MUSHROOM VARIETY 'RO12756'

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 RO12756, exhibits distinct and stable characteristics. Hybrid RO12756 exhibits a light cap color, thicker cap flesh, rounder cap, faster mushroom development, and high yield, especially when compared to available brown mushroom varieties.

Claims

1. A new and distinct variety of Agaricus bisporus Imbach mushroom named RO12756, as shown and described herein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] FIG. 1 shows a representative comparison of Agaricus bisporus variety BR06 (left) and variety RO12756 (right) tray coverage at first break. BR06 displays lower tray coverage, and RO12756 displays higher tray coverage.

[0027] 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.

[0028] 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.

[0029] FIG. 4 shows a representative comparison of cap colors for variety BR06 (left), variety B14528 (center), and variety RO12756 (right) cap colors based on Cap L* Value, Cap a* Value, and Cap b* Value at first break. Mushrooms pictured were grown in the same room, under the same environmental conditions, and were harvested from the same trial in May 2025. All photos have the same scale. Scale bar=1 cm.

[0030] FIG. 5 shows representative comparisons of cap height, relative cap height, and relative cap flesh thickness for variety B14528 (left), variety BR06 (center), and variety RO12756 (right) at first break. Mushrooms pictured were grown in the same room, under the same environmental conditions, and were harvested from the same trial in May 2025. All photos have the same scale. Scale bar=1 cm.

DETAILED BOTANICAL DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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, protoplast, gill, cell, 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.

Trait Determinations

[0036] 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.

[0037] 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.

[0038] Images of mushroom caps and longitudinal cross-sections were captured using a Canon Rebel SL1 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.

[0039] 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), 1st break**, assessed tray-level measurement 2nd break** Cap Color a* Value image analysis 128 to +127 (from pure green to pure 1st break**, (Green-Red) red), individual mushroom-level 2nd break** measurement 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 distance from top of gills to top of cap of 1st break**, Thickness ratio and mushroom, relative to cap height, 2nd break** image analysis individual mushroom-level measurement Cap Edge Flesh calculated sum of distances from outer edge of each 1st break**, Thickness ratio and gill to outer edges of cap of mushroom, 2nd break image analysis relative to cap diameter, individual mushroom-level measurement Cap Flesh Thickness hand- millimeters (mm), distance from top of 1st break**, measurement gill-stipe connection (at center of stipe) to 2nd break** top of cap, individual mushroom-level measurement Cap Height image analysis millimeters (mm), distance from base of 1st break**, cap to top of cap, individual mushroom- 2nd break** level 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 pounds per square foot (lb/ft.sup.2) and 1st break** ratio 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 % of yield harvested by a designated date 17 days after ratio 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 Cap Flesh Thickness Value/Cap Diameter 1st break**, Thickness ratio Value, individual mushroom-level 2nd break** measurement Relative Cap Height calculated Cap Height Value/Cap Diameter Value, 1st break**, ratio individual mushroom-level measurement 2nd break** Relative Stipe Diameter calculated Stipe Diameter Value/Cap Diameter 1st break**, ratio 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 RO12756

[0040] The following detailed description sets forth the breeding procedures and the characteristics of new cultivar designated RO12756. The strain is maintained and propagated as vegetative mycelium. Spawn (inoculum for the substrate compost) is prepared from the vegetative mycelium. The new variety was tested on a scale of hundreds of individual mushrooms. The variety has shown uniformity and stability for the traits, within the limits of expected environmental influence for the traits.

[0041] All homokaryons utilized are strains of Agaricus bisporus.

[0042] In a first cross, a bridging cross strain 4x29 slow grower (sg) was crossed with a commercial off-white hybrid strain protoplast.

[0043] RO12756 was produced through the cross of a homokaryon SSI of the first cross to a homokaryon SSI of strain RO10425.

[0044] RO12756 sets pins and produces harvestable mushrooms as fast or faster than available brown commercial strains. RO12756 also exhibits high tray coverage, high yield, a desirably light brown cap color, medium firmness, and thick stipes and cap flesh relative to cap diameter. RO12756 additionally exhibits rounder cap shape, thicker cap apex flesh, thicker cap edge flesh, and Percent Harvested Speed faster or as fast as other available brown commercial strains.

[0045] Other characteristics of RO12756 distinguishable from other varieties are described below (compared to commercial line BR06 and commercial line B14528).

