MUSHROOM VARIETY 'RO14176'

Abstract

A new and unique brown variety of the mushroom Agaricus bisporus Imbach was produced by crossing the progeny of brown mushrooms and off-white hybrid mushrooms to bridging cross strains. The resultant hybrid, named RO14176, exhibits distinct and stable characteristics. Hybrid RO14176 exhibits a light cap color, thicker cap flesh, rounder cap, faster mushroom development, and higher yield, especially when compared to available brown mushroom varieties.

Claims

1. A new and distinct variety of Agaricus bisporus Imbach mushroom named RO14176, as shown and described herein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

[0028] FIG. 1 shows comparisons between mushrooms of variety RO14176 (top row) and variety RO10425 (bottom row). The top left panel shows caps of 10 representative mushrooms from variety RO14176 and their average L*, a*, and b* values. The top right panel shows a longitudinal slice of each mushroom cap in the top left panel with the 10 mushrooms' average cap diameter, cap height, cap flesh thickness, and stipe diameters reported in millimeters. The bottom left panel shows caps of 10 representative mushrooms from variety RO10425 and their average L*, a*, and b* values. The bottom right panel shows a longitudinal slice of each mushroom cap in the bottom left panel with the 10 mushrooms' average cap diameter, cap height, cap flesh thickness, and stipe diameters reported in millimeters. Mushrooms pictured were grown in the same room, under the same environmental conditions, and were harvested from the same trial in April 2025. All photos have the same scale. Scale bar=1 cm.

[0029] FIG. 2 shows a longitudinal slice of a mushroom of variety RO14176 with markings added to illustrate the calculation of Cap Edge Flesh Thickness, where Cap Edge Flesh Thickness is calculated by ((LG.sub.LCS.sub.L)+(CS.sub.RRG.sub.R))/CS.sub.D. LG.sub.L=left-most x-coordinate of the left gill, CS.sub.L=left-most x-coordinate of the cross-section, CS.sub.R=right-most x-coordinate of the cross-section, RG.sub.R=right-most x-coordinate of the right gill, and CS.sub.D=diameter of the cross-section.

[0030] FIG. 3 shows a longitudinal slice of a mushroom of variety RO14176 with markings added to illustrate the calculation of Cap Apex Flesh Thickness, where Cap Apex Flesh Thickness is calculated by (CS.sub.T((LG.sub.T+RG.sub.T)/2))/CS.sub.H. CS.sub.T=the top-most y-coordinate of the cross-section, LG.sub.T=the top-most y-coordinate of the left gill, RG.sub.T=the top-most y-coordinate of the right gill, and CS.sub.H=the height of the cross-section.

[0031] FIG. 4 shows multiple mushroom caps of variety RO14176 from a top-down view.

[0032] FIG. 5 shows multiple longitudinal slices of mushrooms of variety RO14176.

DETAILED BOTANICAL DESCRIPTION

[0033] In mushroom breeding, mycelial (=vegetative) cultures of two compatible progenitors (typically these are haploid homokaryotic strains called homokaryons) must come into physical contact so that one or more fusion zones can occur between the progenitors. Within those fusion zones nuclei from the two progenitors become associated. In Agaricus bisporus, a novel, hybrid mycelium ultimately containing two compatible haploid nuclear types (one from each of the two progenitors) plus one mitochondrial type (from either one of the progenitors) emerges. This novel hybrid mycelium can be isolated and propagated to provide the new hybrid culture, which can be further subdivided and propagated for commercial or other purposes.

[0034] It should be recognized that the mushrooms of the present disclosure are hybrids (equivalent to crosses), as they are formed by the hybridization of at least two independently derived strains, since they were formed by hybridizing the wild strain AA-0096 with a second, commercialized strain of A. bisporus (with the second strain, in some embodiments, being itself a cross between at least two other identified strains of A. bisporus). The term hybrid mushroom may be used herein to represent the product of a cross between a wild A. bisporus strain and a commercial strain, often including a cross between a brown mushroom strain and an off-white hybrid variety. Differences in terminology (and in the specific techniques used alongside the terminology) are merely indicative of the variety of terminology and techniques used in the mushroom production field.

