Targeted and untargeted metabolomics profiling and evaluation of fungal communities in Italian wheat grains to assess the food security

Introduction With a production of about 6.8 million tons in 2023, Italy is one of the most importan European countries for wheat production. Wheat crops are greatly challenged by climate change and phytopathogenic fungi outbreaks that are evolving and spreading to new geographical areas, posing a significant threat for global food security and environmental sustainability. Furthermore, fungi such as Alternaria, Aspergillus, Fusarium and Penicilliumproduce mycotoxins that contaminate the major cereal crops [1]. Prolonged consumption of mycotoxin contaminated food and feed can lead to acute or chronic health problems to both human and animal health, due to chronic exposure, underestimated dietary intake of masked mycotoxins, and the synergistic threat of co-contaminations by multiple mycotoxins [2]. Experimental In this project, the grain of 13 durum wheat varieties and 11 soft wheat varieties was studied. These samples were harvested in 2023 in different regions of the Italian peninsula in the context of the wheat chain project that lies within the Spoke 9 of the PNRR National Centre CN2 - Agritech. An “untargeted” approach was initially performed using HRAM-Orbitrap and Compound Discoverer™-Thermo Scientific™ to screen for the presence of potential contaminants (chemical, microbiological) in cereals. Afterwards a “targeted” approach was performed using HPLC-MS/MS to screen for the presence of the main mycotoxins in cereals: Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), fumonisins (e.g., fumonisin B1, FB1, fumonisin B2, FB2), trichothecenes (e.g., DON, nivalenol, NIV, 15-acetyldeoxy nivalenol, 15-AcDON, 3-acetyldeoxy nivalenol, 3-AcDON, deoxynivalenol 3-glucoside, D3G, HT-2 toxin, HT-2, T-2 toxin), ochratoxin A (OTA) and zearalenone (ZEA), alternariol (ALT) [3]. To investigate the possibility of contamination we assessed the distribution of endophytic fungal communities through an in vitro selection. Results The “untargeted” approach was initially performed using HRAM-Orbitrap and Compound Discoverer™- Thermo Scientific™ to screen for the presence of potential contaminants (chemical, microbiological) in cereals, which led to the detection of traces of some mycotoxins such as deoxynivalenol (DON). Afterwards a “targeted” approach was performed using HPLC-MS/MS to screen for the presence of the main mycotoxins in cereals: AFB1, AFB2, FB1, FB2, DON, NIV, 15-AcDON, 3-AcDON, D3G, HT-2, T-2 toxin, OTA, ZEA, ALT [3]. The targeted HPLC-MS/MS analysis confirmed the absence of the previous mycotoxins, with only minimal traces detected in a few samples, confirming the quality and safety of these samples of Italian wheat, but it will be necessary to consider other crop seasons to confirm the results. In fact, climate conditions can be conductive or not according to the different crop season. Therefore, to investigate the possibility of contamination we assessed the distribution of endophytic fungal communities through an in vitro selection. This led to the isolation of 452 strains from the caryopses of the 24 wheat varieties considered. The initial phenotypic observation of the mycelia grown on PDA revealed common and specific distributions of the fungal communities: some isolates were abundant in all wheat samples, while others were unique to specific samples. Characterization studies of these fungal isolates are currently ongoing. Conclusions The isolation of pathogenic and/or mycotoxigenic fungi and the metabolomic profile of wheat grains, in relation to the geographical origin and the agronomic practices used for wheat cultivation represents an important step for preserving food safety and security in a warming climate contest. References 1. Alkuwari, Asma, et al. "Occurrence of mycotoxins and toxigenic fungi in cereals and application of yeast volatiles for their biological control." Toxins 14.6 (2022): 404. 2. Latham, Rosie L., et al. "Diverse mycotoxin threats to safe food and feed cereals." Essays in Biochemistry 67.5 (2023): 797-809. 3. Varga, Emese, Péter Fodor, and Csilla Soros. "Multi-mycotoxin LC-MS/MS method validation and its application to fifty-four wheat flours in Hungary." Food Additives & Contaminants: Part A 38.4 (2021): 670-680.