Antonio Prodi

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.

Natural co-occurrence of mycotoxins in wheat grains from Italy and Syria

This article describes the application of an analytical method for the detection of 25 mycotoxins in wheat grain based on simultaneous extraction using matrix solid-phase dispersion (MSPD) followed by liquid chromatography coupled to tandem mass spectrometry, a hybrid triple quadrupole-linear ion trap mass spectrometer (QTrap®). Information Dependent Acquisition (IDA), an extra confirmation tool for samples that contain the target mycotoxins, was used. The analysis of 40 Syrian and 46 Italian wheat grain samples interestingly showed that Syrian samples were mainly contaminated with ochratoxin A and aflatoxins, whereas Italian samples with deoxynivalenol and 15-acetyldeoxynivalenol. Emerging Fusarium mycotoxins were predominant in Italian samples compared to the Syrian. Among the analysed samples, only one was found containing zeralenone with level above the maximum European recommended concentration (100 ppb). These results confirm that climatic differences between Syria and Italy, both in Mediterranean basin, play a key role in the diversity of fungal genera and mycotoxins in wheat grains.

Study of Fungal Colonization of Wheat Kernels in Syria with a Focus on Fusarium Species

Wheat is one of the main crops in Mediterranean countries, and its cultivation has an important role in the Syrian economy. In Syria, Fusarium head blight (FHB) has not been reported so far. Mycological analysis of 48 samples of wheat kernels collected from cultivation areas with different climatic conditions were performed in 2009 and 2010. Fungal isolates were identified at the genus level morphologically; Fusarium species were characterized morphologically and by species-specific PCR. The most frequent fungal genera found were Alternaria spp. and Cladosporium spp., with frequencies of 24.7% and 8.1%, respectively, while the frequency of Fusarium spp. was 1.5% of kernels. Most frequent Fusarium species were F. tricinctum (30% of all Fusarium isolates), F. culmorum (18%), F. equiseti (14%) and F. graminearum (13%). The mycotoxin production potential of selected Fusarium isolates was assessed by HPLC-MS analysis of rice cultures; chemotyping by PCR was carried out for comparison. All six F. graminearum strains tested produced small amounts (<3 mg/kg) of nivalenol (NIV). All ten F. culmorum strains tested produced large amounts of trichothecenes (>100 mg/kg); four strains produced NIV and six strains produced deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3Ac-DON). PCR chemotyping lead to an oversimplified picture, because all 3Ac-DON chemotype strains produced more DON than 3Ac-DON; furthermore, the strongest NIV producers produced significant amounts of DON. All tested strains of F. culmorum, F. graminearum, F. pseudograminearum (two strains) and most F. equiseti strains (five of six strains) produced zearalenone. Grains of durum wheat were more frequently colonized by Fusarium spp. than grains of soft wheat. Incidence of Fusarium spp. in irrigated fields was higher than in rainfed fields. The incidence of Fusarium strains producing mycotoxins raises concerns about the risk of Fusarium head blight to Syria and its consequences for public health.

Validation of a modified Petri-dish test to quantify aggressiveness of Fusarium graminearum in durum wheat

Fusarium graminearum is a common agent causing Fusarium head blight (FHB) on wheat throughout the world. Aggressiveness is crucial for understanding the interaction between host-pathogen in the FHB-wheat system. In this paper, we modified and validated the Petri-dish test originally described by Mesterhazy (Phytopathologische Zeitschrift 93:12–25, 1978) to quantify the aggressiveness of 25 F. graminearum strains using four durum wheat cultivars with different resistance levels for FHB. The results were highly significant and correlated with those obtained using adult plants in the growth chamber and in the field (r = 0.94, P < 0.001 and r = 0.65, P < 0.001, respectively). The Petri-dish test was further investigated for its repeatability and stability in different durum wheat cultivars and highly significant correlation coefficients were obtained (r = 0.90–0.91 (P < 0.001), 0.89–0.95 (P < 0.001), respectively). In this study, we also demonstrated that germination rate reduction and coleoptile length reduction are parameters involved with aggressiveness of F. graminearum. The mean of three disease parameters from the modified Petri-dish method is introduced in this paper as a new parameter for aggressiveness and named “Petri-dish aggressiveness index”. The results obtained reveal that this modified Petri-dish test is rapid, reliable and stable with different durum wheat cultivars, and yields highly significant correlation coefficients with floret and ear inoculations, thus it is suitable to be used for quantification of aggressiveness of F. graminearum.

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.