Comparisons to Commercial Mushroom Varieties

Initial Trials

Tray-Level Comparisons

[0046] In further evidence of the distinctness and superiority of mushroom strain RO12756, 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), yield at each break, and percentage of each break's yield harvested by 17 days post-casing (1st break), 24 days post-casing (2nd break), and 32 days post-casing (3rd break). Characteristics were measured from 74-147 trays of strain RO12756 mushrooms with corresponding measurements taken of 94-166 trays of strain BR06 mushrooms (Table 2A and 2B) and 12-28 trays of strain B14528 mushrooms (Table 3A and 3B). Percent Harvest Speed characteristics were measured for 34-36 trials containing strain RO12756, 33-36 trials containing strain BR06, and 3-4 trials containing strain B14528.

[0047] In addition to the comparisons made below, FIGS. 1 and FIGS. 4-5 visually compare variety RO12756 to other varieties in tray coverage (FIG. 1), cap color (FIG. 4), and qualities regarding cap height and cap flesh thickness (FIG. 5).

[0048] Table 2A below provides qualitative comparisons between mushroom strain RO12756 and strain BR06. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00002 TABLE 2A Characteristic RO12756 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) Flatter Rounder Percent Harvested Speed Similar Similar Yield (lb/ft.sup.2) Similar Similar Second Break Days to 2nd Break Faster Slower Tray Coverage Similar Similar Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Flatter Rounder Percent Harvested Speed Higher Lower Yield (lb/ft.sup.2) Similar Similar Third Break Percent Harvested Speed Higher Lower Yield (lb/ft.sup.2) Higher Lower All Breaks Combined Yield (lb/ft.sup.2) Higher Lower

[0049] Table 2B below provides initial quantitative comparisons between mushroom strain RO12756 and strain BR06. Column 1 lists the tray-level or trial-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, 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 RO12756 BR06 Characteristic Average St. Dev. Average St. Dev. p-value Before First Break Days to Pinset 11.722 0.999 12.316 0.880 4.139E05 First Break Days to 1st Break 17.317 0.752 17.598 0.735 1.153E02 Tray Coverage 4.178 0.799 3.935 0.866 2.900E02 Cap Color Darkness 2.916 0.601 3.872 0.380 6.840E35 Cap Firmness 3.543 0.519 4.154 0.425 4.790E19 Cap Roundness (Visual) 2.848 0.387 2.984 0.127 2.930E04 Percent Harvested Speed 24.105% 23.663 16.868% 19.090 1.241E01 Yield (lb/ft.sup.2) 2.483 0.701 2.523 1.017 6.860E01 Second Break Days to 2nd Break 24.824 0.800 25.274 0.675 1.122E04 Tray Coverage 4.000 0.611 4.150 0.605 6.140E02 Cap Color Darkness 3.009 0.427 3.859 0.349 9.160E45 Cap Firmness 3.509 0.553 3.969 0.435 9.200E12 Cap Roundness (Visual) 2.634 0.484 2.992 0.154 1.030E13 Percent Harvested Speed 42.447% 25.152 25.151% 23.811 1.431E03 Yield (lb/ft.sup.2) 1.606 0.537 1.601 0.617 9.340E01 Third Break Percent Harvested Speed 63.200% 26.702 34.306% 32.035 2.320E05 Yield (lb/ft.sup.2) 1.038 0.313 0.747 0.371 9.180E13 All Breaks Combined Yield (lb/ft.sup.2) 5.127 0.792 4.871 0.918 9.080E03

[0050] Table 3A below provides qualitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00004 TABLE 3A Characteristic RO12756 B14528 Before First Break Compost Colonization Higher Lower First Break Days to 1st Break Similar Similar Tray Coverage Similar Similar Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Similar Similar Percent Harvested Speed Similar Similar Second Break Days to 2nd Break Similar Similar Tray Coverage Higher Lower Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Similar Similar Percent Harvested Speed Similar Similar

