Hybrid mushroom strain B19414 and methods and uses thereof

Abstract

Hybrid mushroom cultures of Agaricus bisporus, designated strain B19414, include representative cultures of the strain deposited under NRRL Accession No. 68095. A method of making a hybrid mushroom culture of Agaricus bisporus, comprising crossing a homokaryon strain designated B12998-s181, a culture of which has been deposited under NRRL Accession No. 68094, with a homokaryon strain designated P2-s203, a culture of which has been deposited under NRRL Accession No. 68093. Additionally provided are dependent varieties, mushrooms, portions of the cultures, products incorporating the cultures, and uses of the cultures.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 322.793, filed March 23, 2022, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a novel class of cultures of the edible cultivated mushroom fungus Agaricus bisporus (Lange) Imbach, and methods for making and using said cultures. More particularly, the present invention relates to a newly developed hybrid strain designated B19414, and cultures that are descendants of or otherwise derived from the Agaricus bisporus B19414 strain, including dependent cultivars. [Background technology]

[0003] The button mushroom, Agaricus bisporus (Lange) Imbach var. bisporus, is a microorganism belonging to the Basidiomycete fungi, widely cultivated as a food product worldwide and is the most widely cultivated mushroom species in Europe and North America. Agaricus bisporus cultivars are dominated by two species, one with a white cap and one with a brown cap. The market is further divided into two marketing categories: medium-sized (25-50 mm) closed cap or button-shaped mushrooms (brown, often called cremini) and open or flat mushrooms at the mature stage of fruiting body formation (flat mushrooms). Two species are well established in the open or flat mushroom marketing category: "Breakfast flat" and "Portobello".

[0004] Cultures of Agaricus, like cultures of other microorganisms, are prepared, maintained, grown, and stored in sterile media using microbiological laboratory methods. Manipulations on cells of pure cultures are performed using sterile tools and techniques in clean rooms or sterile transfer hoods for various purposes including clonal propagation, and for developing new strains using a variety of techniques including germination of spores in sterile growth medium and controlled mating in sterile growth medium. Commercial culture inoculum, including mushroom "spawn" and "casing inoculum", are also prepared using large-scale microbiological production methods, for example, by aseptically introducing an inoculum of a pure culture of a strain of Agaricus bisporus into 1 to 14,000 liters of sterile growth medium under sterile conditions, and provided to the end user as a pure culture in a sterile growth medium contained in a sterile package.

[0005] Commercial cultivation of mushrooms is carried out in dedicated structures on dedicated farms. There are many variations in the method, but a typical one is described below. Finished compost is prepared from lignocellulosic material such as wheat straw, enriched with nitrogenous material, and pasteurized in a suitable facility. A pure culture of one mushroom strain is aseptically incorporated by inoculum, then the mushroom spawn containing the sterilized friable "carrier substrate" on which it is grown is mixed with the pasteurized compost and incubated at a controlled temperature for about 13 to about 19 days, during which the mycelium of the mushroom culture colonizes and begins to digest the entire compost mass. A non-nutritive "casing layer" of material such as peat mixed with lime is then placed on top of the compost to a depth of about 40 to 50 mm. An additional "casing inoculum" incorporating the same mushroom culture can be incorporated into the casing layer to accelerate mushroom formation and harvest, and also to improve the uniformity of the distribution of mycelium and mushrooms within and on the casing surface. Environmental conditions, including temperature and humidity, within the planting facility are then carefully controlled to promote and control the transition from vegetative to reproductive growth of the culture at the casing / air interface. After an additional 13-18 days after casing, the mushrooms develop to a stage suitable for harvest and sale. This is called a flush or break. The first flush or first break of mushrooms containing the original culture can be harvested over a period of 3-4 days. Additional flushes of mushrooms (i.e., second or third flush, or second or third break) occur at approximately one-week intervals. Commercially, two or three flushes or breaks of mushrooms occur within the planting facility, followed by harvest and removal of the compost, which is then replaced.

[0006] Generally speaking, strains may be selected for or against a variety of mushroom-related traits, such as mushroom size (including cap diameter), mushroom shape (e.g., cap roundness, flesh thickness), color (i.e., white cap vs. brown cap), surface texture (e.g., cap smoothness), tissue density and / or hardness, delayed maturation, basidiospore counts of 2 or more, absence of spores, increased dry matter content, improved shelf life, and reduced spoilage, as well as increased crop yield, shift in yield distribution over time, reduced inoculum-to-harvest interval, reduced resistance to bacterial and viral diseases, to name a few. The differentiation can be based on traits associated with the mushroom cultures themselves, and / or the products in which they are incorporated, and / or the crops in which they are incorporated, including resistance to infection, symptoms, or spread of fungal diseases, resistance to insects, resistance to nematodes, ease of crop management, suitability of the crop for mechanical harvesting, and behavioral responses to environmental conditions including stressors, nutritional substrate composition, seasonal effects, farm practices, self / non-self interactions (compatible or incompatible) with different mushroom strains. Strains can also be differentiated based on their genotypic fingerprints (the presence of specific alleles at specially designed marker loci in the nuclear or mitochondrial genome). Strains can have different ancestries, which are reflected directly in the genotype and, in some cases, indirectly in the phenotype.

[0007] Between 5 and 30 percent of Agaricus mushrooms grown in the United States, Europe, and elsewhere have a brown cap, depending on consumer preference for the appearance of the conventional or "old fashioned" product. The "Portobello" mushroom market segment is dominated by cultivation of strains with a brown cap (=brown). Market demand for brown mushrooms in the United States and elsewhere is relatively strict with respect to many observable phenotypic traits, such as size, shape, color, color retention, hardness, and shelf life. Thus, genetically distinct strains of commercially successful brown Agaricus bisporus mushrooms that can be differentiated based on mushroom appearance may be important in some cases.

[0008] Around 1980, the first two white hybrid strains of A. bisporus, developed in a laboratory in Horst, The Netherlands, were introduced into commercial cultivation. These two "Horst" strains, designated U1 and U3, are closely related hybrids created by crossing two existing white cultivars, as described in M. Imbernon et al., Mycologia, 88 (5), 749-761 (1996), which is incorporated herein by reference. The two parents of U1 and U3 are commercial strains belonging to two long-standing taxonomic types of strains known as "smooth-white" (SW) and "off-white" (OW). The original homokaryons (or "lines") obtained from SW and OW strains and used in the cross to create U1 were named H39 and H97, respectively. It is possible that these cultures no longer exist (A. Sonnenberg, personal note).

[0009] However, many laboratories have deheterokaryotized the U1 strain, resulting in neohaplont cultures incorporating either the karyotype corresponding to that contributed by H39 or H97, as well as the mitochondrial type of U1. In the industry, these two types of neohaplonts of U1 are taxonomically referred to as SWNC lineages or homokaryons and OWNC lineages or homokaryons, respectively. The OWNC lineage, designated "H97", was deposited by A. Sonnenberg at the public microbial stock collection Fungal Genetics Stock Center of Kansas, USA under number 10389, and at the public collection American Type Culture Collection of Maryland, USA under number MYA-4626. The genome of H97 has been sequenced and placed in the public domain by the Joint Genome Institute of California, USA (see Morin et al. 2012, incorporated herein by reference).

[0010] One traditional type of brown-capped strain of A. bisporus mushroom, often referred to as the "Old-Fashioned Brown" strain (or "OFB"; examples of OFB strain types are Sylvan strains SB-65, SB-295, and RWK-2042), originated from a wild strain in Europe and was the predominant brown cultivar strain for many decades, and is becoming the only brown cultivar in widespread use during the last years of the 20th century. Several different brown-capped hybrids have been developed since the 1980s, some with some commercial success.

[0011] Heterokaryon spores of the original strain retain the majority of the genotype of the parent (this behavior was shown by R.W. Kerrigan et al. in Genetics, 133, 225-236 (1993), incorporated herein by reference). A group of strains developed from a single ancestor (as opposed to outcrossing between two different ancestors), either by cloning, or by spore culture, or by any other method of "essential derivation" as discussed below, is referred to as a derived lineage. Many commercial mushroom strains developed from OFB stocks meet the criteria for a dependent variety (as the term applies to plant varieties).

[0012] Agaricus bisporus has a reproductive syndrome known as amphithallism, in which two separate life cycles function simultaneously. Like other fungi, reproductive propagules are spores. Agaricus produces spores by meiosis in meiotic sporangia known as basidiophores. In the first life cycle, A. bisporus spores each receive only one haploid post-meiotic nucleus; these spores are capable of mating but not of giving rise to mushrooms. These haploid spores germinate to give rise to homokaryon progeny or lineages that can be crossed with other compatible homokaryons to create novel hybrid heterokaryons capable of giving rise to mushrooms. Heterokaryons generally exhibit a much lower mating ability than homokaryons. This life cycle is called heteromixis and is similar to outbreeding. Although this life cycle is functional, it is not generally predominant in strains of Agaricus bisporus var. bisporus.

[0013] The second life cycle, termed intramixis, is similar to a form of inbreeding and predominates in most strains of Agaricus bisporus var. bisporus. The majority of spores receive two postmeiotic nuclei, and the majority of such nuclei pairs consist of Non-Sister Nuclear Pairs (NSNPs) with heteroallelic genotypes at most or all centromeric linked loci, including the MAT locus. The MAT genotype determines the heterokaryon phenotype of these progeny that are fertile and capable of giving rise to mushroom crops. Unusually among eukaryotes, it has been observed that relatively little (if any) chromosomal crossover occurs in the postmeiotic progeny of A. bisporus; empirically, very little heteroallelicity (analogous to heterozygosity) is lost in the heterokaryon progeny of heterokaryon strains.

[0014] Functionally, in conjunction with intramixis, recombination occurs at a low rate across many of each chromosome, resulting in parental and intramixed progeny heterokaryon genotypes and phenotypes that tend to resemble each other. This provides a preferred method for deriving new cultures from an original culture that have minor, minor genetic changes, but retain the important characteristics of the original culture. Cultures derived by this or alternative methods, either exclusively or primarily from one original culture (as in backcrossing), are referred to as dependent varieties (also known by the acronym EDV). EDVs can be virtually indistinguishable from their original culture. Derivation is a well-known strain development technique in the art and among commercial mushroom seed producers, especially when the commercial objective is to obtain and exploit a "knockoff" of a protected culture, without simply cloning or copying the original culture.

