Aspergillus flavus and Fusarium verticillioides and Their Main Mycotoxins: Global Distribution and Scenarios of Interactions in Maize

Maize is frequently contaminated with multiple mycotoxins, especially those produced by Aspergillus flavus and Fusarium verticillioides. As mycotoxin contamination is a critical factor that destabilizes global food safety, the current review provides an updated overview of the (co-)occurrence of A. flavus and F. verticillioides and (co-)contamination of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) in maize. Furthermore, it summarizes their interactions in maize. The gathered data predict the (co-)occurrence and virulence of A. flavus and F. verticillioides would increase worldwide, especially in European cold climate countries. Studies on the interaction of both fungi regarding their growth mainly showed antagonistic interactions in vitro or in planta conditions. However, the (co-)contamination of AFB1 and FB1 has risen worldwide in the last decade. Primarily, this co-contamination increased by 32% in Europe (2010–2020 vs. 1992–2009). This implies that fungi and mycotoxins would severely threaten European-grown maize.


Introduction
Maize (Zea mays L.) is one of the strategic cereal crops which can be processed into a variety of food, feedstuff, and other industrial products [1].The threat posed to maize production by fungal plant diseases is one of the critical factors that can destabilize global food security and safety.Preharvest losses due to fungal plant diseases are estimated to account for nearly 10-20% of cultivated maize, which can feed about 8.5% of the world's population [2].Among these diseases, Aspergillus Ear Rot and Fusarium Ear Rot, caused by Aspergillus and Fusarium species, respectively, are the most important [3].Both diseases decrease the yield and quality of the maize crop and the safety of maize kernels due to the production of mycotoxins, secondary fungal metabolites toxic to animals and humans [4].
Aspergillus Ear Rot disease is mainly caused by a fungal pervasive maize invader called A. flavus [5].The A. flavus species has been reported in several countries in Africa, America, Asia, and Europe [6][7][8].Toxigenic A. flavus species produce several mycotoxins/secondary metabolites; however, due to their toxicity and widespread contamination, the most studied toxins are aflatoxins (AFs) [9].So far, there are four members of AFs called B1, B, G1, and G2.Aflatoxin B1 (AFB1) is the most potent member of AFs, and several fatal outbreaks have been associated with the consumption of AFB1-contaminated maize in Brazil (60 deaths) and Kenya (317 cases of intoxications and 125 deaths) [10,11].The toxicity of AFB1 has aroused widespread public concern due to its hepatotoxic, immunotoxic, mutagenic, carcinogenic, and teratogenic properties [12].The International Agency for Research on Cancer (IARC) classified AFB1 as a group 1 carcinogen due to the sufficient evidence of causing liver cancer in humans [13].
The other common fungal disease in maize is Fusarium Ear Rot, which is mainly caused by Fusarium verticillioides [14,15].Similarly to A. flavus, the F. verticillioides species has been reported as a worldwide fungal pathogen of maize kernels.The fungus is also considered a major producer of an important group of mycotoxins called fumonisins (FBs) [16].There are three members of FBs called fumonisin B1, FB2, and FB3; however, the main member of FBs is FB1 (FB1) [17].Several foodborne outbreaks due to consumption of FB1-contaminated maize were reported over the years in South Africa (45 cases), Mexico (>100 cases), India (1424 cases), and Brazil (66 cases) [18][19][20].Several studies have shown that FB1 can pose many toxic effects (neurotoxic, hepatotoxic, and nephrotoxic) in humans.The IARC classified FB1 as a class 2B carcinogen (possible human carcinogen) [21,22].
Given the significant negative impacts of these fungi and their mycotoxins on agriculture and human health, this review focuses on the (co-)occurrence of A. flavus and F. verticillioides and the (co-)contamination of AFB1 and FB1 in maize.Furthermore, it highlights how A. flavus and F. verticillioides interact with each other in maize.

