Nanoformulations Use for Control of Fungal Contamination and Mycotoxins Production in Cereal Crops: Advances, Mechanisms and Future Prospects

Prevention of Mold Growth and Toxin Production through Control of Environmental Conditions

Formation and Control of Mycotoxins in Food

A laboratory study was conducted to evaluate the influence of cocultivation of toxigenic Fusarium (F.) and Alternaria (A.) fungi with respect to growth and mycotoxin production. Fusarium culmorum Fc13, Fusarium graminearum Fg23 and two Alternaria tenuissima isolates (At18 and At220) were simultaneously or consecutively co-incubated on wheat kernels in an in vitro test system. Fungal biomass was quantified by determining ergosterol content. Three Fusarium toxins (DON, NIV and ZON) and three Alternaria toxins (AOH, AME and ALT) were analysed by a newly developed HPLC/MS/MS method. In simultaneous cocultures, the fungal biomass was enhanced up to 460% compared with individual cultures; Alternaria toxins were considerably depressed down to <5%. Combining At18 and At220 with Fg23 inhibited the toxin production of both fungal partners. In contrast, Fc13 increased its DON and ZON production in competitive interaction with both A. strains. The interfungal competitive effects aid the understanding of the processes of competition of both fungi in natural environments and the involvement of mycotoxins as antifungal factors. Cocultivation significantly affects fungal growth and mycotoxin production of phytopathogenic Alternaria and Fusarium strains. The impact of mycotoxins on the interfungal competition is highlighted.

<abstract> Mycotoxins are secondary metabolites produced by filamentous fungi in food and feed due to several conditions that affect fungal growth and mycotoxin production in different ways. This review aims to explore the different factors that affect mycotoxin production and their control methods. Environmental conditions such as high temperature and humidity increase the risk of fungal growth and mycotoxin production. Other factors that affect contamination include pH, fungal strain, and substrate. To control mycotoxin contamination an integrated approach that starts in the field prior to planting and continues throughout the whole food chain is required, so good practices help minimize contamination at every step to deliver safe products. Good practices include proper practices on the field before and after planting, good harvest practices, appropriate drying measures, and good storage practices. Mycotoxin contamination is inevitable in food and once present they tend to remain as they are very stable compounds, although several physical, chemical and biological techniques could be applied to help minimize contamination. Food processing may also play a minimal role in controlling mycotoxins. Finally, regulations serve to keep food markets free from highly contaminated products, while proper sampling procedures and analytical methods ensure regulations endorsement. </abstract>

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.

Inhibition of Fusarium Growth and Mycotoxin Production in Culture Medium and in Maize Kernels by Natural Phenolic Acids

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively. Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination(1,2,3,4,5,6). Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (10(6)) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

Reactive oxygen species are the core of host plant defense and also play a vital role in the successful invasion of host plants by pathogenic fungi. Despite its importance, the relevance of oxidative stress response in fungal growth and virulence is poorly understood in P. expansum. In this study, we reveal that the transcription factor PeAP1 acts as a central regulator of oxidative stress response in P. expansum and that there is a major link between PeAP1-mediated oxidative stress response and fungal growth and virulence. To explore the underlying mechanisms, we performed comparative transcriptomic studies and identified a number of H2O2-induced PeAP1 target genes, including four novel ones, PePrx1, PePrx2, PeGST1, and PeTRX2, whose functions were linked to PeAP1 and pathogenicity. These findings provide novel insights into the regulation mechanism of PeAP1 on growth and virulence, which might offer promising targets for control of blue mold and patulin contamination.

Cereal grains are the most important food source for humans. As the global population continues to grow exponentially, the need for the enhanced yield and minimal loss of agricultural crops, mainly cereal grains, is increasing. In general, harvested grains are stored for specific time periods to guarantee their continuous supply throughout the year. During storage, economic losses due to reduction in quality and quantity of grains can become very significant. Grain loss is usually the result of its deterioration due to fungal contamination that can occur from preharvest to postharvest stages. The deleterious fungi can be classified based on predominance at different stages of crop growth and harvest that are affected by environmental factors such as water activity (aw) and eco-physiological requirements. These fungi include species such as those belonging to the genera Aspergillus and Penicillium that can produce mycotoxins harmful to animals and humans. The grain type and condition, environment, and biological factors can also influence the occurrence and predominance of mycotoxigenic fungi in stored grains. The main environmental factors influencing grain fungi and mycotoxins are temperature and aw. This review discusses the effects of temperature and aw on fungal growth and mycotoxin production in stored grains. The focus is on the occurrence and optimum and minimum growth requirements for grain fungi and mycotoxin production. The environmental influence on aflatoxin production and hypothesized mechanisms of its molecular suppression in response to environmental changes are also discussed. In addition, the use of controlled or modified atmosphere as an environmentally safe alternative to harmful agricultural chemicals is discussed and recommended future research issues are highlighted.