[0015] As mentioned above, the first hybrid strains of A. bisporus developed with a heteromixic life cycle in a laboratory in Horst, The Netherlands, were introduced into commercial cultivation around 1980. These two white "Horst" strains, designated U1 and U3, were derived by crossing two existing white cultivars, and after the successful commercial release of the white U1 strain and related dependent varieties (as defined herein), this first commercially successful hybrid brown cultivar entered the market in the early 1990s with the release of Sylvan 600, patented as X618 (U.S. Patent Plant 7,636). Since about 2008, the most widely sold commercial brown cultivar is Heirloom, a strain protected by U.S. Patent No. 7,608,760. In 2014, Sylvan's Tuscan Brown cultivar line (also known as Tuscan-860 and in U.S. Patent No. 9,642,333 as B14528) was also successfully released.

[0016] Also, as noted above, the mushroom market is divided into two broad categories: closed cap and flat mushrooms. While current brown strains, e.g., Heirloom, Tuscan-820 and Tuscan-860, have adequate flat mushroom performance, there is a need for strains with thinner caps and larger cap diameters that tend to produce better portobellos or breakfast flats. Thus, two important mushroom sales niches: the North American portobello market and the UK / Ireland flat mushroom market, desire a wider and flatter mushroom shape with a thinner cap. Flat mushrooms are treated quite differently from button mushrooms in home and restaurant kitchens. For example, in North America, portobellos are commonly marinated and grilled and are commonly consumed as a meat substitute. In the UK and Ireland, flat, open mushrooms are deep-fried and form part of the traditional Irish or British "fried breakfast". For both of these applications, the flatter shape is preferred. For ease of discussion, hereafter references to "pot bella" or "pot bella mushroom" mean any flat mushroom with a closed cap, brownish cap, as distinguished from a button mushroom.

[0017] In the UK and Ireland, 26-30% of mushrooms from the first break are harvested as either "breakfast flats" or pot bellas, with smaller amounts of second break produce also thought to enter the flat mushroom market. In fact, 4.2 million pounds of pot bellas are grown each year in the UK and Ireland, with a market value of €4.2 million. These mushrooms can be brown or white.

[0018] In the US and Canadian markets, there is a trend to use brown stocks to grow larger sized (70-100mm) pot bella. In 2019-2020, 183.7 million pounds of brown mushrooms were produced in the US (USDA NASS). Approximately 35-40% of this total production (64-73.5 million pounds) were pot bella. These larger mushrooms brought in a total value of $99-104 million.

[0019] Smaller mushrooms from such crops, and mushrooms with later breaks, are sold as button mushrooms. For example, about two-thirds of the total brown crop is harvested as closed button mushrooms. From the preceding information, it will be appreciated that any mushroom strain used to produce large open or flat mushrooms must also be capable of producing good quality button mushrooms in sufficient quantities to result in an optimally profitable product mix. Summary of the Invention [Problem to be solved by the invention]

[0020] Hygiene is very important for protected (i.e. enclosed) crops such as mushrooms. Mushroom viruses, especially La France disease and Mushroom Virus X, can be devastating diseases and are spread via infected spores or contaminated compost substrates. When growers switch to a strain that is not vegetatively compatible with the strain previously used, these two main routes of spread can be interrupted by taking advantage of the dramatic reduction in anastomosis (cell fusion) between incompatible heterokaryonous strains. The end result is a "breakthrough" of the disease and a restoration of yield and quality. Thus, one advantage of having more than one strain available is the option to use alternative strains to achieve disease breakthrough against highly transmissible diseases such as mushroom viruses. More generally, it is well established that monoculture of crops poses a significant risk of widespread crop failure due to a common weakness. General diversification of crops in production manages this risk of monoculture. Having multiple genetically diverse commercially acceptable choices provides a solution to this problem.

[0021] Further, there is a need for strains of Agaricus bisporus that produce mushroom crops having brown caps and three break yields at least equivalent to those of accepted commercial strains, particularly the Heirloom strain, a representative culture of which has been deposited with the American Type Culture Collection (ATCC) under the designation BR06 and ATCC Accession No. PTA-6876, and which is disclosed in U.S. Patent No. 7,608,760. A further need exists for generating strains of Agaricus bisporus that produce mushrooms with larger cap diameters than other known commercially available brown cap mushrooms, including Heirloom (see above), Tuscan-860 (a representative culture of strain Tuscan-860 has been deposited with the Agriculture Research Service Culture Collection (NRRL) under NRRL accession number 50900), Tuscan-820 (a representative culture of strain Tuscan-820 has been deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Paris, France under CNCM accession number I-5527), and Brawn (a representative culture of strain Brawn has been deposited with the Agriculture Research Service Culture Collection (NRRL) under NRRL accession number 68258).

[0022] Additionally, there is a need to generate Agaricus bisporus strains that produce mushrooms with a thinner flesh thickness and flatter cap at maturity, or a smaller proportion of stalk tissue, compared to Heirloom. There is a further need for strains that have the aforementioned attributes while also possessing genetic novelty that enhances crop diversification. [Means for solving the problem]

[0023] The present invention is generally directed to a new and distinct Agaricus bisporus mushroom culture designated B19414, which is a hybrid strain obtained by directional mating of two homokaryon cultures. A culture of strain B19414 has been deposited at the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit is March 9, 2022. The deposited culture was obtained from the same culture maintained by the assignee of record, Sylvan Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to satisfy all deposit requirements of the US Patent and Trademark Office, including 37 CFR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. The NRRL accession number is 68095. The deposit will be maintained in the depository institution for 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and will be replaced as necessary during that period. The culture will be irrevocably and without restriction or conditions available to the public upon the filing of a priority application or the issuance of a patent on the strain, in accordance with the patent laws.

[0024] The B19414 strain was obtained by directional mating of two homokaryonous parents, namely, strains B12998-s181 and P2-s203. A culture of strain B12998-s181 has been deposited at the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit is March 9, 2022. The deposited culture was obtained from the same culture maintained by the assignee of record, Sylvan Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to satisfy all deposit requirements of the US Patent and Trademark Office, including 37 CFR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. The NRRL accession number is 68093. The deposit will be maintained in the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be replaced as necessary during that period. The cultures will be irrevocably and without restriction or conditions available to the public upon the filing of a priority application or the issuance of a patent on this strain, in accordance with the patent laws.

[0025] Similarly, a culture of the P2-s203 line has been deposited at the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit is March 9, 2022. The deposited culture was obtained from the same culture maintained by the assignee of record, Sylvan Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions on the deposit are removed, and the deposit is intended to satisfy all deposit requirements of the US Patent and Trademark Office, including 37 CFR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. The NRRL accession number is 68094. The deposit will be maintained in the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be replaced as necessary during that period. The culture will be irrevocably and without restriction or conditionally made available to the public upon filing of a priority application or issuance of a patent relating to this strain in accordance with patent laws.

[0026] Thus, the invention includes a method of producing a mushroom culture of Agaricus bisporus comprising crossing a homokaryon strain designated B12998-s181, a culture of which has been deposited under NRRL Accession No. 68094, with a homokaryon strain designated P2-s203, a culture of which has been deposited under NRRL Accession No. 68093.

[0027] It is noted that cultures of the B19414 strain encompass mushrooms, parts of mushrooms including spores, and parts of the culture including caps, stalks, gills, cells, nuclei, mitochondria, protoplasts, cell walls and membranes, hyphae, and mycelium. It is noted that cultures of the B19414 strain give rise to strains and lineages, which are derived from at least a portion of such cultures, or strains and lineages can be derived from at least a portion of such cultures. Thus, the present invention encompasses cultures of the B19414 strain resulting from the B19414 strain, i.e., mushrooms and parts of mushrooms including spores, subordinate varieties (EDV) (defined below and generally including cultures derived solely or primarily from an initial culture of the B19414 strain [as by repeated backcrossing]), dormant or active growth cultures present in dormant or germinating spores of the B19414 strain, and cultures into which genetic material of the B19414 strain has been incorporated. It is noted that cultures of the B19414 strain can give rise to, or be capable of giving rise to, edible mushroom crops having the characteristics described below. The present invention is also directed to methods of making and using the B19414 strain. Uses of cultures of B19414, and the other cultures mentioned above, include incorporation of the cultures into commercial products such as mushroom spawn and casing inoculants, as well as mushroom production, development of additional new cultures of A. bisporus, and farm hygiene, including crop diversification and "virus breaking."

[0028] With respect to spores, live spores are dormant heterokaryons or homokaryons. Spores are part of the mushroom organism culture and incorporate only the genetic material of the single culture (often conveniently referred to as the "parent") that gave rise to them. The other parts of the culture include the cap, stalk, rhizobia, cells (defined as the mycelial compartment in which the nucleus, mitochondria, protoplasts, cell membranes, and cell walls, including the transverse wall, are incorporated), hyphae, and mycelium. Spores can be aseptically collected on sterile materials, suspended in sterile water at various dilutions, and plated on sterile agar growth medium to produce germinated spores and incorporate the culture into the spores. A preferred technique is to place a live Agaricus culture in a sealed Petri plate, which can stimulate spore germination by diffusing a volatile pheromone. Germinated spores can be isolated on new nutrient agar plates using a sterile microtool such as a steel needle under a microscope. Using this method, it is possible to obtain heterokaryon and homokaryon progeny of the B19414 strain, including spores of the B19414 strain and cultures integrated within the spores.

[0029] The development of novel hybrid varieties by heteromixis involves the controlled physical association and mating of two compatible cultures to obtain novel heterokaryon cultures. Homokaryons (= "lines") are the preferred starting cultures for mating, as they have the greatest capacity to anastomose with other cultures and achieve cytoplasmic fusion. Heterokaryons can also be brought into physical contact, but the probability of a mating resulting in the successful formation of a novel heterokaryon is commercially unreasonably low. Compatibility is determined by the genotype at the MAT locus; two homokaryons with the same MAT alleles cannot establish a heterokaryon after anastomosis, and thus homokaryon compatibility becomes a genetic difference. In the breeding program, homokaryon lines are obtained and related in defined pairwise combinations. In one method, homokaryon pairs are placed in close proximity on the surface of nutrient agar in a petri dish and allowed to grow together (in physical association) until the point where anastomosis occurs between the two cultures. Mating successfully results in heterokaryons. By displacing mycelium from the fusion zone of the dish, novel hybrid heterokaryons can be obtained. Using such a pairwise mating method between B12998-s181 and P2-s203, strain B19414 was obtained.

