The Effect of Aspergillus flavus on Seedling Development in Maize

Academic Editor: Ramon Gerardo Guevara-Gonzalez Received: 6 February 2025 Revised: 26 March 2025 Accepted: 28 March 2025 Published: 2 April 2025 Citation: Mazzoleni, I.; Novarina, E.; Zerlottin, Y.M.; Bardelli, T.; Dal Prà, M.; Zuffada, M.; Cremonesi, M.; Antonietti, L.; Bravi, R.; Bianchi, P.G.; et al. The Effect of Aspergillus flavus on Seedling Development in Maize. Plants 2025,14, 1109. https:// doi.org/10.3390/plants14071109 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article The Effect of Aspergillus flavus on Seedling Development in Maize Isabella Mazzoleni 1,†, Elena Novarina 2,†, Yuki Michelangelo Zerlottin 3, Tommaso Bardelli 2, Mauro Dal Prà4, Mattia Zuffada 1, Matteo Cremonesi 1, Luca Antonietti 1, Romana Bravi 4, Pier Giacomo Bianchi 2 and Anna Pia Maria Giulini 2,* 1Research Centre for Plant Protection and Certification, via Emilia km 307, 26838 Tavazzano con Villavesco, Italy; [email protected] (I.M.) 2Research Centre for Plant Protection and Certification, via G. Venezian 22, 20133 Milan, Italy; [email protected].it (E.N.); [email protected] (T.B.); [email protected] (P.G.B.) 3UniversitàCattolica del Sacro Cuore, Facoltàdi Scienze Agrarie, Alimentari e Ambientali, via Emilia Parmense 84, 29122 Piacenza, Italy; [email protected] 4Research Centre for Plant Protection and Certification, via G. Marconi 2, 36045 Lonigo, Italy; [email protected] (M.D.P.); [email protected] (R.B.) *Correspondence: [email protected] †These authors contributed equally to this work. Abstract: Plant growth and its interaction with microorganisms change yearly. High temperature and humidity have characterized recent seasons in the north of Italy and around the world, increasing the parasitic ability of Aspergillus flavus to colonize maize kernels and aflatoxin levels. These molecules have the highest acute and chronic toxicity of all mycotoxins; the maximal concentration in agricultural food and feed products, and their commodities, are regulated worldwide. In this study we suggest a simple methodology to test the susceptibility of candidate maize varieties to A. flavus before their release onto the market. A panel of 92 inbred lines and 14 hybrids were analysed, disease phenotypes were scored on artificially inoculated kernels using a rolled towel assay, and therefore we observed different responses to fungal infection on the kernels, outlining a high variability among the tested lines characterized by a different effect of the pathogen on seedling development. Even the hybrids responded differently on a statistical basis to A. flavus with regard to the development of coleoptile, allowing their categorization into classes of susceptibility to be used for the varietal registration. Interestingly, the hybrid 6a-A was less susceptible to A. flavus compared to its reciprocal in terms of the length of the coleoptile. The comparison of breeding lines released on the market in different years suggested a poor improvement in genetic resistance against A. flavus in maize so far, opening up a possible topic for future research aimed at mitigating the impact of climate change on agriculture. Keywords: maize; aflatoxins; biotic stress; climate change; new varieties 1. Introduction Maize (Zea Mays L.) is the most widely grown agricultural crop in the world that has become a model organism for basic and applied research in plant biology [ 1 ]. This dual importance of maize is largely due to its complex and diverse genome, which has allowed researchers to understand genetics, cytogenetics and genomics better, and has offered a rich pool of genetic diversity, helping breeders to improve germplasm [2]. Plants 2025,14, 1109 https://doi.org/10.3390/plants14071109 Plants 2025,14, 1109 2 of 18 This agricultural species, mainly used as a global feed, is also important as a food crop, particularly in sub-Saharan Africa and Latin America, in addition to being cultivated for other non-food uses such as industrial and energy crops [ 3 ]. The global maize area (for dry grain) stands at 204 M ha, and the annual production overtakes 1 billion metric tons [ 4 ]. The breeding programs, developed principally by private seed companies, are dynamic, releasing every year numerous maize hybrids characterized by better performance compared with all the material released on the market. The improved germplasm is crucial, not only for raising the potential yield, but also for addressing the emerging challenges which arise continuously due to climate change. Maize is considered one of the crops most susceptible to mycotoxins world-wide [ 5 ]. Specifically, hot and humid climates may have an impact on maize contamination by several fungal species, mostly Aspergillus, Alternaria, Fusarium and Penicillium [ 6 , 7 ], and on the presence of mycotoxins. A recent analysis of European Food Safety Agency (EFSA) data reveals that up to 80% of the crops grown, stored and traded worldwide are contaminated with detectable quantities of secondary fungal metabolites classed as mycotoxins, with 20% being over the legal limit for contamination [ 8 ]. Among mycotoxins, aflatoxin B1 (AFB1) is one of the strongest human carcinogens [ 9 ]. Aspergillus flavus and A. parasiticus are the main producers of aflatoxins and, together with other species of Aspergillus section Flavi, have the capacity to generate this secondary metabolite [ 10 ]. The optimal growth of A. flavus occurs over the range of 19–35 ◦ C [ 11 ], with 28 ◦ C being optimum for aflatoxin production [ 12 , 13 ]. Maize is susceptible to infection and colonization by A. flavus and aflatoxin production during both the preand post-harvest phases of crop growth and storage. The contamination of aflatoxin increases from preto post-harvest [ 14 ], and it is strongly affected by temperature and moisture availability. A.flavus can be divided into two distinct evolutionary lineages designated as 1B and 1C [ 15 ]. Lineage 1B strains, predominantly clonal, are mostly not aflatoxigenic, few of them can produce aflatoxins but in low amount. Conversely, the latter strains vary widely in their ability to produce aflatoxins [ 16 ]. A. flavus is ubiquitous and genetically and phenotypically very diverse [ 17 ]. This fungus can reproduce not