Occurrence of aflatoxin in three maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) hybrids over 5 years in Northern Mississippi

Aflatoxins are produced as secondary metabolites under conducive climatic conditions by Aspergillus flavus. The incidence of aflatoxin varies with environmental conditions, genotype, and location. An expanded understanding of the interaction of the plant, fungus, and weather conditions is needed to further elucidate the field infection process of maize by A. flavus and subsequent aflatoxin contamination. One of the problems in evaluating maize hybrids for resistance to kernel infection and aflatoxin contamination is identifying a time period and environmental conditions that are most advantageous. Three maize genotypes (Pioneer Brand 3223, Mo18W × Mp313E, and Mp313E × Mp420) were evaluated from 1998 to 2002 in response to A. flavus inoculation and aflatoxin contamination and weather conditions favorable for aflatoxin contamination were identified. The highest aflatoxin levels were observed in 1998 and 2000 (1186 and 901 ng g⁻¹; P < 0.0001); while the lowest levels were detected in 1999 (39 ng g⁻¹). Pioneer 3223 had significantly higher levels (1198 ng g⁻¹) than Mp313E × Mp420 (205 ng g⁻¹), and Mo18W ×Mp313E (161 ng g⁻¹; P < 0.0001). The hybrids had six weather-related variables in common that were positively correlated with aflatoxin accumulation. Four of these occurred during 65–85 days after planting and were temperature-related. These results suggest that regardless of the hybrid’s maturity or physiological development, the time from 65 to 85 days after planting may be indicative of a period of stress which leads to greater aflatoxin accumulation at harvest. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Confirming quantitative trait loci for aflatoxin resistance from Mp313E in different genetic backgrounds

The fungus Aspergillus flavus (Link:Fr) causes ear rot of maize (Zea mays L.) and produces the toxic metabolic product aflatoxin. One particularly effective method of controlling the fungus is via host plant resistance, but while several resistant breeding lines have been identified, transferring the resistance genes from these lines into elite cultivars has been less effective than needed. A high number of genes involved with resistance, each with a small effect, and some only found under certain environmental conditions, has hampered resistance breeding. The identification of markers linked to genomic regions associated with resistance would aid in this effort. The goals of this study were to identify and characterize quantitative trait loci (QTL) conferring resistance to aflatoxin accumulation from resistant maize donor Mp313E in a background of the susceptible inbred line Va35; to compare them to the QTL identified from Mp313E in a background of B73; and to test the stability of the QTL identified in Mp313E × Va35 in multiple environments by remapping the phenotypic tails of the Mp313E × Va35 mapping population in new locations. Twenty different QTL were found in this study, 11 of which were also found in different environments using the phenotypic tail subset mapping population, and five of which were likely the same as those reported in the Mp313E × B73 mapping population. This indicates that many of the QTL are stable over the environments and genetic backgrounds tested, which will make them more valuable in breeding efforts.     

Identification of novel QTL contributing resistance to aflatoxin accumulation in maize

The toxic metabolic product aflatoxin produced by the opportunistic fungus Aspergillus flavus (Link:Fr) in maize (Zea mays L.) can cause disease and economic harm when levels exceed very minute quantities. The selection of resistant germplasm has great potential to reduce the problem, but the highly quantitative nature of the trait makes this a difficult endeavor. The identification of aflatoxin accumulation resistance quantitative trait loci (QTL) from resistant donor lines and the discovery of linked markers could speed this task. To identify marker–trait associations for marker-assisted breeding, a genetic mapping population of F_2:3 families was developed from Mp715, a maize inbred line resistant to aflatoxin accumulation, and T173, a susceptible, southern-adapted maize inbred line. QTL, some with large phenotypic effects, were identified in multiple years on chromosomes 1, 3, 5, and 10, and smaller QTL identified in only 1 year were found on chromosomes 4 and 9. The phenotypic effect of each QTL ranged from 2.7 to 18.5%, and models created with multiple QTL could explain up to 45.7% of the phenotypic variation across years, indicating that the variation associated with the trait can be manipulated using molecular markers.     

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 F₁ crosses with 50% IITA and 50% US germplasm. A total of 65 S₄ lines were developed from the backcross populations and 144 S₄ lines were derived from the F₁ 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 S₄ 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 S₄ 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.     

