Mutations of β-tubulin codon 198 or 200 indicate thiabendazole resistance among isolates of Penicillium digitatum collected from citrus in Taiwan

Abstract

Penicillium digitatum causes green mold on citrus, resulting in severe postharvest fruit decay and economic losses in many citrus-producing areas of the world. Forty isolates of P. digitatum were cultured from citrus groves, packinghouses, and local markets in Taiwan, and assessed quantitatively for their sensitivity to thiabendazole (TBZ) fungicide. Sensitivity assays using a 96-well microtiter plate revealed that, of 40 isolates examined, only one isolate collected from fruit produced in Taiwan and two isolates from Florida-imported citrus fruit were sensitive to TBZ. The concentration of TBZ causing a 50% growth reduction (EC(50)) was less than 1 μg/mL. The remaining 37 isolates could tolerate high concentrations of TBZ, with an EC(50) greater than 80 μg/mL. Overall, more than 97% of P. digitatum isolates tested in Taiwan were found to be resistant to TBZ. In vitro assays also revealed the ineffectiveness of TBZ for controlling a TBZ-resistant isolate on sweet oranges. A sequence analysis of β-tubulin genes revealed that all TBZ-resistant isolates displayed a single transversion point mutation, resulting in a change at either amino acid 198 (glutamic acid→glutamine) or 200 (phenylalanine→tyrosine). The repetitive use of a single fungicide over several decades has favored the selection and dominance of TBZ-resistant isolates of P. digitatum.