

Abstract The present chapter gives an up-to-date overview of the presence of fungal and mycotoxin contaminations of grapes and grape-derived products in the Mediterranean area. Mycotoxins represent a high risk for public health in food commodities worldwide. Aspergillus carbonarius and Aspergillus niger “aggregate” are the main fungal species causing grape rot and producing the mycotoxin ochratoxin A (OTA), which can be found in the grape chain. Wine represents the second source of human exposure to OTA after cereals, with red wines generally being more contaminated than rose and white dry wines. These differences can be explained by varietal susceptibility, wine-making techniques, and the latitude and weather conditions of the country producing a certain type of wine. Sweet or special wines from dehydrated grapes as well as grape juices and dried vine fruits result in higher OTA levels. Preventive control measures in pre- and post-harvest are the best tools for reducing OTA contamination. Furthermore, more efforts to develop simple, low cost, harmonized sampling and analytical methods are necessary.

Effect of wheat infection timing on Fusarium head blight causal agents and secondary metabolites in grain

Fusarium head blight (FHB) results in yield loss and damaging contamination of cereal grains and can be caused by several Fusarium species. The objective of the present study was to determine, in a greenhouse experiment on winter wheat, how FHB was affected by timing of infection (0, 3, 6 or 9 days after anthesis, daa) by the aggressive species Fusarium graminearum compared to the relatively weak species Fusarium avenaceum, Fusarium poae and Fusarium acuminatum. Measures of FHB development were: symptoms in spikes (visually assessed), fungal biomass (quantified by real time quantitative PCR) and accumulation of fungal secondary metabolites (quantified by liquid chromatography-tandem mass spectrometry) in kernels. With regard to symptoms, F. graminearum was unaffected by inoculation timing, while the weaker pathogens caused greater disease severity at later timings. In contrast, the accumulation of F. graminearum biomass was strongly affected by inoculation timing (3 daa ≥ 6 daa ≥ 0 daa = 9 daa), while colonization by the weaker pathogens was less influenced. Similarly, F. graminearum secondary metabolite accumulation was affected by inoculation timing (3 daa ≥ 6 daa ≥ 0 daa = 9 daa), while that of the weaker species was less affected. However, secondary metabolites produced by these weaker species tended to be higher from intermediate-late inoculations (6 daa). Overall, infection timing appeared to play a role particularly in F. graminearum colonization and secondary metabolite accumulation. However, secondary metabolites of weaker Fusarium species may be relatively more abundant when environmental conditions promote spore dispersal later in anthesis, while secondary metabolites produced by F. graminearum are relatively favored by earlier conducive conditions.