Development of Maize Host Resistance to Aflatoxigenic Fungi

2.1. Traditional screening techniques

Screening maize for resistance to kernel infection by Aspergillus flavusor for resistance to aflatoxin production is a more difficult task than most disease screening. Successful screening in the past had been hindered [15] by the lack of 1) a resistant control; 2) inoculation methods that yield infection/aflatoxin levels high enough to differentiate among genotypes (natural infection is undependable); 3) repeatability across different locations and years; and, 4) rapid and inexpensive methods for assessment of fungal infection and aflatoxin levels. Several inoculation methods, including the pinbar inoculation technique (for inoculating kernels through husks), the silk inoculation technique, and infesting corn ears with insect larvae infected with A. flavusconidia have been tried with varying degrees of success [9, 16]. These methods can each be useful, however, clarity must exist as to the actual resistance trait to be measured (e.g. husk tightness; silk traits; the kernel pericarp barrier; wounded kernel resistance), before an appropriate technique can be employed. Silk inoculation, however, (possibly more dependent upon the plant’s physiological stage and/or environmental conditions) has proven to be the most inconsistent of the inoculation methods [17].

Plating kernels to determine the frequency of kernel infection and examining kernels for emission of a bright greenish-yellow fluorescence (BGYF) are methods that have been used for assessing A. flavusinfection [15]. While both methods can indicate the presence of A.flavusin seed, neither can provide the kind of accurate quantitative or tissue-localization data useful for effective resistance breeding. Several protocols have been developed and used for separation and relatively accurate quantification of aflatoxins [18].

2.2. Early identification of resistant maize lines

Two resistant inbreds (Mp420 and Mp313E) were discovered and tested in field trials at different locations and released as sources of resistant germplasm [11, 19]. The pinbar inoculation technique was one of the methods employed in the initial trials, and contributed towards the separation of resistant from susceptible lines [11]. Several other inbreds, demonstrating resistance to aflatoxin contamination in Illinois field trials (employing a modified pinbar technique) also were discovered [12]. Another source of resistance discovered was the maize breeding population, GT-MAS:gk. This population was derived from visibly classified segregating kernels, obtained from a single fungus-infected hybrid ear [10]. It tested resistant in trials conducted over a five year period, where a kernel knife inoculation technique was employed.

These discoveries of resistant germplasm may have been facilitated by the use of inoculation techniques capable of repeatedly providing high infection/aflatoxin levels for genotype separation to occur. While these maize lines do not generally possess commercially acceptable agronomic traits, they may be invaluable sources of resistance genes, and as such, provide a basis for the rapid development of host resistance strategies to eliminate aflatoxin contamination.

3.1. Molecular genetic investigations of aflatoxin-resistant lines

Chromosome regions associated with resistance to A. flavusand inhibition of aflatoxin production in maize have been identified through Restriction Fragment Length Polymorphism (RFLP) analysis in three “resistant” lines (R001, LB31, and Tex6) in an Illinois breeding program, after mapping populations were developed using B73 and/or Mo17 elite inbreds as the “susceptible” parents [20, 21]. Chromosome regions associated with inhibition of aflatoxin in studies considering all 3 resistant lines demonstrated that there are some regions in common. Regions on chromosome arms 2L, 3L, 4S, and 8S may prove promising for improving resistance through marker assisted breeding into commercial lines [21]. In some cases, chromosomal regions were associated with resistance to Aspergillusear rot and not aflatoxin inhibition, and vice versa, whereas others were found to be associated with both traits. This suggests that these two traits may be at least partially under separate genetic control. QTL studies involving other populations have identified chromosome regions associated with low aflatoxin accumulation.

