Use of Chitin:DNA ratio to assess growth form in fungal cells

Abstract

Dimorphism, the ability to switch between a ‘yeast-like’ and a hyphal growth form, is an important feature of certain fungi, including important plant and human pathogens. The switch to hyphal growth is often associated with virulence, pathogenicity, biofilm formation and stress resistance. Thus, the ability to accurately and efficiently measure fungal growth form is key to research into these fungi, especially for discovery of potential drug targets. To date, fungal growth form has been assessed microscopically, a process that is both labour intensive and costly. Here, we unite quantification of the chitin in fungal cell walls and the DNA in nuclei to produce a methodology that allows fungal cell shape to be estimated by calculation of the ratio between cell wall quantity and number of nuclei present in a sample of fungus or infected host tissue. Using the wheat pathogenZymoseptoria triticias a test case, with confirmation in the distantly relatedFusarium oxysporum, we demonstrate a close, linear relationship between the chitin:DNA ratio and the average polarity index (length/width) of fungal cells. We show the utility of the method for estimating growth form in infected wheat leaves, differentiating between the timing of germination in two differentZ. triticiisolates using this ratio. We also show that the method is robust to the occurence of thick-walled chlamydospores, which show a chitin:DNA ratio that is distinct from either ‘yeast-like’ blastospores or hyphae.