**4. Current efforts to develop resistant lines**

#### **4.1. Closely-related lines**

Recently, the screening of progeny generated through a collaborative breeding program be‐ tween IITA-Nigeria (International Institute of Tropical Agriculture) and the Southern Re‐ gional 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 signifi‐ cantly in aflatoxin accumulation, and proteome analysis of these lines is being conducted [77, 78]. Investigating corn lines sharing close genetic backgrounds should enhance the iden‐ tification 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 (*via* KSA) [77, 78]. Five elite tropical inbred lines from IITA adapted to the Savanna and mid-altitude ecological zones of West and Cen‐ tral Africa were crossed with four U.S. resistant maize lines in Ibadan, Nigeria. The five Af‐ rican 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 adap‐ tation. Promising S5 lines were screened with the KSA (Table 1). In total, five pairs of close‐ ly-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 dif‐ ferences 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-associ‐ ated proteins (putative regulatory proteins) was identified, including a serine/threonine pro‐ tein 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 re‐ sistance to *A. flavus* infection and aflatoxin production will be further investigated. Conduct‐ ing proteomic analyses using lines from this program not only enhances chances of identifying genes important to resistance, but may have immediate practical value. The II‐ TA-SRRC collaboration has registered and released six inbred lines with aflatoxin-resistance in good agronomic backgrounds, which also demonstrate good levels of resistance to south‐ ern 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 undesir‐ able traits.

maize resistant and susceptible inbred silks suggests that these proteins may contribute to

To investigate gene expression in response to *A. flavus'* infection and to more thoroughly identify factors potentially involved in the regulation of RAP genes, a transcriptomic profile was conducted on maize kernels of two inbred lines that were genetically closely-related [73]. Similar work had previously been performed using Tex6 as the resistant line and B73 as the susceptible [74], however, in the study using closely-related lines, imbibed mature ker‐ nels were used (for the first time) and proved to be a quicker and easier approach than tradi‐ tional approaches. The involvement of certain stress-related and antifungal genes previously shown to be associated with constitutive resistance was demonstrated here; a kinase-bind‐ ing protein, Xa21 was highly up-regulated in the resistant line compared to the susceptible,

Recently, the screening of progeny generated through a collaborative breeding program be‐ tween IITA-Nigeria (International Institute of Tropical Agriculture) and the Southern Re‐ gional 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 signifi‐ cantly in aflatoxin accumulation, and proteome analysis of these lines is being conducted [77, 78]. Investigating corn lines sharing close genetic backgrounds should enhance the iden‐ tification 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 (*via* KSA) [77, 78]. Five elite tropical inbred lines from IITA adapted to the Savanna and mid-altitude ecological zones of West and Cen‐ tral Africa were crossed with four U.S. resistant maize lines in Ibadan, Nigeria. The five Af‐ rican 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 adap‐ tation. Promising S5 lines were screened with the KSA (Table 1). In total, five pairs of close‐ ly-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 dif‐ ferences 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

resistance.

*3.5.4. Transcriptomic analyses*

12 Aflatoxins - Recent Advances and Future Prospects

**4.1. Closely-related lines**

both constitutively and in the inducible state.

**4. Current efforts to develop resistant lines**


**Table 1.** KSA screening of IITA-SRRC maize breeding materials which identified 2 closely related lines (87.5% genetic similarity), #22 and #25, from parental cross (GT-MASgk x Ku1414SR) x GT-MAS:gk; these contrast significantly in aflatoxin accumulation. Values followed by the same letter are not significantly different by the least significant difference test (P = 0.05).

#### **4.2. Recent breeding efforts**

Recent breeding efforts towards the development of aflatoxin-resistant maize lines has re‐ sulted in a number of germplasm releases including the above-mentioned IITA-SRRC in‐ breds. 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].

