Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with Boscalid resistance in Alternaria alternata from California pistachio

Thirty-eight isolates of Alternaria alternata from pistachio orchards with a history of Pristine (pyraclostrobin + boscalid) applications and displaying high levels of resistance to boscalid fungicide (mean EC(50) values >500 microg/ml) were identified following 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 AaSdhB full sequences from sensitive and resistant isolates revealed that a highly conserved histidine residue (codon CAC in sensitive isolates) was converted to either tyrosine (codon TAC, type I mutants) or arginine (codon CGC, type II mutants) at position 277. In other fungal species this residue is involved in carboxamide resistance. In this study, 10 and 5 mutants were of type I and type II respectively, while 23 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 polymerase chain reaction (PCR) primers to facilitate rapid detection. A PCR-restriction fragment length polymorphism (RFLP) assay in which amplified gene fragments were digested with AciI was successfully employed for the diagnosis of type II mutants. The relevance of these modifications in A. alternata AaSdhB sequence in conferring boscalid resistance is discussed.