[0035] After parental homokaryotic spores germinated into mycelia, single spore isolates (SSIs) were selected, preferentially those exhibiting slow mycelial growth. The slowly growing mycelia of SSIs usually contain one nuclear type, are thus homokaryotic, and are suitable for crossing, and thus for breeding. Slow growers are denoted as sg. Some homokaryons were obtained through protoplasting; this technique involves treating the vegetative mycelia of the heterokaryons to damage the fungal cell wall, then selecting colonies of protoclones that are each derived from a single, regenerated protoplast. All crosses were initially cultivated on a small scale, and spores were collected for the parents of the crosses to produce the hybrids described herein.

[0036] One useful feature in the breeding programs for these novel mushroom varieties is the use of a bridging cross strain, which is a hybrid variety produced from crossing a commercial brown variety with a commercial white variety. The particular bridging cross strain of note in these breeding programs is known as the 4x29 strain. Strain 4x29 has been previously deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, Md., USA, ATCC accession No. PTA-6877 (deposited on Jul. 20, 2005). Strain 4x29 is the product of crossing a commercial brown strain with a commercial white strain. The 29 of this strain is a unique homokaryon collected from an off-white hybrid strain.

[0037] The varieties, mushrooms, strains, and cultures disclosed herein are understood to comprise parts of mushrooms, such as the fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, or product produced from the mushroom. In some embodiments, the mushroom part from the hybrid mushroom produced by any method disclosed herein is a spore. In some embodiments, the mushroom part produced from a mushroom strain disclosed herein is a spore. In some embodiments, RO14176 is a mushroom part produced from the hybrid mushroom of RO10425. In some embodiments, the mushroom part produced from the hybrid mushroom of RO10425 is RO14176. FIG. 1 compares caps and longitudinal mushroom slices of RO14176 and RO10425.

Trait Determinations

[0038] In the descriptions and tables that follow, numerous traits are calculated/measured as follows in Table 1. Visual depictions of the measurements used for calculating Cap Apex Flesh Thickness are shown in FIG. 3. Visual depictions of the measurements used for calculating Cap Edge Flesh Thickness are shown in FIG. 2.

[0039] Mushrooms were harvested at Stage 3 as described by Hammond & Nichols (Hammond, J. B. W., & Nichols, R. (1976). Carbohydrate metabolism in Agaricus bisporus (Lange) Sing.: changes in soluble carbohydrates during growth of mycelium and sporophore. Microbiology, 93(2), 309-320), when cap diameters were approx. 3-5 cm with the veil still intact. To reduce confounding effects introduced by different trials, developmental timing and yield traits were compared only from trials where all strains being compared were present together.

[0040] Images of mushroom caps and longitudinal cross-sections were captured using a Canon Rebel SLI camera (Canon, Inc., Toyko, Japan) in a custom-built photo box with standardized lighting conditions. The camera was calibrated for color consistency using a ColorChecker gray balance card (Calibrite LLC, Wilmington, Del.). L*, a* and b* values were captured across the surface of each mushroom cap and cross-section using ImageJ (Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671-675). Images captured in 2024 and 2025 were used for analysis. Extreme outliers were excluded from the data, defined as those falling 3 times the interquartile range (IQR) above the 75th percentile for that trait or more than 3 times the IQR below the 25th percentile for that trait.

[0041] All statistical analyses were carried out in the R programming language (R Core Team, (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. www[dot]R-project[dot]org/). Levene's test was performed using the car package (Fox, J., & Weisberg, S. (2018). An R companion to applied regression. SAGE Publications), Shapiro-Wilk tests and Tukey HSD were performed using the stats package (R Core Team (2013)), and standard ANOVAs, Kruskal-Wallis tests and Dunn's tests were performed using the rstatix package (Kassambara, A (2023). rstatix: Pipe-Friendly Framework for Basic Statistical Tests. R package version 0.7.2, rpkgs[dot]datanovia[dot]com/rstatix/).