only asexually by conidia and sclerotia [ 18 ], but also sexually through the interaction between mating type loci MAT1-1 and MAT1-2 [19], thus generating new genotypes [20]. The primary source of fungus inoculum is soil, specifically in the fields of highly susceptible crops, but also in forest ground without hosts, demonstrating its saprophytic ability [ 18 ]. Under favourable conditions, which include high temperature and water stress [ 21 ], the spores of the fungus, in the form of wind-dispersed conidia released from mycelium and sclerotia on soil surfaces, infect the developing inflorescences of maize. Once deposited on the silks [ 22 ], these spores colonize kernels’ surfaces and invade all the tissues of the seed through the pedicel region, through wounds created by insects or the mechanical injury of the pericarp [23–25], producing aflatoxin. Histological studies performed on maize infected artificially by the conidia of A. flavus with a needle showed the localization of fungal mycelium and its morphological changes during the colonization of the diverse parts of the seed, taking into account that A. flavus preferentially colonizes tissues with high oil content as the germ. From endosperm, the fungus reached the germ through the basal transfer layer (BETL) of the basal endosperm and the scutellum [ 25 ] then, with intact aleurone layers, the fungus grows around the kernel between the pericarp and aleurone with a minimal colonization of the endosperm, reaching the scutellum and the germ from the aleurone layer which forms a barrier limiting the route of the fungus through the endosperm [ 26 , 27 ]. At the endosperm–germ interface, A. flavus often constitutes a biofilm-like structure surrounding the germ, and it is hypothesized that this structure contributes to the germ infection [ 23 ]. Fungal growth was slower or limited Plants 2025,14, 1109 3 of 18 in the resistant hybrid compared to the susceptible ones, specifically in the aleurone layer or at the endosperm–scutellum interface [ 27 ]. Indeed, resistant hybrids may differentiate endosperm-specific antifungal proteins [ 28 ], hydrolytic enzymes [ 29 , 30 ] and antifungal ribosome-inactivation proteins in the scutellum [31]. Several strategies may be combined to produce healthy crops free of aflatoxin, even if increasing resistance seems to be the best solution [32]. The genetic basis of resistance to Aspergillus flavus has been studied using different maize populations, indicating that the resistance is a polygenic character in which additive effects play an essential role. The trait also showed low heritability [ 33 , 34 ], although higher heritability has been reported in the literature [ 35 , 36 ]. Large gene–environment interactions reduce the possibility of identifying good markers to select maize genotypes with a resistance to A. flavus [ 33 , 34 , 36 – 47 ]. Interestingly, Zhang and colleagues [ 46 ] combined linkage-based mapping on Quantitative Trait Locus (QTL) with Genome-Wide Association Studies (GWAS) to resolve a major QTL for A. flavus to identify several candidate genes for resistance. The meta-analysis of QTLs [ 48 ] gave good results in pinpointing candidate genes (and markers) associated with resistance to the fungus. Specifically, three markers close to MQTL2.4, MQTL4.1 and MQTL8.2 were identified for their great potential to be used in marker-assisted selection for A. flavus resistance. Furthermore, the identification of MQTLs allowed the authors to search for candidate genes with a potential involvement in maize–A. flavus interaction. For instance, a polyphenol oxidase (PPO) that plays an important role in plant defence mechanisms against biotic stresses, a plasma membrane intrinsic protein (PIP) that is involved in channels’ functioning, and an RNA-directed RNA polymerase that controls epigenetic changes and could act in disease resistance response have all been identified. Likewise, proteomic and transcriptomic analyses have helped to detect several proteins expressed during the infection of maize seeds by A. flavus. Few proteins are known to have antifungal activity, or to be involved in host defence. These include pathogenesis-related (PR) proteins, lipoxygenases, alpha-amylase inhibitors and ribosome-inactivating proteins [49]. Another possibility in ensuring maize hybrids to be more resistant to the A. flavus on the market is to revise the procedure for varietal registration, introducing new valuations to the final approval of the candidate varieties. Indeed, within the European Union (EU), crop varieties must be included in a Member State’s National List and/or common catalog before being marketed. This requires plant variety testing processes to evaluate whether the variety candidate is distinct, uniform and stable (DUS), and meets the requirement of value for cultivation or use (VCU). So far, the technical protocol for the approval of new maize varieties in Italy and in several European countries does not include the characterization of the resistance to Aspergillus flavus in candidate varieties. In this context, the primary goal of this study was to phenotypically evaluate maize genotypes, part of our reference collection, for resistance to A. flavus using the RTA assay (Rolled Towel Assay), and to consider the effects of the artificial infection on seed germination to disclose a potential mechanism for resistance on mature kernels. The RTA has been previously and successfully applied to both soybean and maize genotypes to evaluate the resistance to Fusarium spp. [ 50 – 53 ]. So far, the genetic architecture of resistance to the A. flavus infection of seedlings has not yet been investigated using the Rolled Towel Assay. In this study, we described the different responses to artificial inoculation of mature maize kernels with spores of A. flavus, showing a variability in susceptibility to the fungus among the selected genotypes. The seedling development was determined between control vs. A. flavus inoculation in 77 maize lines and 14 hybrids using a five-point severity scale and measuring the length of the coleoptile. Plants 2025,14, 1109 4 of 18 As the final aim of this study, we proposed a procedure to categorize the hybrids in specific classes of susceptibility to the fungal infection to apply during the varietal registration process in order to evaluate the resistance of new hybrids. We also suggested measuring officially the resistance to A. flavus, not only in the hybrid but also in its reciprocal, once the preliminary data confirmed and highlighted the potential effect of the parental line on the tolerance to the fungus. Appling the same methodology, we also tested the susceptibility to A. flavus in breeding lines released on the market in different years (2001–2010–2022), disclosing the breeding effort on resistance to A. flavus in maize along the years. 