In a study involving 2 populations from Tex6 x B73, conducted in 1996 and 1997, promising QTLs for low aflatoxin were detected in bins 3.05-6, 4.07-8, 5.01-2, 5.05-5, and 10.05-10.07 [22]. Environment strongly influenced detection of QTLs for lower toxin in different years; QTLs for lower aflatoxin were attributed to both parental sources. In a study involving a cross between B73 and resistant inbred Oh516, QTL associated with reduced aflatoxin were identified on chromosomes 2, 3 and 7 (bins 2.01 to 2.03, 2.08, 3.08, and 7.06) [23]. QTLs contributing resistance to aflatoxin accumulation were also identified using a population created by B73 and resistant inbred Mp313E, on chromosome 4 of Mp313E [24]. This confirmed the findings of an earlier study involving Mp313E and susceptible Va35 [25]. Another QTL in this study, which has similar effects to that on chromosome 4, was identified on chromosome 2 [24]. A recent study to identify aflatoxin-resistance QTL and linked markers for marker-assisted breeding was conducted using a population developed from Mp717, an aflatoxin-resistant maize inbred, and NC300, a susceptible inbred adapted to the southern U.S. QTL were identified on all chromosomes, except 4, 6, and 9; individual QTL accounted for up to 11% of phenotypic variance in aflatoxin accumulation [26]. Lastly, in a study of population of F2:3 families developed from resistant Mp715 and a southern-adapted susceptible, T173, QTL with phenotypic effects up to 18.5% were identified in multiple years on chromosomes 1, 3, 5, and 10 [27].

A number of genes corresponding to resistance-associated proteins (RAPs), that were identified in proteomics studies (see section 3.5.1 below) have been mapped to chromosomal location using the genetic sequence of B73 now available online (http://archive.maizesequence.org/index.html) [28]. Using the DNA sequence of the RAPs and blasting them against the B73 sequence allowed us to place each gene into a virtual bin, allowing us to pinpoint the chromosomal location to which each gene maps. The chromosomes involved include the above-mentioned chromosomes 1, 2, 3, 7, 8 and 10, some in bins closely located to those described above. Another study also mapped RAPs to bins on the above-chromosomes as well as chromosomes 4 and 9 [29].

3.2. Kernel pericarp wax

Kernel pericarp wax of maize breeding population GT-MAS:gk has been associated with resistance to Aspergillus flavusinfection /aflatoxin production. Previously, kernel wax of GT-MAS:gk was compared to that of 3 susceptible genotypes. Thin layer chromatography (TLC) of wax from these genotypes showed a band unique to GT-MAS:gk and a band unique to the three susceptible lines [30]. GT-MAS:gk kernel wax also was shown to inhibit A. flavusgrowth. A later investigation compared GT-MAS:gk wax resistance-associated traits to that of twelve susceptible maize genotypes [31]. TLC results of wax from these lines confirmed findings of the previous investigation, demonstrating both the unique GT-MAS:gk TLC band and the unique ‘susceptible’ band. Gas chromatography/mass spectroscopy (GC/MS) analysis of the whole wax component showed a higher percentage of phenol-like compounds in the resistant genotype than in the susceptibles. Alkylresorcinol content was dramatically higher in GT-MAS:gk wax than in susceptible lines. An alkylresorcinol, 5-methylresorcinol, also inhibited in vitrogrowth of A. flavus. Further research is needed for a clear identification of the component(s) responsible for kernel wax resistance and to determine its expression level in other maize lines.

3.3. Two levels of resistance

The KSA employs a very simple and inexpensive apparatus involving bioassay trays, petri dishes, vial caps as seed containers, and chromatography paper for holding moisture [14]. Kernels screened by the KSA are maintained in 100% humidity, at a temperature favoring A. flavus(31° C) growth and aflatoxin production, and are usually incubated for seven days. Aflatoxin data from KSA experiments can be obtained two to three weeks after experiments are initiated. KSA experiments confirmed GT-MAS:gk resistance to aflatoxin production and demonstrated that it is maintained even when the pericarp barrier, in otherwise viable kernels, is breached [13]. Penetration through the pericarp barrier was achieved by wounding the kernel with a hypodermic needle down to the endosperm, prior to inoculation. Wounding facilitates differentiation between different resistance mechanisms in operation, and the manipulation of aflatoxin levels in kernels for comparison with other traits (e.g. fungal growth; protein induction). The results of this study indicate the presence of two levels of resistance: at the pericarp and at the subpericarp level. The former was supported by the above-studies which demonstrated a role for pericarp waxes in kernel resistance [30], and highlighted quantitative and qualitative differences in pericarp wax between GT-MAS:gk and susceptible genotypes [31, 32].