Fusarium proliferatum and fumonisin B1 co-occur with Fusarium species causing Fusarium Head Blight in durum wheat in Italy

Fusarium Head Blight caused by phytopathogenic Fusarium spp. with Fusarium graminearum as main causal agent is a major disease of durum wheat ( Triticum durum Desf.). Mycotoxins in wheat are dominated by trichothecenes B. Fumonisins have only occasionally been reported from wheat; their occurrence was attributed to Fusarium proliferatum and Fusarium verticillioides . We investigated kernels of durum wheat grown in Italy in 2008 - 2010 for colonization with Fusarium spp. and for the content of Fusarium mycotoxins. Fungal biomass was determined using species-specific qPCR and mycotoxins were quantified by HPLC-MS/MS. Fusarium graminearum and Fusarium culmorum were dominating Fusarium species, followed by Fusarium poae, Fusarium tricinctum, and Fusarium proliferatum . No Fusarium verticillioides DNA was found. Toxicologically relevant levels of deoxynivalenol and nivalenol but no trichothecenes A were detected. Enniatins, fumonisin B1 and beauvericin were present in grain in all three years. Based on these results and on the evaluation of previous published reports, we hypothesize that low levels of fumonisins commonly occur in wheat grains produced in warm climate; they may remain undetected as long as mycotoxin monitoring programs for wheat do not include fumonisins. The only relevant source of fumonisins in wheat grain appears to be Fusarium proliferatum.

Detection of Fumonisins in Fresh and Dehydrated Commercial Garlic

An epidemic fungal disease caused by Fusarium proliferatum, responsible for fumonisin production (FB1, FB2, and FB3), has been reported in the main garlic-producing countries in recent years. Fumonisins are a group of structurally related toxic metabolites produced by this pathogen. The aim of this work was to establish an enzyme-linked immunosorbent assay (ELISA) procedure, mostly applied to cereals, that is suitable for fumonisin detection in garlic and compare these results to those obtained by high-performance liquid chromatography (HPLC) and screening of fresh and dehydrated garlic for toxicological risk. The results show good correlation between the two analytical methods. In fresh symptomatic garlic, fumonisin levels were higher in the basal plates than those in the portions with necrotic spots. Among the 56 commercially dehydrated garlic samples screened, three were positive by ELISA test and only one was above the limit of quantitation. The same samples analyzed by HPLC showed the presence of FB1 in trace amounts that was below the limit of quantitation; FB2 and FB3 were absent. The results are reassuring, because no substantial contamination by fumonisins was found in commercial garlic.

Determination of deoxynivalenol and nivalenol producing chemotypes of Fusarium graminearum isolated from durum wheat in different Italian regions

Determination of deoxynivalenol and nivalenol producing chemotypes of Fusarium graminearum isolated from durum wheat in different Italian regions Durum wheat production in Italy is economically of great importance. Fusarium graminearum is the main fusarium head blight (FHB) causal agent in wheat, reducing both yield and grain quality. F. graminearum produces several mycotoxins and, among trichothecenes, deoxynivalenol (DON) and nivalenol (NIV) are the most studied for their toxicity towards humans and animals. DON-producing isolates can be further distinguished on the basis of the predominant acetyl-DON derivative in 3-acetyldeoxynivalenol (3-ADON) or 15-acetyldeoxynivalenol (15-ADON). In order to evaluate possible mycotoxin contamination risks in food, it is very important to know which chemotype is the prevalent in a F. graminearum population. F. graminearum sensu stricto strains were collected from symptomatic durum wheat heads and grains of several naturally infected fields located mostly in Emilia - Romagna, The Marche, Lazio, Tuscany and Umbria. A multiplex PCR in the region of genes Tri12, located in the terminal gene cluster of trichothecenes, was used to characterize 187 single-spore isolates of F. graminearum as NIV, 3-ADON and 15-ADON chemotypes. All the three chemotypes were present in the F. graminearum population studied. The most frequent chemotype was 15-ADON (83.4%), followed by 3-ADON (10.7%) and NIV (5.9%). NIV-producing isolates were found only in Emilia-Romagna (3.5%), Umbria (33.3%) and The Marche (5.7%).

Development of three fusarium crown rot causal agents and systemic translocation of deoxynivalenol following stem base infection of soft wheat

Fusarium pseudograminearum, F. culmorum and F. graminearum are the most important fusarium crown rot (FCR) causal agents. They have the common ability to biosynthesize deoxynivalenol (DON). To elucidate the behaviour of each of the three species, a comparative study was carried out to investigate symptom progression, fungal systemic growth and translocation of DON following stem base inoculation of soft wheat. FCR symptoms were mainly localized in the inoculated area, which extended up to the second node for all inoculated species. Only the most aggressive strains caused symptoms up to the third node. Real-time quantitative PCR showed that fungal colonization reached the third node for all the tested species, but a low percentage of plants showed colonization above the third node following inoculation with the most aggressive strains. Fungal growth was detected in symptomless tissues but none of the three species was able to colonize as far as the head tissues. However, even if the pathogens were not detected in the heads, DON was detected in head tissues of the plants inoculated with the most aggressive strains. These results demonstrate that F. pseudograminearum, F. culmorum and F. graminearum, under the same experimental conditions, follow a similar pattern of symptom progression, fungal colonization and DON translocation after stem base infection. Differences in the extent of symptoms, fungal colonization and mycotoxin distribution were mainly attributable to strain aggressiveness. These findings provide comparative information on the events following infection of the stem base of wheat by three of the most important FCR casual agents.