These investigations include QTL analyses to locate regions of chromosomes associated with the resistant phenotype, and the discovery of kernel resistance-related traits. We now know that there are two levels of resistance in kernels, pericarp and subpericarp. Also, there is a two-phased kernel resistance response to fungal attack: constitutive at the time of fungal attack and that which is induced by the attack. Thus far, it's been demon‐ strated that natural resistance mechanisms discovered are antifungal in nature as op‐

One of the most important discoveries, thus far, has been that of resistance-associated pro‐ teins or RAPs. Due to the significance of the constitutive response, constitutive RAPs were investigated first, although induced proteins are being studied as well. Investigations of oth‐ er tissues such as rachis and silks begin to provide a more complete picture of the maize re‐ sistance response to aflatoxigenic fungi. RAP characterization studies provide greater evidence that these proteins are important to resistance, although clearly, more investiga‐ tions are needed. Looking at data collectively that's been obtained from different types of studies may enhance the identification of markers for breeding. A good example of this may be the supporting evidence provided by QTL data to proteomic and RAP characterization data suggesting the involvement of 14 kDa TI, water stress inducible protein, zeamatin, heat shock, cold-regulated, glyoxalase I, cupin-domain and PR10 proteins in aflatoxin-resistance. It will be interesting to determine if this marker discovery approach can lead to the success‐ ful transfer of a multigene-based and quantitative phenomenon such as aflatoxin-resistance

Research discussed in this review received support from the USAID Linkage Program-IITA, Nigeria, and the USDA-ARS Office of International Research Programs (OIRP) -USAID Col‐

, Thomas E. Cleveland1

2 Department of Plant Pathology and Crop Physiology, Louisiana State University Agricul‐

, Zhi-Yuan Chen2

Development of Maize Host Resistance to Aflatoxigenic Fungi

http://dx.doi.org/10.5772/54654

15

and

posed to inhibiting the aflatoxin biosynthetic pathway.

to commercially-useful genetic backgrounds.

**Acknowledgements**

laborative Support Program.

Robert L. Brown1\*, Deepak Bhatnagar1

tural Center, Baton Rouge, LA, USA

\*Address all correspondence to: Robert.brown@ars.usda.gov

3 International Institute of Tropical Agriculture, Ibadan, Nigeria

1 USDA-ARS, Southern Regional Research Center, New Orleans, LA, USA

**Author details**

Abebe Menkir3

**Figure 1.** Inoculation of maize ears with *Aspergillus flavus* spores using a 'side needle' wound technique for field eval‐ uations of TZAR lines developed through IITA-SRRC program.

## **5. Conclusion**

The host resistance approach to eliminating aflatoxin contamination of maize has been advanced forward by the identification/development of maize lines with resistance to aflatoxin accumulation. However, to fully exploit the resistance discovered in these lines, markers must be identified to transfer resistance to commercially useful backgrounds. Towards this goal numerous investigations have been undertaken to discover the factors that contribute to resistance, laying the basis for exploiting these discoveries as well. These investigations include QTL analyses to locate regions of chromosomes associated with the resistant phenotype, and the discovery of kernel resistance-related traits. We now know that there are two levels of resistance in kernels, pericarp and subpericarp. Also, there is a two-phased kernel resistance response to fungal attack: constitutive at the time of fungal attack and that which is induced by the attack. Thus far, it's been demon‐ strated that natural resistance mechanisms discovered are antifungal in nature as op‐ posed to inhibiting the aflatoxin biosynthetic pathway.

One of the most important discoveries, thus far, has been that of resistance-associated pro‐ teins or RAPs. Due to the significance of the constitutive response, constitutive RAPs were investigated first, although induced proteins are being studied as well. Investigations of oth‐ er tissues such as rachis and silks begin to provide a more complete picture of the maize re‐ sistance response to aflatoxigenic fungi. RAP characterization studies provide greater evidence that these proteins are important to resistance, although clearly, more investiga‐ tions are needed. Looking at data collectively that's been obtained from different types of studies may enhance the identification of markers for breeding. A good example of this may be the supporting evidence provided by QTL data to proteomic and RAP characterization data suggesting the involvement of 14 kDa TI, water stress inducible protein, zeamatin, heat shock, cold-regulated, glyoxalase I, cupin-domain and PR10 proteins in aflatoxin-resistance. It will be interesting to determine if this marker discovery approach can lead to the success‐ ful transfer of a multigene-based and quantitative phenomenon such as aflatoxin-resistance to commercially-useful genetic backgrounds.