3.4. Comparing fungal growth to toxin production

When selected resistant Illinois maize inbreds (MI82, CI2, and T115) were examined by the KSA, modified to include an A. flavusGUS transformant (a strain genetically engineered with a gene construct consisting of a β-glucuronidase reporter gene linked to an A. flavusbeta-tubulin gene promoter for monitoring fungal growth) [14], kernel resistance to fungal infection in nonwounded and wounded kernels was demonstrated both visually and quantitatively, as was a positive relationship between the degree of fungal infection and aflatoxin levels [14, 33]. This made it possible assess fungal infection levels and to determine if a correlation exists between infection and aflatoxin levels in the same kernels. A. flavusGUS transformants with the reporter gene linked to an aflatoxin biosynthetic pathway gene could also provide a way to indirectly measure aflatoxin levels [34-36], based on the extent of the expression of the pathway gene.

Recently, It was demonstrated, using the KSA and an F. moniliformestrain, genetically transformed with a GUS reporter gene linked to an A. flavusβ-tubulin gene promoter, that the aflatoxin-resistant genotype, GT-MAS:gk, inhibits growth of F. moniliformeas well [37]. This indicates that some resistance mechanisms may be generic for ear rotting/mycotoxigenic fungi.

A more recent use of reporter genes was performed on cotton using a green fluorescent protein reporter; a GFP-expressing A. flavusstrain to successfully monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins [38]. This strain provides for an easy, potentially non-destructive, rapid and economical assay which can be done in real time, and may constitute an advance over GUS transformants.

3.5. Resistance-associated proteins

Developing resistance to fungal infection in wounded as well as intact kernels would go a long way toward solving the aflatoxin problem [17]. Studies demonstrating subpericarp (wounded-kernel) resistance in maize kernels have led to research for identification of subpericarp resistance mechanisms. Examinations of kernel proteins of several genotypes revealed differences between genotypes resistant and susceptible to aflatoxin contamination [39]. Imbibed susceptible kernels, for example, showed decreased aflatoxin levels and contained germination-induced ribosome inactivating protein (RIP) and zeamatin [40]. Both zeamatin and RIP have been shown to inhibit A. flavusgrowth in vitro[40]. In another study, two kernel proteins were identified from a resistant corn inbred (Tex6) which may contribute to resistance to aflatoxin contamination [41]. One protein, 28 kDa in size, inhibited A. flavusgrowth, while a second, over 100 kDa in size, primarily inhibited toxin formation. When a commercial corn hybrid was inoculated with aflatoxin and nonaflatoxin-producing strains of A. flavusat milk stage, one induced chitinase and one ß-1,3-glucanase isoform was detected in maturing infected kernels, while another isoform was detected in maturing uninfected kernels [42].

In another investigation, an examination of kernel protein profiles of 13 maize genotypes revealed that a 14 kDa trypsin inhibitor protein (TI) is present at relatively high concentrations in seven resistant maize lines, but at low concentrations or is absent in six susceptible lines [43]. The mode of action of TI against fungal growth may be partially due to its inhibition of fungal -amylase, limiting A. flavusaccess to simple sugars [44] required not only for fungal growth, but also for toxin production [45]. TI also demonstrated antifungal activity against other mycotoxigenic species [46]. The identification of these proteins may provide markers for plant breeders, and may facilitate the cloning and introduction of antifungal genes through genetic engineering into other aflatoxin-susceptible crops.

