DETECTION AND QUANTIFICATION OF THE FUNGAL PHYTOTOXIN TENUAZONIC ACID PRODUCED BY PYRICULARIA-ORYZAE

The fungal phytotoxin tenuazonic acid (3-acetyl 5-sec-butyl pyrrolidine-2,4-dione) (TA) was detected by HPLC, GC-EIMS and 1H NMR in the culture filtrates of four isolates of the rice pathogen Pyricularia oryzae from different geographic origins. The valine analogue of TA (3-acetyl 5-iso-propyl pyrrolidine-2,4-dione) was also detected in trace amounts by HPLC and GC-EIMS. We developed a simple calorimetric assay of Fe-TA complexes to monitor TA production in liquid cultures as well as a sensitive TLC method (terbium fluorescence) to monitor TA production in solid media. In liquid cultures TA was produced during the exponential growth phase and stayed at a constant level during the stationary growth phase. We studied the effect of different culture media and conditions on TA production. High temperatures (30°) greatly enhanced TA production compared to 20-23°. Glucose, sucrose and trehalose allowed a high TA production but not fructose, whatever the isolate tested. There were some differences between isolates with regard to their ability to produce TA on a particular nitrogen source. Two isolates (Md4, Cd41) produced high TA amounts on all nitrogen sources tested and two others (P2B, Cd2) only on ammonium tartrate. Thus, we used a medium composed of sucrose and ammonium tartrate to compare the production of TA by 15 P. oryzae isolates from different geographic origins. TA was detected in the culture filtrates of all these isolates showing that it is a common metabolite of P. oryzae, but TA production differed greatly from one isolate to another (0.05-0.8 mM). © 1990.