1. Introduction
Toxigenic fungi cause severe economic losses during maize production, and the preharvest or postharvest occurrence of all toxin limits must be measured. When in the field, the preharvest character dominates breeding in terms of resistance, fungicide protection, etc., and provides valuable information toward their control. As in Hungary A. flavus is a new problem of possible preharvest origin, it was the focus of this paper. However, near to no published data are known about the preharvest presence of DON and fumonisins, and they occur regularly, as even farmers know about this problem, and therefore, these toxins have to receive the same attention. This is the reason that all three toxins are controlled, and so epidemiological data of their significance can also be presented.
Christensen and Kaufmann [
1] divided fungi into two categories: fungi isolated from grains for storage and field fungi. Species from
Alternaria,
Cladosporium,
Helminthosporium, and
Fusarium spp. Are classified as field fungi. The EU regulations [
2,
3,
4] indicate the binding limits for food and suggest limits for feeds. As these limits are different for humans and also differ between animals, we used the limits for swine as they are the most sensitive to toxins (suggested EU feed limits for adults: DON 0.9 mg/kg, FUM 5 mg/kg, and aflatoxin 20 μg/kg; As swines are as sensitive to toxins as human beings, for piglets, about 20–25% of the limit defined for adult swine would be sufficient. The regulations do not differentiate between preharvest and postharvest contamination. However, in this paper, we test the preharvest origin as this will help to identify the field originated risks and this is the first point from where field-originated toxin problems can be identified and the best solution to reduce this problem can be found.
Among field-borne toxigenic fungi,
Fusarium species are well-known toxin producers [
1,
5,
6]. Past research has also shown that their toxins are also field-borne, so no additional proof is provided [
1,
5,
7]. The proposition that field-borne infection decreases during storage could be true, but this is not necessarily right for all mycotoxins [
5,
6].
In Hungary, artificially inoculated wheat spikes of
F. graminearum showed a concentration as high as 432 mg/kg of DON, but zearalenone was never found [
8]. Munkvold and White [
7] noted that aflatoxins have both field and storage significance. Moreover, aflatoxin production in the field in the USA is so important that
A. flavus was classified among the field ear rots [
7]. For this reason, we treat
A. flavus in the preharvest group and included into the resistance studies. However, for preharvest aflatoxin occurrence, no reliable data have been published from Hungary, and thus this problem needed a solution.
Prior to 2004, the European literature did not recognize aflatoxins in the Mediterranean area. The U.S. literature has reported [
4] high aflatoxin contamination in the USA under southern tropical and subtropical climatic conditions. Shotwell [
9] reported that, in 1964 and 1965, 2.3%of 1311 corn samples were contaminated by aflatoxins. The samples were commodities, and the preharvest origin of the aflatoxin was not demonstrated. The first evidence of aflatoxins in preharvest maize was published in 1975. Anderson et al. [
10] reported aflatoxin levels exceeding 400 mg/kg in individual kernels under artificial inoculation, thereby demonstrating the possibility that aflatoxins can be produced under field conditions. Lillehoj et al. [
11] were the first to collect 3600 ears of maize from fields and found that 120 had aflatoxin levels higher than 20 μg/kg. Subsequently, aflatoxin has also been considered a field-borne mycotoxin in the USA [
12,
13,
14]. Lillehoj [
15] was among the first to report differences in aflatoxins in various maize hybrids.
Abbas et al. [
16] performed tests following natural infection from fumonisins and aflatoxin, a rare case for evaluating visual symptoms, but the relation between symptoms and toxin contamination was not analyzed. In some experiments, a positive correlation was found between aflatoxin and fumonisin contamination, but the correlated value was only r=0.298, with no statistical significance. Further, Abbas et al. [
16] concluded that the same cultural practices may influence differently aflatoxin and fumonisin contamination. In Turkey [
17], 19.3% of the isolates from freshly harvested maize grains belonged to
Aspergillus flavus. In another study, aflatoxins were detected in 17 out of the 73 samples collected (0.7–50 μg/kg). In another test, 46% of the samples contained 3–70 μg/kg of aflatoxins [
17], but visual notes were not reported. Between 2000 and 2003, Abbas et al. [
18] found significantly lower natural aflatoxin levels (1 to 2 μg/kg), with the maximum being 9.2 μg/kg. Abbas et al. [
19] found that the common smut infection by
Ustilago maydis can increase aflatoxin contamination by 45-fold, whereas the concentration of fumonisins increased only 5.2-fold. Lillehoj [
20] reported that earlier studies concentrated on stored commodities, as
A. flavus and
A. parasiticus were classified as storage fungi. The discovery of preharvest infection and aflatoxin contamination in the field opened a new avenue of research in mycotoxicology. This shift caused a radical reorientation in scientific thinking. The consequence was the research on aflatoxin production and its conditions [
21,
22].