[0051] Table 3B below provides initial quantitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the tray-level or trial-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, 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 RO12756 B14528 Characteristic Average St. Dev. Average St. Dev. p-value Before First Break Compost Colonization 4.350 0.737 3.643 1.026 6.920E05 First Break Days to 1st Break 17.317 0.752 17.000 0.649 8.599E02 Tray Coverage 4.178 0.799 4.000 0.471 2.630E01 Cap Color Darkness 2.916 0.601 4.000 0.272 4.050E16 Cap Firmness 3.543 0.519 3.786 0.418 2.400E02 Cap Roundness (Visual) 2.848 0.387 2.929 0.262 2.990E01 Percent Harvested Speed 24.105% 23.660 19.000% 11.247 4.392E01 Second Break Days to 2nd Break 24.824 0.800 24.417 0.669 9.848E02 Tray Coverage 4.000 0.611 3.250 0.550 1.150E06 Cap Color Darkness 3.009 0.427 3.929 0.378 2.670E19 Cap Firmness 3.509 0.553 3.857 0.448 2.470E03 Cap Roundness (Visual) 2.634 0.484 2.536 0.508 3.430E01 Percent Harvested Speed 42.447% 25.152 39.000% 34.438 8.380E01

Individual Mushroom-Level Comparisons

[0052] In additional evidence of the distinctness and superiority of mushroom strain RO12756, 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 390 first break and 100 second break RO12756 mushrooms were compared to 400 first break and 120 second break BR06 mushrooms (Tables 14A and 14B) and 150 first break and 80 second break B14528 mushrooms (Tables 15A and 15B).

[0053] Table 4A below provides qualitative comparisons between mushroom strain RO12756 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 RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00006 TABLE 4A Characteristic RO12756 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 Smaller Larger Cap Height Shorter Taller Cap Flesh Thickness Similar Similar Stipe Diameter Smaller Larger Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Smaller Larger 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 Similar Similar Cap Height Shorter Taller Cap Flesh Thickness Similar Similar Stipe Diameter Similar Similar Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Similar Similar

[0054] Table 4B below provides initial quantitative comparisons between mushroom strain RO12756 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 RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, 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 RO12756 BR06 Characteristic Average St. Dev. Average St. Dev. p-value First Break Cap L* Value (dark-light) 74.237 3.524 64.335 5.707 5.943E128 Cap a* Value (green-red) 3.899 1.581 7.605 1.770 1.242E138 Cap b* Value (blue-yellow) 24.770 2.559 28.453 2.451 3.905E76 Cap Diameter 40.571 3.829 42.200 4.005 7.566E09 Cap Height 24.199 1.994 25.455 2.059 1.797E17 Cap Flesh Thickness 13.069 1.395 13.130 1.313 5.270E01 Stipe Diameter 18.258 2.139 19.546 2.585 1.130E13 Relative Cap Height 0.598 0.033 0.605 0.031 3.410E03 Relative Cap Flesh Thickness 0.323 0.028 0.312 0.026 3.060E08 Relative Stipe Diameter 0.451 0.040 0.464 0.054 9.166E05 Second Break Cap L* Value (dark-light) 73.318 3.497 66.428 4.274 1.03E28 Cap a* Value (green-red) 4.282 1.216 6.359 1.461 1.17E23 Cap b* Value (blue-yellow) 24.796 3.168 27.600 2.440 2.72E12 Cap Diameter 42.833 5.946 43.005 4.063 8.00E01 Cap Height 24.156 2.112 25.313 2.234 1.19E04 Cap Flesh Thickness 12.359 1.048 12.526 1.163 2.71E01 Stipe Diameter 16.115 1.904 15.878 2.022 3.75E01 Relative Cap Height 0.569 0.053 0.590 0.035 6.90E04 Relative Cap Flesh Thickness 0.293 0.040 0.292 0.025 9.06E01 Relative Stipe Diameter 0.379 0.038 0.370 0.035 5.72E02

[0055] Table 5A below provides qualitative comparisons between mushroom strain RO12756 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 RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00008 TABLE 5A Characteristic RO12756 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 Smaller Larger Cap Height Shorter Taller Cap Flesh Thickness Similar Similar Stipe Diameter Larger Smaller Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Larger Smaller 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 Similar Similar Cap Height Shorter Taller Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Larger Smaller

