Adventures with BIOLOG®: Phenotype MicroArray™ plates as a

measurement tool for the functional diversity of ectomycorrhizal fungal hyphae Jennifer KM Walker, Valerie Ward, and Melanie D Jones

University of British Columbia Okanagan, Biology and Physical Geography Unit Background 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 (CWD) 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.

Experimental Methods 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. Seven different colony types could be distinguished; three of each were chosen for DNA extraction, PCR, and sequencing.

The objective of the study was to determine if there are differences in ECM fungal physiological activity between CWD+ and CWD- plots.

The objective of this experiment was to determine if BIOLOG®

plates are an appropriate tool for assessing the physiological capabilities of hyphae from an ectomycorrhizal fungal assemblage.

!!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, and white 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 common contaminant Cladosporium and the plant pathogen Phoma, among other non-mycorrhizal genera, were also detected.

Decision 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.

Three replicate 10 ha clearcut blocks were investigated at the Sicamous Creek Silvicultural Systems Trial in Sicamous, BC. In each of these three blocks, there are two 1 ha treatment plots that were established post-harvest in 1994/95: CWD retention (+) and CWD removal (-). In order to answer questions at the plot scale, ten 10 yr.-old operationally planted spruce trees were randomly chosen at all CWD+ and CWD- plots, and three hyphae trap bags were buried around each.

1 ha.

3 mesh bags

CWD+

CWD-

10 ha. cutblock

10 ha cutblock replanted with Engelmann spruce in 1996 showing the relative position of 1 ha coarse woody debris retention (CWD+) or removal (CWD-) plots and mesh bag sampling details.

Representative ECM fungi from randomly selected root tips on lateral roots of 10 yr.-old spruce in both CWD+ and CWD- plots identified at Sicamous Creek by both morphological and molecular methods:

Bags were opened and visually assessed for hyphal colonization; the contents of all three bags at one tree were combined.

Identities of fungal cultures sampled after 21-day growth on BIOLOG microplates. Colony colour was initially used to distinguish groups of fungi; samples were randomly selected from nitrogen (N)- or phosphorus (P)- based plates, coarse woody debris (CWD) retention (+) or removal (-) plots, and cutblocks A, B, or C. % similarities were derived from NCBI Blastn returns via UNITE.

Outcome

10 yr.-old spruce

Hyphae-trap mesh bag

10 yr.-old Picea engelmannii surrounded by three sand-filled mesh bags; the bags were buried in fall 2006 and excavated in fall 2007.

Bag contents were shaken in water with detergent, then washed through sieves; fragmented hyphae was collected by floating.

Supernatant was mixed with an agar/glucose substrate, plated, and incubated for three weeks.

Day 1

Day 21

Hyphae

collected from surface

Colony colour on plate Sample source % similarity Putative identity

Dark Green Block C CWD- N plate 99 Gyoerffyella sp.

Light Green Block C CWD+ P plate 98 Cladophialophora minutissima

Purple Block C CWD+ N plate 99 Cladophialophora minutissima

Blue Block B CWD- P plate 98 Phoma herbarum

Blue Block A CWD- N plate 100 Cladosporium cladosporioides

Dark Green Block C CWD- N plate 99 Cladosporium cladosporioides

Pink Block A CWD- N plate 99 Tolypocladium inflatum

Pink Block A CWD- P plate 100 Tolypocladium inflatum

Pink Block A CWD- P plate 100 Tolypocladium inflatum

White Block A CWD- N plate 100 Tolypocladium inflatum

Brown Block A CWD- P plate 98 Thelephora terrestris

Brown Block B CWD+ N plate 99 Phialophora sp.

White Block C CWD+ P plate 96 uncultured Tricholomataceae

White Block C CWD+ N plate 99 Ophiocordyceps sinensis

Agaricales

Amphinema spp.

Atheliaceae

Cenococcum geophilum

Cortinarius/Dermocybe spp.

Hygrophorus spp.

Inocybe spp.

Lactarius spp.

Meliniomyces bicolor

Pezizales

Piloderma spp.

Pyronemataceae

Russula aeruginea

Thelephora terrestris

Tylospora spp.

Ascomycetes

References Altschul SF, TL Madden, AA Schaffer, JH Zhang, Z Zhang, W Miller, and DJ Lipman. 1997. Gapped BLAST and PSI-BLAST – a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402. BIOLOG 3938 Trust Way, Hayward CA, 510 785-2564, www.biolog.com. Dobranic JK, and JC Zak. 1999. A microtitre plate procedure for evaluating fungal functional diversity. Mycologia 91: 756-765. Kõljalg U, Larsson K-H, Abarenkov K, Nilsson RH, Alexander IJ, Eberhardt U, Erland S, Hoiland K, Kjoller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo L, Vralstad T, Ursing BM. 2005. UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi. New Phytologist 166: 1063-1068. Grizzle HW, and JC Zak. 2006. A microtitre plate procedure for evaluating fungal functional diversity on nitrogen substrates. Mycologia 98: 353-363 Sobek EA, and JC Zak. 2003. The Soil FungiLog procedure: method and analytical approaches toward understanding fungal functional diversity. Mycologia 95: 590-602. Wallander H, LO Nilsson, D Hagerberg, E Bååth. 2001. Estimation of the biomass and seasonal growth of external mycelium of ectomycorrhizal fungi in the field. New Phytologist 151: 753-760. Wallander, H., Göransson,H., Rosengren,U. 2004. Production, standing biomass, and natural abundance of 15N and 13C in ectomycorrhizal mycelia collected at different soil depths in two forest types. Oecologia 139: 89-97.