[0030] Advantageously, the B19414 strain tends to produce better pot bellies or breakfast flats due to the thin cap flesh. The B19414 strain produces mushrooms with thin cap flesh, which results in a flat shape. Furthermore, the B19414 strain is advantageously a mushroom strain that can be used to produce large open or flat mushrooms, but also produce good quality button mushrooms in sufficient quantities to produce an optimally profitable product mix.

[0031] Moreover, strain B19414 has been found to advantageously produce potbellied mushrooms at first break that are larger in diameter than any other commercially available brown mushroom at the same stage of maturity, including those selected from the group consisting of Heirloom, Tuscan-820, Tuscan-860 and Brawn. It will be understood that cap diameter is measured at the time of membrane rupture of the mushroom.

[0032] Mushroom morphology can be divided into four broad categories; cap, gill, stalk and membrane. For all Agaricus mushrooms, both brown and white, the goal is to harvest the mushrooms before the membrane ruptures. There are two reasons for this. First, mushrooms with open membranes will lose moisture and lose weight, thereby affecting harvest weight and ultimately profit. Second, mushrooms with ruptured membranes may begin to release spores, which can inhibit the growth of new mushrooms. More importantly, released spores have been found to spread several mushroom viruses, which can have devastating effects on yield and quality.

[0033] Additionally, if the mushrooms are left on the bed past the point of membrane rupture, the cap edge will begin to curl upwards as the spores are released. Growers prefer to harvest when the underside of the cap edge is "rolled under", which occurs when the membrane is partially or fully attached.

[0034] Further advantage is found in B19414 strains that produce mushrooms with a flesh thickness ratio of less than 0.49, and in some embodiments, less than 0.40.

[0035] B19414 strain can also give rise to homokaryons and heterokaryons, and thus homokaryons and heterokaryons can be obtained from the strain and EDV defined thereunder. Several homokaryons have been obtained from B19414 strain, including B19414-s9 and B14919-s19. Such homokaryons can be suitable for use in the strain development method described immediately above, i.e., the directed pair mating method, in which two compatible preselected homokaryons are used to obtain F1 hybrid strains. In this method, the homokaryon can be used as the first parent and crossed with the second homokaryon. In such a case, the first parent is a lineage or homokaryon derived from B19414 strain or EDV of B19414 strain.

[0036] In contrast, an EDV is derived directly from only or primarily a single primary culture (e.g., strain), and all such derivations give rise to an EDV. This definition is consistent with the broadly understood term. Methods that result in cultures that are, by definition, EDVs of a single primary culture of A. bisporus, i.e., "strain development methods," include somatic selection, tissue culture selection, single spore germination, multiple spore germination, selfing, repeated backcrossing to the primary culture, mutagenesis, and transformation, to give some examples. DNA-mediated transformation of A.bisporus has been reported by Velcko, AJ Jr., Kerrigan, RW, MacDonald, LA, Wach, MP, Schlagnhaufer, C., and Romaine, CP 2004, Expression of novel genes in Agaricus bisporus using an Agrobacterium-mediated transformation technique. Mush. Sci. 16: 591-597, which is incorporated herein by reference, and references therein. Transformation can introduce a single new gene or allele into the genome of the original culture. Furthermore, recent reports on other closely related fungi have raised the possibility of CRISPR gene editing.

[0037] EDVs can be clearly recognized by their genotypes, which are primarily or more often entirely a subset of a single initial culture. The percentage of the initial genotype that will be present in Agaricus bisporus EDVs ranges from 100% or virtually 100% in the case of single spore cultures and somatic selection, to 99.x% in the case of strains modified by DNA-mediated transformation, to 90-99.x% in the case of some single or multiple spore selections or some mutagenesis, to an average of at least 75-85% in the case of sib-offspring mating (=selfing) and backcrossing to the initial culture. Many well-known genotyping methods can be used to determine the percentage of DNA from the initial culture present in another culture, and to make an unequivocal determination of the relatedness of the two cultures and any method used to manipulate or exploit the initial culture, including the methods described below and others known in the art.

[0038] Repeated backcrossing to the initial culture also results in the EDV of the initial culture. In one hypothetical example, in the first successive iterations of this process, the resulting strains of this generation have, on average, about 75% of the DNA of the initial strain, while about 25% of the DNA is contributed by the second strain or lineage. As this process is repeated, the DNA corresponding to the initial strain increases to an average of at least 87% after one further backcross, to an average of at least 94% after an additional backcross, and approaches an average of 97% after three further successive iterations. It should be understood that any culture that is a descendant of (as opposed to derived from) the initial culture has only 50% genotypic identity to the initial culture, so any culture that has 75-100% genotypic identity to the initial culture will exhibit the EDV of the initial culture. The EDV of the EDV is also established to be the EDV of the initial strain, given that both the first EDV and the second EDV have very close genetic identity to the initial strain. Finally, because Agaricus bisporus alternates between heterokaryon and homokaryon lineages, the essential criteria of derivation apply equally to cultures of both strains and lineages.

[0039] Thus, the invention further includes a culture of Agaricus bisporus directly derived from an initial culture of strain B19414, which culture has been deposited under NRRL Accession No. 68095, and thus all of the genome or genotype of the directly derived culture of Agaricus bisporus is present in the genome or genotype of the original culture of strain B19414. Further, the invention includes a culture of Agaricus bisporus derived from an initial culture, which initial culture is strain B19414, and thus at least 75% of the genome or genotype of the derived culture of Agaricus bisporus is present in the genome or genotype of the original culture of strain B19414, which culture of strain B19414 has been deposited under NRRL Accession No. 68095.

[0040] A genotypic fingerprint is a description of a genotype at a specially prepared locus, where the presence of a characterized allele is recorded. Such fingerprints provide a powerful and effective technique for recognizing clones of the original strain and all types of EDV, as well as for recognizing ancestors that fall within the range of outbred lineages. Many techniques are available for defining and characterizing genotypic loci and alleles. The most detailed approach is provided by whole genome sequencing (WGS), which allows direct characterization and comparison of DNA sequences across the entire genome. Using this technique to generate robust genotypic fingerprints that incorporate a large number of marker loci, it is possible to establish the nature of the relatedness between two strains, including strains that are related by phylogenetic lineage over several generations. Applicant has tracked genetic markers over four to six generations of strain development pedigrees. If a sufficient number of rare markers are present in the original strain or lineage, it becomes possible to identify lineages from the original strain or lineage after several outbred generations without undue experimentation. In one hypothetical example, at the fourth outbred generation, the average expected genome percentage corresponding to the original haploid lineage in the F4 hybrid is 3.1% (50% / 24), which corresponds to about 1 Mb of nuclear genomic DNA of A. bisporus. Based on experimental and mathematical analysis, the amount of DNA from two unrelated strains of A. bisporus can generally contain about 10,000 to about 20,000 single nucleotide polymorphisms (SNPs), any one of which may provide an identification marker linking the F4 hybrid to the original lineage. By using a large number of independent markers, the origin of the lineage can be identified with a very high success rate and with a reasonable degree of confidence.

[0041] As mentioned above, one of the biologically and commercially interesting traits is heterokaryon incompatibility. The genetics of these self / non-self recognition systems in basidiomycetes such as Agaricus are not fully understood, but in other groups of fungi, multiple alleles at multiple independent loci are known to be involved. The presumed genotypic differences at the incompatibility loci prevent successful anastomosis and cytoplasmic continuity between physical mixtures of two or more heterokaryons. One consequence of such antagonistic responses is retarded growth and development, and reduced crop yield. Partial crop failure of this kind is well known and self-evident to growers with experience. Another consequence of heterokaryon incompatibility is limited opportunities for intracellular viruses to move freely throughout or between the hyphal network. Viral diseases, such as those caused by La France virus or MVX virus, can have a significant negative impact on the productivity of facilities and must be remedied using sanitation practices that can be assisted by rotation of strains. The method of improving mushroom farm hygiene, called "virus breakthrough", is carried out by replacing the planting materials (compost, seed culture, casing inoculant) that are incorporated into the original strain with inoculants and planting materials that are incorporated into another different strain that is incompatible with the original strain. The most effective implementation of the virus breakthrough method is to replace all biological material of the original strain in the mushroom farm with biological material of a second, incompatible strain. Strain incompatibility creates an effective, if not absolute, barrier to the transfer of viruses from the biological reservoir in the facility into the new crop. The infection and introduction cycle of exogenous pests and pathogens can also be interrupted by rotating the cultivation usage between genotypically different mushroom strains.

[0042] The strains currently available to the mushroom industry are those that allow growers to successfully and usually profitably produce mushroom crops. There are several factors that influence the degree of success and profitability that is realized. For example, the strain must be capable of producing at least equivalent crop yields over two to three breaks or flushes as compared to strains currently sold and grown commercially. Also, some physical characteristics of the resulting mushrooms, such as cap color categories, as well as general sizes and dimensions such as cap diameter, allow the mushrooms to be sold in familiar product categories.

[0043] Thus, another aspect of the present invention includes providing a culture that produces a first crop of Breakpot Bella mushrooms having a brown cap and a cap diameter greater than the cap diameter of a cultivar strain selected from the group consisting of Heirloom, Tuscan-820, Tuscan-860 and Brawn at the same stage of maturity. The culture may also produce a mushroom crop having a brown cap and a flesh thickness ratio of less than 0.49.

[0044] On the other hand, improving certain traits can confer a higher market value to the mushrooms and / or to the plants that produce those mushrooms, examples of such traits include cap shape and the proportion of less desirable stalk tissue in the mushroom.

[0045] Existing pot-bowl style mushrooms, which have a relatively rounded cap shape at maturity, are suitable for certain cooking methods (e.g., stuffing into a mushroom cap), whereas mushrooms with a flat cap shape are more suitable for general processing such as baking and are prepared as a traditional "breakfast flat" meal item, and such use is more desirable. Mushrooms with a smaller proportion of stalk tissue are more highly valued by consumers because less wasteful trimmings can be made, an important consideration when mushrooms are sold by weight. Thus, mushrooms and mushroom strains may have all of the above improvements and still fit into the established pot-bowl commercial product category.

[0046] The B19414 strain has been shown to produce mushroom crops with brown caps and three break yields at least equivalent to those of the Heirloom strain, which is a representative culture of a recognized commercial strain, in particular the Heirloom strain deposited under ATCC Accession No. PTA-6876 (US7,608,760) as BR06. The B19414 strain produces mushrooms with a thinner flesh thickness and flatter cap at maturity, or with a smaller proportion of stalk tissue, compared to Heirloom. More specifically, the B19414 strain has brown caps, a three break crop yield not less than the Heirloom strain, and a cap diameter that is at least as long (i.e., not significantly different) from the cultivar Heirloom strain at the same stage of maturity. The B19414 strain has the aforementioned attributes, while also possessing genetic novelty that enhances crop diversification. Thus, the B19414 strain is believed to meet a market need and resolve the current problem of non-availability of such strains in the market. Furthermore, the B19414 strain is genotypically distinct when compared to other current brown cultivars (see Tables I-IV below).