TABLE-US-00001 TABLE 1 Trait Tool Measurement Scale Timing Cap Color Darkness visually 0 to 5 (from white to dark brown), tray-level 1st break**, assessed measurement 2nd break** Cap Color a* Value image analysis 128 to +127 (from pure green to pure red), 1st break**, (Green-Red) individual mushroom-level measurement 2nd break** Cap Color b* Value image analysis 128 to +127 (from pure blue to pure 1st break**, (Blue-Yellow) yellow), individual mushroom-level 2nd break** measurement Cap Color L* Value image analysis 0 to 100 (from pure black to pure white), 1st break**, (Dark-Light) individual mushroom-level measurement 2nd break** Cap Diameter image analysis millimeters (mm), individual mushroom- 1st break**, level measurement 2nd break** Cap Firmness manual 1 to 5 (from very soft to very firm), 1st break**, squeeze test tray-level measurement 2nd break** Cap Apex Flesh calculated ratio distance from top of gills to top of cap of 1st break**, Thickness and image mushroom, relative to cap height, individual 2nd break** analysis mushroom-level measurement Cap Edge Flesh calculated ratio sum of distances from outer edge of each 1st break**, Thickness and image gill to outer edges of cap of mushroom, 2nd break** analysis relative to cap diameter, individual mushroom-level measurement Cap Flesh Thickness hand- millimeters (mm), distance from top of gill- 1st break**, measurement stipe connection (at center of stipe) to top of 2nd break** cap, individual mushroom-level measurement Cap Height image analysis millimeters (mm), distance from base of cap 1st break**, to top of cap, individual mushroom-level 2nd break** measurement Cap Roundness image analysis Unitless ratio (0-1), 1st break**, (Calculated) calculated as 4 * CS.sub.A/( * CS.sub.D.sup.2), where 2nd break** CS.sub.A = area of the cross-section, and CS.sub.D = diameter of the cross-section; assessed with image analysis, individual mushroom-level measurement Cap Roundness (Visual) visually 1 to 5 (from very flat to spherical), 1st break**, assessed tray-level measurement 2nd break** Compatibility calculated ratio pounds per square foot (lb/ft.sup.2) and 1st break** kilograms per square meter (kg/m.sup.2), weight of 1st break mushrooms yielded relative to area of tray, tray-level measurement Compost Colonization visually 0 to 5 (from no colonization to robust before 1st break assessed colonization), tray-level measurement (10 days after spawning) Days to 1st Break visually number of days elapsed after casing at 1st break** assessed which at least half of tray is of harvestable maturity for 1st break, tray-level measurement Days to 2nd Break visually number of days elapsed after casing at 2nd break** assessed which at least half of tray is of harvestable maturity for 2nd break, tray-level measurement Days to Pinset visually number of days elapsed after casing at before 1st break assessed which pins first reach 5 mm, tray-level measurement Percent Harvested Speed calculated ratio % of yield harvested by a designated date 17 days after within break, casing (1st break), yield at date within break/total yield for 24 days after break, trial-level measurement casing (2nd break), 32 days after casing (3rd break) Relative Cap Flesh calculated ratio Cap Flesh Thickness Value/Cap Diameter 1st break**, Thickness Value, individual mushroom-level 2nd break** measurement Relative Cap Height calculated ratio Cap Height Value/Cap Diameter Value, 1st break**, individual mushroom-level measurement 2nd break** Relative Stipe Diameter calculated ratio Stipe Diameter Value/Cap Diameter 1st break**, Value, individual mushroom-level 2nd break** measurement Stipe Diameter hand- millimeters (mm), stipe diameter at base of 1st break**, measurement cap, individual mushroom-level 2nd break* measurement Tray Coverage visually 0 to 5 (from no coverage to full coverage), 1st break**, assessed tray-level measurement 2nd break** Yield (Each Break) weighing kg/m.sup.2, tray-level measurement; or Assessed lb/ft.sup.2, tray-level measurement cumulatively throughout 1st, 2nd, and 3rd breaks Yield (Total) weighing kg/m.sup.2, tray-level measurement; Assessed or lb/ft.sup.2, tray-level measurement cumulatively across 1st, 2nd, and 3rd breaks **at date when at least half of tray is of harvestable maturity in break

Objective Description of the Variety RO14176

[0042] The following detailed description sets forth the breeding procedures and the characteristics of new cultivar designated RO14176. The strain is maintained and propagated as vegetative mycelium. Spawn (inoculum for the substrate compost) is prepared from the vegetative mycelium. The variety has shown uniformity and stability for the traits, within the limits of expected environmental influence for the traits.

[0043] All homokaryons utilized are strains of Agaricus bisporus.

[0044] RO14176 was produced as a heterokaryon SSI derived from strain RO10425.

[0045] RO14176 sets pins and produces harvestable mushrooms at first break and second break as fast or faster than available brown commercial strains. RO14176 additionally exhibits higher yield at third break, and higher overall yield, than available brown commercial strains. RO14176 also exhibits rounder cap shape, thicker cap apex flesh, thicker cap edge flesh, and a new cap color compared to available brown commercial strains. RO14176 additionally exhibits percent harvested speed faster or as fast as other available brown commercial strains.

