Sabrina Locatelli1*, Francesca Fumagalli1, Stefania Mascheroni1, Fabrizio Facchinetti1, Chiara Lanzanova1 1 Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria. Cerealicoltura e colture industriali. (CREA-CI). Via Stezzano, 24, 24126,
Bergamo (BG).
*[email protected] Abstract
Maize is subjected to infection by a variety of toxigenic fungi. This study is focused in Northern Italy, the main area of maize production (Piemonte, Lombardia, Veneto, Friuli Venezia Giulia, Emilia Romagna), to monitor the occurrence and accumulation of principal mycotoxins in the early stages of storage. A total of 1076 grain samples from about 50 storage centers distributed in the principal maize cultivation areas, were collected over a 3-year period (2014-2016). Fumonisins (FBs) were present in all samples and the percentage with a FBs content over 4000 µg/kg ranged from a minimum of 27% in 2015 to a maximum of 54% in 2014. Climatic conditions strongly affect growth of different fungal species. In 2015, the growing season of maize was characterized by prolonged high temperatures and low rainfall. These conditions have greatly promoted the development of Aspergillus flavus: 18% of maize samples were found over 20 µg/kg of aflatoxin B1 (AFB1).
In 2014 spring was extremely rainy. This delayed sowing, development and harvest and favoured the growth of Fusarium
graminearum; in fact, in 2014, 49% of the samples had a content of deoxynivalenol (DON) exceeding 1750 µg/kg and 40%
exceeded 350 µg/kg for zearalenone (ZEA). In 2016climate conditions (temperatures and precipitation) were favourable to maize until the third week of August; subsequently, an abrupt rise in temperatures caused a fall in yields, placing them between 2014 and 2015. Therefore, analysis carried out in 2016 showed that 12% of maize samples showed AFB1 content greater than 20 µg/kg.
Keywords: maize; mycotoxins; fumonisins; aflatoxin B1; deoxynivalenol; zearalenone; storage.
Parole chiave: mais; micotossine; fumonisine; aflatossina B1; deossinivalenolo; zearalenone; stoccaggio.
Introduction
Maize is a major crop in Italy, where it plays an important role in animal feed, for direct human consumption and as source of many commercial products. A number of studies have documented that maize is subjected to infection by a variety of toxigenicity fungi. In Italy mainly Fusarium verticillioides, F. graminearum, and Aspergillus flavus are responsible for the presence of the most common toxins fumonisins (FBs), deoxynivalenol (DON), zearalenone (ZEA), and aflatoxins (AFB) respectively. The presence of different fungi and their mycotoxins is influenced by environments and years. Indeed, the development of maize fungi and the consequent accumulation of mycotoxins are strongly influenced by: i) climatic factors (temperature, humidity), ii) biotic factors (insect attacks), iii) abiotic factors (hail, mechanical damage), iv) field stress conditions in the field (drought).
Available data for the incidence of mycotoxins on maize production in Italy are limited and irregular; additionally, no national database for collecting information to predict annual risk exposure is active (Berardo et al., 2011). Therefore, a systematic effort to monitor the levels of mycotoxin contaminants in maize grain production is needed. Since 1999, CREA coordinates a network of about 50 storage centers (Figure 1), stable in the years of investigation, distributed in Northern Italy, the main area of maize production (Piemonte, Lombardia, Veneto, Friuli Venezia Giulia, Emilia Romagna), to monitor the occurrence and levels of the main mycotoxins in the early stages of storage and preservation.
Figure 1: Italian Mycotoxin Maize Monitoring Network Figura 1: Rete di monitoraggio delle micotossine in mais in Italia.
Maize grain samples were collected over a 3-year period (2014–2016). This study shows the results of a survey on mycotoxin occurrence in Italian maize produced from in this period.
Materials and Methods
Maize samples. A total of 1076 representative grain samples were collected, after dry processing, over a 3-year period
(2014–2016) from about 50 storage centers. From each storage center, 5–10 samples were collected each year. A dynamic grain sampling strategy (Munkvold 2003) was performed on the product in motion to obtain a representative sample (Council for Agricultural Science and Technology (CAST) 2003). Accordingly, we took a pre-sample of 15–20 kg from all production components (whole and broken grains and small parts). From these pre-samples, by applying standard procedures, sorter samples of 1–1.5 kg each were obtained for laboratory analyses. These samples were placed in sealed bags and stored in a cool room for 1 day until milling, according to European recommendations (Commission Regulation 2006).
Chemical analyses. The grain samples were milled with Retsch - ZM 200 mill with 0.5 mm sieve. Mycotoxin concentration
levels were determined by the Enzyme-Linked Immunoassorbent Assay (ELISA). The Ridascreen® R-Biopharm kit tests were performed using the Chemwell Automatic Awareness Engineer (inc.).