2. Results 2.1. Germination Scores and Disease Severity The germination scores revealed that 41 out of 92 inbred lines had more than 90% of the germination in the control assay (Figure 1). The lines with less than 50% germination were discarded, leaving 77 lines for further analysis. No frequencies of maize lines were detected in either the 10% or 20% classes of germination (Figure 1). Plants 2025, 14, x FOR PEER REVIEW 4 of 19 determined between control vs. A. flavus inoculation in 77 maize lines and 14 hybrids using a five-point severity scale and measuring the length of the coleoptile. As the final aim of this study, we proposed a procedure to categorize the hybrids in specific classes of susceptibility to the fungal infection to apply during the varietal registration process in order to evaluate the resistance of new hybrids. We also suggested measuring officially the resistance to A. flavus, not only in the hybrid but also in its reciprocal, once the preliminary data confirmed and highlighted the potential effect of the parental line on the tolerance to the fungus. Appling the same methodology, we also tested the susceptibility to A. flavus in breeding lines released on the market in different years (2001–2010–2022), disclosing the breeding effort on resistance to A. flavus in maize along the years. 2. Results 2.1. Germination Scores and Disease Severity The germination scores revealed that 41 out of 92 inbred lines had more than 90% of the germination in the control assay (Figure 1). The lines with less than 50% germination were discarded, leaving 77 lines for further analysis. No frequencies of maize lines were detected in either the 10% or 20% classes of germination (Figure 1). Figure 1. The percentage of germination on maize lines tested in the control assay. The low germination observed in some inbred lines could be attributed mainly to two factors: the long-term storage of the kernels [54] and the sterilization procedure applied in the RTA protocol. Indeed, more than half of the selected lines were stored in a cold room for more than 10 years, and several lines displayed a reduction on the germination scores only if treated with ethanol and bleach that may affect the vitality of the kernel with non-visible damage on the surface. Comparing the control and A. flavus inoculation of the 77 maize lines, we observed a reduction in percentage of the score 1 (healthy and germinated seedling with no visible sign of colonization; 67% vs. 8%), showing a relevant effect of the fungus infection that also appears with an increase in proportion of the score 2 (germinated seedling with slight colonization of the kernel near the pedicel; 11% vs. 59%) and 4 (germinated seedling with reduced development with complete colonization of the kernel; 0% vs. 4%). However, the 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Frequencies of lines's number Germination (%) Figure 1. The percentage of germination on maize lines tested in the control assay. The low germination observed in some inbred lines could be attributed mainly to two factors: the long-term storage of the kernels [ 54 ] and the sterilization procedure applied in the RTA protocol. Indeed, more than half of the selected lines were stored in a cold room for more than 10 years, and several lines displayed a reduction on the germination scores only if treated with ethanol and bleach that may affect the vitality of the kernel with non-visible damage on the surface. Comparing the control and A. flavus inoculation of the 77 maize lines, we observed a reduction in percentage of the score 1 (healthy and germinated seedling with no visible sign of colonization; 67% vs. 8%), showing a relevant effect of the fungus infection that also appears with an increase in proportion of the score 2 (germinated seedling with slight colonization of the kernel near the pedicel; 11% vs. 59%) and 4 (germinated seedling with reduced development with complete colonization of the kernel; 0% vs. 4%). However, the percentage of score 5 (no germination; 19% vs. 26%) was quite similar between the control and inoculation (Figure 2), probably due to the damage of kernels or their contamination Plants 2025,14, 1109 5 of 18 also recorded in the control assays, indicating a presence of infection occurring inside the kernel [55]. Plants 2025, 14, x FOR PEER REVIEW 5 of 19 percentage of score 5 (no germination; 19% vs. 26%) was quite similar between the control and inoculation (Figure 2), probably due to the damage of kernels or their contamination also recorded in the control assays, indicating a presence of infection occurring inside the kernel [55]. All the raw data concerning the severity of the infection (score), together with the phenotypic measurement determined in maize lines as the length of the coleoptile, were reported in Table S1. Figure 2. Disease severity rate in the control vs. inoculation rolled towel assays of kernels in 77 maize lines. The seedling development was determined using a five-point severity scale as previously described in [52] (1 = healthy and germinated seedling; 2 = germinated seedling with slight colonization of the kernel near pedicel; 3 = germinated seedling with widespread colonization of the kernel and browning of the coleoptile; 4 = germinated seedling with reduced development and complete colonization of the kernel; 5 = no germination due to complete rotting of the kernel). 