The preharvest origin of the aflatoxin has not been considered a central problem [
23]. In Hungary [
24], until 2006, no aflatoxins were reported in maize. Masic et al. [
25] reported on maize samples from Hungary, but the status of the samples (preharvest or stored) was not provided. In Hungary, data on 17,011 maize samples regarding mycotoxins was published from 2012 to 2017 [
26]. Aflatoxins were found every year, with maximums between 0.44 and 115 μg/kg. As the data originated mostly from stored mixed corn samples, no conclusions could be drawn about the preharvest or postharvest origins of the contamination or the possible role of resistance [
26]. Therefore, aflatoxin was included into the tested toxins. When preharvest occurrence is continuous and significant, breeding, agronomy, etc. may have a role in its control.
In areas where aflatoxin was demonstrated to have a field origin, such as in the southeastern parts of the USA and tropical regions, the search for sources of resistance started decades before [
15,
20,
27,
28,
29]. In these areas, Munkvold and White [
7] maintain that preharvest aflatoxin contamination is more important than storage-borne contamination. Unsurprisingly, nearly everywhere, following artificial inoculation, aflatoxin contamination has been accompanied by studies on the development of the disease resistance. However, from these cannot help to demonstrate the preharvest natural contamination and its significance.
The ecological conditions supporting the toxigenic diseases and the regulation of the toxin contamination are roughly known [
7,
30,
31,
32,
33,
34]. The data provide the variety differences to the diseases, but there is no general knowledge in this field. The existing risk is sufficiently enough to receive attention, but increasing temperatures in west and north Europe will cause increasing DON contamination [
35], and higher fumonisin and aflatoxin contamination is also forecast. Monitoring is important in order to detect the problem before it spreads to the more jeopardized regions.
We should not forget that the forecasting refers to the preharvest toxin contamination. As
A. flavus and
F. verticillioides need a higher optimum temperature than the ruling weather conditions secured in middle and western Europe, warmer and dryer summer conditions will increase the risk of aflatoxin and fumonisin contamination [
36,
37]. The different forecasting models [
35,
38,
39,
40,
41,
42] do not consider the resistance of the hybrids but use only the increasing temperatures and other non-plant traits. Accordingly, significant aflatoxin contamination is forecast in middle, west, and towards northern European regions. However, on the other hand, a number of papers provided adequate proof on the differences in resistance for each important toxigenic fungus [
43]. Other authors [
29,
30,
44,
45,
46] have recognized that there is higher toxin contamination in warmer and drier years. However the possibility of the higher resistance has not been considered as a possible control method. Rather, authors think that biocontrol using an atoxic
A. flavus strain [
46,
47] can be more successful. In Hungary, Mesterhazy et al. (2022) [
48] published artificial inoculation result for
F. graminearum, F. verticillioides, and
A. flavus. In the non-inoculated sample, DON, FUM B
1+B
2, and AFB1 were also controlled. For the natural toxin contamination, only the mean values were presented (2017–2020), but for the entire period (2014-2021), the yearly data are important to gain a better understanding of the nature of the natural toxin contamination. The conclusion is that the resistance level or contamination level of the hybrids should be considered to see how far a forecasting can be valid for all hybrids, and how relevant it is to forecast toxin contamination without resistance data. The work in [
41] is important because it showed that beside the temperature also humidity is needed for disease spread and toxin contamination. The toxin contamination for a percentage of the visual scores was recognized in artificial inoculation tests [
48]; in this paper, we test this for natural infection.
Storage is a key problem. In Hungary, regularly stored and moldy corn samples were compared in 1993–1999 [
24]. The data are important (
Table 1), because the assumption that field-originated toxins do not increase during storage [
1] seems to be false. In a bad storage environment, there is, on average, a two- to eight-fold increase in the levels of different mycotoxins. During regular storage, a lower level of mycotoxin contamination increase was observed. In bad storage conditions, a sharp increase in toxins was documented. In other words, it appears storability is better when the starting mycotoxin conditions of the grain are healthier. Unfavorable storage conditions can considerably increase the mycotoxin contamination caused by field fungi.
