Extraction of fungal fatty acids for gas chromatography analysis

Abstract

The standard method for the extraction of fatty acids from fungi recommended in association with commercially produced fatty acid analysis systems (MIDI Inc., Newark, Delaware) was compared with methods utilizing a Polytron homogenizer and vortexing with glass beads for disruption of fungal mycelia. The extraction efficiencies and variability were compared utilizing two isolates of Ceratocystis fagacearum, the causal agent of oak wilt. Using a Polytron homogenizer significantly reduced the fatty acid levels (61%) when compared to the standard method. Vortexing with glass beads (425–600 μm) increased the fatty acid levels (49%) and reduced variability (20%) when compared to the standard method. Different sizes of glass beads (150–212 μm, 425–600 μm, 710–1180 μm) did not yield significant differences in extraction efficiency with the fatty acid levels with 150–212 μm and 710–1180 μm being 71% and 59% compared to the use of 425–600 μm glass beads. The addition of 1 g glass beads/sample followed by vortexing for 2 min is easily adapted in any laboratory conducting fatty acid analysis without additional equipment. The reduction in the variability of samples would be extremely important when looking for minor changes in fatty acid percentages and composition. The standard recommended growth temperature (28 C) was decreased (22 C) for maximal growth of C. fagacearum. No significant differences were noted between the use of SDB and PDB except in the level of 14:0 fatty acid which was a minor component in the total levels of fatty acids (0.7 and 0.3%, respectively). However, growth of C. fagacearum on potato dextrose broth was 51.5% that obtained on Saubouraud dextrose broth. No significant differences were observed between the fatty acid profiles of C. fagacearum isolates from South Carolina, West Virginia, Ohio, Wisconsin and Texas utilizing the amended method.

Key Words:

• Ceratocystis fagacearum
• FAME analysis
• oak wilt