Chlorociboria aeruginascens SR003, originally isolated from a decaying Populus sp. log in Keweenaw County, MI was grown on plates containing varying amounts and sizes of ground wood mixed into agar media. Following inoculation, the diameter of each colony was recorded.
Preliminary testing: suspension squares versus ground wood
Several sizes and dispersion methods were utilized when making the wood-media plates. Aspen (Populus deltoides Bartr.), sugar maple (Acer saccharum Marsh.), tree of heaven (Ailanthus altissima (Mill.) Swingle), and spalted aspen (previously spalted with Scytalidium cuboideum (Sacc. & Ellis)) were chopped into roughly 5 mm × 5 mm × 2 mm thick squares. These squares were suspended in 2% malt extract agar media (100 mm × 15 mm Fisher brand polystyrene petri dishes) in either a high concentration (15-20 squares) or a low concentration (five to seven squares). Suspension was achieved by first pouring the hot media into the Petri plates, allowing the media to cool for approximately ten minutes in a laminar flow hood, then using tweezers to drop the sterilized wood squares into the media plates.
The same wood species were also ground using a Wiley mill, using both the 20 and 30 mesh screens (generating particles roughly 0.9 mm2 and 0.6 mm2, respectively). The wood was stored in glass jars with screw caps and was autoclaved just prior to being mixed with 2% malt agar media. Two different types of wood dust plates were made: plates with a high concentration of wood and plates with a low concentration of wood. Plates containing a high amount of wood were made by adding 7.5 ml of ground wood into each Petri plate, after which the hot malt agar was poured on top and then agitated by hand using a sterilized spatula for 10 seconds. Plates containing a low amount of wood were made by mixing 2.5 ml of ground wood directly into the pre-autoclaved media (with each batch of media containing 2.5 g of malt and 1.875 g of agar and 125 mL of water), autoclaving for 15 minutes, then pouring the sterilized media into the Petri plates. Plates containing only the agar media with no wood added acted as controls. Plates were left to cool in a laminar flow hood for 24 hours prior to inoculation.
The prepared plates were inoculated with C. aeruginascens using a four-point inoculation. In the first round of testing, only colonies that were producing xylindein were inoculated onto the test plates. These plates were evaluated after two weeks.
Preliminary testing: high amount versus low amount of ground wood, 20 and 30 mesh
In the second round of testing, the square piece suspension was dropped from testing, and only ground wood plates were tested. In this second round of testing, two points on each plate were inoculated from the C. aeruginascens isolate that produced xylindein, while the other remaining two points were taken from copies which had stopped producing the pigment. The plates were maintained in the dark at room temperature (21 C). After five days of initial growth, the widest diameter of each colony was measured using a caliper. This size was measured as the base size of the colony. Each colony was then re-measured every three to four days, and the new width of the colony was recorded. Recording was completed when inoculation points on the same media plate touched (22 days). Six replicate plates were made per wood species, per mesh size, per wood amount for all preliminary tests.
Preliminary testing: white colonies to green
The size of white colonies was also measured, as was any green pigment produced by the white colonies during the incubation cycle. Final colony color was recorded after three weeks. Colonies were counted as green if the green pigment (assumed to be xylindein) appeared anywhere within the colony.
Secondary testing: additional isolates
Based upon the results from the primary testing, aspen, spalted aspen, sugar maple, and tree of heaven were ground on a Wiley Mill using both a 20 and 30 mesh screen. For the secondary testing, the sterilized wood was poured directly into an empty Petri plate until the plate was half-filled with wood. Autoclaved 2% malt agar was then poured onto the wood so that the agar just covered the bottom of the Petri plate.
Three additional isolates of C. aeruginascens
(DT8315, isolated from a decaying Populus
log in Ontario, Canada; UAMH7614, isolated in Lake District, UK; UAMH 7615, isolated in Lake District, UK) were tested, including two that had been kept previously in refrigerated storage and were not producing xylindein on 2% malt agar plates (UAMH 7614, UAMH 7615). A two-point inoculation was used, and the same recording system as mentioned above was utilized, and data collection continued until colonies began to touch at 26 days. Three replicate plates were used per wood species per mesh size per isolate. A simple sugar analysis of test wood was conducted following the methods presented in Robinson et al. (Robinson et al. 2011
), with three tested replicates per wood species. Final green colony diameters for all plates were recorded again after four months.
After two weeks, colony diameter in square section suspension was compared against colony diameter on 20 and 30 ground wood plates using a 1-way ANOVA with size (square, 20 mesh, 30 mesh, no wood) as the independent variables. Follow-up testing on mesh-only plates was analyzed using a 4-way ANOVA, with wood species, wood amount (high or low), mesh size (20 or 30), and time as independent variables, and the diameter of the colony as the dependent variable. Tukey's HSD was then used to discern the location of the differences.
Data analysis on the white colonies was done using a 3-way ANOVA, with wood species, wood amount (high or low), and mesh (20 or 30) as the independent variables. Time was not used as a variable, instead readings were based upon final colony color at 45 days incubation.
For secondary testing, strains were compared using a 1-way ANOVA, followed by Tukey's HSD. Both UAMH strains were then combined into a single strain and run in a 4-way ANOVA, with strain, wood species, mesh size, and time as independent variables and colony diameter as the dependent variable. This test was followed by Tukey's HSD.