Effects of Helminthosporium carbonum Toxin on Nitrate Uptake and Reduction by Corn Tissues

Abstract

Susceptible corn tissues exposed to the host-specific toxin of Helminthosporiuna carbonum race 1 reduced more nitrate to nitrite than did control tissues, as measured by an in vivo method. There were no differences in nitrate reductase activities extracted from treated and control tissues and assayed by an in vitro method. Toxin-treated susceptible roots (15,16). This "host-specific toxin" is required by the fungus for pathogenicity and for initial colonization of susceptible plant tissues (1, 16). Among its described physiological effects on susceptible tissues, HC toxin increases electrolyte leakage, respiration, dark fixation of CO2, and the incorporation of uridine and amino acids. Most of these effects are not evident unless tissues are exposed to toxin for 8 to 12 hr (9, 17); they could be secondary results of an initial toxic effect.The original aim of this work was to determine whether or not toxin has a direct effect on protein synthesis. An inducible enzyme, nitrate reductase, was chosen to study this question. Zielke and Filner (23) MATERIALS AND METHODSPlants. H. carbonum-resistant (Prl X K61) or susceptible (Pr X K61) corn hybrids were used in all experiments. Roots were produced by two methods. (a) Seeds (250) were incubated 8 to 12 hr in water, then were washed and placed embryo side down on a cheesecloth surface in a beaker above 4 liters of 0.2 mm CaCl2. A second layer of cheesecloth was placed over the seeds, the beaker was covered with plastic film, and the solution was aerated. Primary roots were used 4 days later when they were 8 to 12 cm long. (b) Seeds (150) were placed embryo side down on an agar medium (100 ml of 0.9% agar in a 15-cm Petri dish) containing White's solution (20) minus nitrate. After 60 to 84 hr of incubation, the germinated seeds had 4-to 8-cm roots which were used as experimental tissues.Toxin Preparation. Toxin was isolated from H. carbonum race 1 by previously described procedures (15, 21). Culture filtrates were concentrated, deproteinized by methanol precipitation, and extracted with chloroform. The chloroform solution was taken to dryness in vacuo, and the residue was dissolved in ethanol, mixed with 20 volumes of diethyl ether, and stored at 5 C for 24 hr. The precipitate was discarded, and the solution was taken to dryness. A second ethanol-ether extract was taken to dryness, dissolved in water, and passed through a BioGel P-2 column. Fractions with host-specific toxicity were dried, and the residues were stored under nitrogen over CaCl2 at -20 C. This preparation caused 50% inhibition of susceptible corn roots at 0.2 ug/ml, and of resistant roots at 20 ug/ml, when assayed by the seedling root growth method (16). Thus, the toxin preparation was active at 0.295 ,uM (mol wt = 679), which is as active as the best crystalline preparations (14, 15). Reaction mixtures were assayed before and after each experiment; there were no losses in host-specific toxicity.Nitrate Reductase Assays. Nitrate reductase activity was determined in vivo by the method of Ferr...

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

“…Aseptic conditions were not maintained for the experiments reported here. However, stimulation of nitrate reductase activity by toxin occurred under aseptic conditions (31 …”

“…Methods of growing plants, preparing toxin, and assaying for nitrate and nitrite in tissues are described elsewhere (31 In some experiments, the final rinse was followed by a brief desorption period at 24 C; the usual desorption at 0 to 5 C (4) was not used because the purpose was to determine effects on efflux under physiological conditions.…”

“…Nitrate uptake usually was measured along with other test solutes, because NO3-uptake was known to be affected by toxin (31). Isotopes were used to measure absorption of all solutes except nitrate and nitrite.…”

“…Increased respiration is secondary to initial lesions occurring elsewhere; this is evident from data showing no effects of toxin on mitochondria or chloroplasts isolated from toxin-sensitive cells (28). Likewise, the apparent effect on nitrate reductase is secondary, because the increase in activity was traced to toxin-stimulated uptake of substrate, not to increased synthesis of the enzyme (31). Some of the other stimulatory effects of HC toxin may be secondary to initial toxic lesions.…”

“…Nitrite, C1-, SO,-, Na+, K+, Ca2+, phosphate ion (H2PO-and HP042-), 3-o-methylglucose, leucine, and glutamic acid were selected for further study. HC toxin is selective for susceptible corn in its effects on NO3-uptake, and the increase in uptake is the earliest effect of toxin reported to date (31). An abstract describing some of the work has been published (30).…”