Peanut (Arachis hypogaea L.) is one of the most susceptible host crops to Aspergillus flavus (Link) invasion and subsequent aflatoxin production before and after harvest. The extent of aflatoxin contamination varies with geographic location, agricultural and agronomic practices, storage and processing period and conditions (Stoloff, 1985). In China, the severity of aflatoxin contamination gradually decreases as latitude increases. Contamination is more serious in southern peanut production areas than in northern areas (Xiao, 1989). The Chinese government pays great attention to the aflatoxin contamination problem and the research projects in this field receive high priority. Adopting new cultural, curing and storage practices can minimize aflatoxin contamination. However, these practices may not be suited to small-scale farming in developing countries, especially in tropical areas. Chemical control and removal of toxins have not yet been completely successful. An effective solution to the problem may be the use of peanut varieties that are resistant to infection by the aflatoxin-producing fungi, or varieties that suppress aflatoxin production if colonized by the fungus (Anderson et al., 1995). Advances in genomic tools and information through the international Peanut Genomics Initiative should accelerate the development of agronomically acceptable peanut cultivars with reduced aflatoxin contamination.
In this review, recent advances are highlighted concerning host resistance against aflatoxin contamination and genomics research activities conducted at the Crops Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China.
Host Resistance to Aflatoxin Contamination
Identification of resistant germplasm and development of resistant cultivars
Through screening of over 2,000 germplasm accessions of peanuts, 20 genotypes were identified as highly resistant to A. flavus invasion in seeds (Zhou et al., 2002). These resistant genotypes include local landraces and some genotypes introduced from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). PI 337494F, J-11, Zhanqiu 48, UF71513 and Meixianhonhyi have also been used in the aflatoxin resistance breeding program. Two improved cultivars Yueyou 9 and Yueyou 20 with high resistance to A. flavus invasion and acceptable agronomic traits were released in 2004 by the National Peanut Varieties Approval Committee and Guangdong Crops Varieties Approval, respectively. Some promising advanced breeding lines with resistance to preharvest aflatoxin contamination (50 to 66 reduction in aflatoxin contamination compared to susceptible accessions in the fields) also have been developed. Zhou et al. (1999, 2002) studied the inheritance of resistance to seed infection by A. flavus and found the resistant characters were controlled by a pair of major genes with additive value 0.38 and a pair of minor genes with additive value 0.12. The additive gene actions were important for resistant, and the percent of recombination between parent genotypes was estimated at 43.22. Heritability was estimated at 58.
Identification of resistance traits
A better understanding of mechanisms of resistance to fungal infection and aflatoxin production should accelerate the development of resistant cultivars. Mechanisms of peanut resistance toinvasion and aflatoxin production have been studied at the Guangdong Academy since 1999 and several significant factors have been observed.
Structure of seed coat
Liang et al. (2003b) studied the role of wax and cutin layers in peanut seed coats in resistance to invasion and colonization by. Results showed that wax contents of resistant genotypes were significantly higher than susceptible cultivars. The resistant kernels had a thicker and coarser waxy deposit on seed coat surfaces than susceptible genotypes as observed by scanning electron microscope. Removal of wax with chloroform or removing of cutin with KOH and cutinase can increase the susceptibility of peanut seeds. The bioassays of wax in vitro showed that there were no significant test-by-treatment interactions. These results indicated that the wax and cutin layers of peanut seed coat might only be a physical barrier to A. flavus invasion and colonization.
Active oxygen and membrane lipid peroxidation
Liang et al (2002) observed differences in the changes of active oxygen species including superoxide anion (O2), hydrogen peroxide (H2O2), and hydroxyl radial (OH), lipoxygenase (LOX) activity and membrane lipid peroxidation levels between resistant and susceptible seeds after inoculation with A. flavus. In resistant genotypes, the levels of malondialdehyde and the degree of membrane lipid peroxidation were significantly increased (78) at 23 d after inoculation. Moreover, the generation rates of O2, H2O2, and LOX were also increased markedly at an early stage after inoculation. O2 and H2O2 were increased rapidly and quickly reached a maximum level. However, no significant increases in the activities of superoxide dismutase (SOD) or catalase (CAT) were observed, which implies that there was not enough SOD and CAT to scavenge active oxygen. The accumulation of active oxygen and the increased activity of LOX can cause changes in membrane lipid peroxidation, cell wall strength, synthesis of phytoalexin and hypersensitive cell death (Dixion and Harrison, 1994; Baker and Orlandi, 1995). In contrast, the MDA(Malondialdehyde)level was increased at 56 d, and the production rates of O2, content of H2O2, and activity of LOX in susceptible genotypes was increased much later than in the resistant genotypes (Liang et al., 2001, 2002).
Phytoalexins are antibiotic secondary metabolites produced by plants in response to injury and invasion by some pathogens and appear to be involved in disease resistance (Keen, 1986; Sobolev et al., 2007). Resveratrol is one of the phytoalexin compounds found in peanut seeds (Sanders et al., 2000). Liang et al. (2006b) compared the synthesis capacity for resveratrol between the resistant and susceptible seeds after inoculation with A. flavus. The results showed that the accumulation of resveratrol in resistant genotypes was increased 3 at 3 d after inoculation, but susceptible genotypes did not reach the same levels until 4 d after inoculation.
Protein profiles of 15 peanut genotypes revealed that the trypsin inhibitor and lipid transfer protein were present at relatively high concentration in resistant genotypes (Liang et al., 2003a, 2003c, 2004b). Both proteins exhibited strong bioactivity against the growth of A. flavus. In another investigation, the difference of total seed protein in resistant and susceptible peanut genotypes was investigated by proteomic approaches (Liang et al., 2004a). The major qualitative difference between resistant genotypes and susceptible genotypes is that resistant genotypes contained three unique proteins P1 (22.5 kD, pI 4.1), P2 (22.5 kD, pI 8.2) and P3 (23.8 kD, pI 5.9), while susceptible genotypes contained one unique protein P4 (23.5 kD, pI 7.0). Another protein P5 (22.5 kD, pI 7.3) also was found in concentration tenfold more in resistant vs. susceptible genotypes. The peptide sequences of spots P-1 and P-3 are identical to legumin A precursor from Vicia narbonensis L. The peptide sequences of spots P-2 and P-4 are the same and identical to glycinin from A. hypogaea. The polymorphic protein peptides distinguished by 2-D PAGE may be used as markers for identification of resistant peanut lines. In addition to constitutive seed antifungal proteins, there were significant differences of two pathogenesis-related proteins (chitinase and -1-3-glucanase) between the resistant and susceptible genotypes after inoculation with A. flavus (Liang et al., 2003a, 2005). The activities of endo-chitinase and -1-3-glucanase increased earlier in resistant than in susceptible genotypes after invasion by A. flavus, while more isoform bands of -1-3-glucanase were observed in resistant than in susceptible genotypes. The purified chitinase can significantly inhibit spore germination and hypha growth of A. flavus in vitro, while thin-layer chromatography analysis of the hydrolytic product from -1-3-glucanase and hypha of A. flavus revealed the presence of enzymatic hydrolytic oligomer products (Liang et al., 2005).