[0056] Table 5B below provides quantitative comparisons between mushroom strain RO12756 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 RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, 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 RO12756 B14528 Characteristic Average St. Dev. Average St. Dev. p-value First Break Cap L* Value (dark-light) 74.237 3.524 67.579 5.925 1.560E47 Cap a* Value (green-red) 3.899 1.581 5.987 1.854 3.850E34 Cap b* Value (blue-yellow) 24.770 2.559 27.637 2.363 3.510E29 Cap Diameter 40.571 3.829 43.218 3.827 6.590E13 Cap Height 24.199 1.994 26.568 1.872 2.430E33 Cap Flesh Thickness 13.069 1.395 13.057 1.012 9.210E01 Stipe Diameter 18.258 2.139 17.787 2.018 1.790E02 Relative Cap Height 0.598 0.033 0.616 0.030 2.260E09 Relative Cap Flesh Thickness 0.323 0.028 0.303 0.024 3.050E14 Relative Stipe Diameter 0.451 0.040 0.413 0.042 1.900E21 Second Break Cap L* Value (dark-light) 73.318 3.497 68.288 5.217 8.20E13 Cap a* Value (green-red) 4.282 1.216 5.256 1.241 3.51E07 Cap b* Value (blue-yellow) 24.796 3.168 26.285 2.185 4.48E04 Cap Diameter 42.833 5.946 41.712 3.417 1.35E01 Cap Height 24.156 2.112 25.062 2.039 4.14E03 Cap Flesh Thickness 12.359 1.048 11.868 1.370 7.02E03 Stipe Diameter 16.115 1.904 14.725 1.442 2.04E07 Relative Cap Height 0.569 0.053 0.602 0.033 4.09E06 Relative Cap Flesh Thickness 0.293 0.040 0.286 0.036 2.14E01 Relative Stipe Diameter 0.379 0.038 0.355 0.044 1.48E04

Additional Trials

Tray-Level Comparisons

[0057] Table 6A below provides qualitative comparisons between mushroom strain RO12756 and strain BR06. Traits of particular interest are bolded. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00010 TABLE 6A Characteristic RO12756 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 Higher Lower Third Break Higher Lower All Breaks Combined Higher Lower

[0058] Table 6B below provides quantitative comparisons between mushroom strain RO12756 and strain BR06.

[0059] The first part of analyzing trait measurements in the additional trials herein included making comparisons between all three of instant strain RO12756, 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.

[0060] 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.

[0061] 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 RO12756 11.4 11.0 1.00 8 a BR06 12.6 13.0 1.00 8 b First Break Days to 1st Break RO12756 17.5 17.0 1.00 8 a BR06 18.5 18.0 1.00 8 b RO12756 11.91 12.40 4.88 8 a BR06 11.96 12.69 9.52 8 a Second Break Days to 2nd Break RO12756 24.8 25.0 2.00 7 a BR06 25.5 25.0 1.00 7 b RO12756 8.54 8.15 2.64 8 a BR06 7.37 5.96 4.39 8 b Third Break RO12756 4.78 4.83 1.86 8 a BR06 3.66 2.78 2.78 8 b All Breaks Combined RO12756 25.29 24.95 4.15 8 a BR06 23.00 23.48 5.81 8 b Significance Percent Harvested Speed Group (measured i nFirst Break) SD * 100 (Tukey HSD) RO12756 48.2% 50.9% 16.30 8 b BR06 28.6% 27.4% 20.90 8 a

[0062] Table 7A below provides qualitative comparisons between mushroom strain RO12756 and strain B14528. Traits of particular interest are bolded. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00012 TABLE 7A Characteristic RO12756 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

[0063] Table 7B below provides additional quantitative comparison between mushroom strain RO12756 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 RO12756 11.40 11.00 1.00 8 a B14528 11.40 11.00 1.00 8 a First Break Days to 1st Break RO12756 17.50 17.00 1.00 8 a B14528 17.50 17.00 1.00 8 a RO12756 11.91 12.40 4.88 8 a B14528 12.55 12.25 5.27 8 a Second Break Days to 2nd Break RO12756 24.80 25.00 2.00 7 a B14528 25.00 25.00 0.50 7 a RO12756 8.54 8.15 2.64 8 a B14528 8.45 8.01 2.39 8 a Third Break RO12756 4.78 4.83 1.86 8 a B14528 5.52 5.57 2.88 8 a All Breaks Combined RO12756 25.29 24.95 4.15 8 a B14528 26.51 27.10 5.76 8 a Percent Harvested Speed Significance (measured in Group First Break) Mean Median SD * 100 (Tukey HSD) RO12756 48.2% 50.9% 16.30 8 b B14528 53.5% 55.7% 14.00 8 b

Individual Mushroom-Level Comparisons

[0064] In additional evidence of the distinctness and superiority of mushroom strain RO12756, 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.