Quantitative trait loci (QTL) for reducing aflatoxin accumulation in corn

Aflatoxin produced by Aspergillus flavus in corn poses significant health risks to both humans and livestock. Exploitation of host-plant resistance in breeding programs is a sustainable way to minimize aflatoxin contamination. Identification of quantitative trait loci (QTL) associated with resistance to aflatoxin accumulation in kernels can accelerate development of aflatoxin-resistant corn using marker-assisted selection. An F_2:3 mapping population, developed from a cross involving a resistant inbred Mp715 and a susceptible inbred B73, was evaluated in replicated field trials with developing ears artificially inoculated with A. flavus for 2 years to identify QTL for reduced aflatoxin accumulation. Using composite interval mapping, 6 to 7 QTL for aflatoxin content were identified in both years with contribution of individual QTL ranging from <1 to 10% of phenotypic variation. More QTL were detected for husk coverage with phenotypic variance range of <1 to 16% explained by individual QTL. Both B73 and Mp715 alleles at these QTL loci contributed toward resistance. Husk coverage and aflatoxin levels were significantly correlated in both years. Our findings were further supported by overlapping of QTL for husk coverage ratings in four genomic regions on chromosomes 4, 8, and 10, where aflatoxin resistance QTL were reported in previous studies. Since most of the QTL were of low to moderate effects, pyramiding of these QTL may lead to enhanced resistance to aflatoxin accumulation in corn.     

Southwestern corn borer (Lepidoptera: Crambidae) damage and aflatoxin accumulation in maize

Aflatoxin, a potent carcinogen, is produced by the fungus Aspergillus flavus Link: Fr. Drought, high temperatures, and insect damage contribute to increased levels of aflatoxin contamination in corn, Zea mays L. Plant resistance is widely considered a desirable method of reducing aflatoxin contamination. Germplasm lines with aflatoxin resistance have been developed. This investigation was undertaken to determine whether crosses among these lines exhibited resistance to southwestern corn borer, Diatraea grandiosella Dyar, and to assess the effects of southwestern corn borer feeding on aflatoxin accumulation. Differences in ear damage among southwestern corn borer infested hybrids were significant. Estimates of general combining ability effects indicated that the lines Mp80:04, Mp420, and Mp488 contributed to reduced ear damage, and SC213 and T165 contributed to greater damage when used in hybrids. Mean aflatoxin levels were 254 ng/g for hybrids infested with southwestern corn borer larvae and 164 ng/g for noninfested hybrids in 2000 when environmental conditions were conducive to aflatoxin production. In contrast, the overall mean aflatoxin level for southwestern corn borer infested hybrids was only 5 ng/g in 1999 when environmental conditions did not favor aflatoxin accumulation. Crosses that included lines selected for aflatoxin resistance as parents (Mp80:04 and Mp313E) exhibited lower levels of aflatoxin contamination both with and without southwestern corn borer infestation in 2000. Only the experimental line Mp80:04 contributed significantly to both reduced southwestern corn borer damage and reduced aflatoxin contamination.     

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?C94% 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.     

Genetic variation within maize population GT-MAS:gk and the relationship with resistance to Aspergillus flavus and aflatoxin production

Aspergillus flavus (Link:Fr.) infection and aflatoxin contamination of maize (Zea mays L.) grain are an extremely serious problem. Maize genotypes resistant to A. flavus attack are needed. Maize breeders and plant pathologists must identify resistance sources and incorporate resistance into adapted breeding material. Maize population GT-MAS:gk has been released for use as a resistance source. In this study, we surveyed the genetic variation in this population and made the breeders/plant pathologists aware of the heterogeneous nature in this maize population by using RAPD analysis and correlated the RAPD marker association with the resistance to A. flavus and aflatoxin production. Of 40 RAPD primers, only 15 gave sufficient numbers of reproducible and readily scored polymorphic bands suggesting that this population was highly homogeneous. However, genetic distances, ranging from 0.08 to 0.28 and averaging 0.17, suggest that there is variation within the population. Cluster analysis distinguished three major polymorphic groups. Laboratory bioassay revealed that group I contained the most resistant individuals, i.e., those with less aflatoxin production. Group II had the least resistance, and group III was intermediate. This study showed that the maize population GT-MAS:gk is heterogeneous and individuals are different in resistance to A. flavus and aflatoxin production. Resistance should be confirmed through progeny testing before further development. The RAPD marker OPX-04, which may be associated with the resistance trait, has been cloned and further characterization will be pursued. Received: 10 May 2000 / Accepted: 12 January 2001     

Proteomic analysis of the maize rachis: potential roles of constitutive and induced proteins in resistance to Aspergillus flavus infection and aflatoxin accumulation

Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses.     