Objectives. The main task of this study is to monitor the significance of the natural preharvest contamination of mycotoxins, including aflatoxin, DON, and fumonisins, in South Hungary. The assessment of the differences among possible hybrids was also conducted. The relations between infection severity and toxin contamination are mostly unknown at this low infection level. The data can also contribute to the epidemiological knowledge of the three toxins. We also considered the possibilities of identifying hybrids that adapt better to different ecological situations, and we expect the study to contribute to a more extended use of preharvest control methods. Similarly, we expect to see an increased role of preharvest contamination in the plant production process.
4. Discussion
4.1. Mycotoxins and Their Preharvest Character
Aflatoxin B
1 occurred every year. The maximum value varied greatly every year but was only lower than the EU limit of 20 μg/kg in 2016. In 2014, it was 121 μg/kg; in 2015, it was 1030 μg/kg; in 2017, it was 385 μg/kg; in 2018, it was 70 μg/kg; in 2019, it was 65 μg/kg; and in 2020, it peaked at 2286 μg/kg. The yearly means were higher than 20 μg/kg in 2015 (87 μg/kg), 2017 (51 μg/kg), and 2020 (316 μg/kg). These results are significantly higher than the EU limits [
2,
3,
4]. Therefore, in Hungary, aflatoxin B
1 should be considered a regularly occurring mycotoxin in maize before harvest. This is also true for the mean values of DON (epidemics in 2014 and 2019) and fumonisins (epidemics in 2014 (20.79 mg/kg) and 2015 (4.03 mg/kg) but not in 2016 (2.16 mg/kg). Different weather patterns induce different epidemics; in some years, more rain can increase toxin contamination. Genotype differences seem to be large. We do not discuss resistance differences, but it is possible that these differences could be an explanation. This agrees well with the conclusions of Munkvold and White [
7]. This does not mean that postharvest control is not of great significance [
24]. On the contrary, it is. However, excellent quality at harvest must be preserved during a long storage period. The conclusion is that, without effective preharvest control methods, the problem cannot be solved. The weather data and the mycotoxin contamination show a loose, mostly non-significant, correlation matrix. In most years, the same weather conditions allowed very large toxin differences and the differences were from toxin to toxin. Aflatoxin accumulation prefers high temperatures. In 2020, when the temperature did not reach values above 35 °C on any day, the largest aflatoxin contamination was recorded for several hybrids, and for others, the largest aflatoxin contamination was recorded for several hybrids. For others, the AFB1 content was below the detection limit. We do not think that meteorological data are not important; many other traits influence toxin contamination and, therefore, the resistance differences should also be considered responsible for the results.
One conclusion seems to be that the resistance to toxin accumulation for all three toxins and for most of the hybrids differs significantly and this can cause the highly different toxin production rates for a percent of visual infection. For a hybrid, the rates for different toxins in different seasons can also differ significantly. At present, we can only say that it is hardly possible to forecast toxin contamination on the basis of visual symptom severity alone; therefore, all samples should be tested for toxins.
4.2. Reasons for Controversial Visual Ear Rot and Toxin Data
Rachis can play a significant role in the extension of infection and aflatoxin contamination.
A. flavus can infect the whole depth of the ear [
58,
59,
60] and may invade the kernels through the rachilla [
61]. Rachis resistance is, therefore, also considered a component of ear rot resistance [
60]. Such a situation was found in
F. graminearum in 2014 in Hungary. The germ part was severely infected, but the dent part was mostly healthy. The reason is that the rachilla contains 12–20% more water than the grain at different developmental stages [
62]. As fungal growth on the ear surface stops at 23% grain moisture [
1], on the surface of the cob (rachilla), its growth is possible for about 2 weeks longer depending on ecology, drydown, and genetic factors. Additionally, the fungus spreads at a higher speed in susceptible rachises, as compared to more resistant rachises [
59]. When examining unshelled ears, such an infection remains hidden. The systemic infection of a maize plant by
A. flavus could be one explanation for the presence of aflatoxins in symptomless ears, as aflatoxins might translocate within the plant [
63]. Drought and high temperatures are almost always initiators of aflatoxin outbreaks [
64], even when existing infections cannot spread [
21]. Drought stress indicates proline accumulation [
65], which enhances aflatoxin production [
66].
