Performance of testers with contrasting provitamin A content to evaluate provitamin A maize for resistance to Aspergillus flavus infection and aflatoxin production

{"Background": "In sub-Saharan Africa (SSA), maize is a primary staple providing approximately 30% of total calories for over 4.5 billion people. However, white maize, the most common type in SSA, is devoid of provitamin A (PVA) carotenoids (average 2 \u03bcg/g), leading to widespread vitamin A deficiency (VAD) affecting up to 5.7 million preschool-age children and 9.7 million pregnant women. Concurrently, maize consumers in SSA face exposure to aflatoxins\u2014highly toxic secondary metabolites produced by Aspergillus species, particularly Aspergillus flavus. Aflatoxin B1 is classified as a Group 1 carcinogen by IARC and is associated with impaired food conversion, stunted growth, immune suppression, and hepatocellular carcinoma. Previous research suggested that carotenoids, especially \u03b2-carotene and \u03b2-cryptoxanthin, may downregulate aflatoxin biosynthesis by quenching reactive oxygen species. This study aimed to evaluate aflatoxin resistance among PVA biofortified maize germplasm, investigate the impact of testers with contrasting PVA content on identifying superior inbreds, and identify parental lines with desirable combining ability for both high PVA and reduced aflatoxin accumulation.", "Methods": "Sixty PVA maize inbreds (PVA range 5.4 to 51.7 \u00b5g/g) were crossed with two inbred testers having contrasting PVA content: T1 (high PVA, 25.0 \u00b5g/g) and T2 (low PVA, 14.4 \u00b5g/g), generating 120 PVA hybrids. Four checks were included: T1\u00d7T2 hybrid and three commercial hybrids (Oba Super 2, Ife Hybrid-3, Ife Hybrid-4). The testcrosses were evaluated in four environments: Ikenne and Saminaka in Nigeria during the 2020 and 2021 main rainy seasons, arranged in a 31 \u00d7 4 alpha-lattice design with two replications. Carotenoids were extracted and quantified using HPLC. For aflatoxin assessment, 5 g kernel samples were surface sterilized, inoculated with 0.5 ml of A. flavus La3228 suspension (4 \u00d7 10^6 conidia/ml), and incubated for 7 days at 31\u00b0C. Aflatoxin was quantified using thin-layer chromatography with scanning densitometry, and spores were quantified using a turbidimeter. Statistical analyses included combined ANOVA, genetic correlation, GCA and SCA effects estimation, Baker's ratio, K-means clustering, and eta-squared calculations.", "Key Results": "The combined ANOVA showed significant effects (p < 0.01\u20130.001) for environment, hybrids, GCA effects of inbreds and testers, and SCA effects for most traits. Baker's ratios were >0.5 for all traits (0.96\u20130.99), indicating predominance of additive gene action. The contribution of Line, Tester, and Line \u00d7 Tester to total variance was 5.9%, 90.7%, and 3.4% for aflatoxin; and 2.9%, 96.7%, and 0.4% for PVA content. Testcross means ranged from 3.9 to 5.5 ppb for aflatoxin, 1.93 to 2.57 NTU for spore count, and 6.2 to 18.4 \u00b5g/g for PVA. K-means analysis classified 30 testcrosses as tolerant, 34 as moderately susceptible, and 56 as susceptible to aflatoxin. Among tolerant testcrosses, 63.3% involved T1. Aflatoxin had a significant negative genetic correlation with \u03b2-carotene (r = \u22120.29, p < 0.0001) and PVA (r = \u22120.23, p < 0.0001), and positive correlation with spore count (r = 0.24, p < 0.0001). Eta-squared analysis showed PVA effect (\u03b7\u00b2 = 0.36) was larger than spore count (\u03b7\u00b2 = 0.13) on aflatoxin resistance. T1 had significant negative GCA for aflatoxin and significant positive GCA for PVA, \u03b2-carotene, and lutein. Eight inbreds (including TZI2071 and TZI1715) combined significant negative GCA for aflatoxin and spore count with significant positive GCA for PVA. Standard heterosis ranged from \u221212.5% to 23.1% for aflatoxin and \u221234.1% to 95.9% for PVA. Among 26 testcrosses with negative heterosis for aflatoxin, 21 involved T1.", "Clinical Implications": "This study demonstrates that PVA biofortified maize can serve a dual purpose: alleviating vitamin A deficiency while reducing aflatoxin exposure in populations dependent on maize as a staple food. The identification of eight inbred lines with desirable combining ability for both high PVA and reduced aflatoxin accumulation provides breeders with valuable genetic resources. The high-PVA tester T1 was particularly effective at identifying superior inbreds and can be used as a suitable tester in breeding programs. The findings support integrating PVA biofortification into integrated aflatoxin management programs alongside pre- and post-harvest practices, biocontrol agents, improved drying, and optimal storage. However, the relatively modest correlation between PVA and aflatoxin (r = \u22120.23) indicates that high PVA alone is not sufficient for complete aflatoxin control, and further research is needed to confirm these findings across more environments and with other mycotoxin-producing fungi. The commercial hybrid Oba Super 2, despite low PVA content, accumulated low aflatoxin, suggesting additional resistance mechanisms (e.g., protein expression, kernel architecture) warrant investigation."}