[0047] These and other advantages of the present invention over the existing prior art relating to Agaricus bisporus mushrooms and cultures will become apparent from the following description and are realized by the present invention as hereinafter described and claimed.

[0048] One or more aspects of the invention may be realized by a hybrid mushroom culture of Agaricus bisporus designated strain B19414, a representative culture of which has been deposited under NRRL Accession No. 68095. Strain B19414 may include various parts of the culture, including mycelia, spores, and cells and parts of cells, including nuclei, mitochondria, protoplasts, cell membranes, and cell walls, present in both the vegetative mycelium of the culture and in the mushrooms resulting from the culture. The spores may be dormant or germinated spores and may include heterokaryons and homokaryons incorporated therein.

[0049] One or more products can be made incorporating the hybrid mushroom culture of Agaricus bisporus designated strain B19414. Such products include mycelium, seed cultures, inoculants, casing inoculants, raw mushrooms, processed mushroom products, mushroom extracts and fractions, mushroom pieces, and isolated colonized substrates selected from grains, compost, and friable particulate matter. It will be understood that mushroom pieces refer to stalks, caps, and other larger parts of the mushroom itself. Mushroom spores can be dormant or germinated spores and can include heterokaryons and homokaryons incorporated therein.

[0050] One or more other aspects of the present invention can be realized by an EDV of a culture of the hybrid mushroom strain B19414. In one or more embodiments, the Agaricus bisporus culture produced by essential derivation has at least one of the essential characteristics of the B19414 strain, such as the same heterokaryon compatibility phenotype and / or other characteristics of cap roundness, flesh thickness, yield performance, and yield timing compared to the commercial strains Heirloom, Tuscan-820, and Tuscan 860. Here, the culture of the B19414 strain has been deposited under NRRL Accession No. 68095.

[0051] Another aspect of the present invention can be realized by a method for making a hybrid culture of Agaricus bisporus, comprising the step of crossing B12998-s181 (deposited under NRRL Accession No. 68093) with a second homokaryon, i.e., line P2-s203, a culture of which has been deposited under NRRL Accession No. 68094. Such crossing results in mushroom culture B19414, which exhibits antagonism against a panel of commercial cultivars; Heirloom, Tuscan-820, Tuscan-860, and Brawn. This antagonism demonstrates that the B19414 strain is genetically distinct. In one or more embodiments, the method further comprises providing a mushroom culture of the invention consisting of mycelium, seed culture, inoculum, casing inoculum, raw mushroom, processed mushroom, mushroom parts, mushroom extracts and fractions, mushroom pieces, and an isolated culture substrate selected from grains, compost, and friable particulate matter. In other embodiments, the method may comprise providing the mushroom culture as a derivative culture selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue explant cultures, protoplasts, resting spores, germinating spores, inbred and outbred progeny, transgenic cultures, gene edited cultures, and cultures having a genome with a single locus transposition.

[0052] Yet another aspect of the invention can be realized by a hybrid mushroom culture of Agaricus bisporus having a genotypic fingerprint having traits at the marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AF, and FF, all of the traits of the fingerprint being present in the genotypic fingerprint of strain B19414. In one or more embodiments, the culture has a genotypic fingerprint having traits at the marker loci set forth in Table III, all of the traits of the fingerprint being present in the genotypic fingerprint of strain B19414.

[0053] One or more further aspects of the invention may be realized by a culture, cell, or culture comprising the cell produced by the above method. Thus, one or more embodiments may include a method further comprising growing the hybrid mushroom culture to produce hybrid mushrooms and mushroom parts. Other embodiments may provide a method in which the hybrid mushroom culture, or cell produced comprises a marker profile having traits at the marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AF, and FF, all of the traits of the marker profile also present in the marker profile of the B19414 strain. Still other embodiments may provide a method in which the hybrid mushroom culture, or cell produced comprises a marker profile having traits at the marker loci listed in Table I, all of the traits of the marker profile also present in the marker profile of the B19414 strain.

[0054] Finally, another aspect of the invention can be realized by a method of using a hybrid mushroom culture selected from strain B19414 or a dependent variety of strain B19414, a representative culture of which has been deposited under RRL Accession No. 68095. In one embodiment, the method further comprises growing an edible mushroom crop by carrying out the steps described herein above. In another embodiment, the method can comprise using strain B19414 or a dependent variety of strain B19414 in a crop rotation to reduce pathogen pressure and pathogen reservoirs in a mushroom growing facility as described herein above. In yet another embodiment, the method can comprise using EDVs of strains 19414 and B19414 to generate homokaryons and progeny as described herein above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] First, in order to provide a clear and consistent understanding of the specification and claims, including the scope to which such terms are given, the following definitions are provided.

[0056] Allele: One or more alternative forms of a gene arising by mutation and found at the same location on a chromosome; a genetic unit of the genome at a specifically designated locus that is conclusively identified by its DNA sequence (or by other means).

[0057] Amphitheaterism: a reproductive behavioral pattern in which both heteromixis and intramixis are active.

[0058] Anastomosis: the fusion of two or more hyphae achieving cytoplasmic continuity.

[0059] Basidiomycota monophyletic group of fungi that produce meiospores in basidium; members of the corresponding subphylum of Fungi such as Basidiomycetales or Basidiomycotina.

[0060] Basidium: meiosporangial cell in which karyofusion and meiosis occur, resulting in the formation of basidiospores.

[0061] Bioefficiency: For mushroom crops, the net fresh weight of crop harvested for any given sampled area or compost weight divided by the dry weight of the compost substrate at the time of inoculation.

[0062] Breeding: The development of strains, lines, or varieties using methods that emphasize sexual mating.

[0063] Cap: fungal pileus; part of a mushroom, a gilled structure.

[0064] Cap Flatness: A measure of the shape or thickness of the cap of a mature, open mushroom.

[0065] Cap roundness: strictly speaking, the ratio of the maximum distance between the top and bottom of the cap, measured by bisecting the mushroom lengthwise, divided by the maximum distance across the cap; generally averaged over many specimens; subjectively, the "rounded" quality of the cap shape.

[0066] Carrier Substrate: A medium having both nutritional and physical properties suitable for both the growth and dispersion of cultures; examples are formulated substrates for mushroom spawn, casing inoculants, and other inoculants.

[0067] Casing layer, casing: A layer of non-nutritious material such as peat or soil applied to the upper surface of a colonized compost mass to allow the emergence of a mushroom crop.

[0068] Casing Inoculum (CI): An inoculum formulation suitable for mixing into the casing layer, generally incorporating specially prepared heterokaryonous mushroom cultures.

[0069] Cloning: Somatic proliferation without selection.

[0070] Combining ability: the ability of an individual to transmit superior performance to its offspring. Generally, combining ability is the average performance of an individual in a particular series of matings.

[0071] Compatibility: See heterokaryon compatibility, vegetative compatibility, sexual compatibility; incompatibility is the opposite of compatibility.

[0072] CRISPR: (Clustered Regularly Interspaced Short Palindromic Repeats) A genetic engineering technique whereby the genome of a living organism is altered.

[0073] Culture: substantial living organisms; organisms grown in various growth media and substrates; part or whole of a single physical strain, lineage, homokaryon or heterokaryon; the collection of all parts of a culture, including hyphae, mushrooms, spores, cells, nuclei, mitochondria, protoplasts, cell membranes and cell walls.

[0074] Cultivar: commercially grown variety or strain

[0075] Derivation: The development or derivation of a culture solely or primarily from an original strain or culture; see dependent variety. The terms "derive" and "derived" refer to this process or its results.

[0076] Descent clade: a set of subordinate varieties derived from a single original stock and including the original stock.

[0077] Pedigree: a genealogical lineage spanning a limited number of generations (e.g., 10 or fewer).

[0078] Diploid: having two sets of haploid chromosomes within a single nuclear envelope.

[0079] Directed mutagenesis: The process of altering the DNA sequence at at least one specific gene locus.

[0080] Dependent Variety (EDV): A culture derived exclusively or primarily from an original strain or culture; a culture in which 75% or more of its genotypes have the genotype of the original strain and whose condition is the result of its derivation.

[0081] Flesh thickness: the ratio of the greatest distance between the top of the stalk and the top of the cap, measured by bisecting the mushroom lengthwise, divided by the greatest distance across the cap; generally an average over many specimens; subjectively referred to as "flesh thickness."

[0082] Flush: A period within a crop cycle during which mushrooms occur, separated by intervals during which mushrooms do not occur; the term flush encompasses the terms "break" and "wave" and can be read as either of these terms.

[0083] Fungi: microorganisms classified as members of the kingdom Fungi.

[0084] Gene editing: The process of altering specific genes, typically by CRISPR-Cas9 or similar enzyme systems, which alter the sequence of a functional gene, rendering it inactive. Other uses can introduce new sequences (including genes) into a genome.

[0085] Genealogical relatedness: descent from one or more ancestors, e.g., genealogical relationship between parents and descendants.

[0086] Genetic Identity: Genetic information that identifies an individual, including, for example, a representation of that genetic information, including genotype, genotype fingerprint, genomic sequence, genetic marker profile; "genetically identical" = 100% genetic identity, "X% genetically identical" = having X% genetic identity, etc.

[0087] Genotypic Fingerprint: description of the genotype at a specially prepared set of marker loci; a known genotype.

[0088] gill: lamella; part of a mushroom, the structure containing the stenophylla and basidiophores.

[0089] Haploid: Having only a single set of nuclear chromosomes; see homokaryon.

[0090] Heteroallelic: Having two different alleles at a locus; similar to heterozygosity.

[0091] Heteroallelic: Difference between homologous chromosomes in a heterokaryon genotype; similar to heterozygosity.

[0092] Heterokaryon: technically, sexual heterokaryon: a culture that has two complementary (i.e., necessarily heteroallelic at the Mat locus) types of haploid nuclei in a common cytoplasm and is therefore functionally and physiologically similar to a diploid individual (but cytogenetically represented as N+N instead of 2N), is fertile (in the absence of any rare interfering genetic defects at loci other than Mat), and exhibits a nutritionally incompatible response to other heterokaryons; in the context of strain development, it is also referred to as a strain or stock.

