Kernel Screening Assay Research Articles

Identification of Early and Extra-Early Maturing Tropical Maize Inbred Lines with Multiple Disease Resistance for Enhanced Maize Production and Productivity in Sub-Saharan Africa.

Maize, a staple for millions across sub-Saharan Africa (SSA), faces major biotic constraints affecting production and safety of the crop. These include northern corn leaf blight (NCLB), southern corn leaf blight (SCLB), Curvularia leaf spot (CLS), and aflatoxin contamination by Exserohilum turcicum, Bipolaris maydis, Curvularia lunata, and Aspergillus flavus, respectively. Farmers in SSA would benefit tremendously if high-yielding maize hybrids with multiple disease resistance (MDR) were developed and commercialized. In all, 49 early-maturing (EM; 90 to 95 days to physiological maturity) and 55 extra-early-maturing (EEM, 80 to 85 days to physiological maturity) inbred lines developed by the International Institute of Tropical Agriculture were identified as resistant to NCLB in field evaluations in multiple agroecologies of Nigeria in 2017 and 2018. From each maturity group, the 30 most resistant inbreds were selected for evaluation for resistance to SCLB and CLS using a detached-leaf assay. Additionally, the inbreds were screened for resistance to kernel rot and aflatoxin contamination using a kernel screening assay. In all, 7 EM and 6 EEM maize inbreds were found to be highly resistant to the three foliar pathogens while 10 inbreds were resistant to the foliar pathogens and supported significantly less (P = 0.01) aflatoxin accumulation than other inbreds. Inbreds having MDR should be tested extensively in hybrid combinations and commercialized. Large-scale use of maize hybrids with MDR would (i) increase maize production and productivity and (ii) reduce losses caused by aflatoxin contamination. Overall, planting of EM and EEM maize hybrids with MDR would contribute to food security, reduced aflatoxin exposure, and increased incomes of maize farmers in SSA.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Host Induced Gene Silencing Targeting Aspergillus flavus aflM Reduced Aflatoxin Contamination in Transgenic Maize Under Field Conditions.

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to the Food and Drug Administration (FDA) regulations on permissible levels of aflatoxins in food and feed. Although biocontrol has yielded some successes in managing aflatoxin contamination, enhancing crop resistance is still the preferred choice of management for long-term sustainability. Hence, host induced gene silencing (HIGS) strategy was explored in this study. The A. flavus gene aflM encoding versicolorin dehydrogenase, a key enzyme involved in the aflatoxin biosynthetic pathway, was selected as a possible target for suppression through HIGS. An RNAi vector containing a portion of the aflM gene was constructed and introduced into immature B104 maize zygotic embryos through Agrobacterium transformation. PCR analysis of the genomic DNA from T0 leaf tissue confirmed the presence of the transgene in six out of the seven events. The seeds from the lines that showed reduced aflatoxin production in laboratory aflatoxin kernel screening assay (KSA) have been increased from T1 to T4 generation in the past four years. Changes in aflatoxin resistance in these transgenic kernels have been evaluated under both field and laboratory conditions. The T2 generation kernels containing the transgene from two events out of four examined had less aflatoxin (P ≤ 0.01 and P ≤ 0.08) than those without the transgene. Field-inoculated homozygous T3 and T4 transgenic kernels also revealed lower levels of aflatoxins (P ≤ 0.04) than kernels from the null (segregated non-transgenic samples) or B104 controls. A similar result was observed when the harvested T3 and T4 homozygous transgenic kernels were evaluated under KSA conditions without inoculation (P ≤ 0.003–0.05). These two events were crossed with LH195, LH197, LH210, and PHW79 elite breeding lines and the resulting crosses supported less aflatoxin (P ≤ 0.02) than the crosses made with non-transgenic lines. In addition, significantly higher levels of aflM gene-specific small RNAs were detected in the transgenic leaf and kernel tissues, indicating that the enhanced aflatoxin resistance in the homozygous transgenic kernels is likely due to suppression of aflM expression through HIGS.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of α-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. α-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus α-amylase enzyme and belongs to the lectin-arcelin-α-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus α-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values. The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%-72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%-88% reduction in aflatoxin levels.

The role of ear environment in postharvest susceptibility of maize to toxigenic Aspergillus flavus

AbstractA kernel screening assay (KSA) was used to assess the genetic and environmental effects on the vulnerability of maize to aflatoxin accumulation. Kernels of 26 inbred lines that had been grown in seven environments, and 190 lines of the Intermated B73xMo17 (IBM) population grown in one location in the United States, were inoculated with a toxigenic strain of A. flavus and incubated in the dark at 30°C for 6 days. Percent kernel colonization (PKC), sporulation and aflatoxin were influenced by the maize genotypes (G), the location (“ear environment” or E) and the GxE interactions. Overall, low broad‐sense heritabilities were observed for PKC, sporulation and aflatoxin. PKC was significantly correlated with sporulation in all environments. Aflatoxin was positively correlated with colonization for two and with sporulation for all ear environments. Higher grain sulphur or magnesium in IBM was associated with less colonization or aflatoxin. Postharvest susceptibility of maize to aflatoxin is thus influenced by factors that are modulated by the ear environment. In a KSA, sporulation could be a proxy test for aflatoxin accumulation.

Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182

Aspergillus flavus is an opportunistic, saprophytic fungus that infects maize and other fatty acid-rich food and feed crops and produces toxic and carcinogenic secondary metabolites known as aflatoxins. Contamination of maize with aflatoxin poses a serious threat to human health in addition to reducing the crop value leading to a substantial economic loss. Here we report designing a tachyplesin1-derived synthetic peptide AGM182 and testing its antifungal activity both in vitro and in planta. In vitro studies showed a five-fold increase in antifungal activity of AGM182 (vs. tachyplesin1) against A. flavus. Transgenic maize plants expressing AGM182 under maize Ubiquitin-1 promoter were produced through Agrobacterium-mediated transformation. PCR products confirmed integration of the AGM182 gene, while RT-PCR of maize RNA confirmed the presence of AGM182 transcripts. Maize kernel screening assay using a highly aflatoxigenic A. flavus strain (AF70) showed up to 72% reduction in fungal growth in the transgenic AGM182 seeds compared to isogenic negative control seeds. Reduced fungal growth in the AGM182 transgenic seeds resulted in a significant reduction in aflatoxin levels (76–98%). The results presented here show the power of computational and synthetic biology to rationally design and synthesize an antimicrobial peptide against A. flavus that is effective in reducing fungal growth and aflatoxin contamination in an economically important food and feed crop such as maize.

Identifying and developing maize germplasm with resistance to accumulation of aflatoxins

Efforts to identify maize germplasm with resistance toAspergillus flavus infection and subsequent accumulation of aflatoxins were initiated by the US Department of Agriculture, Agricultural Research Service at several locations in the late 1970s and early 1980s. Research units at four locations in the south-eastern USA are currently engaged in identification and development of maize germplasm with resistance toA. flavus infection and accumulation of aflatoxins. The Corn Host Plant Resistance Research Unit, Mississippi State, MS, developed procedures for screening germplasm for resistance toA. flavus infection and accumulation of aflatoxins. Mp313E, released in 1990, was the first line released as a source of resistance toA. flavus infection. Subsequently, germplasm lines Mp420, Mp715, Mp717, Mp718, and Mp719 were released as additional sources of resistance. Quantitative trait loci associated with resistance have also been identified in four bi-parental populations. The Crop Protection and Management Research Unit and Crop Genetics and Breeding Research Unit, Tifton, GA, created a breeding population GT-MAS:gk. GT601, GT602, and GT603 were developed from GT-MAS:gk. The Food and Feed Safety Research Unit, New Orleans, LA, in collaboration with the International Institute for Tropical Agriculture used a kernel screening assay to screen germplasm and develop six germplasm lines with resistance to aflatoxins. The Plant Science Research Unit, Raleigh, NC, through the Germplasm Enhancement of Maize (GEM) Project provides to co-operators diverse germplasm that is a valuable source of resistance toA. flavus infection and accumulation of aflatoxins in maize.

Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressingAspergillus flavus strain

Resistance or susceptibility of maize inbreds to infection byAspergillus flavus was evaluated by the kernel screening assay. A green fluorescent protein-expressing strain ofA. flavus was used to measure fungal spread and aflatoxin levels in real-time following fungal infection of kernels. Among the four inbreds tested, MI82 showed the most resistance and Ga209 the least. TZAR101 was also resistant to fungal infection, whereas Va35 was susceptible to fungal infection. However, Va35 produced lower aflatoxin levels compared to the susceptible line Ga209. Fluorescence microscopy indicated that the site of entry of the fungus into the kernel was consistently through the pedicel. Entry through the pericarp was never observed in undamaged kernels. In view of these results, incorporation or overexpression of antifungal proteins should be targeted to the pedicel and basal endosperm region in developing kernels. Once the fungus has entered through the pedicel, it spreads quickly through the open spaces between the pericarp and the aleurone layer, ultimately colonising the endosperm and scutellum and, finally, the embryo. A clear correlation was established between fungal fluorescence and aflatoxin levels. This method provides a quick, reliable means of evaluating resistance toA. flavus in undamaged kernels and provides breeders with a rapid method to evaluate maize germplasm.

Transcriptional profiles uncover Aspergillus flavus-induced resistance in maize kernels.

Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and through storage. Previous studies have highlighted the constitutive production of proteins involved in maize kernel resistance against A. flavus’ infection. However, little is known about induced resistance nor about defense gene expression and regulation in kernels. In this study, maize oligonucleotide arrays and a pair of closely-related maize lines varying in aflatoxin accumulation were used to reveal the gene expression network in imbibed mature kernels in response to A. flavus’ challenge. Inoculated kernels were incubated 72 h via the laboratory-based Kernel Screening Assay (KSA), which highlights kernel responses to fungal challenge. Gene expression profiling detected 6955 genes in resistant and 6565 genes in susceptible controls; 214 genes induced in resistant and 2159 genes induced in susceptible inoculated kernels. Defense related and regulation related genes were identified in both treatments. Comparisons between the resistant and susceptible lines indicate differences in the gene expression network which may enhance our understanding of the maize-A. flavus interaction.

Identification of resistance-associated proteins in closely-related maize lines varying in aflatoxin accumulation

Aspergillus flavus infection of maize and subsequent contamination with carcinogenic aflatoxins poses serious health concerns, especially in developing countries. Maize lines resistant to A. flavus infection have been identified; however, the development of commercially-useful aflatoxin-resistant maize lines has been hindered due to a lack of breeding markers. To identify maize resistance-associated proteins (RAPs) as potential markers for breeding, 52 BC1S4 lines developed from crosses between five African maize inbreds and five temperate aflatoxin-resistant lines were screened using the kernel screening assay. Five pairs of closely-related lines that had 75–94% genetic similarity within each pair and which varied within each pair in aflatoxin accumulation were selected for proteomic investigation. Kernel embryo and endosperm protein profile differences within the pair and across pairs were compared using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed (≥1.5-fold) RAPs were sequenced through tandem mass spectrometry and were identified as antifungal, stress-related, storage or regulatory proteins. Sequence homology analysis highlighted several proteins in maize that confer resistance to A. flavus infection and/or aflatoxin production.

A USA–Africa collaborative strategy for identifying, characterizing, and developing maize germplasm with resistance to aflatoxin contamination

Aflatoxin contamination of maize by Aspergillus flavus poses serious potential economic losses in the US and health hazards to humans, particularly in West Africa. The Southern Regional Research Center of the United States Department of Agriculture, Agricultural Research Service (USDA-ARS-SRRC) and the International Institute of Tropical Agriculture (IITA) initiated a collaborative breeding project to develop maize germplasm with resistance to aflatoxin accumulation. Resistant genotypes from the US and selected inbred lines from IITA were used to generate backcrosses with 75% US germplasm and F(1) crosses with 50% IITA and 50% US germplasm. A total of 65 S(4) lines were developed from the backcross populations and 144 S(4) lines were derived from the F(1) crosses. These lines were separated into groups and screened in SRRC laboratory using a kernel-screening assay. Significant differences in aflatoxin production were detected among the lines within each group. Several promising S(4) lines with aflatoxin values significantly lower than their respective US resistant recurrent parent or their elite tropical inbred parent were selected for resistance-confirmation tests. We found pairs of S(4) lines with 75-94% common genetic backgrounds differing significantly in aflatoxin accumulation. These pairs of lines are currently being used for proteome analysis to identify resistance-associated proteins and the corresponding genes underlying resistance to aflatoxin accumulation. Following confirmation tests in the laboratory, lines with consistently low aflatoxin levels will be inoculated with A. flavus in the field in Nigeria to identify lines resistant to strains specific to both US and West Africa. Maize inbred lines with desirable agronomic traits and low levels of aflatoxin in the field would be released as sources of genes for resistance to aflatoxin production.

Proteomics to identify resistance factors in corn-a review

The host resistance strategy for eliminating aflatoxins from corn has been advanced by the discovery of natural resistance traits such as proteins. This progress was aided by the development of a rapid laboratory-based kernel screening assay (KSA) used to separate resistant from susceptible seed, and for investigating kernel resistance.A. flavus GUS transformants have also been used, in conjunction with the KSA, to assess the amount of fungal growth in kernels and compare it with aflatoxin accumulation. Several proteins associated with resistance (RAPs) have been identified using 1 D PAGE. However, proteomics is now being used to further the discovery of RAPs. This methodology has led to the identification of stress-related RAPs as well as other antifungals. Characterization studies being conducted, including RNAi gene silencing experiments, may confirm roles for RAPs in host resistance.