Global Distribution of A. flavus and F. verticillioides in Maize
A general overview of the number of studies per country reporting the (co-)occurrence of A. flavus and F. verticillioides in different continents between 1980 and 2020 is shown in Figure 1.Furthermore, their sampling years, sample numbers, and the percentage of occurrence are shown in Table 1.Most of these studies were on A. flavus occurrence, followed by F. verticillioides, while fewer studies on the co-infection were published.The research of A. flavus was highest in Africa, with 74 scientific papers, followed by Asia (39 studies), Europe (35 studies), and the Americas (14 studies).However, Europe has a high awareness of studying the contamination of F. verticillioides that was reported in 51 papers, followed by Africa (40 studies), Asia (37 studies), and the Americas (27 studies).These data show that people are paying more attention to the contamination of both fungi [4], which reflect that the mycotoxin pollution problem of these two is increasing during these years.Figure 1 shows that more research of A. flavus and F. verticillioides is related to hot and rainy climates in African countries, which favored the growth of the two fungi.Apart from Africa, it was noticeable that southern European countries (Italy, Portugal, Spain, and Romania), some Asian countries (China, India, Iran, and Pakistan), and other countries in Latin and northern Americas (Brazil, Argentina, and United States) had a considerable number of publications.Gradually, those areas face an increased risk of A. flavus and F. verticillioides co-contamination [23].In total, there are 30 papers in the literature reporting the co-occurrence of A. flavus and F. verticillioides in maize from all continents: Africa (12 studies); Europe (8 studies); Asia (6 studies); and the Americas (4 studies).It is noticeable that all these previous surveys were conducted in countries with hot climates.However, there is an increasing number of studies in European cold climate countries due to global warming.Global warming is no longer a trend but a reality because many countries have excessive emissions of CO 2 [24].To mitigate the threat of climate change, 195 countries agreed to limit the emission of CO 2 by adopting new rules [25].The co-occurrence of A. flavus and F. verticillioides will likely increase worldwide, especially in Europe.Americas (4 studies).It is noticeable that all these previous surveys were conducted in countries with hot climates.However, there is an increasing number of studies in European cold climate countries due to global warming.Global warming is no longer a trend but a reality because many countries have excessive emissions of CO2 [24].To mitigate the threat of climate change, 195 countries agreed to limit the emission of CO2 by adopting new rules [25].The co-occurrence of A. flavus and F. verticillioides will likely increase worldwide, especially in Europe.+: occurrence without percentage.The occurrence percentage of A. flavus or F. verticillioides alone or together in maize per continent between 1980 and 2020 is depicted in Figure 2 and their sampling years, sample numbers, and the percentage of occurrence are shown in Table 1.Over the period between 1980 and 2020, there was a considerable variation in the occurrence percentage for A. flavus and F. verticillioides in maize in all the continents, which does not provide a consistent trend.Such variation shows that predicting the contamination of these fungi is difficult.Indeed, co-founding factors such as sample size, sampling strategies, fungal isolation, and identification methods affect the reported results in these papers.Comparing the median values for A. flavus and F. verticillioides occurrence percentages among the four continents shows that the occurrences in Europe are the lowest.On the other hand, the Americas (North and Latin America) had the highest occurrence percentages for both fungi in the surveyed maize samples (Figure 2).
This matches with the increasing awareness of global warming, which is expected to impact the presence of mycotoxins in food and feed severely.Battilani et al. reported that AFB1 is predicted to become a food safety issue in maize cultivated in Europe, especially under the +2 • C scenario, the most probable climate change scenario for the following years [23,247].A similar scenario applies to F. verticillioides and FB1 in maize [104].However, after considering the publications focused on isolating both fungi in maize, it is seen that the median values for the occurrences of both fungi in Africa and Europe are close.Different overview of the occurrence percentages for both fungi in America in which the median value for the occurrence percentage of A. flavus is three times higher than F. verticillioides which is the opposite situation in Asia.This also shows the significant variation in the detection of both fungi in maize samples and the difficulty in drawing a consistent conclusion.This matches with the increasing awareness of global warming, which is expected to impact the presence of mycotoxins in food and feed severely.Ba ilani et al. reported that AFB1 is predicted to become a food safety issue in maize cultivated in Europe, especially under the +2 °C scenario, the most probable climate change scenario for the following years [23,247].A similar scenario applies to F. verticillioides and FB1 in maize [104].However, after considering the publications focused on isolating both fungi in maize, it is seen that the median values for the occurrences of both fungi in Africa and Europe are close.Different overview of the occurrence percentages for both fungi in America in which the median value for the occurrence percentage of A. flavus is three times higher than F. verticillioides which is the opposite situation in Asia.This also shows the significant variation in the detection of both fungi in maize samples and the difficulty in drawing a consistent conclusion.
On the other hand, researchers investigated the possible interactions between A. flavus and F. verticillioides and their toxins in maize, which is presented in the following section of this review.Furthermore, a global summary is provided on the (co-)occurrence of the commonly produced mycotoxins (AFB1 and FB1) in maize.