Another source can be the seemingly healthy grain that cans experience severe
Aspergillus infection (
Figure 1, left). In such grains, a high AFB1 can be present in healthy-looking whole kernels, which could lead to a better understanding of the source of high toxin contamination without a visually detectable infection. As atoxigenic lines also occur among the
A. flavus strains, their presence does not automatically indicate aflatoxin contamination [
67,
68]. These data support the view that disease and toxin regulation, even though they have common features, can be contradictory.
The correlations between symptoms and toxin contamination are mostly weak. The connection between symptoms and toxin content is better in artificial inoculation [
52,
53]. We have to relate the visual symptom severity found in this paper (0–2%) to the official maize hybrid tests from 2010 where the maximum
Fusarium ear rot incidence (%) was 85% in the observed ears and 27% ear coverage. The most resistant had 27% incidence and 11% coverage (52). Furthermore, it can be seen that toxin contamination for 1% visual ear infection can be variable in different hybrids, different pathogens, and different years. We do not know much about the effects of genetics and the environment, so this is a major research objective for the future. Arid areas increase the danger of aflatoxin [
69] and fumonisin [
70] contamination. Rainy periods can also increase toxin contamination, as in this study, when the rainy months of September and October favored severe toxin contamination by DON and FUM but less so by AFB1.This was also observed for
Fusarium; it seems that toxin and disease regulation do not necessarily agree [
43,
52,
53]. Our data working with the yearly means show mostly not significant correlation among temperature, hot days, and precipitation. In the eight years of the study’s duration, only two years (2015 and 2021) had lower mean values than the EU limits for all three toxins; three epidemics were caused by DON, two by fumonisins and two by aflatoxin. In addition, a very high variability was detected between hybrids in the different years. This explains why the low natural infection below 1–2% does not provide information about suitability for food and feed safety, and sometimes high toxin contamination can be found where there is no visible infection, which is mostly characteristic for
A. flavus. We agree that the relationships between symptoms and toxin contamination are poorly understood [
34]. Our conclusion is that, with a lack of close correlations between symptoms and toxin contamination for natural infection regimes, the measurement of the toxin is the only way to receive reliable information about the food and feed safety value of a given maize lot. An indirect way to estimate toxin contamination based on natural infection does not seem reliable. As there may be many toxins in a sample, multitoxin tests are recommended.
There is another source for the mostly non-significant correlations. We showed that, for artificial inoculation tests [
48], the toxin production for a percentage of visual infection can have very high differences and can lead to toxin overproduction and underproduction [
48]. From this paper, it seems that this is true also for the natural infection regime.
4.3. Toxin Forecasting
The data clearly show that, in most cases, the correlations between visual ear rot and toxin contamination are not significant. For this reason, it is not possible to estimate toxin contamination on the basis of visual infection data. As in most cases, multitoxin contamination occurs, making the problem even more complicated. The forecasting of toxin contamination is a complex activity [
38,
45,
46,
48,
69,
70,
71,
72,
73,
74,
75,
76], and the resistance level is not included as an influencing factor. As data about resistance differences have been proved for all three pathogens [
43,
52,
53,
77,
78,
79,
80,
81], it is clear that, compared with natural data regimes, the same ecological nursery conditions result in highly different infection rates and toxin data. This clearly indicates that resistance data should be considered. There is a problem, as such data do not exist (Battilani, personal comm. 2019).Therefore, forecast scientists cannot be blamed. They simply do not have support from the various governmental organizations or plant breeders. Additionally, looking at the general occurrence of multitoxin presence in most corn samples, we need forecasting programs that can handle the three or four most important toxins at the same time. Our conclusion is that it is better to focus on the toxin contamination directly as a useful result since toxin contamination from infection data is not possible with the present knowledge. Therefore, the forecast procedure correctly concentrates on the toxins and not the symptoms. The paper showed evidence that the decision to focus on toxin forecasting was correct. For this reason, not only resistance to disease, but also a toxin regulation in hybrids being independent from resistance, should also be considered.
4.4. What Is the Usefullness of Natural Toxin Data?
All toxin regulations refer to natural toxin contamination [
2,
3,
4], independently from their origin, preharvest, postharvest, or combined.
