Effect of null mutations in the AbNIK1 gene on saprophytic and parasitic fitness of Alternaria brassicicola isolates highly resistant to dicarboximide fungicides

Alternaria brassicicola field isolates highly resistant to dicarboximide fungicides (DCF) and carrying non-sense codons or micro-deletions in the AbNIK1 gene encoding a group III histidine kinase were characterized at the protein level using specific antibodies. These isolates, as well as laboratory mutants expressing the same resistant phenotype, were shown to correspond to null mutants. The fitness of these isolates was compared to that of wild-type isolates by measuring i) mycelial growth, ii) sporulation and iii) conidial germination under standard conditions. None of these factors were affected in the resistant isolates. Mycelial growth was also measured under stress conditions. Unlike suboptimal incubation temperatures or exposure to the superoxide ion generator menadione, osmotic stress generated by high sorbitol concentrations in the culture medium significantly affected the growth of AbNIK1-null mutants compared to wild-types. The effects of these mutations on parasitic fitness were estimated under both controlled and field conditions after inoculation of radish with individual isolates, to measure their aggressiveness, or with mixed conidial suspensions, to measure their competitive ability against wild-type isolates. These tests on radish seedlings revealed that DCF-resistant isolates were still aggressive. Despite this, the data obtained in the competition experiments showed that the loss of the ability to synthesize AbNIK1p in A. brassicicola DCF-resistant mutants was linked to a severe decrease in their competitiveness.