[0093] Heterokaryon compatibility: the absence of observed antagonism between two genetically non-identical heterokaryons in physical proximity or contact; see heterokaryon incompatibility.

[0094] Heterokaryon incompatibility: phenomenon of antagonism observed in physical proximity or contact between two genetically non-identical heterokaryons; multilocus self / non-self recognition system; i.e., a genetic system that operates to limit anastomosis (hyphal fusion) and cytoplasmic contact in basidiomycete heterokaryons, allowing one heterokaryon culture to discriminate and recognize another culture as either self or non-self; nutritional incompatibility.

[0095] Heterokaryosis: Possession of heterokaryon traits.

[0096] Heteromixis: life cycle involving mating between two different non-sib haploid individuals or gametes; similar to outcrossing.

[0097] Homoallelic: Having not more than one allele at a locus. For diploid organisms the equivalent term is "homozygous." Haploid strains are by definition completely homoallelic at all non-overlapping loci.

[0098] Homokaryon: a haploid culture that has a single type (or somatic lineage) of haploid nucleus (cytogenetically designated N), is normally sterile, does not exhibit the typical self / non-self incompatibility reaction to heterokaryons, and can function as a gamete in a sexually complementary anastomosis; a "lineage" that transmits a uniform genotype to offspring, as in inbred plant lines; a predominantly homoallelic lineage that mates well and forms poor fruiting bodies is a putative homokaryon for strain development; see discussion below.

[0099] Homokaryosis: Possessing the trait of homokaryons; haploid.

[0100] Hybrid: of biparental origin, usually applied to heterokaryonous strains and cultures produced by controlled mating.

[0101] Hybridize: physical association of two cultures, usually homokaryons, for example on a Petri dish containing a sterile agar-based nutrient medium, in an attempt to achieve anastomosis, cytoplasmic fusion, and formation of sexual heterokaryons (=mating); success as previously mentioned.

[0102] Hyphae: thread-like elements of the mycelium, composed of cell-like compartments.

[0103] Inbreeding: mating including sib-crossing, backcrossing to a parent line or strain, and intramixis; reproduction involving genetically related parents.

[0104] Incompatibility: See heterokaryon incompatibility.

[0105] Induced mutagenesis: A non-spontaneous process that alters the DNA sequence of at least one gene locus.

[0106] Initial Culture: A culture used as starting material in the strain development process; more specifically, the strain from which a dependent variety is derived.

[0107] Inoculum: A culture in a form that allows for the transfer and growth of a culture, e.g., in fresh medium; specialized commercial types of inoculum include seeds and CIs.

[0108] Intramixis: uniparental sexual life cycle involving the formation of complementary "mated" pairs of post-meiotic nuclei within the basidium or individual spores.

[0109] Lamella: See gill.

[0110] Line: culture used for crossing to produce hybrid strains; usually homokaryons and therefore homoallelic, or otherwise highly homoallelic non-heterokaryotic (non-NSNPP) cultures; in fact, functionally homokaryonic and completely or mainly homoallelic cultures; similar to the mainly or completely homozygous inbred lines in plant breeding.

[0111] Clade: See "clone." A set of EDV derivatives derived from a single original strain or variant.

[0112] locus: specifically reserved contiguous portions of the genome that are homologous but often vary among different genotypes; plural: loci.

[0113] Marker-assisted selection: The use of linked genetic markers, including molecular markers, to trace trait-determining loci of interest among offspring and through pedigrees.

[0114] MAT: mating type locus determining sexual compatibility and heterokaryon status.

[0115] Mating: sexual union of two cultures by anastomosis and cytoplasmic fusion; methods for obtaining matings between mushroom cultures are well known in the art.

[0116] Mycelium: the vegetative body or thallus of the mushroom organism, composed of thread-like hyphae.

[0117] Mushroom: reproductive structure of a fungus in the Agaricaceae family; agaric; cultivated food of the same name.

[0118] Neohaploid: haploid culture or line obtained by physically deheterokaryosis (reduction to haploid components) of heterokaryons; somatically derived homokaryons.

[0119] Progeny: descendants of parent heterokaryons within a single generation, e.g.; often used to describe cultures obtained from spores from one mushroom of one strain.

[0120] Outbreeding: mating between unrelated or distantly related individuals; similar to heteromixis in mushrooms.

[0121] Parent: The immediate ancestor of an individual; parental strains are heterokaryons and parental lines are homokaryons; heterokaryons can be the parents of F1 heterokaryons through intermediate parental lines.

[0122] Pedigree-assisted strain development: The use of phylogenetic information to identify desirable strain combinations in a controlled breeding program.

[0123] Phenotype: The observable characteristics of a strain or lineage that are expressed and manifested in the environment.

[0124] Cytoplasmic fusion: establishment of cytoplasmic continuity by anastomosis, leading to the formation of sexual heterokaryons.

[0125] Ancestor: Ancestors, including parents (direct ancestors).

[0126] Selfing: mating between sibling lines; similar to intramixis.

[0127] Sexual compatibility: the condition between different lineages with non-identical alleles at the Mat locus, in which the two lineages can mate to produce stable and fertile heterokaryons. The opposite condition, sexual incompatibility, occurs when two lineages have the same allele at the Mat locus.

[0128] Single Nucleotide Polymorphism (SNP): A marker having a locus position that varies between individuals and can distinguish two sequences or individuals.

[0129] Somatic cell: A cell of the vegetative hyphae.

[0130] Seed: Mushroom culture, generally a pure culture of heterokaryons on a sterile substrate, generally a friable, dispersible particulate material, in some cases a grain; commercial inoculant for compost; reference to seed includes reference to the culture on the substrate.

[0131] Spore: part of a mushroom, the reproductive propagule.

[0132] Stem: Stipe; part of a mushroom, the structure that supports the cap.

[0133] Axenic Growth Medium: A nutrient medium, sterilized by autoclaving or other methods, that supports the growth of an organism; examples include agar-based solid nutrient media, such as potato dextrose agar (PDA), nutrient broth, and many other materials.

[0134] Stipe: See stem.

[0135] Strain: heterokaryon having specially reserved characteristics or a specific identity or ancestry; similar to a variety.

[0136] Targeted mutagenesis: The process of altering the DNA sequence at at least one specific gene locus.

[0137] Tissue culture: dedifferentiated vegetative mycelium obtained from differentiated tissues of a mushroom.

[0138] Trait transformation: A method for selectively introducing the genetic determinants of one (single-locus transformation) or more desirable traits into the genetic background of an original strain, while retaining the majority of the genetic background of the original strain.

[0139] Transformation: The process of altering the genetic material of an individual cell by incorporating foreign (exogenous) DNA into its genome; methods for obtaining transformation, including single-locus transformation.

[0140] vegetative compatibility: absence of the phenomenon of antagonism observed in conditions of physical proximity or contact between two genetically non-identical heterokaryons, as determined by a multilocus self / non-self recognition system that operates to limit anastomosis (hyphal fusion) and cytoplasmic contact in basidiomycete heterokaryons; heterokaryon compatibility; the converse of vegetative incompatibility.

[0141] vegetative incompatibility phenomenon of antagonism observed in physical proximity or contact between two genetically non-identical heterokaryons determined by a multilocus self / non-self recognition system that operates to limit anastomosis (hyphal fusion) and cytoplasmic contact in basidiomycete heterokaryons; heterokaryon incompatibility.

[0142] Virus Breakdown: The sequential use of multiple incompatible strains, i.e. strains exhibiting heterokaryon incompatibility, in a planned program of strain rotation within a mushroom production facility to reduce transmission of viruses from on-site virus reservoirs to newly planted crops.

[0143] Whole-genome sequencing (WGS): the DNA sequence of an organism such as Agaricus

[0144] Yield: The net fresh weight of a harvested crop, usually expressed in pounds per square foot.

[0145] Yield patterns: distribution of yield within each flush and among all flushes; affects crop and product size, quality, harvesting costs, and relative disease pressure.

[0146] Regarding the above definition of homokaryons, homokaryons and homoallelic lineages are subject to technical and practical considerations: note that homokaryons are haploid cultures that are self-evidently completely homoallelic in classical terms. In practical terms, for fungal strain development purposes, this definition is somewhat broadened to accommodate both technical limitations and cytological variations by treating all primarily homoallelic lineages as homokaryons. Technical limitations include the fact that genomes contain duplicated DNA regions, including repeated elements such as transposons, and may also contain large duplications of chromosomal segments due to past translocation events, and such regions do not appear to be homoallelic by the majority of genotyping methods. Two distinct A. bisporus genomes sequenced by the Joint Genome Institute, a US federal agency, differ in estimated length by 4.4% and gene number by 8.2%, suggesting a significant amount of DNA duplication or rearrangement within the different strains of the species. The currently available genomes of A. bisporus cannot fully account for the physical arrangement of such elements and translocations, and therefore assembled genome sequences of haploid lineages may have regions that appear heteroallelic using currently available genotyping methods. Cytologically, homokaryon progeny will usually be spores that receive one haploid postmeiotic nucleus. However, spores that receive two third division nuclei from the basidium will be genetically equivalent to homokaryons. Spores that receive second division "sister" postmeiotic nuclei will be functional homokaryons, although some terminal "islands" of heteroallelicity may be present due to crossing over during meiosis. Also, meiosis in which homologs segregate asymmetrically may result in aneuploid functionally homokaryon spores with an extra chromosome that gives rise to regions of heteroallelicity. All of these cultures are highly homoallelic and all function as homokaryons. Scientific and technological limitations make it impractical to differentiate such cultures, and also to exclude duplication of DNA segments as an explanation for limited and isolated regions of genome sequence assemblies that appear heteroallelic.Thus, in this application, the use of the term "homoallelic" to characterize a lineage encompasses completely or predominantly homoallelic lineages, and cultures so described are functional homoallelic and putatively homoallelic, all of which are defined as homoallelic in this application.

[0147] With respect to the present invention, as described above, the present invention relates to cultures of the hybrid Agaricus bisporus strain B19414 and cultures derived or descended from the B19414 strain. Such cultures are used to produce mushrooms and mushroom parts. Thus, the present invention further relates to methods of making and using the B19414 strain and dependent varieties (EDV) of the B19414 strain.