2.2. Effect of A. flavus Inoculation on the Length of Coleoptile in Selected Inbred Maize Lines of the Panel The effect of inoculation with Aspergillus flavus on the length of coleoptile was performed on selected maize lines (public lines) of the panel, characterized by the higher percentage of germination and seed vigour. The length of the coleoptile, computed as the mean of ten kernels for each inbred line for the two replications carried out as a function of treatment (control vs. inoculation with A. flavus), is reported in Table S2a. Significant differences were found among lines and treatment, but not between replications, confirming the reproducibility of the RTA assay (Table S2b,c). According to the treatment (control vs. inoculation), specific and different responses were observed among the selected maize lines (Figure 3). The length of the coleoptile was negatively affected by the inoculation in five genotypes (Table S3a,b). In particular, the greatest reduction was observed in line B73 followed by Hi53, TSU-CHIAO-HSI-WU 102, INBRED 39-1546 and F252 (Figure 3). No significant difference between control and inoculation was recorded for the following lines: W22 BRINK, T143, CML182 and Mp705 (Figure 3). Among the lines studied, the length of the coleoptile in B73 was highly inhibited by A. flavus (Figure 4A), whereas a promoting growth in the inoculated kernels was found in CE-777 (Figure 4B). This latter result was previously observed by several authors [56,57], with F. verticillioides boosting the growth in germinating seeds as a defence mechanism. Figure 2. Disease severity rate in the control vs. inoculation rolled towel assays of kernels in 77 maize lines. The seedling development was determined using a five-point severity scale as previously described in [ 52 ] (1 = healthy and germinated seedling; 2 = germinated seedling with slight colonization of the kernel near pedicel; 3 = germinated seedling with widespread colonization of the kernel and browning of the coleoptile; 4 = germinated seedling with reduced development and complete colonization of the kernel; 5 = no germination due to complete rotting of the kernel). All the raw data concerning the severity of the infection (score), together with the phenotypic measurement determined in maize lines as the length of the coleoptile, were reported in Table S1. 2.2. Effect of A. flavus Inoculation on the Length of Coleoptile in Selected Inbred Maize Lines of the Panel The effect of inoculation with Aspergillus flavus on the length of coleoptile was performed on selected maize lines (public lines) of the panel, characterized by the higher percentage of germination and seed vigour. The length of the coleoptile, computed as the mean of ten kernels for each inbred line for the two replications carried out as a function of treatment (control vs. inoculation with A. flavus), is reported in Table S2a. Significant differences were found among lines and treatment, but not between replications, confirming the reproducibility of the RTA assay (Table S2b,c). According to the treatment (control vs. inoculation), specific and different responses were observed among the selected maize lines (Figure 3). The length of the coleoptile was negatively affected by the inoculation in five genotypes (Table S3a,b). In particular, the greatest reduction was observed in line B73 followed by Hi53, TSU-CHIAO-HSI-WU 102, INBRED 39-1546 and F252 (Figure 3). No significant difference between control and inoculation was recorded for the following lines: W22 BRINK, T143, CML182 and Mp705 (Figure 3). Among the lines studied, the length of the coleoptile in B73 was highly inhibited by A. flavus (Figure 4A), whereas a promoting growth in the inoculated kernels was found in CE-777 (Figure 4B). This latter result was previously observed by several authors [ 56 , 57 ], with F. verticillioides boosting the growth in germinating seeds as a defence mechanism. Plants 2025,14, 1109 6 of 18 Plants 2025, 14, x FOR PEER REVIEW 6 of 19 Figure 3. Effect of the treatment (inoculation with A. flavus vs. control) on the length of the coleoptile along different maize lines. Figure 4. Length of the coleoptile observed in the control vs. inoculated kernels in line B73 (A) and in line CE-777 (B). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the treatment. Figure 3. Effect of the treatment (inoculation with A. flavus vs. control) on the length of the coleoptile along different maize lines. Plants 2025, 14, x FOR PEER REVIEW 6 of 19 Figure 3. Effect of the treatment (inoculation with A. flavus vs. control) on the length of the coleoptile along different maize lines. Figure 4. Length of the coleoptile observed in the control vs. inoculated kernels in line B73 (A) and in line CE-777 (B). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the treatment. Figure 4. Length of the coleoptile observed in the control vs. inoculated kernels in line B73 (A) and in line CE-777 (B). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the treatment. Plants 2025,14, 1109 7 of 18 Afterwards, significant differences among maize lines were further analysed only in the inoculated kernels to underline their susceptibility to A. flavus (Table S2a; Figure 5). Plants 2025, 14, x FOR PEER REVIEW 7 of 19 Afterwards, significant differences among maize lines were further analysed only in the inoculated kernels to underline their susceptibility to A. flavus (Table S2a; Figure 5). A more pronounced length of coleoptile was recorded in line Mp705, which seems to be less susceptible to A. flavus compared to the other genotypes (Figure 5). On the other hand, lines B73 and F252 showed the strongest reduction in seedling development, confirming the susceptibility of the B73 inbred line as described by several authors [58,59]. Furthermore, lines W22 BRINK, TSU-CHIAO-HSI-WU 102 and INBRED 39-1546 clustered together, resulting in no difference with lines T143, CML182 and CE-777. Line Hi53 was in between, with maize lines clustering in bc and d (Figure 5). Figure 5. Comparisons between selected maize lines with regard to A. flavus, considering the length of the coleoptile. Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of maize lines. 