[0046] Other characteristics of RO14176 distinguishable from other varieties are described below (compared to commercial line BR06 and commercial line B14528).

[0047] FIG. 4 and FIG. 5 show mushroom caps and longitudinal slices, respectively, of strain RO14176.

Comparisons to Commercial Mushroom Varieties

Initial Trials

Tray-Level Comparisons

[0048] In further evidence of the distinctness and superiority of mushroom strain RO14176, provided below are the results of trials conducted across multiple strains' yielded trays of mushrooms. These trials compare timing for trays to reach pinset and harvestable maturity, tray coverage, cap color darkness, cap firmness, cap roundness (visual), and yield at each break. Characteristics were measured from 46 trays of strain RO14176 mushrooms for yield traits and from 26 trays of strain RO14176 mushrooms for other traits. Corresponding measurements were taken from 38 trays of strain BR06 mushrooms for yield traits and 19 trays of strain BR06 mushrooms for other traits (Table 2A and 2B), and corresponding measurements were taken from 17 trays of strain B14528 mushrooms (Table 3A and 3B).

[0049] Table 2A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00002 TABLE 2A Characteristic RO14176 BR06 Before First Break Days to Pinset Faster Slower First Break Days to 1st Break Faster Slower Tray Coverage Higher Lower Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Rounder Flatter Yield (lb/ft.sup.2) Similar Similar Second Break Days to 2nd Break Similar Similar Tray Coverage Similar Similar Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Flatter Rounder Yield (lb/ft.sup.2) Higher Lower Third Break Yield (lb/ft.sup.2) Similar Similar All Breaks Combined Yield (lb/ft.sup.2) Higher Lower

[0050] Table 2B below provides initial quantitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the tray-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00003 TABLE 2B RO14176 BR06 Characteristic Average St. Dev. Average St. Dev. p-value Before First Break Days to Pinset 11.23 0.82 12.68 1.42 8.25E05 First Break Days to 1st Break 17.46 1.03 18.53 1.71 1.27E02 Tray Coverage 3.88 0.91 3.16 1.30 3.27E02 Cap Color Darkness 3.85 0.37 4.21 0.42 3.46E03 Cap Firmness 3.69 0.47 4.68 0.48 1.57E08 Cap Roundness (Visual) 3.38 0.50 2.95 0.23 9.07E04 Yield (lb/ft.sup.2) 2.47 0.80 2.16 1.36 2.00E01 Second Break Days to 2nd Break 24.42 0.86 24.95 0.91 5.48E02 Tray Coverage 3.81 0.49 3.42 1.22 1.49E01 Cap Color Darkness 3.58 0.50 4.05 0.23 4.15E04 Cap Firmness 3.81 0.40 4.44 0.51 3.61E05 Cap Roundness (Visual) 2.69 0.47 2.94 0.24 4.24E02 Yield (lb/ft.sup.2) 1.93 0.42 1.47 0.63 1.68E04 Third Break Yield (lb/ft.sup.2) 1.00 0.33 0.87 0.47 1.66E01 All Breaks Combined Yield (lb/ft.sup.2) 5.39 0.92 4.51 1.44 9.79E04

[0051] Table 3A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00004 TABLE 3A Characteristic RO14176 B14528 Before First Break Days to Pinset Similar Similar First Break Days to 1st Break Similar Similar Tray Coverage Similar Similar Cap Color Darkness Similar Similar Cap Firmness Similar Similar Cap Roundness (Visual) Rounder Flatter Yield (lb/ft.sup.2) Similar Similar Second Break Days to 2nd Break Faster Slower Tray Coverage Higher Lower Cap Color Darkness Similar Similar Cap Firmness Similar Similar Cap Roundness (Visual) Similar Similar Yield (lb/ft.sup.2) Similar Similar Third Break Yield (lb/ft.sup.2) Similar Similar All Breaks Combined Yield (lb/ft.sup.2) Similar Similar