1. The entire food and feed industry is based on toxin contamination data. For this reason, the preharvest toxin data have a much higher significance than is often thought [
43]. As no toxin data can be forecast from visual scores, toxins should be measured. We stress the significance of the preharvest toxin data as these provide the first possibility to act. A rapid test should be performed for every truck from the field to separate shipments with excellent quality from low-quality ones and store them later separately. Cooperativa Agraria (Guarapuara, Brazil) work according to this rule, treating more than one million tons of grain yearly (Mesterhazy, 2022, pers. communication). We have a similar experience in Hungary (Bonafarm Inc., Dalmand, Hungary) at a smaller size.
2. As hybrids arrive from many trucks, their toxin data are very useful for the grower to withdraw hybrids from production where the rate of highly contaminated lots are more frequent. This must be treated as a risk factor.
3. The mixing of grain from different hybrids and fields with different toxin contamination happens often deteriorating the entire storage content. By separate storage, this problem can be avoided. From a highly inhomogeneous grain mass, a reliable toxin contamination level is not possible, and even five-fold differences among the regular sampled muster occur (Tanyi, 2015, personal communication).
4. If growers receive feedback quickly about the value of the variety, risky cultivars can be withdrawn from production, as is the case with Cooperativa Agrária Agroindustrial, Guarapuara, Brazil. Preharvest data can provide information about the various epidemics. Based on data on natural toxin contamination, the breeders can decide whether a breeding program should be taken forward considering the pathogen and its toxin(s). At the same time, the breeders can have feedback regarding whether the hybrid they produced fits to the resistance class that was previously suggested.
5. Regular preharvest toxin controls can contribute to identifying the location, amount, and quality of maize lots in silos, which could be marketed within the country or be exported, and can provide information about the losses caused by mycotoxins.
4.5. Adaptation to Environmental Stresses
Climate models forecast variable warming scenarios with locally lower or higher precipitation levels [
36]. There are many types of
Fusaria-causing ear rot [
82,
83], but the two most damaging are
F. graminearum and
F. verticillioides. Resistance to them is not connected and supposedly their present significance is moderate, but the species structure may change. Therefore, the
Fusarium spp. population structure should be checked to identify emerging mycotoxins in time. As many hybrids had very low or high to very high toxin contamination under the same environmental conditions, it is clear that, without knowing the resistance classification of the hybrid, its toxin behavior can hardly be forecasted. The forecasts shows higher toxin contamination in the northern hemisphere [
35,
37,
42,
84,
85]. As the differences in hybrid resistance are very large in artificial inoculation tests [
48,
52,
53] and also at natural infection and contamination verify this hypothesis, the combination of the two test regimes can lead to a decrease in toxin contamination. Therefore, the increasing resistance to toxigenic fungi can be an effective and excellent tool against the negative effects of warming climate. Under these conditions, hybrids with good resistance to heat, drought, and ear rot pathogens can be competitive and safer in middle Europe or further north, but also in regions where they cause severe problems now.
The correlations between infection, toxin contamination, and meteorological data show that, alone, warmer seasons do not interfere significantly with the production of toxin disease symptoms. For this, precipitation is also needed, and extra hot days also influence the results significantly, but differently on a monthly scale. The expression “global warming” simplifies the situation; therefore, it is better to avoid it. The forecast models use these data, so there is no problem in this respect.
4.6. Control Measurements
Artificial inoculation results clearly show resistance differences and the high deviations within season in natural toxin contamination support the view that higher resistance levels have a significant role in improving food and feed safety. Higher resistance levels can make fungicide control more efficient as it did in wheat [
86,
87]. The use of atoxic
A. flavus isolates significantly reduces aflatoxin contamination [
47,
88]. We think that a higher resistance could help to further reduce aflatoxin contamination by a possible additive effect. This is a future research task. Higher resistance could help with the successful application of Bt maize hybrids. This could be supported by conservation agriculture [
88,
89], which could stabilize the resistance of maize to climatic stresses and indirectly reduce aflatoxin contamination. Similarly, it is supposed that plants with higher resistance to toxigenic fungi have a better tolerance to the higher disease pressure when cereals or maize were the previous crops, so the tillage without plugging could be realized with less toxin risks [
90,
91,
92,
93].
The harmful consequences of climate change can be significantly balanced both in regions where toxins are a daily problem now and in regions that are exposed to these threats in these years and later. For this reason, we need to apply different approaches combining them to have a better control, higher yield, and improved food and feed safety. The key is integrated plant management with increased resistance supported by a field-specific mix of the best possible optimizing of management practices for each field.Further, susceptible hybrids should be withdrawn from production and the variety registration should ban the registration and production of the susceptible hybrids.