Antifungal proteins and other mechanisms in the control of sorghum stalk rot and grain mold.

Research on antifungal proteins and other mechanisms that provide the biochemical basis for host-plant resistance to stalk rot and grain molds is reviewed in this paper. Stalk rot caused by Fusarium species leads to substantial yield loss due to poor grain filling and/or lodging. A transgenic sorghum expressing high levels of chitinase exhibited less stalk rot development when exposed to conidia of F. thapsinum. Grain mold of sorghum is associated with warm humid environments and results from colonization by several fungi (F. thapsinum, Curvularia lunata, and Alternaria alternata) of the developing caryopsis. The roles of several biochemical mechanisms (tannins, phenolic compounds, red pericarp, proteins, hard endosperm, and antifungal proteins) on grain mold resistance are discussed. Resistance mechanisms related to these compounds appear to be additive, and pyramiding of genes is a feasible approach to limit grain deterioration. Several experimental approaches are proposed to extend current findings.

Activity of antifungal proteins against mold in sorghum caryopses in the field.

Sorghums were stressed with pathogenic fungi and sprinkling to determine relationships between changes in chitinase and sormatin in caryopses and grain mold resistance. Panicles of 10 cultivars differing in mold resistance and accumulation of antifungal proteins (AFPs) were inoculated at anthesis with Fusarium moniliforme and Curvularia lunata spores. Panicles were sampled at 30 and 50 days after anthesis, and caryopses were evaluated for chitinase and sormatin using western blots. Sprinkling panicles (to mimic rainfall) decreased sormatin and chitinase in most cultivars. Inoculation decreased AFPs in susceptible cultivars, but resistant cultivars maintained or increased AFPs in caryopses. Grain mold resistance corresponded to induction of AFP synthesis in response to sprinkling, fungal stress, and/or adverse field conditions. Sormatin and chitinase appear to be an active part of the defense mechanism of the caryopsis against grain mold.

Antifungal proteins and grain mold resistance in sorghum with nonpigmented testa.

Levels of four antifungal proteins (AFPs) were determined in mature caryopses (40-45 days after anthesis) of eight grain mold resistant (GMR) and eight susceptible (GMS) sorghum lines using the immunoblot technique. These 16 lines came from the same cross and were selected for high and low grain mold resistance. The 16 lines were grown in eight environments over three years. In the environments with grain mold incidence, levels of sormatin, chitinases, and ribosomal inactivating proteins (RIP) in the GMR group were higher than those in the GMS group. In a grain mold-free environment, the GMR group had higher RIP and lower beta-1,3-glucanase levels than the GMS group. Unlike the GMS group, chitinase, sormatin, and RIP levels in the GMR group were higher in the environments with grain mold than in the mold-free environment. AFPs correlated among themselves and with grain mold resistance. Grain mold infection pressure caused GMR lines to induce and/or retain more AFPs compared to GMS lines. The coexpression of these four AFPs may be a necessary prerequisite for resistance to grain mold in sorghums without a pigmented testa.

Starch hydrolysis by the ruminal microflora.

The effects of grain type and processing on ruminal starch digestion are well documented but poorly understood at the biochemical and molecular levels. Waxy grains have starches high in amylopectin and are more readily digested than nonwaxy grains. However, the composition of the endosperm cell matrix and the extent to which the starch granules are embedded within it also affect starch digestion rates. Continued work is needed to determine the influence of specific cell matrix proteins, protein-starch interactions and cell wall carbohydrates on starch availability. The microbial populations that metabolize starch are diverse, differing in their capacities to hydrolyze starch granules and soluble forms of starch. Surveys show that the amylases are under regulatory control in most of these organisms, but few studies have addressed the types of amylolytic enzymes produced, their regulation and the impact of other plant polymers on their synthesis. Research in these areas, coupled with the development and use of isogeneic or near-isogeneic grain cultivars with biochemically defined endosperm characteristics, will enhance our ability to identify mechanisms to manipulate ruminal starch digestion.

Microbiological Characteristics and Shelf Life of Corn Tortillas with and without Antimicrobial Agents.

Commercial corn tortillas were assayed for pH, moisture, aw, aerobic plate count (APC) and mold and yeast count (MYC). The shelf life of commercial tortillas ranged from 3 to 60 days, depending on the amount and type of preservative added. High initial APCs of commercial tortillas were associated with poor sanitary conditions at the plants, airborne contamination and improper storage practices. Shelf life of tortillas was greatly enhanced by refrigerated storage with or without the use of antimicrobials. For tortillas stored at 25°C, substantial increases in shelf life were attained by acidification (0.45% fumaric acid) plus addition of K-sorbate or Ca-propionate, but particularly by combination of these two antimicrobials. For tortillas stored at 4°C, shelf life of non-acidified samples was somewhat enhanced by addition of parabens and combinations of K-sorbate and Ca-propionate. Shelf life of acidified tortillas at 4°C was somewhat enhanced by addition of parabens and K-sorbate but increased substantially by addition of Ca-propionate and combinations of K-sorbate and Ca-propionate.

Preparation of maltosyl, beta-cyclodextrinyl, glucosaminyl, and glucosamineoctaosyl derivatives of beta-lactoglobulin.

beta-Lactoglobulin was modified to various degrees with maltose or beta-cyclodextrin using the cyclic carbonate method and with glucosamine or glucosamine-octaose using the carbodiimide method. Up to 65% of the amino or the carboxyl groups of b-LG were glycosylated using the two methods, respectively. Up to 32 maltose residues were coupled to b-LG using the cyclic carbonate method while up to 16.6 glucosamine residues were coupled to b-LG using the carbodiimide method. This resulted because more than one disaccharide was attached to each group of b-LG that was modified. Even so, the coupling efficiency of the active compound was higher for the carbodiimide derivative than for the cyclic carbonate derivative. The electrophoretic analyses of the glycosylated proteins indicated that a heterogeneous population of protein molecules was formed during modification. The electrophoretic mobility of maltosyl-beta-lactoglobulin derivatives was essentially unchanged while the mobility of glucosaminyl-beta-lactoglobulin derivatives decreased as the extent of modification increased. The glycosylated proteins were suitable for studying their structural and functional properties.

Physicochemical properties of maltosyl and glucosaminyl derivatives of beta-lactoglobulin.

Maltosyl and glucosaminyl derivatives of beta-lactoglobulin (b-LG) were analyzed for their physicochemical properties: reduced viscosity, ultraviolet difference spectra, intrinsic fluorescence, hydrophobicity and circular dichroism. The viscosity of these derivatives increased as the mass of the carbohydrates covalently linked to b-LG increased. The ultraviolet difference spectra and the intrinsic fluorescence of these proteins revealed that the microenvironments of aromatic amino acid residues of b-LG were increasingly exposed to the surface of the protein as the extent of modification increased; and the polarity of these residues also increased as modification increased. The hydrophobicities of M-b-LG derivatives decreased as the extent of modification increased while the hydrophobicities of G-b-LG derivatives were relatively unchanged. The circular dichroic analysis of these proteins indicated that the order secondary structures of the extensively modified derivatives of b-LG were partially unfolded. Thus, the carbohydrates covalently linked to b-LG altered many physiochemical properties. These physicochemical changes of b-LG apparently resulted from an alteration of forces stabilizing the structure of the protein.