[0052] Table 3B below provides initial quantitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the tray-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain B14528, column 5 shows the standard deviation of the characteristic for mushroom strain B14528, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00005 TABLE 3B RO14176 B14528 Characteristic Average St. Dev. Average St. Dev. p-value Before First Break Days to Pinset 11.23 0.82 11.41 0.87 4.92E01 First Break Days to 1st Break 17.46 1.03 17.47 0.51 9.73E01 Tray Coverage 3.88 0.91 3.59 0.87 2.94E01 Cap Color Darkness 3.85 0.37 3.82 0.39 8.49E01 Cap Firmness 3.69 0.47 3.88 0.33 1.56E01 Cap Roundness (Visual) 3.38 0.50 2.88 0.33 7.05E04 Yield (lb/ft.sup.2) 2.47 0.80 2.39 0.54 7.11E01 Second Break Days to 2nd Break 24.42 0.86 25.12 0.86 1.30E02 Tray Coverage 3.81 0.49 3.47 0.51 3.67E02 Cap Color Darkness 3.58 0.50 3.76 0.56 2.60E01 Cap Firmness 3.81 0.40 3.82 0.39 8.99E01 Cap Roundness (Visual) 2.69 0.47 2.82 0.39 3.47E01 Yield (lb/ft.sup.2) 1.93 0.42 1.74 0.38 1.09E01 Third Break Yield (lb/ft.sup.2) 1.00 0.33 1.18 0.38 6.16E02 All Breaks Combined Yield (lb/ft.sup.2) 5.39 0.92 5.31 0.76 7.45E01

Individual Mushroom-Level Comparisons

[0053] In additional evidence of the distinctness and superiority of mushroom strain RO14176, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Diameter, Cap Height, Cap Flesh Thickness, Stipe Diameter, Relative Cap Height, Relative Cap Flesh Thickness, and Relative Stipe Diameter (as described in Table 1). Measurements from 210 first break and 130 second break RO14176 mushrooms were compared to 350 first break and 130 second break BR06 mushrooms (Tables 4A and 4B) and 90 first break and 40 second break B14528 mushrooms (Tables 5A and 5B).

[0054] Table 4A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00006 TABLE 4A Characteristic RO14176 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Larger Smaller Cap Height Taller Shorter Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Taller Shorter Relative Cap Flesh Thicker Thinner Thickness Relative Stipe Diameter Similar Similar Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Smaller Larger Cap Height Similar Similar Cap Flesh Thickness Thicker Thinner Stipe Diameter Smaller Larger Relative Cap Height Taller Shorter Relative Cap Flesh Thicker Thinner Thickness Relative Stipe Diameter Smaller Larger

[0055] Table 4B below provides initial quantitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00007 TABLE 4B RO14176 BR06 St. St. Characteristic Average Dev. Average Dev. p-value First Break Cap L* Value (dark-light) 65.73 4.23 63.49 6.52 1.00E05 Cap a* Value (green-red) 7.64 1.00 8.53 1.85 2.45E10 Cap b* Value (blue-yellow) 26.84 1.43 28.15 2.48 7.83E12 Cap Diameter 43.93 3.61 42.78 4.41 1.45E03 Cap Height 28.15 2.18 25.75 2.35 1.27E29 Cap Flesh Thickness 15.37 1.96 13.25 1.46 1.88E44 Stipe Diameter 19.92 2.43 19.25 2.47 1.19E03 Relative Cap Height 0.64 0.03 0.60 0.04 4.25E33 Relative Cap Flesh 0.35 0.04 0.31 0.03 3.34E39 Thickness Relative Stipe Diameter 0.45 0.05 0.45 0.06 5.18E01 Second Break Cap L* Value (dark-light) 68.87 3.95 64.27 3.81 5.37E20 Cap a* Value (green-red) 6.66 0.95 8.20 1.14 1.67E27 Cap b* Value (blue-yellow) 26.77 1.64 28.31 1.65 1.08E13 Cap Diameter 42.13 3.35 43.26 4.15 1.32E02 Cap Height 26.36 2.27 25.80 2.61 5.55E02 Cap Flesh Thickness 14.15 1.77 13.46 1.52 6.12E04 Stipe Diameter 17.80 2.58 18.97 3.67 2.54E03 Relative Cap Height 0.63 0.05 0.60 0.04 1.01E07 Relative Cap Flesh 0.34 0.04 0.31 0.03 1.18E07 Thickness Relative Stipe Diameter 0.42 0.05 0.44 0.08 3.93E02

[0056] Table 5A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00008 TABLE 5A Characteristic RO14176 B14528 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Similar Similar Cap Height Taller Shorter Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Taller Shorter Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Larger Smaller Second Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Smaller Larger Cap Height Similar Similar Cap Flesh Thickness Thicker Thinner Stipe Diameter Similar Similar Relative Cap Height Taller Shorter Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Similar Similar

