Wheat grain discoloration, a worldwide disease that lowers grain quality and decreases grain yield, does not have a single etiology. It has been proposed that it is a consequence of an abiotic mechanism, a response to environmental conditions or enzymatic activity. It has also been suggest that it is a biotic mechanism, a fungal infection principally by Alternaria spp. and Bipolaris sorokiniana. The present work was carried out to analyze the possible etiology of this disease in nine durum wheat genotypes from two localities of southern Buenos Aires province (Argentina) on two sowing dates. Incidence (percentage of grain discoloration) was recorded and mycobiota associated with this pathology was registered following ISTA rules. Peroxidase activity in an extract obtained from grains belonging to genotypes of the locality that showed the highest incidence was measured.
The incidence among genotypes, localities and sowing dates varied, although the genotypes with the higher and lower values of incidence were the same for all the variables tested. The fungus Alternaria spp. was isolated the most frequently followed by Fusarium spp., while Bipolaris sorokiniana was found the least frequently. Peroxidase activity showed that all the treatments had similar levels of enzymatic activity, but there was no clear differentiation between controls either between genotypes with the lowest or the highest incidence values. This suggests that peroxidase activity did not have a clear relationship with grain discoloration. In this research, it is presumed that fungal infection is the main cause of this disease.
Plant derived α-amylase inhibitors are proteinaceous molecules that regulate the enzyme activity in plants and also protect plants from insect attack. In the current study, 28 accessions of 19 plant species were screened for their α-amylase inhibitory activity. The durum wheat varieties, Beni Suef-1 and Beni Suef-5, showed strong α-amylase inhibitory activity and were subjected to further purification studies using ammonium sulfate fractionation and DEAE-Sephadex G-25 column. The isolated inhibitors were found to be stable at temperatures below 80°C with maximum activity obtained at 40−50°C. Also, they were stable in a wide pH range (2−12). The ion exchange products of purified α-amylase inhibitors from Beni Suef-1 and Beni Suef-5 varieties showed a molecular weight of 16 and 24 kDa, respectively. The purified α-amylase inhibitors were tested against Tribolium castaneum and Callosobruchus maculatus both in vitro and in vivo. There was linear inhibition of α-amylase activity with increasing inhibitor concentration until saturation was reached. Beni Suef-5 α-amylase inhibitor was more potent against α-amylase with lower IC50 values than Beni Suef-1 α-amylase inhibitor except in the case of T. castaneum larva. Kinetics analysis revealed that Beni Suef-1 and Beni Suef-5 α-amylase inhibitors are non-competitive types of inhibitors with high affinity toward α-amylase of T. castaneum and C. maculatus. Results of the in vivo studies demonstrated that α-amylase inhibitors isolated from durum wheat, Beni Suef-1 and Beni Suef-5 varieties, were very effective in inhibiting the development of T. castaneum and C. maculatus and could be used for future studies in developing insect resistant transgenic plants approaching α-amylase inhibitor genes.