Prevention of preharvest aflatoxin contamination through genetic engineering of crops

Current practices on prevention of aflatoxin contamination of crop species include time consuming, expensive agronomic practices. Of all the methods available to-date, conventional breeding and/or genetic engineering to develop host plant-based resistance to aflatoxin-producing fungi appear to be valuable for several reasons. However, breeding for disease-resistant crops is very time consuming, especially in tree crops, and does not lend itself ready to combat the evolution of new virulent fungal races. Moreover, availability of known genotypes with natural resistance to mycotoxin-producing fungi is a prerequisite for the successful breeding program. While it is possible to identify a few genotypes of corn or peanuts that are naturally resistant toAspergillus we do not know whether these antifungal factors are specific toA. flavus. In crops like cotton, there are no known naturally resistant varieties toAspergillus. Availability of transgenic varieties with antifungal traits is extremely valuable as a breeding tool. Several antifungal proteins and peptides are available for genetic engineering of susceptible crop species, thanks to the availability of efficient modern tools to understand and evaluate protein interactions by proteomics of host, and genomics and field ecology of the fungus. Transgenic approaches are being undertaken in several industry and academic laboratories to prevent invasion byAspergillus fungi or to prevent biosynthesis of aflatoxin. Recent trends in reducing aflatoxin contamination through genetic engineering of cultivated crop species with antifungal proteins are summarized in this report. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005     

Evaluating Tripsacum‐introgressed maize germplasm after infestation with western corn rootworms (Coleoptera: Chrysomelidae)

Maize (Zea mays L.) is a valuable commodity throughout the world, but corn rootworms (Chrysomelidae: Diabrotica spp.) often cause economic damage and increase production costs. Current rootworm management strategies have limitations, and in order to create viable management alternatives, researchers have been developing novel maize lines using Eastern gamagrass (Tripsacum dactyloides L.) germplasm, a wild relative of maize that is resistant to rootworms. Ten maize Tripsacum‐introgressed inbred lines derived from recurrent selection of crosses with gamagrass and teosinte (Zea diploperennis Iltis) recombinants and two public inbred lines were assessed for susceptibility to western corn rootworm (Diabrotica virgifera virgifera LeConte) and yield in a two‐year field study. Two experimental maize inbred lines, SDG11 and SDG20, had mean root damage ratings that were significantly lower than the susceptible public line B73. Two other experimental maize inbred lines, SDG12 and SDG6, appeared tolerant to rootworm damage because they exhibited yield increases after rootworm infestation in both years. In the majority of cases, mean yield per plant of experimental maize lines used in yield analyses was equal to or exceeded that of the public inbred lines B73 and W64A. Our study indicates that there is potential to use Tripsacum‐introgressed maize germplasm in breeding programs to enhance plant resistance and/or tolerance to corn rootworms, although further research on insect resistance and agronomic potential of this germplasm needs to be conducted in F₁ hybrids.     

Studies on the biocontrol of aflatoxin in maize in Thailand

Aflatoxins in maize and peanuts remain a major cause of liver cancer and other human and animal health issues. The principal causal fungi are Aspergillus flavus and A. parasiticus. Relatively little attention has been paid to reducing aflatoxin formation before harvest. The most promising approach is biocontrol by competitive exclusion. This project aimed to demonstrate the efficacy of locally isolated strains of A. flavus for biocontrol of aflatoxin in maize in Thailand. After a rigorous process utilising molecular methods was used to select non-toxigenic A. flavus strains, field inoculum was produced by using hulled rice coated with A. flavus spores in molasses. Field experiments were conducted over two years in two districts, one of light sandy soil (Chokchai), the other a heavy, close textured, soil (Pakchong). Postharvest treatments representative of local practice were also undertaken. Crops 1 and 2 were not significantly contaminated with aflatoxin at the time of harvest, so any impact of biocontrol could not be assessed. However, wet shelling plus storage before drying resulted in increased aflatoxin contamination; biocontrol had no impact on this increase. In crops 3 and 4, biocontrol had a beneficial impact in some freshly harvested maize. Biocontrol treatments also significantly reduced aflatoxin contamination in samples from some treatments stored for two or four days after shelling, but had minimal effect in others. These experiments demonstrated that biocontrol can be highly effective in reducing aflatoxin contamination in maize in Thailand, both at harvest and during poor postharvest crop handling. However, results were inconsistent.     

