Revealing TCA Cycle Flow Dynamics in Fungicide-Adapted <i>Botrytis cinerea</i> through Anionic Chromatographic Simultaneous Separation

The exploration of new strategies to monitor <i>Botrytis cinerea</i> populations is critical for disease management. This study achieved efficient and simultaneous separation of all TCA cycle intermediates in mycelia treated with pyraclostrobin (PYR), tebuconazole (TEB), and carbendazim (CAR) using anionic chromatography. Resolutions of nine organic acids and six inorganic anions, including all TCA cycle intermediates, exceeded 1.5. The fungicides inhibited mycelial growth concentration-dependently, but prolonged exposure reduced the level of inhibition. TEB decreased fumarate and malate levels at 48 h, increased citrate, isocitrate, and oxalate levels and reduced α-ketoglutarate levels at 96 h. PYR showed biphasic effects, elevating fumarate, malate, and succinate at both time points but reducing citrate and succinate at 96 h. CAR attenuated TCA cycle flow, with isocitrate and α-ketoglutarate declining continuously. Moreover, TEB promoted oxalate accumulation, PYR had no effect, and CAR suppressed it. These findings enable precise TCA cycle assessment for fungicide adaption in <i>B. cinerea</i>, aiding Botrytis management.