Background/Question/Methods Ectomycorrhizal (ECM) fungi play important roles in ecosystem function.  Investigations of ECM physiology have been hampered by a paucity of methods for studying the external hyphal portion of the symbiosis. In order to investigate the effect of coarse woody debris on hyphal function, ECM hyphae-trapping bags were buried adjacent to roots of Picea engelmannii. We endeavoured to test community physiological functioning by plating fragmented hyphae extracted from these bags on BIOLOG^® phenotype microtitre plates. We hypothesized that ECM communities from plots with CWD retained would show a different pattern of substrate usage than those from plots with CWD removed.
Sand from colonized bags was shaken in water, sieved, and washed into a beaker where hyphal fragments floated. This extract was mixed with liquid media and added to wells of BIOLOG^® PM3 and PM4 plates. Visual observations of growth and colourimetric measurements were made at 24 hour intervals for 9 days, and again at 3 weeks. Nine different colony types could be distinguished; three of each were chosen for DNA extraction, PCR, and sequencing.
The objective of this study was to determine if BIOLOG^® plates are an appropriate tool for assessing the physiological capabilities of hyphae from an ectomycorrhizal fungal assemblage.

Results/Conclusions Visual assessment of mesh bag colonization and estimation of mycelial volume in extraction flasks correlated well with overall growth on plates. Aliquots of extracted, fragmented mycelium yielded an identifiable portion of hyphae with a frequency of 0.4 to 0.8. Visual observations did not show a relationship with absorbance measurements. Purple colour in colonies was attributed to the dye reduction reaction, but green, blue, pink, brown, yellow, white, and black colonies were also observed. Growth originating in individual wells continued to appear between 216 hr and 3 week observations. After 3 weeks all new growth appeared to be due to contamination from adjacent wells. Sequencing results revealed the presence of Thelephora terrestris, a known ectomycorrhizal fungus, but the allergen Cladosporium and the plant pathogen Phoma, among other non-mycorrhizal genera, were also detected.
We conclude that BIOLOG^® Phenotype MicroArray™ plates fail as a way to test the physiological functioning of ECM fungal hyphae because (i) mycelia cannot be adequately fragmented and evenly distributed among wells; (ii) the purple colour attributed to respiration was confounded by fungal pigments; (iii) growth observed visually was not detected by the plate reader; and (iv) the method is cultivation-dependant, which is not suitable for most ECM fungi.