Worldwide Co-Occurrence of AFB1 and FB1 in Maize and Maize-Based Products
The simultaneous occurrence of several mycotoxins in a single product is a common situation, with the natural co-contamination of AFB1 and FB1 in maize and maize products as an example.An overview of the surveys conducted on AFB1 and FB1 is summarized in Table 2, which contains the sampling years, sample numbers, detection methods, and concentrations of both toxins between 1991 and 2020.The most common On the other hand, researchers investigated the possible interactions between A. flavus and F. verticillioides and their toxins in maize, which is presented in the following section of this review.Furthermore, a global summary is provided on the (co-)occurrence of the commonly produced mycotoxins (AFB1 and FB1) in maize.

Worldwide Co-Occurrence of AFB1 and FB1 in Maize and Maize-Based Products
The simultaneous occurrence of several mycotoxins in a single product is a common situation, with the natural co-contamination of AFB1 and FB1 in maize and maize products as an example.An overview of the surveys conducted on AFB1 and FB1 is summarized in Table 2, which contains the sampling years, sample numbers, detection methods, and concentrations of both toxins between 1991 and 2020.The most common analytical technique (up to 66.7%) used for detecting and quantifying AFB1 and FB1 in the last decade was liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).This is owing to the essential strengths of HPLC-MS/MS, including potentially high analytical specificity, a wide range of applicability to small and large molecules, the capability of multi-and mega-parametric tests, and the opportunity to develop robust assays with a high degree of flexibility within a short time frame [248].
In Africa, high concentrations of FB1 were 10,447 µg/kg and 18,184 µg/kg, and AFB1 concentrations that co-occur with high FB1 were 6738 µg/kg and 1081 µg/kg, respectively (see Table 2) [249,250].Based on this, it was found that there can be a positive relationship between AFB1 and FB1 under this co-existence condition with the collected data in Africa: the concentration of AFB1 is correspondingly high/low in the presence of high/low concentrations of FB1 according to the correlation coefficient (r > 0.8).However, Sangare-Tigori et al., Kpodo et al., and Kimanya et al. contradicted this positive relationship, which can be the selection of detection methods.In the Americas, 70% of FB1 were higher than 2000 µg/kg, and the highest was up to 53,000.0 µg/kg [251], almost ten times more than in Africa under the co-occurrence of AFB1 and FB1.In Asia, the highest concentrations of FB1 and AFB1 were 37,000 µg/kg and 4030 µg/kg in the analyzed samples [18,252].Moreover, since 2010, AFB1 concentration was significantly decreased compared with before 2010.However, there was no apparent interaction between AFB1 and FB1 in the samples in the Americas (r < 0.1) and Asia (r < 0.1).In Europe, with the co-occurrence of AFB1 and FB1, FB1 contamination was severe, and 85.7% of cases exceeded 2000 µg/kg.There were even 57.1% of cases higher than 10,000 µg/kg; the highest was up to 51,690 µg/kg [253].However, AFB1 concentrations are lower than in other continents, and the increase in FB1 hardly affects AFB1 concentrations under their co-existence.It was found that the co-occurrence of both toxins was detected in Serbia and Spain by 2012, which can be a portent of the co-contamination of AFB1 and FB1 threatening to Europe [254,255].The mean of AFB1 and FB1 levels in studies from different continents is shown in Figure 3.As more than 70% of the produced maize was primarily used for animal feed in the world [286], the EU maximum limits for feed maize FB1(2000 µg/kg) and AFB1 (20 µg/kg) were selected as thresholds to interpret the collected data.From 1991 to 2020, 38% of AFB1 and 61% of FB1 studies exceeded the EU maximum limits separately.However, these excess issues have not happened in all continents under the co-occurrence of AFB1 and FB1.In Africa, the co-occurrence of both mycotoxins has risen to 53.8% since 2012.From 2012, 30.0% of survey studies are out of the AFB1 threshold, but all cases are below the FB1 threshold.In the Americas, 44.4% of AFB1 was higher than 20 µg/kg, and 33.3% of FB1 was higher than 2000 µg/kg.In Asia, 62.5% of studies exceeded the AFB1 limit, and only one study reported FB1 contamination exceeding the FB1 limit.There were no cases exceeding the EU maximum limits for both toxins in the last decade year.In Europe, the co-contamination of AFB1 and FB1 has increased to 31.4% since 2012.Over the period 2012 until 2020, 25.0% of AFB1 was beyond its limit, which never happened before 2012, and 58.3% of FB1 was beyond its limit, which decreased to 13.1% compared with before 2012.UK Climate has reported that the most recent decade (2011-2020) has been, on average, 0.5 • C warmer than the 1981-2010 average, and the 21st century so far has gradually been warmer, which is roughly consistent with the observed rate of global mean temperature warming [287].Therefore, it can predict that the co-contamination of AFB1 and FB1 will become more serious worldwide due to global warming, and the risk of human co-exposure to both toxins will increase.The outcome of the interactions between A. flavus and F. verticillioides differs depending on the applied laboratory conditions for each experiment.This includes the substrate, culture media (in vitro) or maize (in vivo), and the related incubation conditions.Fakhrunnisa and Ghaffar have proved that A. flavus inhibited the growth of F.