[0148] The morphological and physiological characteristics of strain B19414 under culture in standard culture medium, Difco brand PDA medium, are presented as follows. Strain B19414 grown in PDA medium in 8.5 cm diameter Petri dishes gave rise to colonies that were white or light brownish yellow or "tan" or darker brown irregularly lobed, approximately circular in overall outline, and expanded in diameter at (0.79-0.88-)0.92 (-0.93-1.11) mm / day during dynamic equilibrium growth between days 14 and 24 after inoculation of the culture into PDA using a 3.5 mm diameter circular plug as inoculum. Strains were allowed to expand by transferring pure inoculum to larger volumes of sterile culture medium. There are no variant traits observed or predicted in strain B19414.

[0149] The hybrid strain B19414 is the product of two generations of controlled lineage crossing by the applicant. To obtain the strain B19414, homokaryon lines derived from spores of each heterokaryon parent were selected and crossed. The two lines crossed are B12998-s181 and P2-s203. The resulting hybrid culture, i.e., the heterokaryon, was named strain B19414.

[0150] One use of cultures of strain B19414 is the production of edible mushroom crops for sale. Another use is improved facility sanitation through strain rotation and the "virus-busting" effect, with a related use being improved production crop diversification. A third use is the incorporation of genetic material of strain B19414 into progeny and derived or descendent cultures, including dormant and germinating spores and protoplasts. Additional uses exist as noted above.

[0151] Crossing of Agaricus bisporus cultures of the present invention can be accomplished by growing two different cultures, one of which is the genetic lineage present in the B19414 spore, together, preferably on axenic medium, in close proximity until anastomosis (i.e., mycelium or cell fusion) occurs. In a successful cross, the resulting fusion culture is a first generation outbred hybrid culture incorporating the genetic lineage present in the mushroom spore that is part of one embodiment of the present invention. Protoplasts derived from the basidium, or other parts of the organism, are another part of the B19414 mushroom that can be used to transfer the genetic material of B19414 to new cultures.

[0152] Methods for obtaining, manipulating, and breeding the cultures of the present invention to generate the progeny, inoculants, products, and crops of the present invention, to improve mushroom farm hygiene using strain rotation programs, and to obtain genotypic fingerprints of mushroom cultures have been described herein above and are well known to those skilled in the art. Although the present invention has been described with respect to specific embodiments in this application, those skilled in the art can generate additional embodiments and modifications in light of the teachings herein without departing from the spirit and beyond the scope of the claimed invention. It is therefore understood that the description herein is provided merely to facilitate understanding of the present invention and should not be construed as limiting the scope of the present invention.

[0153] The four commercially available brown controls used in this study are cultivar cultures that were commercially grown in Europe, the United States, and other markets in 2020 and 2021; the test data was collected in France, the Netherlands, Belgium, and the United States by the assignee of record, Sylvan Inc. The control strains were Heirloom (Amycel), Tuscan-820 (Sylvan), Tuscan-860 (Sylvan), and Brawn (Amycel). Heirloom is the subject of US 7,608,760, where it is named BRO6 and deposited under ATCC Accession No. PTA-6876. Tuscan-820 is the subject of a pending PCT application published as WO2022023290A1, where it is named LA3782 and deposited under CNCM Accession No. I-5528. Tuscan-860 is known in some commercial territories as Tuscan Brown, and also known as B14528 in US 9,017,988, and has NRRL accession number 50900. Brawn is a sister strain of Heirloom and is commercially sold in North America by Amycel. To ensure availability, Brawn has been re-deposited by Sylvan with the NRRL under accession number 68258.

[0154] For the purposes of the present invention, the whole genomic DNA sequence of strain B19414 and the whole genomic DNA sequences of cultures of its parental strains B12998-s181 and P2-s203 were obtained by applicants using the following method: Homokaryon parental strain cultures were grown in sterile broth growth medium after dissociation. After 2-4 weeks, mycelial cells were collected by filtration, frozen at -80°C, and lyophilized until dry. Umbrella tissue was obtained from mushrooms resulting from cultures of heterokaryonous strain B19414, frozen, and lyophilized. DNA was extracted from the lyophilized samples using the CTAB protocol, followed by ribonuclease (RNAse) treatment and gel purification. DNA libraries were prepared from the DNA of each culture by contractor Genewiz (New Brunswick, New Jersey), and the libraries were sequenced using Illumina technology. To obtain the whole genome sequence, Illumina 250 bp reads aligned to the H97 version 2 reference genome using DNAstar version 18 were utilized. Assembly of the reads into genome sequence using the H97 version 2.0 public domain reference genome sequence (Morin et al. 2012; PNAS 109 (43): 17501, incorporated herein by reference) was performed by Applicant. Thus, Sylvan, Inc. knows with certainty approximately 93% to approximately 95% of the entire genotype of the B19414 strain and its parental homokaryons. The reference genome is important because the precise location of each SNP in the genome can be numerically determined using the base pair positions known for H97.

[0155] Tables I and II present a SNP-based comparison of the genotypes of related cultures. SNPs represent single nucleotide polymorphisms, which vary between individuals and provide markers together with the position of a locus that can distinguish two sequences or individuals.

[0156] The data in Tables I and II are presented as 9mers, and in most cases the central base (position 5) carries the SNP. Occasionally there is more than one SNP within a 9mer locus; each such 9mer is treated as a single SNP. Robust markers were selected at loci aligned across each of the first 19 scaffolds. SNP alleles at those loci were determined in Table I for the homokaryon parents of B19414 (P2-s203 and B12998-s181) and in Table II for the other four commercial strains included for comparison (Tuscan-820, Heirloom, Tuscan-860, and Brawn).

[0157] [Table 1] TIFF2025509479000002.tif249160TIFF2025509479000003.tif249160TIFF2025509479000004.tif249160TIFF20255094790 00005.tif249160TIFF2025509479000006.tif249160TIFF2025509479000007.tif249161TIFF2025509479000008.tif203160

[0158] [Table 2] TIFF2025509479000010.tif244170TIFF2025509479000011.tif246170TIFF2025509479000012.tif246170 TIFF2025509479000013.tif245170TIFF2025509479000014.tif245170TIFF2025509479000015.tif244170

[0159] A total of 199 SNP loci were utilized in each of Tables I and II. Note that there are two alleles (whether homoallelic or heteroallelic) at each marker locus for the five heterokaryon strains and a single allele per locus for the three homokaryon cultures. The IUPAC nucleotide and ambiguity codes are used to represent the individual or composite 9-base DNA allele marker sequences reported above, each of which represents an allele or composite pair of alleles at the genomic marker locus at the indicated genomic location. The first two columns present the location information for each marker locus from the haploid / homokaryon reference genome H97 version 2.0 published by the USDepartment of Energy Joint Genome Institute (Morin et al. 2012). H97 was originally derived from a white cultivar strain. Genotypic differences are evident among the strains. Combining the SNP patterns at each locus for B12998-s181 and P2s-203, it is also clear that the compound is a perfect match for the B19414 heterokaryon. Also, marker scaffold 4, 753116, is not a classical 9-mer. This marker was included in the table simply because it was used in prior patent applications, including PCT Publication No. WO2022023290A1.

[0160] Using the SNP (single nucleotide polymorphism) data in Table I, it is possible to calculate an estimate of the genotypic differences between B19414 and the four other brown cultivar strains. The data in Table II show that Tuscan-820 has 58 / 199 or 29.1% variance from B19414, Heirloom has 99 / 199 or 49.7% variance from B19414, Tuscan-860 has 51 / 199 or 25.6% variance from B19414, and Brawn follows Heirloom closely with 109 / 199 or 54.8% variance from B19414. Thus, the B19414 strain is substantially different from the other available brown cultivar strains, providing both genetic diversification and commercially acceptable performance in terms of crop and product characteristics.

[0161] Using the complete set of SNP data captured for scaffolds 1 to 19, it was also possible to estimate how similar the four brown strains were to the H97 reference genome and to each other using a process provided by the Lasergene software package. This estimation process has some limitations, notably that all data is generated in comparison to a reference genome, in this case the H97 reference genome. These tabulations provided by the software showed that B19414, Heirloom, Tuscan-820, Tuscan-860 and Brawn all had over 380,000 SNP differences from H97. Furthermore, by subtracting the total SNPs reported for B19414 from the total SNPs for each of the other strains, the number of SNPs in Heirloom, Tuscan-820, Tuscan-860 and Brawn that differ from the SNPs in B19414 can be calculated. Based on the absolute value of each difference, an estimate was obtained of the minimum number of SNP differences that would distinguish B19414 from each strain. These calculations show that Tuscan-820 has 269,416 SNPs that are different from the corresponding SNPs in B19414, Tuscan-860 has 157,704 SNPs that are different from the corresponding SNPs in B19414, Heirloom has 272,938 SNPs that are different from the corresponding SNPs in B19414, and Brawn has 128,212 SNPs that are different from the corresponding SNPs in B19414. These very large numbers demonstrate that B19414 has a unique and substantially different genotype compared to the available brown cultivars. There are some caveats to these calculations due to factors such as software limitations and DNA sequence read depth. Therefore, it is emphasized that these numbers are approximate.

[0162] Tables III and IV report data from six genetic marker loci, as reported by the standard, for the same panel of strains utilized in Tables I and II. As in Tables I and II, there are two alleles at each marker locus for the five heterokaryon strains, and a single allele per locus for the three homokaryon cultures. Data were generated by using targeted polymerase chain reaction (PCR) to amplify genomic regions spanning specially designed markers from each of the culture DNA. Data were generated using PCR primers that flank the specially designed marker regions at the locations indicated by the location information provided below; methods for designing appropriate primers are well known in the art. DNA was sequenced from the amplified PCR products by contractor Eurofins (Louisville, Kentucky) using selected methods, and genotypes were determined by directly examining these sequences against Sylvan's database of reference marker / allele sequences. In most cases, sequences were further confirmed by directly examining the corresponding whole genome sequences for the cultures.

[0163] [Table 3]

[0164] [Table 4]

[0165] The data in Tables III and IV demonstrate that the B19414 strain has a unique combination of alleles at this group of six standard marker loci. In particular, the genotypes at three of the six marker loci, FF, AN and AS, are not present in any of the other cultivars. Further analysis of these six markers is described below.

[0166] Description of p1n150-G3-2 marker: The 5' end of this marker segment begins at position 1 with the first "T" of the sequence TCCCAAGT, corresponding to H97 JGI V2.0 scaffold 1 position 868615 (Morin et al. 2012), and in the reverse orientation (relative to the scaffold orientation), spans approximately 600 nt in the majority of alleles; longer segments result from insertions of allele 1T into the DNA. Currently, nine alleles incorporating at least 30 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain repository.