2.3. Effect of A. flavus Inoculation on the Length of Coleoptile in Maize Hybrids The Rolled Towel Assay (RTA) was also performed on maize hybrids, with the final aim being to develop a methodology to apply within the registration process to evaluate the resistance to this fungus on the new candidates’ varieties. Seven hybrids and their reciprocal (a total of 14 hybrids) were counted, and the length of coleoptile computed as the mean of ten kernels per each hybrid, determined in two replications carried out as a function of treatment (control vs. inoculation), as shown in Table S4a. Significant differences were found among hybrids and treatment (Table S4b), but not between replications, confirming the reproducibility of RTA assay (Table S4b,c). The seven hybrids, labelled with a specific code, responded statistically differently to A. flavus based on the development of coleoptile (Tabel S4c; Figure 6). Even if the number of studied hybrids is limited, it still allowed variations in terms of susceptibility to the fungal infection to be identified, pointing out three clusters. The hybrid labelled with code 6a showed the highest value, appearing to be more tolerant to the infection, followed by the hybrid 7a, and then both hybrids 4a and 5a as sensitive to A. flavus. The remaining three hybrids (1a, 2a and 3a) are not clearly distinguishable in specific groups, although they exhibit their own responses to the infection. Figure 5. Comparisons between selected maize lines with regard to A. flavus, considering the length of the coleoptile. Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of maize lines. A more pronounced length of coleoptile was recorded in line Mp705, which seems to be less susceptible to A. flavus compared to the other genotypes (Figure 5). On the other hand, lines B73 and F252 showed the strongest reduction in seedling development, confirming the susceptibility of the B73 inbred line as described by several authors [ 58 , 59 ]. Furthermore, lines W22 BRINK, TSU-CHIAO-HSI-WU 102 and INBRED 39-1546 clustered together, resulting in no difference with lines T143, CML182 and CE-777. Line Hi53 was in between, with maize lines clustering in bc and d (Figure 5). 2.3. Effect of A. flavus Inoculation on the Length of Coleoptile in Maize Hybrids The Rolled Towel Assay (RTA) was also performed on maize hybrids, with the final aim being to develop a methodology to apply within the registration process to evaluate the resistance to this fungus on the new candidates’ varieties. Seven hybrids and their reciprocal (a total of 14 hybrids) were counted, and the length of coleoptile computed as the mean of ten kernels per each hybrid, determined in two replications carried out as a function of treatment (control vs. inoculation), as shown in Table S4a. Significant differences were found among hybrids and treatment (Table S4b), but not between replications, confirming the reproducibility of RTA assay (Table S4b,c). The seven hybrids, labelled with a specific code, responded statistically differently to A. flavus based on the development of coleoptile (Table S4c; Figure 6). Even if the number of studied hybrids is limited, it still allowed variations in terms of susceptibility to the fungal infection to be identified, pointing out three clusters. The hybrid labelled with code 6a showed the highest value, appearing to be more tolerant to the infection, followed by the hybrid 7a, and then both hybrids 4a and 5a as sensitive to A. flavus. The remaining three hybrids (1a, 2a and 3a) are not clearly distinguishable in specific groups, although they exhibit their own responses to the infection. Plants 2025,14, 1109 8 of 18 Plants 2025, 14, x FOR PEER REVIEW 8 of 19 Figure 6. Length of the coleoptile measured in the inoculated kernels of selected maize hybrids labelled with a specific code (from 1 to 7). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the hybrids. 2.4. Effect of Parental Line on the Susceptibility to A. flavus in Maize Hybrid To explore the impact of parental lines on the susceptibility of seven hybrids to A. flavus, we evaluated the effect of the inoculation in each hybrid and its reciprocal in terms of the length of the coleoptile. The analysis showed significant differences only in one hybrid 6a-A and its reciprocal 6a-B (Table S5a,b, Figure 7), highlighting a possible effect of parental lines on the susceptibility to the fungus on the hybrids. Figure 7. Length of the coleoptile measured in the inoculated kernels in a specific maize hybrid (6aA) and its reciprocal (6a-B). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the hybrid. 2.5. Variations in the Inoculated Maize Lines According to the Year Three years (2001 vs. 2010 vs. 2022) were considered to unravel possible variations in the inoculated maize lines with A. flavus (Table S6a). In detail, sixteen maize lines registered in the Italian National list were selected in each year, and the length of the coleoptile was determined as the mean of ten kernels per each line in two replications (Table S6b). The length of the coleoptile significantly changed across years (Table S6c), recording higher values in the control compared to the inoculation maize lines (2010 and 2022) (FigFigure 6. Length of the coleoptile measured in the inoculated kernels of selected maize hybrids labelled with a specific code (from 1 to 7). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the hybrids. 2.4. Effect of Parental Line on the Susceptibility to A. flavus in Maize Hybrid To explore the impact of parental lines on the susceptibility of seven hybrids to A. flavus, we evaluated the effect of the inoculation in each hybrid and its reciprocal in terms of the length of the coleoptile. The analysis showed significant differences only in one hybrid 6a-A and its reciprocal 6a-B (Table S5a,b, Figure 7), highlighting a possible effect of parental lines on the susceptibility to the fungus on the hybrids. Plants 2025, 14, x FOR PEER REVIEW 8 of 19 Figure 6. Length of the coleoptile measured in the inoculated kernels of selected maize hybrids labelled with a specific code (from 1 to 7). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the hybrids. 2.4. Effect of Parental Line on the Susceptibility to A. flavus in Maize Hybrid To explore the impact of parental lines on the susceptibility of seven hybrids to A. flavus, we evaluated the effect of the inoculation in each hybrid and its reciprocal in terms of the length of the coleoptile. The analysis showed significant differences only in one hybrid 6a-A and its reciprocal 6a-B (Table S5a,b, Figure 7), highlighting a possible effect of parental lines on the susceptibility to the fungus on the hybrids. Figure 7. Length of the coleoptile measured in the inoculated kernels in a specific maize hybrid (6aA) and its reciprocal (6a-B). