Molecular characterization of Alternaria alternata field isolates highly resistant to the carboxamide fungicide boscalid

Thirty-eight isolates of Alternaria alternata isolated from pistachio orchards with a history of Pristine+ (pyraclostrobin + boscalid) applications displayed high levels of resistance to boscalid fungicide (mean EC sub(50) values > 100 kg/ml) as determined with mycelial growth tests. A cross-resistance study revealed that the same isolates were also resistant to carboxin, a known inhibitor of succinate dehydrogenase (Sdh). To determine the genetic basis of boscalid resistance in A. alternata, the entire iron sulphur gene (AaSdhB) was isolated from a fungicide-sensitive isolate. The deduced amino-acid sequence showed high similarity with iron sulphur proteins (Ip) from other organisms. Comparison of full sequences of AaSdhB from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, in ten resistant isolates, type I mutants) or arginine (codon CGC, five resistant isolates type II mutants) at position 277. This finding is consistent with previously published reports, highlighting the crucial role of the conserved histidine residue. Twenty-three other resistant isolates (type III mutants) had no mutation in the histidine codon. The point mutation detected in type I mutants was used to design a pair of allele-specific PCR primers to facilitate rapid detection. A PCR-RFLP assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. These molecular assays will greatly improve the detection and efficient monitoring of boscalid resistant A. alternata from pistachio.