Ultrastructural effects of AAL-toxin T A from the fungus Alternaria alternata on black nightshade ( Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity

Abstract

Ultrastructural effects of AAL-toxin T A from Alternaria alternata on black nightshade ( Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity. In black nightshade ( Solanum nigrum L.) leaf discs floating in solutions of AAL-toxin T A (0.01–200 μM) under continuous light at 25°C, electrolyte leakage, chlorophyll loss, autolysis, and photobleaching were observed within 24 h. Electrolyte leakage, measured by the conductivity increase in the culture medium, began after 12 h with 200 μM AAL-toxin T A, but was observed after 24 h with 0.01 to 50 μM AAL-toxin T A, when it ranged from 25% to 63% of total releasable electrolytes, respectively. After 48 h incubation, leakage ranged from 39% to 79% of total for 0.01 to 200 μM AAL-toxin T A, respectively, while chlorophyll loss ranged from 5% to 32% of total, respectively. Ultrastructural examination of black nightshade leaf discs floating in 10 μM AAL-toxin T A under continuous light at 25°C revealed cytological damage beginning at 30 h, consistent with the time electrolyte leakage and chlorophyll reduction were observed. After 30 h incubation chloroplast starch grains were enlarged in control leaf discs, but not in AAL-toxin T A-treated discs, and the thylakoids of treated tissue contained structural abnormalities. After 36–48 h incubation with 10 μM AAL-toxin T A, all tissues were destroyed with only cell walls, starch grains, and thylakoid fragments remaining. Toxicity was light-dependent, because leaf discs incubated with AAL-toxin T A in darkness for up to 72 h showed little phytotoxic damage. Within 6 h of exposure to ≥0.5 μM toxin, phytosphingosine and sphinganine in black nightshade leaf discs increased markedly, and continued to increase up to 24 h exposure. Thus, physiological and ultrastructural changes occurred in parallel with disruption of sphingolipid synthesis, consistent with the hypothesis that AAL-toxin T A causes phytotoxicity by interrupting sphingolipid biosynthesis, thereby damaging cellular membranes.