QTL mapping for European corn borer resistance (<Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> Hb.), agronomic and forage quality traits of testcross progenies in early-maturing European maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) germplasm

In hybrid breeding the performance of lines in hybrid combinations is more important than their performance per se. Little information is available on the correlation between individual line and testcross (TC) performances for the resistance to European corn borer (ECB, Ostrinia nubilalis Hb.) in maize (Zea mays L.). Marker assisted selection (MAS) will be successful only if quantitative trait loci (QTL) found in F₂ derived lines for ECB resistance are still expressed in hybrid combinations. The objectives of our study were: (1) to identify and characterize QTL for ECB resistance as well as agronomic and forage quality traits in a population of testcrossed F_2:3 families; (2) to evaluate the consistency of QTL for per se and TC performances; and (3) to determine the association between per se and TC performances of F_2:3 lines for these traits. Two hundred and four F_2:3 lines were derived from the cross between maize lines D06 (resistant) and D408 (susceptible). These lines were crossed to D171 and the TC progenies were evaluated for ECB resistance and agronomic performance in two locations in 2000 and 2001. Using these TC progenies, six QTL for stalk damage rating (SDR) were found. These QTL explained 27.4% of the genotypic variance in a simultaneous fit. Three QTL for SDR were detected consistently for per se and TC performance. Phenotypic and genotypic correlations were low for per se and TC performance for SDR. Correlations between SDR and quality traits were not significant. Based on these results, we conclude that MAS will not be an efficient method for improving SDR. However, new molecular tools might provide the opportunity to use QTL data as a first step to identify genes involved in ECB resistance. Efficient MAS procedures might then be based on markers designed to trace and to combine specific genes and their alleles in elite maize breeding germplasm.Communicated by G. Wenzel     

Aspergillus Colonization and Aflatoxin Contamination of Maize and Sesame Kernels in Two Agro‐ecological Zones in Senegal

Aflatoxin contamination of major food crops is a serious problem in Senegal. Maize and sesame samples were collected during a survey in five districts located in two agro‐ecological zones in Senegal to determine levels of aflatoxin contamination and the distribution and toxigenicity potential of members of Aspergillus section Flavi. Maize samples from the Guinea Savannah zone (SG) exhibited lower aflatoxin content and colony‐forming units (cfu) than those collected from the Sudan Savannah (SS) zone. In maize, aflatoxin concentration and cfu of A. flavus varied with cultivars, shelling practices and storage methods. The maize variety ‘Jaune de Bambey’ had high aflatoxin levels in both agro‐ecological zones. Aflatoxin content in machine‐shelled maize (120 ng/g) was more than 10‐fold higher than that in manually shelled (8 ng/g) or unshelled maize. Aflatoxin content (between 0.1 and 1.2 ng/g) and cfu values (between 13 and 42 000 cfu/g) of sesame were low, suggesting a low susceptibility to A. flavus. In both agro‐ecological zones, and in all storage systems, aflatoxin contamination was lower in sesame than in maize. In this study, only three species of Aspergillus section Flavi (A. flavus, A. tamarii and the unnamed taxon S_BG) were observed with the frequency of toxigenic strains remaining below 50% in maize from the SG zone compared with 51% of isolates from samples collected in Sedhiou district in SS zone. The proportion of toxigenic strains isolated from sesame was variable. For both crops, L‐strains were the most prevalent in the two agro‐ecological zones. Some of the atoxigenic strains collected could be valuable microbial resources for the biological control of aflatoxin in Senegal.     