[0167] Alleles 2 and 5 are alleles present in the B19414 pedigree across three generations and are characterized as follows (format: nucleotide base symbol @ alignment position):

[0168] Allele 1T: 320nt ABR transposon insertion @206^207, "A" @321; "T" @327; "C" @374; "G" @378; "G" @422, "C" @431; "G" @472; etc.

[0169] Allele 2: no Abr1 insertion; "A"@321; "C"@327, "C"@374; "C"@378; "G"@422; "T"@431; "G"@472; etc.

[0170] Allele 5: no Abr1 insertion; "G"@321; "C"@327, "C"@374; "C"@378; "G"@422; "T"@431; "G"@472; etc.

[0171] Due to linkage to the MAT locus, which is obligately heteroallelic in fertile heterokaryons, all known and predicted heterokaryon genotypes in p1n150-G3-2 are also heteroallelic.

[0172] The genotype of the B19414 heterokaryon at the p1n150-G3-2 marker locus is "2 / 5" (heteroallelic), indicating the presence of alleles 2 and 5. Allele 2 was contributed by P2-s203, and allele 5 was transmitted from B12998-s181. The "2 / 5" genotype is also shared by Tuscan-820 and Tuscan-860. Heirloom and Brawn have alleles 1T and 5.

[0173] Description of ITS (=ITS 1+2 region) markers: The ITS segment is part of the nuclear rDNA region located on chromosome 9 (scaffold 10 in JGI H97 V2.0). The rDNA is a cassette that is repeated in tandem up to an estimated 100 times in the haploid genome of A. bisporus. Therefore, there is no single exact positioning of this sequence in the assembled H97 genome, and in fact it is difficult or impossible to assemble the sequence precisely across all the tandem repeats. Three copies of the cassette were included on scaffold 10 of the H97 JGI V2.0 assembly, starting at position 1612110; a partial copy is also assembled on scaffold 29 (Morin et al. 2012). The 5' end of this marker segment starts at position 1 with the first "G" of the sequence GGAAGGAT and spans approximately 703-704 nt in the majority of alleles in the forward direction (relative to the orientation of the scaffold). To date, more than nine alleles incorporating at least 11 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial stock collection.

[0174] The alleles present in B19414 are I1 and I2, characterized as follows (format: nucleotide base symbol @ alignment position, based on alignment of 9 alleles):

[0175] Allele I1: "C"@52; "T"@461; "T"@522; "T"@563; etc.

[0176] Allele I2: "T"@52; "T"@461; "T"@522; "T"@563; etc.

[0177] Allele I5: "G"@52; "A"@461; "C"@522; "C"@563; etc.

[0178] The genotype of B19414 heterokaryons at the ITS marker locus is "I1 / I2" (heteroallelic), indicating the presence of alleles I1 and I2. This pattern is consistent with Tuscan-860 and distinct from Heirloom, Tuscan-820 and Brawn.

[0179] Description of MFPC-1-ELF marker: The 5' end of this marker segment begins at position 1 with the first "G" of the sequence GGGAGGGT, corresponding to H97 JGI V2.0 scaffold 8 position 829770 (Morin et al. 2012), and spans approximately 860 nt in the forward direction (relative to the orientation of the scaffold) in the majority of alleles. Currently, seven alleles incorporating at least 40 polymorphic positions have been demonstrated from diverse strains in at least the Sylvan microbial strain repository.

[0180] The alleles present in B19414 are E3 and E4.

[0181] Allele E1: "A"@63; "A"@77; "A"@232; "A"@309; "T"@334; "A"@390; "A"@400; "T"@446, "A"@481; etc.

[0182] Allele E3: "A"@63; "A"@77; "A"@232; "G"@309; "T"@334; "A"@390; "A"@400; "C"@446, "G"@481; etc.

[0183] Allele E4: "G"@63; "A"@77; "A"@232; "G"@309; "T"@334; "A"@390; "A"@400; "C"@446; "G"@481; etc.

[0184] The E3 / E4 genotype is consistent with Heirloom, Tuscan-860 and Brawn, but Tuscan-820 has a different genotype.

[0185] AN Marker Description: The 5' end of this marker segment begins at position 1 with the first "G" of the sequence GGGTTTGT, corresponding to H97 JGI V2.0 scaffold 9 positions 1701712 (Morin et al. 2012), and spans approximately 1660 (H97 genome) to 1700 nt (alignment interval) in the known alleles (relative to the orientation of the scaffold); several insertions / deletions have created polymorphisms of length that characterize the alleles in addition to point mutations at individual nucleotides. Currently, five alleles incorporating more than 70 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain repository.

[0186] The alleles present are as follows (format: nucleotide base symbol @ alignment position, based on alignment of alleles N1 through N4):

[0187] Allele N1: "G"@640; [deletion]@844~846; "C"@954; "T"@882; "A"@954, etc.

[0188] Allele N2: "A"@640; [deletion]@844~846; "C"@954; "T"@882; "T"@954, etc.

[0189] Allele N3: "A"@640; [deletion]@844~846; "T"@954; "C"@882; "G"@954, etc.

[0190] Allele N4: "A"@640; [deletion]@844~846; "C"@954; "C"@882; "G"@954, etc.

[0191] The "N1 / N3" genotype is unique to B19414; all other genotypes tested, including Heirloom, Tuscan-860, Tuscan-820 and Brawn, had different allele combinations at the AN locus.

[0192] AS Marker Description: The 5' end of this marker segment begins at position 1 with the first "G" of the sequence GG(T / N)GTGAT, corresponding to H97 JGI V2.0 scaffold 4 position 752867 (Morin et al. 2012), and spans approximately 1620 (H97 genome) to 1693 nt (alignment interval) in the known alleles (relative to the orientation of the scaffold); several insertions / deletions have created polymorphisms of length that characterize the alleles in addition to point mutations at individual nucleotides. Currently, seven alleles incorporating more than 80 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain repository.

[0193] The alleles present in the B19414 pedigree are alleles SA and SD. In the later umbrella diameter experiment described below, Brawn was added as an additional commercial strain, which has allele SB, also described below. The markers are characterized in part as follows (format: using nucleotide base symbol @ alignment position, based on alignment of alleles SA to SG):

[0194] Allele SA: "T"@28; [deletion]@258~263; "G"@275; [insertion]+"TTTC C CAGC" + [insert] @ 309 ~ 249; "C" @ 404, etc.

[0195] Allele SB: "T" @ 28; [deletion] @ 258 ~ 263; "T" @ 275; [insertion] + "TTTC C CAGC" + [insert] @ 309 ~ 249; "C" @ 440, etc.

[0196] Allele SC: "T" @ 28; "GATATC" @ 258 ~ 263; "G" @ 275; [insert] + "TTTCT CAGC" + [insert] @ 309 ~ 249; "C" @ 404, etc.

[0197] Allele SD: "C"@28; [deletion]@258~263; "T"@275; [deletion]@309~249; "T"@275; [deletion]@309~349, "T"@404, etc.

[0198] The "SA / SC" genotype is unique to B19414 and is not found in Heirloom, Tuscan-820, Tuscan-860, or Brawn.

[0199] FF Marker Description: The 5' end of this marker segment begins at position 1 with the first "T" of the sequence TTCGGGTG, corresponding to H97 JGI V2.0 scaffold 12 position 281999 (Morin et al. 2012), and spans approximately 570 nt in the forward direction (relative to the orientation of the scaffold) in the majority of alleles. Currently, seven alleles incorporating at least 20 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain repository.

[0200] Both alleles of the B19414 heterokaryon are of the "FF1" genotype.

[0201] Aller FF1: "CCG" @48~50, "C" @91, etc.

[0202] Aller FF3: "TTC" @48~50; "T" @91, etc.

[0203] B19414 was the only strain with the "FF1 / FF1" genotype in this study.

[0204] A unique genotype, or genetic fingerprint, allows for unambiguous identification of strain B19414 (and its EDV and direct descendants). Agronomically, genetic diversity among cultivated strains is a desirable objective, since it is well established that genetic monoculture among agricultural crop species can lead to costly failures due to specific diseases, pests, or environmental pressures. Thus, any otherwise desirable commercial strain that possesses genetic novelty would be beneficial. Strain B19414 meets these criteria.

[0205] [Table 5]

[0206] For the data in Table V and the following tables, significance is indicated with asterisks: * for p=0.05 or less; ** for p=0.01 or less; *** for p=0.001 or less; **** for p=0.0001 or less.

[0207] Mushrooms were collected at a European experimental farm using the latest Dutch techniques for mushroom growth. In particular, Phase III compost incubation was utilized, supplementary materials were added to the casings, and the same parameters as those used for Heirloom were used, in particular the same temperature regime, CO2 management and timing of water application. On the fourth day the crop was aerated and mushrooms were collected over a three-day harvest for each of the three flushes.

[0208] As noted above, there is a clear need for new brown strains of Agaricus bisporus that have equivalent yields to current cultivars such as Heirloom. The data in Table V show that B19414 is comparable to Heirloom in terms of total yield. B19414 had significantly higher yields at the third break. The trend toward equilibrating break yields shown by B19414 will result in corresponding improvements in product quality and value.

[0209] [Table 6]

[0210] The color of the mushroom caps was measured using a Minolta Chroma Meter CR-200. The sample size was 20 medium-sized mushrooms with diameters of 30-40 mm. The L*a*b method was used, where "L" is a scale of lightness, with 100 being completely white and 0 being completely black. For the other two measurements, "a" is the green / red axis and "b" is the yellow / blue axis. For "a", the red values ​​line up on the positive side of the common axis and the green values ​​line up on the negative value side. Similarly, for the "b" axis, the yellow values ​​are positive and the blue values ​​are negative.

[0211] B19414 had similar "L" values ​​to Tuscan-860 and was less L (significantly darker) than Heirloom and Tuscan-820.

[0212] The "a" measurements show that B19414 is less red than Heirloom and Tuscan-860. There was no significant difference between B19414 and Tuscan-820.

[0213] The most informative data was collected for the "b" parameter: strain B19414 had a b value significantly smaller than the other three strains.

[0214] [Table 7]

[0215] Applicant's marketing research has shown that darker brown mushrooms are often preferred by retailers and consumers. It should be noted that even if two colors are statistically different based on chromameter measurements, they are not necessarily perceptible as different by the human eye. Perceptibility can be estimated by calculating the Delta E value between the colors (Sharma, Wu & Dalal; Color Research and Application: 2004, pp 21-30). The results of the Delta E equation range from 0 to 100. Values ​​between 2 and 10 are similar but perceptibly different at a glance, while values ​​below 2 require close observation to distinguish, and values ​​above 10 are significantly different. Delta E can be calculated using one of several formulas, with the CIEDE2000 published by the CIE in 2001 being the most accurate.