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the hybrid. 2.5. Variations in the Inoculated Maize Lines According to the Year Three years (2001 vs. 2010 vs. 2022) were considered to unravel possible variations in the inoculated maize lines with A. flavus (Table S6a). In detail, sixteen maize lines registered in the Italian National list were selected in each year, and the length of the coleoptile was determined as the mean of ten kernels per each line in two replications (Table S6b). The length of the coleoptile significantly changed across years (Table S6c), recording higher values in the control compared to the inoculation maize lines (2010 and 2022) (FigFigure 7. Length of the coleoptile measured in the inoculated kernels in a specific maize hybrid (6a-A) and its reciprocal (6a-B). Different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of the hybrid. 2.5. Variations in the Inoculated Maize Lines According to the Year Three years (2001 vs. 2010 vs. 2022) were considered to unravel possible variations in the inoculated maize lines with A. flavus (Table S6a). In detail, sixteen maize lines registered in the Italian National list were selected in each year, and the length of the coleoptile was determined as the mean of ten kernels per each line in two replications (Table S6b). Plants 2025,14, 1109 9 of 18 The length of the coleoptile significantly changed across years (Table S6c), recording higher values in the control compared to the inoculation maize lines (2010 and 2022) (Figure 8). In the year 2001, we did not observe any effect between the control and the inoculation (Table S6d). Lower values were recorded in the year 2001 compared to in 2010 and 2022, when less fluctuation among the means of detected maize lines (identified with points in Figure 9) was observed. Significant differences were found between 2001 and the remaining years (Figure 9), whereas the length of the coleoptile was similar between the years 2010 and 2022. Plants 2025, 14, x FOR PEER REVIEW 9 of 19 ure 8). In the year 2001, we did not observe any effect between the control and the inoculation (Table S6d). Lower values were recorded in the year 2001 compared to in 2010 and 2022, when less fluctuation among the means of detected maize lines (identified with points in Figure 9) was observed. Significant differences were found between 2001 and the remaining years (Figure 9), whereas the length of the coleoptile was similar between the years 2010 and 2022. Figure 8. The violin plots show the length of the coleoptile determined in the control vs. inoculated kernels in 32 selected maize lines (16 genotypes × 2 replications) in 2010 (A) and 2022 (B). The length of the coleoptile was computed as the mean of ten kernels in each replication; different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of treatment. Figure 8. The violin plots show the length of the coleoptile determined in the control vs. inoculated kernels in 32 selected maize lines (16 genotypes × 2 replications) in 2010 (A) and 2022 (B). The length of the coleoptile was computed as the mean of ten kernels in each replication; different letters indicate significant differences (p ≤ 0.05; ANOVA followed by the Fisher post-hoc test) as a function of treatment. Plants 2025,14, 1109 16 of 18 8. Eskola, M.; Kos, G.; Elliott, C.T.; Hajšlová, J.; Mayar, S.; Krska, R. Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited ‘FAO estimate’ of 25%. Crit. Rev. Food Sci. Nutr. 2020,60, 2773–2789. [CrossRef] [PubMed] 9. Stutt, R.O.J.H.; Castle, M.D.; Markwell, P.; Baker, R.; Gilligan, C.A. An integrated model for preand post-harvest aflatoxin contamination in maize. Sci. Food 2023,7, 60. [CrossRef] 10. Ahmad, M.M.; Ahmad, M.; Ali, A.; Hamid, R.; Javed, S.; Abdin, M.Z. Detection of Aspergillus flavus and Aspergillus parasiticus from aflatoxin-contamined peanuts and their differentiation using PCR-RFLP. Ann. Microbiol 2014,64, 1597–1605. [CrossRef] 11. Northolt, M.D.; van Egmond, H.P. Limits of water activity and temperature for the production of some mycotoxins. In Proceedings of the 4th Meeting Mycotoxins in Animal Disease, Weybridge, UK, 1–3 April 1981; pp. 106–108. 12. Scott, P.M.; Lawrence, J.W.; Van Walbeek, W. Detection of mycotoxins by thin layer chromatography: Application to screening of fungal extracts. Appl. Microbiol. 1970,20, 839–842. [CrossRef] 13. Sanchis, V.; Magan, N. Environmental conditions affecting mycotoxins. In Mycotoxins in Food: Detection and Control; Woodhead Publishing: Sawston, UK, 2004; pp. 174–189. [CrossRef] 14. Assaye, M.A.; Gemeda, N.; Weledesemayat, G.T. Aspergillus species and aflatoxins contamination of pre and post-harvest maize grain in West Gojam, Ethiopia. J. Food Sci. Nutr. 2016,2, 013. [CrossRef] 15. Geiser, D.M.; Dorner, J.W.; Horn, B.W.; Taylor, J.W. The phylogenetics of Mycotoxin and Sclerotium production in Aspergillus flavus and Aspergillus oryzae.Fungal Genet. Biol. 2000,31, 169–179. [CrossRef] [PubMed] 16. Carbone, I.; Jakobek, Y.L.; Ramirez-Prado, J.H.; Horn, B.W. Recombination, balancing selection and adaptative evolution in the aflatoxin gene cluster of Aspergillus parasiticus.Mol. Ecol. 2007,16, 4401–4417. [CrossRef] 17. Sweany, R.R.; Damann Jr, K.E.; Kaller, M.D. Comparison of soil and corn kernel Aspergillus flavus populations: Evidence for niche specialization. Phytopathology 2011,101, 952–959. [CrossRef] 18. Horn, B.W. Ecology and population biology of aflatoxigenic fungi in soil. J. Toxicol. 2003,22, 351–379. [CrossRef] 19. Horn, B.W.; Gell, R.M.; Singh, R.; Sorensen, R.B.; Carbone, I. Sexual reproduction in Aspergillus flavus sclerotia: Acquisition of novel alleles from soil populations and uniparental mitochondrial inheritance. PLoS ONE 2016,11, e0146169. [CrossRef] [PubMed] 20. Gell, R.M.; Horn, B.W.; Carbone, I. Genetic map and heritability of Aspergillus flavus.Fungal Genet. Biol. 2020,144, 103478. [CrossRef] 21. Hill, R.A.; Blankenship, P.D.; Cole, R.J.; Sanders, T.H. Effects of soil moisture and temperature on preharvest invasion of peanuts by the Aspergillus flavus group and subsequent aflatoxins development. Appl. Environ. Microbiol. 