Impact of Aspergillus section Flavi community structure on the development of lethal levels of aflatoxins in Kenyan maize (Zea mays)

Aims: To evaluate the potential role of fungal community structure in predisposing Kenyan maize to severe aflatoxin contamination by contrasting aflatoxin‐producing fungi resident in the region with repeated outbreaks of lethal aflatoxicosis to those in regions without a history of aflatoxicosis. Methods and Results: Fungi belonging to Aspergillus section Flavi were isolated from maize samples from three Kenyan provinces between 2004 and 2006. Frequencies of identified strains and aflatoxin‐producing abilities were assessed, and the data were analysed by statistical means. Most aflatoxin‐producing fungi belonged to Aspergillus flavus. The two major morphotypes of A. flavus varied greatly between provinces, with the S strain dominant in both soil and maize within aflatoxicosis outbreak regions and the L strain dominant in nonoutbreak regions. Conclusions: Aspergillus community structure is an important factor in the development of aflatoxins in maize in Kenya and, as such, is a major contributor to the development of aflatoxicosis in the Eastern Province. Significance and Impact of the Study: Since 1982, deaths caused by aflatoxin‐contaminated maize have repeatedly occurred in the Eastern Province of Kenya. The current study characterized an unusual fungal community structure associated with the lethal contamination events. The results will be helpful in developing aflatoxin management practices to prevent future outbreaks in Kenya.     

Environmental distribution and genetic diversity of vegetative compatibility groups determine biocontrol strategies to mitigate aflatoxin contamination of maize by Aspergillus flavus

Maize infected by aflatoxin‐producing Aspergillus flavus may become contaminated with aflatoxins, and as a result, threaten human health, food security and farmers' income in developing countries where maize is a staple. Environmental distribution and genetic diversity of A. flavus can influence the effectiveness of atoxigenic isolates in mitigating aflatoxin contamination. However, such information has not been used to facilitate selection and deployment of atoxigenic isolates. A total of 35 isolates of A. flavus isolated from maize samples collected from three agro‐ecological zones of Nigeria were used in this study. Ecophysiological characteristics, distribution and genetic diversity of the isolates were determined to identify vegetative compatibility groups (VCGs). The generated data were used to inform selection and deployment of native atoxigenic isolates to mitigate aflatoxin contamination in maize. In co‐inoculation with toxigenic isolates, atoxigenic isolates reduced aflatoxin contamination in grain by > 96%. A total of 25 VCGs were inferred from the collected isolates based on complementation tests involving nitrate non‐utilizing (nit^?) mutants. To determine genetic diversity and distribution of VCGs across agro‐ecological zones, 832 nit^? mutants from 52 locations in 11 administrative districts were paired with one self‐complementary nitrate auxotroph tester‐pair for each VCG. Atoxigenic VCGs accounted for 81.1% of the 153 positive complementations recorded. Genetic diversity of VCGs was highest in the derived savannah agro‐ecological zone (H = 2.61) compared with the southern Guinea savannah (H = 1.90) and northern Guinea savannah (H = 0.94) zones. Genetic richness (H = 2.60) and evenness (E₅ = 0.96) of VCGs were high across all agro‐ecological zones. Ten VCGs (40%) had members restricted to the original location of isolation, whereas 15 VCGs (60%) had members located between the original source of isolation and a distance > 400 km away. The present study identified widely distributed VCGs in Nigeria such as AV0222, AV3279, AV3304 and AV16127, whose atoxigenic members can be deployed for a region‐wide biocontrol of toxigenic isolates to reduce aflatoxin contamination in maize.     

Peanut Resistance Gene Expression in Response to Aspergillus flavus Infection During Seed Germination

Aspergillus flavus produces potent mutagenic and carcinogenic polyketide‐derived secondary metabolites known as aflatoxins. Development of host plant resistance in peanut and other crops is the most environmentally friendly and cost‐effective method to eliminate the serious problem of aflatoxin contamination in grains. To confirm that putative peanut genes identified in a previous microarray study were involved in peanut resistance to A. flavus infection, 14 genes were selected for further investigation through real‐time PCR. The results revealed diverse patterns of gene expression during seed germination after A. flavus inoculation. Based on the expression levels and the relative‐expression patterns over a 7‐day period, the 14 host genes could be classified into six different groups belonging to three main biochemical and genetic defence processes of lipid metabolism, oxidative signalling and cell‐wall synthesis during counter‐attack. A network of gene expression patterns was activated in sequential order in response to A. flavus invasion in both resistant and susceptible peanut lines during seed germination. Understanding gene expression patterns in peanut will be useful to breeders and other scientists interested in incorporating genetic resources of resistance against A. flavus into peanut germplasm and/or commercial cultivars via conventional and/or molecular methods.     

Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.