In vitro activity of imazalil against Penicillium expansum: comparison of the CLSI M38-A broth microdilution method with traditional techniques.

Penicillium expansum is one of the most important pathogens that cause blue mold in stored apples and is regarded as the major producer of the mycotoxin patulin. Imazalil is one of the fungicides used in Spain to control postharvest blue mold, but development of fungal resistance has been reported in P. digitatum and P. italicum. The most common used methods to detect antifungal susceptibility of fungal crop pathogens in vitro, are direct-plating isolates in media amended with various concentrations of fungicide and determining inhibition of growth and/or spore germination. These techniques are time- and labor-intensive and are not suitable if a large number of isolates has to be evaluated. On the other hand, the broth microdilution method M38-A is the reference method developed by the Clinical and Laboratory Standards Institute (CLSI) for antifungal susceptibility testing in some clinical fungi, but Penicillium spp. are not included. Due to the lack of a standard method, the aim of this work is to evaluate the suitability of an adaptation of the CLSI M38-A method to monitor P. expansum susceptibility to imazalil in comparison with other techniques. A total of 128 P. expansum strains have been studied (118 isolates from apples and pears, 5 from grapes and 5 reference strains). Imazalil has shown to be highly active in vitro against all the P. expansum isolates tested, as all the evaluated parameters were in the range reported for imazalil sensitive Penicillium spp. The mean minimum inhibitory concentration determined by broth microdilution method and by agar dilution method (48-72 h readings) was 0.0625 microg/ml and 0.11-0.12 microg/ml respectively. The mean concentration that inhibited the size of colonies (48-72 h) and spore germination by 50% was 0.05-0.06 and 0.04 microg/ml respectively. Our results highlight that the broth microdilution method CLSI M38-A is a good alternative to be used in screening the in vitro activity of imazalil against a large number of isolates.