[0057] Table 5B below provides initial quantitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain B14528, column 5 shows the standard deviation of the characteristic for mushroom strain B14528, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00009 TABLE 5B RO14176 B14528 St. St. Characteristic Average Dev. Average Dev. p-value First Break Cap L* Value (dark-light) 65.73 4.23 64.48 5.25 2.98E02 Cap a* Value (green-red) 7.64 1.00 8.03 1.65 1.30E02 Cap b* Value (blue-yellow) 26.84 1.43 29.38 1.50 3.68E34 Cap Diameter 43.93 3.61 43.84 3.80 8.49E01 Cap Height 28.15 2.18 26.60 1.97 1.91E08 Cap Flesh Thickness 15.37 1.96 12.91 0.93 2.05E27 Stipe Diameter 19.92 2.43 17.58 1.44 3.66E17 Relative Cap Height 0.64 0.03 0.61 0.03 1.59E15 Relative Cap Flesh 0.35 0.04 0.30 0.03 2.71E28 Thickness Relative Stipe Diameter 0.45 0.05 0.40 0.04 2.36E18 Second Break Cap L* Value (dark-light) 68.87 3.95 68.52 4.46 6.36E01 Cap a* Value (green-red) 6.66 0.95 7.88 1.49 7.36E09 Cap b* Value (blue-yellow) 26.77 1.64 28.55 1.65 1.30E08 Cap Diameter 42.13 3.35 44.86 3.59 1.67E05 Cap Height 26.36 2.27 26.29 1.72 8.51E01 Cap Flesh Thickness 14.15 1.77 12.60 1.32 8.12E07 Stipe Diameter 17.80 2.58 18.11 3.29 5.33E01 Relative Cap Height 0.63 0.05 0.59 0.04 1.36E05 Relative Cap Flesh 0.34 0.04 0.28 0.03 9.66E13 Thickness Relative Stipe Diameter 0.42 0.05 0.40 0.07 9.03E02

Additional Trials

Tray-Level Comparisons

[0058] Several additional trials were conducted for characterizing RO14176. In further evidence of the distinctness and superiority of mushroom strain RO14176, provided below are the results of trials conducted across multiple strains' yielded trays of mushrooms. These trials compare timing for trays to reach pinset and harvestable maturity, and yield at each break.

[0059] Table 6A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Traits of particular interest are bolded. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00010 TABLE 6A Characteristic RO14176 BR06 Before First Break Days to Pinset Faster Slower First Break Days to 1st Break Faster Slower Percent Harvested Speed Faster Slower Similar Similar Second Break Days to 2nd Break Faster Slower Similar Similar Third Break Higher Lower All Breaks Combined Higher Lower

[0060] Table 6B below provides further quantitative comparisons between mushroom strain RO14176 and strain BR06.

[0061] The first part of analyzing trait measurements in the additional trials herein included making comparisons between all three of instant strain RO14176, comparative strain BR06, and comparative strain B14528. For these three strains, a Shapiro-Wilk test and a Levene test were performed first to assess if the data satisfy the assumptions of normality and equal variances among groups, respectively, of a standard ANOVA. The Shapiro-Wilk test and Levene test determined that most of these traits satisfied neither assumption. Therefore, a Kruskal-Wallis test (Kruskal, W. H., & Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. Journal of the American statistical Association, 47(260), 583-621), a non-parametric variation of ANOVA that does not assume normality, equal variances among groups, or equal sample sizes among groups, was performed. Medians and interquartile ranges (IQR) were reported, as these metrics are robust to non-normal distributions. Means are also reported for consistency with normally-distributed traits.

[0062] The Shapiro-Wilk test and Levene test determined that most developmental timing traits (Days to Pinset, Days to 1st Break, and Days to 2nd Break) and yield traits were not normally distributed and variances among groups were not equal. The Kruskal-Wallis test and Dunn post-hoc test (Dunn's Test; Dunn, O. J. (1964). Multiple comparisons using rank sums. Technometrics, 6(3), 241-252) were used in these cases. When the Kruskal-Wallis test results were significant based on a threshold of p<0.05, Dunn's test was performed post-hoc to make pairwise strain comparisons (Dunn, 1964). P-values were adjusted using the Bonferroni correction to minimize risk of Type 1 error (false significance). An exception to this trend was Percent Harvested Speed, which was normally distributed and exhibited equal variance for all strains. For assessing Percent Harvested Speed, a standard ANOVA was used and a Tukey Honestly Significant Difference (HSD) test was performed post-hoc for pairwise strain comparisons. Standard deviations multiplied by 100 for consistency alongside percentages (SD*100) were reported for this trait. This testing was performed on groups of three strains, but the results show a pairwise subset of the tests for consistency with the initial trials.