Interactions between A. flavus and F. verticillioides and Their Toxins in Maize
The outcome of the interactions between A. flavus and F. verticillioides differs depending on the applied laboratory conditions for each experiment.This includes the substrate, culture media (in vitro) or maize (in vivo), and the related incubation conditions.Fakhrunnisa and Ghaffar have proved that A. flavus inhibited the growth of F. verticillioides (inhibition rate 16.67%) by producing a zone of inhibition in the dual agar culture plate assay [288].In case the incubation conditions are changed (e.g., temperature, CO 2 , and humility), the interaction between A. flavus and F. verticillioides can also change, as reported by Camardo Leggieri et al. [289].In their study, the growth of A. flavus was affected by the co-inoculum of F. verticillioides, and colony diameter was significantly lower than that measured in pure colonies if the incubation was between 20 • C and 25 • C. On the contrary, at 35 • C, A. flavus growth was enhanced by the presence of F. verticillioides [289].Consistently, Giorni et al. reported that the co-existence of A. flavus and F. verticillioides was influenced by the temperature and water activity [290].They reported that with the presence of both fungi, F. verticillioides nutritionally dominated all the strains of A. flavus at 20 • C and 0.95 aw, while A. flavus always nutritionally dominated F. verticillioides at 30 • C with either high aw (0.98 aw) or reduced aw (0.87 aw) [290].In a recent study, the effect on fungal growth and the production of their main mycotoxins (AFs and FBs) on co-inoculation were also investigated by another group [291].It was demonstrated that the growth rate of A. flavus and F. verticillioides, when grown in dual or mixed culture, was slower compared with the growth rate in a single culture, and average growth rate reductions of 10% and 11% were observed for A. flavus and F. verticillioides, respectively.When A. flavus and F. verticillioides were mixed, the production of AFB1 and FB1 significantly decreased.Likewise, Lanubile et al. showed that in the co-occurrence of A. flavus and F. verticillioides, both mycotoxins resulted in the reduction compared with the amount produced with single inoculation, and these findings were independent of temperature [292].
The interaction between A. flavus and F. verticillioides under in vivo environment is also highly dynamic.It depends on the experimental conditions, the variable measured, and how they colonize the host.Chen et al. observed the symptoms of the lesion and mycotoxin production to evaluate the interaction of A. flavus and F. verticillioides in maize [290].The dual inoculation resulted in reduced lesions of A. flavus.In contrast, the lesion size and toxin production of F. verticillioides were unaffected in the presence of A. flavus in maize at 25 • C. In contrast, their mixed inoculation resulted in more extensive lesions than a single A. flavus inoculation and higher FB production than a single F. verticillioides inoculation [290].The study indicates that A. flavus can be more affected by F. verticillioides in maize.A previous study underlined the different abilities of A. flavus and F. verticillioides to grow simultaneously on maize since they usually occupy different niches regarding carbon sources [290].It is stated that F. verticillioides seems to be dominant because it can use more carbon sources at the lowest temperatures (15 • C) and the highest aw levels (> 0.95 aw), while A. flavus becomes dominant at higher temperatures (>25-30 • C) and dry conditions (0.87 aw) [290,293].However, Lanubile et al. reported that in the co-occurrence of A. flavus and F. verticillioides, mycotoxin production has no significant differences among three different temperature regimes (20, 25, and 30 • C) for maize kernel contamination.However, FBs and AFs decreased compared with single inoculation at all the tested temperatures [292].It is worth mentioning that Lanubile et al. tested maize kernels as the in vivo host, different from the above baby maize tested by Chen et al. [291], which can be the cause of different interactions of A. flavus and F. verticillioides.Overall, the interaction of A. flavus and F. verticillioides is manifested in the resistance to the growth of each other both in vitro and in vivo.At the same time, mycotoxin production is highly dependent on the temperature and the tested inoculation host.