Results and Discussion
An overview of mycotoxin contamination found in maize in the three years is presented in Table 1.
Table 1: Mycotoxins detected from 2014 to 2016 and percentage of maize samples resulted positive for contamination; fumonisins (FBs), aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEA).
Tabella 1: Micotossine rilevate dal 2014 al 2016 e percentuale di campioni di mais risultati positive alla contaminazione; fumonisine (FBs), aflatossina B1 (AFB1), deossinivalenolo (DON), zearalenone (ZEA).
Years N° of samples % of samples with FBs > 4000 µg/kg % of samples with AFB1 > 20 µg/kg % of samples with DON > 1750 µg/kg % of samples with ZEA > 350 µg/kg 2014 356 54% 0% 49% 40% 2015 400 27% 18% 1% 0% 2016 320 39% 12% 11% 0%
Fumonisins (FBs) are the most common mycotoxins found in the Italian maize area and the distribution of their concentration classes frequency is, in the different years, the most homogeneous and constant within all mycotoxins considered (Locatelli et al., 2016a). Fumonisins are produced by Fusarium verticillioides which, as Aspergillus flavus is ubiquitous, but is not able to tolerate drought and high temperatures. It prefers a milder humid weather conditions (optimum temperature between 22 and 27 °C), especially in post flowering. During 2016 (Locatelli et al., 2017), 39% of maize samples from storage centers showed FBs content above 4000 µg/kg, the limit value for food use (EC Regulation, 2007). This percentage ranged from 27% in 2015 (Locatelli et al., 2016b) to a 54%, in 2014 (Locatelli et al., 2015), as shown in Table 1.
Aflatoxin B1 (AFB1) is produced by Aspergillus flavus, whose growth is favored by high temperatures (optimum 32 °C - 36 °C, T min 12 °C, T max 42 °C) and low moisture content. The physiological phases in which maize cultivation is more susceptible to fungal attack are flowering and ripening; in particular, when silks are senescent (yellow-brown) and the grain moisture drops below 28%. An irregular evolution for maize cultivation was observed during 2016. The weather data provided by meteorological station of CREA Bergamo indicate that after a relatively cool and rainy period from June to August, during the period between August 20 and September 20, there was an exceptionally warm temperature with average temperatures higher than 3 °C over the reference period. This anomalous trend, observed in different regions affected: an anticipation of phenotypes of senescence and physiological maturation, a shortening of the accumulation period with consequent decrement production compared to forecasts until the beginning of August and finally allowed favorable conditions to the accumulation of aflatoxins (Mazzinelli et al., 2017). Therefore, analysis carried out in 2016 showed that 12% of maize samples showed AFB1 content greater than 20 µg/kg (Table 1), the reference value for maize destined for feed materials (EC Regulation, 2011). This result observed is lower than 18% obtained in 2015, year during which a hot and droughty summer, with average temperatures lower than in 2016 in the second half of August and September, involved the entire maize cycle. The maize field during 2014, characterized by mild and rainy summer, had not reported the presence of aflatoxins in the analysed samples.
Fusarium graminearum prefers a rainy climate condition and low temperatures from flowering to harvest. Better
temperature for its growth is between 24 and 26 °C. As for moisture, a greater need for Fusarium verticillioides was observed. During 2014: 49% of samples analysed showed deoxynivalenol (DON) values above 1750 µg/Kg (1% in 2015, 11% in 2016) and 40% of maize samples showed zearalenone (ZEA) above 350 µg/Kg, reference value for maize intended for human consumption (EC Regulation, 2007). In 2015 and 2016, none of the samples tested showed zearalenone (ZEA) content above 350 µg / Kg.
Conclusions
This study provides data on incidence and distribution of mycotoxin contamination of maize by sampling a large fraction of Italian maize grain production. The findings allow an assessment of the status of Italian production affected by mycotoxin contamination. The number of samples tested encompassed a large number of storage centers. Although variations in toxin levels is highly dependent on weather and growing conditions, the number of samples was sufficiently large to give a picture of the level of contamination in Italian maize grain production.
Fusarium verticillioides is endemically present in Italy, because of specific adaptation to the environmental and climatic
conditions. Particular climatic anomalies favour the presence of Aspergillus flavus or Fusarium graminearum. The problem related to mycotoxin contamination has reached levels of attention that cannot longer be considered less important e in relation to its use in human food and livestock. Monitoring activities conducted through a network of sampling stable over the years, is an essential tool for the management of domestic stocks and to highlight new mycotoxin alerts.
Acknowledgments
The research was carried out within the RQC-Mais research project, funded by the Ministry of Food and Forestry Policies (MiPAAF, DD 88666 of 03/12/2014).
Special thanks to storage centers belonging to the Mycotoxin Maize Monitoring Network.
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