Determination of Mycotoxins and Characterization of Aflatoxin-Producing Aspergillus Section Flavi in Maize from North-West Nigeria

The effects of different ecophysiological factors on ochratoxin A production

Herbal medicines have been applied in clinical treatment worldwide, whose significant curative effect attracts considerable global research attention. However, as the herbal medicine industry develops continuously in recent years, new challenges including monitoring the quality and safety of herbal medicines appear in this industry. Numerous cases of fungal and mycotoxin contamination have been reported that affected the quality and safety of herbal medicines. The main mycotoxins found in herbal medicines include aflatoxin, ochratoxin A, and fumonisin B, which cause substantial harm to human health. They are mainly produced by species from Aspergillus, Penicillium, and Fusarium. Various reports have focused on studying the conditions for fungal growth and mycotoxin synthesis to provide references for prevention. The chemical compounds and antagonism microorganisms were also explored to inhibit fungal growth, and decrease mycotoxin accumulation. This review discusses natural occurrence of three main fungal genera (Aspergillus, Penicillium, and Fusarium) and three main mycotoxins (aflatoxin, ochratoxin A, and fumonisin B) in herbal medicines, analyzing the endogenous and exogenous factors that affect fungal growth and mycotoxin production. Moreover, the prevention methods of fungal contamination are included.

This study evaluates the impact of traditional sun drying and modern industrial drying techniques on fungal contamination and mycotoxin production in dried figs. A total of 80 samples (40 per drying method), collected from various retail sources in Upper Egypt, were analyzed. Fungal isolation was performed on Dichloran Rose Bengal Chloramphenicol agar medium and incubated at 28°C. Sun-dried figs exhibited significantly higher fungal loads (1395 colony-forming unit [CFU]/g) compared with industrially dried figs (750 CFU/g). Mycobiota analysis identified 33 fungal species across 12 genera in sun-dried figs, whereas industrial drying yielded 21 species. Internal transcribed spacer sequencing facilitated species identification, with accession numbers PV065865 to PV065896 deposited in GenBank. Aspergillus spp. were dominant in both drying methods, with Aspergillus welwitschiae, A. flavus, and A. niger being the most prevalent. Mycotoxin analysis revealed aflatoxin contamination in 37.5% of sun-dried and 15% of industrially dried figs, while ochratoxin A was detected in 57.5% and 27.5% of samples, respectively, for sun-dried and industrially dried figs. Total fumonisins were present in 12.5% of sun-dried and 5% of industrially dried figs. These findings highlight the efficacy of industrial drying techniques in mitigating fungal contamination and mycotoxin accumulation, thereby improving the microbiological safety of dried figs.

Maize is a possible host of many fungi, some of them able to produce different mycotoxins. Few studies exist on co-occurring fungi and resulting multi-mycotoxin contamination in field; for this reason, in field trials were conducted in two consecutive years to verify fungal incidence and mycotoxin production in the case of the co-occurrence of the three main mycotoxigenic fungi of maize in Italy: Aspergillus flavus, Fusarium verticillioides, and Fusarium graminearum able to produce, respectively, aflatoxin B1 (AFB1), fumonisins (FBs), and deoxynivalenol (DON). Artificial inoculation was done after silk emergence of maize and samples were collected with a 2 week schedule up to harvest time (four samplings). Fungal interaction resulted as playing a role for both fungal incidence and mycotoxins production, as did weather conditions too. Main interactions were noted between A. flavus and F. verticillioides, and between F. verticillioides and F. graminearum. In particular, as a result of fungal co-occurrence, AFB1 resulted stimulated by F. graminearum presence while no effects were noted in FBs and DON in case of F. verticillioides–F. graminearum co-occurrence. Interestingly, the co-presence of A. flavus significantly reduced both FB and DON production.

Effects of infestations of the storage mite Tyrophagus putrescentiae (Acaridae) on the presence of fungal species and mycotoxin production in stored products