1983,45, 628–633. [CrossRef] [PubMed] 22. Jones, R.K.; Duncan, H.E.; Payne, G.A.; Leonard, K.J. Factors influencing infection by Aspergillus flavus in silk-inoculated corn. Plant Dis. 1980,64, 859–863. [CrossRef] 23. Dolezal, A.L.; Obrian, G.R.; Nielsen, D.M.; Woloshuk, C.P.; Boston, R.S.; Payne, G.A. Localization, morphology and transcriptional profile of Aspergillus flavus during seed colonization. Mol. Plant Pathol. 2013,14, 898–909. [CrossRef] 24. Dolezal, A.L.; Shu, X.; Obrian, G.R.; Nielsen, D.M.; Woloshuk, C.P.; Boston, R.S.; Payne, G.A. Aspergillus flavus infection induces transcriptional and physical changes in developing maize kernels. Front. Microbiol. 2014,5, 384. [CrossRef] 25. Shu, X.; Livingston, D.P.; Franks, R.G.; Boston, R.S.; Woloshuk, C.P.; Payne, G.A. Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides.Mol. Plant Pathol. 2015,16, 662–674. [CrossRef] 26. Brown, R.L.; Cleveland, T.E.; Payne, G.A.; Woloshuk, C.P.; Campbell, K.W.; White, D.G. Determination of resistance to aflatoxin production in maize kernels and detection of fungal colonization using an Aspergillus flavus transformant expressing Escherichia coli β-glucuronidase. Phytopathology 1995,85, 983–989. [CrossRef] 27. Windham, G.L.; Williams, W.P.; Mylroie, J.E.; Reid, C.X.; Womack, E.D. A histological study of Aspergillus flavus colonization of wound inoculated maize kernels of resistant and susceptible maize hybrids in the field. Front. Microbiol. 2018,9, 799. [CrossRef] [PubMed] 28. Chen, G.; Wang, X.; Hao, J.; Yan, J.; Ding, J. Genome-wide association implicates candidate genes conferring resistance to maize rough dwarf disease in maize. PLoS ONE 2015,10, e0142001. [CrossRef] 29. Casacuberta, J.M.; Puigdomenech, P.; San Segundo, B. A gene coding for a basic pathogenesis-related (PR-like) protein from Zea mays. Molecular cloning and induction by a fungus (Fusarium moniliforme) in germination maize seeds. Plant Mol. Biol. 1991,16, 527–536. [CrossRef] 30. Casacuberta, J.M.; Raventos, D.; Puigdomenech, P.; San Segundo, B. Expression of the gene encoding the PR-like protein PRms in germination maize embryos. Mol. Genet. Genom. 1992,234, 97–104. [CrossRef] [PubMed] 31. Guo, B.Z.; Cleveland, T.E.; Brown, R.L.; Widstrom, N.W.; Lynch, R.E.; Russin, J.S. Distribution of antifungal proteins in maize kernel tissue using immunochemistry. J. Food Prot. 1999,87, 295–299. [CrossRef] 32. Cary, J.W.; Rajasekaran, K.; Brown, R.L.; Luo, M.; Chen, Z.Y.; Bhatnagar, D. Developing Resistance to aflatoxins in maize and cottonseed. Toxins 2011,3, 678–696. [CrossRef] Plants 2025,14, 1109 17 of 18 33. Busboom, K.N.; White, D.G. Inheritance of resistance to aflatoxin production and aspergillus ear rot of corn from the cross of inbreds B73 and Oh516. Phytopathology 2004,10, 1107–1115. [CrossRef] 34. Brooks, T.D.; Williams, W.P.; Windham, G.L.; Willcox, M.C.; Abbas, H.K. Quantitative trait loci contributing resistance to aflatoxin accumulation in the maize inbred Mp313E. Crop Sci. 2005,1, 171–174. [CrossRef] 35. Maupin, L.M.; Clements, M.J.; White, D.G. Evaluation of the M182 corn line as a source of resistance to aflatoxin in grain and use of BGYF as a selection tool. Plant Dis. 2003,87, 1059–1066. [CrossRef] [PubMed] 36. Mideros, S.X.; Warburton, M.L.; Jamann, T.M.; Windham, G.L.; Williams, W.P.; Nelson, R.J. Quantitative trait loci influencing mycotoxin contamination of maize: Analysis by linkage mapping, characterization of near-isogenic lines and met-analysis. Crop Sci. 2014,54, 127–142. [CrossRef] 37. Paul, C.; Naidoo, G.; Forbes, A.; Mikkilineni, V.; White, D.; Rocheford, T. Quantitative trait loci for low aflatoxin production in two related maize populations. Theor. Appl. Genet. 2003,107, 263–270. [CrossRef] [PubMed] 38. Widstrom, N.W.; Burton, A.; Guo, B.Z.; Wilson, D.M.; Snook, M.E.; Cleveland, T.E.; Lynch, R.E. Control of preharvest aflatoxin contamination in maize by pyramiding QTL involved in resistance to ear-feeding insects and invasion by Aspergillus spp. Eur. J. Agron. 2003,19, 563–572. [CrossRef] 39. Alwala, S.; Kimbeng, C.A.; Williams, W.P.; Kang, M.S. Molecular markers associated with resistance to Aspergillus flavus in maize grain: QTL and discriminant analyses. J. New Seeds 2008,9, 1–18. [CrossRef] 40. Warburton, M.L.; Brooks, T.D.; Krakowsky, M.D.; Shan, X.; Windham, G.L.; Williams, W.P. Identification and mapping of new sources of resistance to aflatoxin accumulation in maize. Crop Sci. 2009,49, 1403–1408. [CrossRef] 41. Mayfield, K.L.; Murray, S.C.; Rooney, W.L.; Isakeit, T.; Odvody, G.A. Confirmation of QTL reducing aflatoxin in maize testcrosses. Crop Sci. 2011,51, 2489–2498. [CrossRef] 42. Warburton, M.L.; Brooks, T.D.; Windham, G.L.; Williams, W.P. Identification of novel QTL contributing resistance to aflatoxin accumulation in maize. Mol. Breed. 2011,27, 491–499. [CrossRef] 43. Willcox, M.C.; Davis, G.L.; Warburton, M.L.; Windham, G.L.; Abbas, H.K.; Betran, J.; Holland, J.B.; Williams, W.P. Confirming quantitative trait loci for aflatoxin resistance from Mp313E in different genetic backgrounds. Mol. Breed. 2013,32, 15–26. [CrossRef] 44. Yin, Z.; Wang, Y.; Wu, F.; Gu, X.; Bian, Y.; Wang, Y.; Deng, D. Quantitative trait locus mapping of resistance to Aspergillus flavus infection using a recombinant inbred line population in maize. Mol. Breed. 2014,33, 39–49. [CrossRef] 45. Dhakal, R.; Windham, G.L.; Williams, W.P.; Subudhi, P.K. Quantitative trait loci (QTL) for reducing aflatoxin accumulation in corn. Mol. Breed. 2016,36, 164. [CrossRef] 46. Zhang, Y.; Cui, M.; Zhang, J.; Zhang, L.; Li, C.; Kan, X. Confirmation and fine mapping of a major QTL for aflatoxin resistance in maize using a combination of linkage and association mapping. Toxins 2016,8, 258. [CrossRef] [PubMed] 47. Womack, E.D.; William, W.P.; Windham, G.L.; Xu, W. Mapping quantitative trait loci associated with resistance to aflatoxin accumulation in maize inbred Mp719. Front. Microbiol. 