Glufosinate-Ammonium Reduces Growth and Aflatoxin B1 Production by Aspergillus flavus

The herbicide glufosinate-ammonium (GA) [butanoic acid, 2-amino-4-(hydroxymethylphosphinyl)-ammonium salt] was tested at concentrations from 2 to 2,000 g GA per ml for activity against growth and aflatoxin B1 (AFB1) production by the mycotoxigenic fungus Aspergillus flavus Link:Fr. The highest concentration (2,000 μg GA per ml) reduced colony diameter of A. flavus strain AF13 by 80%. AFB1 production was inhibited by 90% at this concentration. Reduction in mycelial dry weight and AFB1 production in response to GA application ranged from 17.2 to 97.1% and from 39.1 to 90.1%, respectively. Of four concentrations tested, 2 μg GA per ml was weakly inhibitory. In the kernel screening assay, AFB1 production was inhibited 60 to 91% when kernels were preimmersed or immersed 5 days after incubation in 200 μg GA per ml. Both concentrations (2 and 200 μg GA per ml) reduced seed germination by 25 to 50%. Results indicate that GA has an inhibitory effect on growth and AFB1 production by A. flavus.

Resistance to Aflatoxin Accumulation in Kernels of Maize Inbreds Selected for Ear Rot Resistance in West and Central Africa

Thirty-six inbred lines selected in West and Central Africa for moderate to high resistance to maize ear rot under conditions of severe natural infection were screened for resistance to aflatoxin contamination using the previously established kernel screening assay. Results showed that more than half the inbreds accumulated aflatoxins at levels as low as or lower than the resistant U.S. lines GT-MAS:gk or MI82. In 10 selected aflatoxin-resistant or aflatoxin-susceptible inbreds, Aspergillus flavus growth, which was quantified using an A. flavus transformant containing a GUS-β-tubulin reporter gene construct, was, in general, positively related to aflatoxin accumulation. However, one aflatoxin-resistant inbred supported a relatively high level of fungal infection, whereas two susceptibles supported relatively low fungal infection. When kernels of the 10 tested lines were profiled for proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, significant variations from protein profiles of U.S. lines were observed. Confirmation of resistance in promising African lines in field trials may significantly broaden the resistant germplasm base available for managing aflatoxin contamination through breeding approaches. Biochemical resistance markers different from those being identified and characterized in U.S. genotypes, such as ones inhibitory to aflatoxin biosynthesis rather than to fungal infection, may also be identified in African lines. These discoveries could significantly enhance the host resistance strategy of pyramiding different traits into agronomically useful maize germplasm to control aflatoxin contamination.

Advances in the Development of Host Resistance in Corn to Aflatoxin Contamination by Aspergillus flavus

ABSTRACT Aflatoxins are toxic, highly carcinogenic secondary metabolites of Aspergillus flavus and A. parasiticus, which when produced during fungal infection of a susceptible crop in the field or after harvest contaminate food and feed and threaten human and animal health. Although there are several management strategies that may reduce aflatoxin contamination of corn, the preeminent strategy for elimination of aflatoxin is to develop preharvest host resistance to aflatoxin accumulation. This strategy has gained even greater prominence due to recent discoveries of natural resistance in corn that can be exploited in plant-breeding strategies. The ability to identify resistant corn genotypes has been enhanced by the development of a laboratory kernel-screening assay and by a strain of A. flavus genetically engineered to produce beta-glucuronidase, an enzyme whose activity can be monitored to assess the degree of fungal infection in kernels. Investigations of resistant corn genotypes have associated kernel pericarp wax characteristics with resistance, identified kernel proteins associated with resistance to and inhibition of fungal growth or aflatoxin biosynthesis, and identified chromosome regions associated with resistance to Aspergillus ear rot and aflatoxin production. Such research advances could lead, in the near future, to commercially available, agronomically acceptable corn lines with multiple preharvest resistances to aflatoxin contamination.

Determination of Resistance to Aflatoxin Production in Maize Kernels and Detection of Fungal Colonization Using anAspergillus flavusTransformant ExpressingEscherichia colibeta-Glucuronidase

Thirty-one maize inbreds and the highly resistant GT-MAS :gk maize population were screened for resistance to aflatoxin production by Aspergillus flavus using a kernel-screening laboratory assay. Intact kernels from each line were evaluated in three trials. Significant differences among genotypes for resistance to aflatoxin production were found. Certain genotypes, previously shown to be resistant in field trials, demonstrated resistance in the kernel-screening assay. Twenty-two genotypes had intact-kernel resistance that was comparable to GT-MAS :gk. A separate experiment was done to visualize fungal colonization of internal tissue in susceptible and resistant maize kernels and to further elucidate the relationship between fungal colonization and aflatoxin production. Five genotypes screened in the inbred evaluation, were inoculated with an A. flavus aflatoxin-producing strain containing the Escherichia coli β-D-glucuronidase (GUS) reporter gene linked to an A. flavus β-tubulin gene promoter. Histochemical staining of nonwounded and wounded kernels detected differences in GUS expression among genotypes, and there was a relationship between GUS expression and the amount of aflatoxin detected in kernels. Minimal GUS expression was related to low aflatoxin production in wounded kernels of two inbreds previously identified in field trials as having moderate-to-high levels of resistance to aflatoxin production. These results suggest that resistance to aflatoxin production is directly related to resistance to fungal colonization in certain genotypes.