Development of Spontaneous Hygromycin B Resistance in Monilinia fructicola and Its Impact on Growth Rate, Morphology, Susceptibility to Demethylation Inhibitor Fungicides, and Sporulation

Abstract

Agrobacterium tumefaciens-mediated transformation with plasmids carrying the hygromycin B resistance gene hph frequently is being used for inserting genes into fungal spores and mycelial cells and for conducting insertional mutagenesis to identify genes connected to a particular phenotype. In this article, we report that stable hygromycin B resistance can develop spontaneously in germinating conidia from Monilinia fructicola and that the mutants exhibit altered phenotypes. One spontaneously developing hygromycin B-resistant colony developed per 2.5 × 105 germinating conidia. Mutants grew significantly slower on potato dextrose agar, were 2.4- to 3.1-fold more sensitive to demethylation inhibitor fungicides, lacked melanization, and did not produce spores. The mode of action of hygromycin B resistance in the mutants seemed to be different from the hph transgene-mediated hygromycin B resistance based on different phenotypic characters. The ability of M. fructicola and possibly other fungi to spontaneously develop hygromycin B resistance associated with an altered phenotype may interfere with the selection of true transformants if hygromycin B is used as selective agent. This is particularly confounding if the hph gene is used as selectable marker in insertional mutagenesis experiments conducted for the identification of genes involved in melanization, sporulation, or fungicide resistance.