[0063] In Table 6B, column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test. Traits of particular interest are bolded.

TABLE-US-00011 TABLE 6B Significance Number of Group Variety Mean Median IQR Trials (Dunn's Test) Before First Break Days to Pinset RO14176 11.4 12.0 1.00 7 a BR06 12.7 12.5 1.00 7 b First Break Days to 1st Break RO14176 17.7 18.0 1.00 7 a BR06 18.8 18.0 2.00 7 b RO14176 12.40 12.25 4.69 6 a BR06 10.99 11.03 8.10 6 a Second Break Days to 2nd Break RO14176 25.3 25.0 2.00 7 a BR06 25.9 26.0 2.00 7 b RO14176 8.45 8.50 3.52 6 a BR06 7.37 6.59 5.13 6 a Third Break RO14176 4.20 3.71 2.69 6 a BR06 3.27 2.34 3.03 6 b All Breaks Combined RO14176 25.05 23.88 3.66 6 a BR06 21.58 22.02 4.39 6 b Significance Percent Harvested Speed Group (measured in First Break) SD * 100 (Tukey HSD) RO14176 38.7% 39.9% 13.10 5 b BR06 14.5% 12.9% 16.90 5 a

[0064] Table 7A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Traits of particular interest are bolded. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00012 TABLE 7A Characteristic RO14176 B14528 Before First Break Days to Pinset Similar Similar First Break Days to 1st Break Similar Similar Percent Harvested Speed Similar Similar Similar Similar Second Break Days to 2nd Break Similar Similar Similar Similar Third Break Similar Similar All Breaks Combined Similar Similar

[0065] Table 7B below provides additional quantitative comparison between mushroom strain RO14176 and strain B14528. Column 1 shows the strains compared for each trait, column 2 shows the mean value for that strain's measurements, column 3 shows the median value of the same measurements, column 4 shows either the interquartile range (IQR) or the standard deviation multiplied by 100 for consistency alongside percentages (SD*100) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to either Dunn's Test or Tukey HSD.

TABLE-US-00013 TABLE 7B Number Significance of Group Characteristic Mean Median IQR Trials (Dunn's Test) Before First Break Days to Pinset RO14176 11.4 12.0 1.00 7 a B14528 11.2 11.0 1.50 7 a First Break Days to 1st Break RO14176 17.7 18.0 1.00 7 a B14528 17.6 18.0 1.00 7 a RO14176 12.40 12.25 4.69 6 a B14528 11.91 12.21 4.35 6 a Second Break Days to 2nd Break RO14176 25.3 25.0 2.00 7 a B14528 25.5 25.0 1.00 7 ab RO14176 8.45 8.50 3.52 6 a B14528 7.47 7.76 2.93 6 a Third Break RO14176 4.20 3.71 2.69 6 a B14528 4.49 3.95 2.59 6 a All Breaks Combined RO14176 25.05 23.88 3.66 6 a B14528 23.88 23.97 6.69 6 a Percent Harvested Significance Speed (measured Group in First Break) Mean Median SD * 100 (Tukey HSD) RO14176 38.7% 39.9% 13.10 5 b B14528 41.8% 40.3% 7.60 5 b

Individual Mushroom-Level Comparisons

[0066] In additional evidence of the distinctness and superiority of mushroom strain RO14176, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Apex Flesh Thickness, Cap Edge Flesh Thickness, and Cap Roundness (Calculated) (as described in Table 1). Measurements were taken for hundreds of mushrooms per trait, noted as sample size (n) in Tables 8B and 9B.

[0067] Table 8A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Traits of particular interest are bolded. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00014 TABLE 8A Characteristic RO14176 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter

[0068] Table 8B below provides additional quantitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of mushrooms assessed (n), and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test.

