Genetic polymorphisms associated to SDHI fungicides resistance in selected Aspergillus flavus strains and relation with aflatoxin production

Aspergillus flavus is a common and ubiquitous fungal species able to colonize several agricultural commodities, in both pre- and post-harvest conditions. This species represents a very harmful plant pathogen for its ability to synthesize aflatoxin B1, responsible for human primary hepatocellular carcinoma and classified as a group I (human carcinogenic) by the International Agency for Research on Cancer. Several approaches have been proposed to control A. flavus development and related aflatoxin production in field and storage conditions. The Succinate Dehydrogenase Inhibitor (SDHI) fungicide boscalid has been shown to control A. flavus growth and aflatoxin contamination both in vitro and in field experiments. However, this compound is classified as medium-high risk fungicide for triggering fungal resistance and, indeed, resistant strains can occur on crops treated with boscalid. In this paper, we selected laboratory A. flavus strains resistant to boscalid grown on agar medium containing 50 mg/L of boscalid. In order to investigate the molecular mechanism responsible for the resistant phenotype, specific primer pairs were designed to amplify the whole SdhB, SdhC and SdhD genes. By amino acid sequence analysis, two point mutations, Tyrosine replacing Histidine at codon 249 of SdhB (H249Y) and Arginine replacing Glycine at codon 91 of SdhC (G91R), were identified. The effect of SDHI boscalid and isopyrazam on mycelial growth and conidial germination was evaluated. Both resistant genotypes showed high resistance (MIC and EC50 > 1000 mg/L) to boscalid. A positive cross-resistance was found between boscalid and isopyrazam. Specific sub-lethal doses of both fungicides (0.5 mg/L of boscalid and 0.01 mg/L of isopyrazam) interfered with the mechanisms associated to pigmentation of colonies. In particular, fungal colonies appeared depigmented lacking the typical A. flavus green colour shown on un-amended fungicide medium. A strict correlation between lack of pigmentation and increasing aflatoxin production was also observed.