An investigation into maize kernel resistance [47] determined that both constitutive and induced proteins are required for resistance to aflatoxin production. It also showed that one major difference between resistant and susceptible genotypes is that resistant lines constitutively express higher levels of antifungal proteins compared to susceptible lines. The real function of these high levels of constitutive antifungal proteins may be to delay fungal invasion, and consequent aflatoxin formation, until other antifungal proteins can be synthesized to form an active defense system.

4.1. Closely-related lines

Recently, the screening of progeny generated through a collaborative breeding program between IITA-Nigeria (International Institute of Tropical Agriculture) and the Southern Regional Research Center of USDA-ARS in Center (SRRC) of USDA-ARS in New Orleans facilitated the identification of closely-related lines from the same backcross differing significantly in aflatoxin accumulation, and proteome analysis of these lines is being conducted [77, 78]. Investigating corn lines sharing close genetic backgrounds should enhance the identification of RAPs without the confounding effects experienced with lines of diverse genetic backgrounds. The IITA-SRRC collaboration has attempted to combine resistance traits of U.S. resistant inbred lines with those of African lines, originally selected for resistance to ear rot diseases and for potential aflatoxin-resistance (viaKSA) [77, 78]. Five elite tropical inbred lines from IITA adapted to the Savanna and mid-altitude ecological zones of West and Central Africa were crossed with four U.S. resistant maize lines in Ibadan, Nigeria. The five African lines were originally selected for their resistance to ear rot caused by Aspergillus, Botrydiplodia, Diplodia, Fusarium, and Macropomina[77, 78]. The F1 crosses were backcrossed to their respective U.S. inbred lines and self-pollinated thereafter. The resulting lines were selected through the S4 generation for resistance to foliar diseases and desirable agronomic characteristics under conditions of severe natural infection in their respective areas of adaptation. Promising S5 lines were screened with the KSA (Table 1). In total, five pairs of closely-related lines were shown to be significantly different in aflatoxin resistance, while sharing as high as 97% genetic similarity [79]. Using these lines in proteomic comparisons to identify RAPs has advantages: (1) gel comparisons and analyses become easier; and (2) protein differences between resistant and susceptible lines as low as twofold can be identified with confidence. In addition, the likelihood of identifying proteins that are directly involved in host resistance is increased. In a preliminary proteomics comparison of constitutive protein differences between those African closely-related lines, a new category of resistance-associated proteins (putative regulatory proteins) was identified, including a serine/threonine protein kinase and a translation initiation factor 5A [29, 79]. The genes encoding these two resistance associated regulatory proteins are being cloned and their potential role in host resistance to A. flavusinfection and aflatoxin production will be further investigated. Conducting proteomic analyses using lines from this program not only enhances chances of identifying genes important to resistance, but may have immediate practical value. The IITA-SRRC collaboration has registered and released six inbred lines with aflatoxin-resistance in good agronomic backgrounds, which also demonstrate good levels of resistance to southern corn blight and southern corn rust [80]. Resistance field trials for these lines on U.S. soil is being conducted; the ability to use resistance in these lines commercially will depend on having identified excellent markers, since seed companies desire insurance against the transfer of undesirable traits into their elite genetic backgrounds. The fact that this resistance is coming from good genetic backgrounds is also a safeguard against the transfer of undesirable traits.

4.2. Recent breeding efforts

Recent breeding efforts towards the development of aflatoxin-resistant maize lines has resulted in a number of germplasm releases including the above-mentioned IITA-SRRC inbreds. In 2008, TZAR 101-106, derived from a combination of African and southern-adapted U.S. lines are being field-tested in different parts of the Southern U.S. (Figure 1) [80]. These have also exhibited resistance to lodging and common foliar diseases. GT-603 was released in 2011, after having been derived from GT-MAS:gk [81], while Mp-718 and Mp-719 were released as southern adapted resistant lines which are both shorter and earlier than previous Mp lines [82, 83]. These lines are also being tested as inbreds and in hybrid combinations in the southern U.S. [83].

Acknowledgments