TABLE-US-00015 TABLE 8B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO14176 65.76 65.82 5.82 469 a BR06 63.30 62.74 9.83 797 b Cap a* Value (green-red) RO14176 7.45 7.47 1.58 469 a BR06 8.67 8.96 2.58 787 b Cap b* Value (blue-yellow) RO14176 28.17 28.03 2.64 469 a BR06 28.72 29.04 3.17 776 b Cap Apex Flesh Thickness RO14176 0.54 0.54 0.06 453 a BR06 0.52 0.52 0.05 541 b Cap Edge Flesh Thickness RO14176 0.27 0.26 0.07 449 a BR06 0.21 0.21 0.09 537 b Cap Roundness (Calculated) RO14176 0.64 0.64 0.05 455 a BR06 0.61 0.61 0.04 541 b Second Break Cap L* Value (dark-light) RO14176 68.92 68.86 6.39 263 a BR06 65.68 65.49 5.90 340 b Cap a* Value (green-red) RO14176 6.37 6.43 1.60 261 a BR06 7.21 7.31 2.42 340 b Cap b* Value (blue-yellow) RO14176 27.36 27.35 2.11 261 a BR06 28.15 28.49 2.86 340 b Cap Apex Flesh Thickness RO14176 0.54 0.54 0.04 235 a BR06 0.51 0.52 0.07 362 b Cap Edge Flesh Thickness RO14176 0.31 0.31 0.06 235 a BR06 0.23 0.23 0.09 362 b Cap Roundness (Calculated) RO14176 0.61 0.60 0.05 235 a BR06 0.60 0.59 0.06 363 b

[0069] Table 9A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00016 TABLE 9A Characteristic RO14176 B14528 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter Second Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Redder Greener Cap b* Value (blue-yellow) Similar Similar Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter

[0070] Table 9B shows additional quantitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the characteristic, column 4 shows the interquartile range (IQR) for the characteristic, column 5 shows the number of mushrooms assessed (n), and column 6 shows the statistical significance group calculated according to Dunn's Test.

TABLE-US-00017 TABLE 9B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO14176 65.76 65.82 5.82 469 a B14528 64.06 64.13 6.56 340 c Cap a* Value (green-red) RO14176 7.45 7.47 1.58 469 a B14528 8.26 8.31 2.13 340 c Cap b* Value (blue-yellow) RO14176 28.17 28.03 2.64 469 a B14528 30.31 30.16 3.39 340 c Cap Apex Flesh Thickness RO14176 0.54 0.54 0.06 453 a B14528 0.47 0.46 0.06 387 c Cap Edge Flesh Thickness RO14176 0.27 0.26 0.07 449 a B14528 0.22 0.22 0.08 387 b Cap Roundness (Calculated) RO14176 0.64 0.64 0.05 455 a B14528 0.61 0.61 0.05 389 b Second Break Cap L* Value (dark-light) RO14176 68.92 68.86 6.39 263 a B14528 68.56 69.02 6.54 170 a Cap a* Value (green-red) RO14176 6.37 6.43 1.60 261 a B14528 6.09 5.96 2.05 170 c Cap b* Value (blue-yellow) RO14176 27.36 27.35 2.11 261 a B14528 27.37 27.78 3.22 170 a Cap Apex Flesh Thickness RO14176 0.54 0.54 0.04 235 a B14528 0.46 0.46 0.05 198 c Cap Edge Flesh Thickness RO14176 0.31 0.31 0.06 235 a B14528 0.23 0.23 0.06 198 b Cap Roundness (Calculated) RO14176 0.61 0.60 0.05 235 a B14528 0.60 0.60 0.05 198 b

[0071] One of the advantages of the strain RO14176 is its incompatibility with other commercial strains. Table 10 below provides results of compatibility tests between strain RO14176 and commercial strain BR06, commercial strain B14528, or related strain RO10425. These results indicate that strain RO14176 is incompatible with strain BR06 and strain B14528, and compatible with related strain RO10425. This was true regardless of which tested strain served as the compost variety and which tested strain served as the casing variety.

TABLE-US-00018 TABLE 10 Strains (compost variety/ Mean Median SD Significance casing variety) lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 Trials Group RO14176/RO14176 2.66 12.99 2.41 11.77 0.61 2.98 3 a RO10425/RO10425 2.71 13.23 2.86 13.94 0.50 2.44 3 a BR06/BR06 2.62 12.79 2.46 11.99 1.15 5.61 2 a B14528/B14528 2.93 14.31 2.92 14.23 0.34 1.66 1 a RO10425/RO14176, 2.43 11.86 2.75 13.40 0.77 3.76 1 a RO14176/RO10425 RO14176/BR06, 1.19 5.81 1.20 5.86 0.37 1.81 1 b BR06/RO14176 RO14176/B14528, 0.70 3.42 0.76 3.69 0.21 1.03 1 b B14528/RO14176