Conclusions and Outlook
Throughout the last 30 years, the virulence of A. flavus and F. verticillioides and the co-occurrence of AFB1 and FB1 is also gradually contaminating Africa, the Americas, and

Figure 1 .
Figure 1.A world map showing the number of studies that surveyed the (co-)occurrence of A. flavus and F. verticillioides.The number of studies is represented as a bar chart for A. flavus (red color), F. verticillioides (green color), and both fungi (blue color).

Figure 1 .
Figure 1.A world map showing the number of studies that surveyed the (co-)occurrence of A. flavus and F. verticillioides.The number of studies is represented as a bar chart for A. flavus (red color), F. verticillioides (green color), and both fungi (blue color).

Toxins 2023 , 24 Figure 2 .
Figure 2. Boxplots show the percentage of contaminated maize samples with A. flavus, F. verticillioides, and both fungi in survey studies from Africa, the Americas, Asia, and Europe.The data points are colored according to the year of sampling.

Figure 2 .
Figure 2. Boxplots show the percentage of contaminated maize samples with A. flavus, F. verticillioides, and both fungi in survey studies from Africa, the Americas, Asia, and Europe.The data points are colored according to the year of sampling.

Figure 3 .
Figure 3.The reported mean values of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) in studies from different continents.The red lines show the EU maximum limit for AFB1 (20 µg/kg) for cereals and FB1 (2000 µg/kg) for unprocessed maize.4. Interactions between A. flavus and F. verticillioides and Their Toxins in Maize

Figure 3 .
Figure 3.The reported mean values of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) in studies from different continents.The red lines show the EU maximum limit for AFB1 (20 µg/kg) for cereals and FB1 (2000 µg/kg) for unprocessed maize.

Table 1 .
Reported (co-)occurrences of A. flavus and F. verticillioides in maize worldwide.

Table 1 .
Reported (co-)occurrences of A. flavus and F. verticillioides in maize worldwide.