Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some Fusarium and Aspergillus spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi Aspergillus flavus and Fusarium proliferatum, both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by A. flavus up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.
Keywords: aflatoxin B1; combination treatment; mycotoxigenic fungi; pomegranate peel extract; prochloraz; synergistic interaction.