Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

• Published in: Fungal biology 2013-11, Vol.117 (11-12), p.731-743
• Main Authors: Timsina, Brinda A., Sorensen, John L., Weihrauch, Dirk, Piercey-Normore, Michele D.
• Format: Article
• Language: English
• Subjects: agar; Ascomycota - enzymology; Ascomycota - growth & development; Ascomycota - physiology; Chromatography, Thin Layer; culture media; Culture Media - chemistry; DNA, Fungal - chemistry; DNA, Fungal - genetics; environmental factors; gene expression; Gene Expression Profiling; Gene Expression Regulation, Fungal; genes; glucose; Growth medium pH; high performance liquid chromatography; Hydrogen-Ion Concentration; malt; Molecular Sequence Data; Mycobiont; Photomicrography; PKS gene expression; PKS paralog; Polyketide; Polyketide Synthases - biosynthesis; Polyketide Synthases - genetics; Polyketide Synthases - metabolism; polyketides; quantitative polymerase chain reaction; Ramalina; Ramalina dilacerata; Secondary Metabolism; secondary metabolites; Sequence Analysis, DNA; spores; Symbiosis; texture; thin layer chromatography; yeasts
• ISSN: 1878-6146; 1878-6162
• DOI: 10.1016/j.funbio.2013.09.003

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions. •Growth of Ramalina dilacerata in culture followed a standard growth pattern.•Colony diameter was largest when grown on media containing yeast extracts.•Colony diameter was inversely related to number of secondary metabolites.•Expression of wA and MSAS PKS genes was induced by high pH and non-nitrogen media.•Number of secondary metabolites correlated with PKS gene expression.