2020,11, 45. [CrossRef] 48. Baisakh, N.; Da Silva, E.A.; Pradhan, A.K.; Rajasekaran, K. Comprehensive meta-analysis of QTL and gene expression studies identify candidate genes associated with Aspergillus flavus resistance in maize. Front. Plant Sci. 2023,14, 1214907. [CrossRef] 49. Liu, H.; Wu, H.; Wang, Y.; Wang, H.; Chen, S.; Yin, Z. Comparative transcriptome profiling and co-expression network analysis uncover the key genes associated withearly-stage resistance to Aspergillus flavus in maize. BMC Plant Biol. 2021,21, 2016. [CrossRef] 50. Ellis, M.L.; Broders, K.D.; Paul, P.A.; Dorrance, A.E. Infection of soyabean seed by Fusarium graminearum and effect of seed treatments on disease under controlled conditions. Plant Dis. 2011,95, 401–407. [CrossRef] 51. Lanubile, A.; Muppiral, U.K.; Severin, A.J.; Marocco, A.; Munkvold, G.P. Transcriptome profiling of soyabean (Glycine max) roots challenged with pathogenic and non-pathogenic isolates of Fusarium oxysporum.BMC Genom. 2015,16, 1089. [CrossRef] 52. Bernardi, J.; Stagnati, L.; Lucini, L.; Rocchetti, G.; Lanubile, A.; Cortellini, C.; De Poli, G.; Busconi, M.; Marocco, A. Phenolic profile and susceptibility to Fusarium infection of pigmentated maize cultivars. Front. Plant Sci. 2018,9, 1189. [CrossRef] 53. Stagnati, L.; Lanubile, A.; Samayoa, L.F.; Bragalanti, M.; Giorni, P.; Busconi, M.; Holland, J.B.; Marocco, A. A genome wide association study reveals markers and genes associated with resistance to Fusarium verticilloides infection of seedlings in maize diversity panel. G3 2019,9, 571–579. [CrossRef] 54. Xing, M.; Long, Y.; Wang, Q.; Tian, X.; Fan, S.; Zhang, C.; Huang, W. Physiological alterations and nondestructive test methods of crop seed vigor: A comprehensive review. Agriculture 2023,13, 527. [CrossRef] 55. Munkvold, G.P.; Desjardins, A.E. Fumonisins in maize: Can we reduce their occurrence? Plant Dis. 1997,81, 556–565. [CrossRef] 56. Yates, I.E.; Bacon, C.W.; Hinton, D.M. Effects of endophytic infection by Fusarium moniliforme on corn growth and cellular morphology. Plant Dis. 1997,81, 723–728. [CrossRef] 57. Stagnati, L.; Rahjoo, V.; Samayoa, L.F.; Holland, J.B.; Borrelli, V.M.G.; Busconi, M.; Lanubile, A.; Marocco, A. A genome wide association study to understand the effect of Fusarium verticilloides infection on seedling of a maize diversity panel. G3 2020,10, 1685–1696. [CrossRef] [PubMed] Plants 2025,14, 1109 18 of 18 58. Campbell, K.W.; White, D.G. Evaluation of corn genotypes for resistance to Aspergillus ear rot, kernel infection, and aflatoxin production. Plant Dis. 1995,79, 1039–1045. [CrossRef] 59. Chalivendra, S.; Huang, F.; Busman, M.; Williams, W.P.; Ham, J.H. Low aflatoxin levels in aspergillus flavus-resistant maize are correlated with increased corn earworm damage and enhanced seed fumonisin. Front. Plant Sci. 2020,11, 565323. [CrossRef] [PubMed] 60. Mideros, S.X.; Windham, G.L.; Williams, W.P.; Nelson, R.J. Tissue-specific components of resistance to Aspergillus ear rot of maize. Phytopathology 2012,102, 787–793. [CrossRef] [PubMed] 61. Gorman, D.P.; Kang, M.S. Preharvest Aflatoxin Contamination in Maize: Resistance and Genetics. Plant Breed. 1991,107, 1–10. [CrossRef] 62. Gembeh, S.V.; Brown, R.L.; Grimm, C.; Cleveland, T. Identification of chemical components of corn kernel pericarp wax associated with resistance to Aspergillus flavus infection and aflatoxin production. J. Agric. Food Chem. 2001,49, 4635–4641. [CrossRef] 63. Yin, G.; Xin, X.; Song, C.; Chen, X.; Zhang, J.; Wu, S.; Li, R.; Liu, X.; Lu, X. Activity levels and expression of antioxidant enzymes in the ascorbate-glutathione cycle in artificially aged rice seed. Plant Physiol. Biochem. 2014,80, 1–9. [CrossRef] 64. Yao, W.H.; Zhang, Y.D.; Kang, M.S.; Chen, H.; Li, L.; Yu, L.J.; Fan, X. Diallel analysis models: A comparison of certain genetic statistics. Crop Sci. 2013,53, 1481–1490. [CrossRef] 65. Fan, X.M.; Zhang, Y.D.; Yao, W.H.; Bi, Y.Q.; Liu, L.; Chen, H.M.; Kang, M.S. Reciprocal diallel crosses impact combining ability, variance estimation, and heterotic group classification. Crop Sci. 2014,54, 89–97. [CrossRef] 66. Fan, X.M.; Bi, Y.; Zhang, Y.; Jeffers, D.; Yin, X.F.; Kang, M. Improving breeding efficiency of a hybrid maize program using a three heterotic-group classification. J. Agron. 2018,110, 1209–1216. [CrossRef] 67. Dosho, B.M.; Ifie, B.E.; Asante, I.K.; Danquah, E.Y.; Zelekhe, H. Combining ability of quality protein maize inbred lines under low and optimum soil nitrogen environments in Ethiopia. Afr. J. Plant Sci. 2021,15, 237–249. [CrossRef] 68. Bonipas, A.J.; Rajashekhar, M.K.; Gopalakrishna, N.; Sidramappa, C.T.; Zerka, R.; Bindiganavile, S.V.; Nagesh, P.; Shiddappa, R.S.; Prema, G.U. Maternal effects, reciprocal differences and combining ability study for yield and its component traits in maize (Zea mays L.) through modified diallel analysis. PeerJ 2024,12, 17600. [CrossRef] 69. Sheridan, W.F.; Clark, J.K. Fertilization and Embryogeny in Maize. In The Maize Handbook; Freeling, M., Walbot, V., Eds.; Springer Lab Manuals; Springer: New York, NY, USA, 1994; pp. 3–10. [CrossRef] 70. Walbot, V.; Evans, M.M.S. Unique features of the plant life cycle and their consequences. Nat. Rev. Genet. 2003,4, 369–379. [CrossRef] [PubMed] 71. Evans, M.M.S.; Kermicle, J.L. Interaction between maternal effect and zygotic effect mutations during maize seed development. Genetics 2001,159, 303–315. [CrossRef] [PubMed] 72. Gutieérrez-Marcos, J.F.; Costa, L.M.; Biderre-Petit, C.; Khbaya, B.; O’Sullivan, D.M.; Wormald, M.; Perez, P.; Dickinson, H.G. Maternally expressed gene1 is a novel maize endosperm transfer cell-specific gene with a maternal parent-of-origin pattern of expression. Plant Cell 2004,16, 1288–1301. [CrossRef] 73. Costa, M.L.; Yuan, J.; Rouster, J.; Paul, W.; Dickinson, H.; Gutiérrez-Marcos, J.F. Maternal control of nutrient allocation in plant seeds by genomic imprinting. Curr. Biol. 2012,22, 160–165. [CrossRef] 74. Bai, F.; Daliberti, M.; Bagadion, A.; Xu, M.; Li, Y.; Baier, J.; Tseung, C.-W.; Evans, M.M.S.; Settles, A.M. Parent-of-origin-effect rough endosperm mutants in maize. Genetics 2016,204, 221–231. [CrossRef] 75. White, T.J.; Bruns, T.D.; Lee, S.B.; Taylor, J.W. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press: New York, NY, USA, 1990; pp. 315–322. [CrossRef] 76. Septiani, P.; Lanubile, A.; Stagnati, L.; Busconi, M.; Nelissen, H.; Pè, M.E.; Dell’Acqua, M.; Marocco, A. Unravelling the genetic basis of Fusarium seedling rot resistance in the MAGIC maize population: Novel targets for breeding. Sci. Rep. 2019,9, 5665. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.