[0065] Table 8A below provides qualitative comparisons between mushroom strain RO12756 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 RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00014 TABLE 8A Characteristic RO12756 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 Similar Similar Cap Roundness (Calculated) 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 Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Flatter Rounder

[0066] Table 8B below provides additional quantitative comparisons between mushroom strain RO12756 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) RO12756 72.12 72.29 4.81 580 a BR06 63.30 62.74 9.83 797 b Cap a* Value (green-red) RO12756 5.62 5.66 1.88 580 a BR06 8.67 8.96 2.58 787 b Cap b* Value (blue-yellow) RO12756 26.98 27.06 4.11 580 a BR06 28.72 29.04 3.17 776 b Cap Apex Flesh Thickness RO12756 0.55 0.55 0.06 575 a BR06 0.52 0.52 0.05 541 b Cap Edge Flesh Thickness RO12756 0.22 0.23 0.10 569 a BR06 0.21 0.21 0.09 537 a Cap Roundness (Calculated) RO12756 0.61 0.61 0.04 579 a BR06 0.61 0.61 0.04 541 a Second Break Cap L* Value (dark-light) RO12756 73.02 72.87 4.35 200 a BR06 65.68 65.49 5.90 340 b Cap a* Value (green-red) RO12756 4.84 4.95 1.64 200 a BR06 7.21 7.31 2.42 340 b Cap b* Value (blue-yellow) RO12756 26.55 26.83 3.24 200 a BR06 28.15 28.49 2.86 340 b Cap Apex Flesh Thickness RO12756 0.54 0.54 0.04 198 a BR06 0.51 0.52 0.07 362 b Cap Edge Flesh Thickness RO12756 0.27 0.27 0.10 199 a BR06 0.23 0.23 0.09 362 b Cap Roundness (Calculated) RO12756 0.56 0.56 0.05 199 a BR06 0.60 0.59 0.06 363 b

[0067] Table 9A below provides qualitative comparisons between mushroom strain RO12756 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 RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00016 TABLE 9A Characteristic RO12756 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 Similar Similar Cap Roundness (Calculated) 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 Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Flatter Rounder

[0068] Table 9B shows additional quantitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1) and each strain, column 2 shows the mean value of the characteristic, 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) RO12756 72.12 72.29 4.81 580 a B14528 64.06 64.13 6.56 340 b Cap a* Value (green-red) RO12756 5.62 5.66 1.88 580 a B14528 8.26 8.31 2.13 340 c Cap b* Value (blue-yellow) RO12756 26.98 27.06 4.11 580 a B14528 30.31 30.16 3.39 340 c Cap Roundness (Calculated) RO12756 0.61 0.61 0.04 579 a B14528 0.61 0.61 0.05 389 a Cap Apex Flesh Thickness RO12756 0.55 0.55 0.06 575 a B14528 0.47 0.46 0.06 387 c Cap Edge Flesh Thickness RO12756 0.22 0.23 0.10 569 a B14528 0.22 0.22 0.08 387 a Second Break Cap L* Value (dark-light) RO12756 73.02 72.87 4.35 200 a B14528 68.56 69.02 6.54 170 c Cap a* Value (green-red) RO12756 4.84 4.95 1.64 200 a B14528 6.09 5.96 2.05 170 c Cap b* Value (blue-yellow) RO12756 26.55 26.83 3.24 200 a B14528 27.37 27.78 3.22 170 c Cap Roundness (Calculated) RO12756 0.56 0.56 0.05 199 a B14528 0.60 0.60 0.05 198 b Cap Apex Flesh Thickness RO12756 0.54 0.54 0.04 198 a B14528 0.46 0.46 0.05 198 c Cap Edge Flesh Thickness RO12756 0.27 0.27 0.10 199 a B14528 0.23 0.23 0.06 198 b

[0069] One of the advantages of the strain RO12756 is its incompatibility with other commercial strains. Table 10 below provides results of compatibility tests between strain RO12756 and commercial strain BR06, commercial strain B14528, or related strain RO10425. These results indicate that strain RO12756 is incompatible with strain BR06, strain B14528, and 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 RO12756/RO12756 2.62 12.79 2.61 12.74 0.41 2.00 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 RO12756/BR06, 0.70 3.42 0.62 3.03 0.25 1.22 2 b BR06/RO12756 RO12756/B14528, 0.68 3.32 0.60 2.91 0.43 2.10 1 b B14528/RO12756 RO12756/RO10425, 0.91 4.44 1.04 5.08 0.35 1.71 1 b RO10425/RO12756