Introduction

Since the yeast Saccharomyces cerevisiae was sequenced in 1996 (1), the number of available fungal genome sequences has increased by one order of magnitude. Over 100 complete fungal genomes have been publicly released or are currently being sequenced, thus representing the widest sampling of genomes from any eukaryotic kingdom (2). This fungal genomics boom has greatly expanded our view of the genetic and physiological diversity of these organisms. However, the function of many proteins encoded by the genome has not yet been experimentally determined. Targeted inactivation of the corresponding genes and subsequent phenotypic characterization of resulting mutants are frequently the first important step toward determining the cellular role of proteins. Deletion strain collections of all genes in the genome are now available for model organisms such as S. cerevisiae (EUROSCARF, Frankfurt, Germany) and the filamentous ascomycete Neurospora crassa (Fungal Genetics Stock Center, Kansas City, MO, USA). These initiatives have paved the way to the development of phenomics: large-scale quantitative phenotypic analysis of genotypes on a genome-wide scale (3). Procedures based on microscale liquid cultivation and optical recording of growth in automated microplate systems have been developed for high-throughput quantitative pheno-typic profiling of yeasts (4,5). Concerning filamentous fungi, assays based on the analysis of colony expansion rates on solid media or on microscopic measurement of the length of hyphae can generate quantitative phenotypic data. However, such screenings are cumbersome and time-consuming, and therefore not suitable for large-scale phenotypic analysis. Moreover, the specific effects of a particular mutation and/or environment on different fungal growth features (such as the lag phase, rate of growth, and growth yield) cannot be deduced from data obtained on solid media. Despite the fact that they are theoretically only applicable for unicellular organisms (6), spectrophotometric assays have also been developed to monitor the growth of filamentous fungi in microbroth systems over time (7,8). However, the accuracy of spectrophotometric readings may be hampered by the presence of clumps of hyphae and the nonhomogeneous growth of filamentous fungi in liquid media. Moreover, a major drawback of spectro-photometric methods, irrespective of the kind of cells under analysis, is that extinction in cell suspensions is proportional to the cell density only at rather low values. Usually, it is considered that this lack of proportionality is significant at OD values >0.5 and that only corrected photometric readings should be used for values beyond the range of proportionality (7,9,10). In contrast to spectrophotometry, which measures the transmission of light, nephelometry (another light-based technique for measuring medium opacity) uses light scattering. This parameter is directly proportional to the cell density and is suitable for a broad range of cell densities. Since its first description for counting yeast suspensions (11), nephelometry has been routinely used to study the growth of S. cerevisiae, and a microbroth kinetic system was recently developed to monitor the growth of some Candida yeast species (12,13). As it has also been successfully used for quantifying particles in nonhomogeneous suspensions, we assumed that nephelometry might be suitable for plotting accurate growth curves for filamentous fungi. In this study, we took advantage of the commercial availability of a laser-based microplate nephelometer to develop a method based on microscale liquid cultivation for automated recording of fungal growth. This procedure was evaluated with phytopathogenic (Alternaria brassicicola) or human pathogenic (Aspergillus fumigatus and A. terreus) filamentous fungi and the yeast Candida glabrata.

Materials and methods

Fungal strains and cultivation media

The A. brassicicola wild-type strain Abra43 used in this study was isolated from Raphanus sativus seeds. Two disruptants—Abhog1Δ1 and Abnik1Δ3—were obtained from the wild-type strain Abra43 as previously described (14). Strains of A. terreus and C. glabrata were recovered from clinical samples (15) and deposited at the Institute of Hygiene and Epidemiology Mycology section (IHEM; Brussels, Belgium) culture collection. The reference strain 18963 of A. fumigatus, used for sequencing the genome of this species, was obtained from IHEM. A. brassicicola and Aspergillus strains were cultivated at 24°C on potato dextrose (PD) agar medium (Cat. no. 213200; Becton Dickinson, Franklin Lakes, NJ, USA). C. glabrata strains were grown at 30°C on yeast peptone dextrose (YPD) (Cat. no. Y1500, Sigma-Aldrich, St. Louis, MO, USA) agar.