[0216] As shown in Table VII, the mushrooms obtained from the B19414 strain have delta E values ​​between 2 and 10 when compared to Heirloom and Tuscan-820, meaning that their brown color is distinctive and can be distinguished at a glance. The B19414 mushrooms are significantly darker than Heirloom and Tuscan-820, as they have lower L, a, and b values.

[0217] [Table 8]

[0218] These are direct measurements of the terms defined above. These measurements were used to define the percentage measurements in Table IX below.

[0219] [Table 9]

[0220] The mushroom cap measurements in Table VIII were obtained using a Storm 3C301 digital caliper. A sample size of 20 medium-sized mushrooms at commercial maturity (35-40 mm diameter, membrane closed) were harvested and measured to obtain cap diameter and cap height values. The mushrooms were then cut in half lengthwise to measure flesh thickness and stalk width. Ratios between these values ​​were calculated to obtain cap roundness (cap height / cap diameter), flesh thickness (meat thickness / cap diameter), and stalk thickness (stalk width / cap diameter). The data in Table IX show that B19414 has similar cap roundness ratios to Tuscan-860, Heirloom / , and Tuscan-820.

[0221] Concerning the meat thickness ratio, a statistically significant difference was observed when comparing the meat thickness of the B19414 strain with that of Heirloom, Tuscan-820 and Tuscan-860. In Table IX, the meat thickness ratio of B19414 was 0.39, significantly less than the other three strains. In other words, B19414 had proportionally thinner cap meat. This is an important difference since mushrooms tend to flatten as they mature rather than retaining a rounded shape. As mentioned above, there is a lucrative market in North America and Europe for brown strains that produce good breakfast flats or pot bellies.

[0222] Additionally, strain B19414 has a smaller stalk thickness ratio than Tuscan-860, Heirloom, and Tuscan-820. A mushroom with a similar cap diameter and a thinner stalk means that more of the harvested mushroom will be composed of cap tissue, resulting in less stalk waste during preparation in the kitchen.

[0223] [Table 10]

[0224] Weight loss over an 8-day period at 4°C was compared between strain B19414 and Heirloom. Only mushrooms with closed caps at commercial maturity (approximately 40 mm diameter, membranes closed) during the first break were used.

[0225] The containers were Styrofoam wrapped in plastic film and each contained 300 g of mushrooms. These tests replicated what would occur in a supermarket situation, where mushrooms may be kept in a refrigerated display case for periods of up to 10 days.

[0226] The data in Table X clearly show that the B19414 strain lost less moisture than the Heirloom control on days 6, 7, and 8 of Trial 1, and days 7 and 8 of Trial 2. This information is important because moisture loss can lead to drying of the mushrooms, condensation in the overlapped mushroom containers, and loss of product weight at sale. These data further demonstrate one route by which the B19414 strain produces closed button mushrooms with unexpectedly superior quality.

[0227] In conflict cropping trials between strain B19414 and other commercial brown strains, typical heterokaryon incompatibility behavior was observed, including delayed timing of first break and reduced yield.

[0228] In Table XI below, a test was conducted to determine the difference in maximum cap diameter between five different strains of brown mushroom grown as pot bellies. Bulk Phase II compost was purchased from a commercial supplier (Kennett Square, Pennsylvania), mixed with inoculum, and a 13 day spawn run was completed. A total of four 4 ft x 4 ft trays for each strain were inoculated with inoculum. Upon completion of spawn, a layer of casing (peat buffered with CaCO3, pH approx. 7.5, approx. 75% H2O) mixed with the associated casing inoculum was applied to each tray.

[0229] To grow potbellied mushrooms, growers can make a few slight changes to their normal growing conditions. In the example data shown in Table XI, on day 6, the CO in the growth chamber was increased. 2 The CO was flushed by lowering the CO concentration from 4000 ppm to 1400 ppm over a 24 hour period. 2 In addition to the changes in temperature, the room temperature was decreased from 22°C to 16.5°C and the relative humidity was decreased from 95% to 82%. Water was also applied to the casing layer of all plants on the day of flushing, and as needed as the crop developed. Taken together, these growing conditions are described as a mild flush. Small mushrooms were ready for harvest 15 days after casing.

[0230] Trays were managed to produce pot bella mushrooms. Trained harvesters spaced the mushrooms apart to reduce mushroom density on the trays so that maximum size was achieved in all trays. The key metric was to harvest large mushrooms on the day the membrane separates from the cap edge, which is when commercial growers typically harvest pot bella mushrooms. If the mushrooms remain on the bed past the point of membrane rupture, the cap edge will begin to curl upwards and spores will begin to accumulate on the surface of the bed. Growers do not want to harvest mushrooms with cap edges that curl upwards, preferring those with the underside of the cap edge "rolled down". Additionally, open mushrooms present a risk for the spread of mushroom viruses, and spores on the mushroom bed have been shown to reduce yields.

[0231] For potbellied mushrooms, growing the mushrooms is a balancing act between manipulating the number of pinheads on the surface of the bed and encouraging the growth of larger mushrooms that retain intact or nearly intact membranes at harvest. Thus, for the data in Table XI below, the key metric was harvesting large mushrooms on the day the membranes start to separate from the cap edge. All harvested mushrooms in Table XI below had intact membranes on the day of harvest. However, the membranes had begun to stretch and could not be left in the mushroom house for additional days.

[0232] [Table 11]

[0233] For the data in Table XI, five strains were used for the cap diameter test. In Table XI, N is the number of mushrooms harvested on the date of data capture. Mean is the mean (average) cap diameter in millimeters for all N mushrooms harvested, while sd is the standard deviation (a measure of variation within each data set). For p-values, significance is indicated with an asterisk: *** for p=0.001 or less, **** for p=0.0001 or less. The column labeled Class in Table XI indicates the results of an exact t-test comparison between all five strains. Four statistically distinct classes were observed. Note that Brawn and Tuscan-820 were not significantly different from each other and are therefore both listed as class b.

[0234] For Table XI, statistical treatment with p-values ​​was performed by pairwise t-tests comparing B19414 with each of the other five strains separately. These data show clear and highly significant differences between B19414 and the other five strains. Therefore, we conclude that B19414 can be harvested at a larger size than all other brown commercial strains of Agaricus bisporus.

[0235] In summary, it can be clearly seen in Table XI that strain B19414 produced the largest pot bella mushrooms. Large pot bella are premium grade mushrooms that generally command a higher price. Thus, B19414 has a clear market advantage over other cultivars.

[0236] Although the present invention has been described with respect to specific embodiments in this application, those skilled in the art can generate additional embodiments and modifications in light of the teachings herein without departing from the spirit and scope of the claimed invention. It is therefore understood that the description herein is provided merely to facilitate understanding of the invention and should not be construed as limiting the scope of the invention.

Claims

1. A mushroom culture of Agaricus bisporus, referred to as strain B19414, wherein a representative culture of the B19414 strain is deposited under NRRL accession number 68095.

2. A culture of Agaricus bisporus having a genotype fingerprint having alleles at the marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AS, and FF, wherein all of the alleles at the marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AS, and FF of the fingerprint are present in the genotype fingerprint of strain B19414, and a culture of strain B19414 is deposited under NRRL accession number 68095.

3. A culture of Agaricus bisporus directly derived from the first culture of strain B19414, wherein the culture of strain B19414 is deposited under NRRL accession number 68095, and the entirety of its genome or genotype is that of the genome or genotype of the first culture of strain B19414.

4. A culture of Agaricus bisporus derived from an initial culture, wherein the initial culture is the culture described in claim 1, and at least 75% of the genome or genotype of the derived Agaricus bisporus culture is present in the genome or genotype of the initial culture of strain B19414, and the culture of strain B19414 is deposited under NRRL accession number 68095.

5. A culture according to any one of claims 1 to 4, which produces a pot bella mushroom crop at its first break having a brown cap, the diameter of which is greater than the diameter of the cap of a cultivar selected from the group consisting of Heirloom, Tuscan-820, Tuscan-860, and Brown, at the same stage of maturity.

6. A culture according to any one of claims 1 to 4, which produces a mushroom crop having a brown cap and a flesh thickness ratio of less than 0.

49.

7. A portion of a mushroom culture according to any one of claims 1 to 4, wherein the portion is selected from the group consisting of mycelium, spores, cells, and a portion of cells selected from nuclei and protoplasts, and if the portion of the culture is cells, the cells are formed on basidia cells of a mushroom culture of Agaricus bisporus referred to as strain B19414, where a representative culture of strain B19414 is deposited under NRRL accession number 68095, and if the portion of the culture is spores, the spores are selected from resting spores and germinating spores, and the resting spores and germinating spores include heteronuclear symbiotes and homonuclear symbiotes.

8. A product comprising a mushroom culture according to any one of claims 1 to 4, wherein the product comprises a mushroom culture according to any one of claims 1 to 4, selected from the group consisting of mycelium, spawn, inoculant, casing inoculant, raw mushroom, processed mushroom, mushroom pieces, and isolated culture substrate containing grain, compost, and easily crushable particulate matter.

9. The mushroom culture according to claim 2, wherein the culture has a genotype fingerprint having alleles at the marker locus indicated in the column labeled B19414 in Table I, all of the alleles of the fingerprint are present in the genotype fingerprint of strain B19414, and the culture of strain B19414 is deposited under NRRL accession number 68095.

10. A culture obtained from a portion of the mushroom culture described in claim 7.

11. A strain incorporating a portion of the mushroom culture described in claim 7.

12. Heteronuclear symbiotes and heteronuclear symbiotes obtained from mushroom cultures according to any one of claims 1 to 4.

13. Spores obtained from a mushroom culture according to any one of claims 1 to 4.

14. Mushrooms obtained from a mushroom culture according to any one of claims 1 to 4.

15. A method for producing a mushroom culture of Agaricus bisporus, comprising crossing a herniated symbiotic strain called B12998-s181, whose culture is deposited under NRRL accession number 68094, with a herniated symbiotic strain called P2-s203, whose culture is deposited under NRRL accession number 68093.

16. A mushroom culture according to any one of claims 1 to 4, or cells of a mushroom culture prepared by the method described in claim 15.

17. A culture containing the cells described in claim 16.

18. Use of the mushroom culture according to any one of claims 1 to 4 to obtain a second mushroom culture by using the strain development method.

19. Use of a mushroom culture according to any one of claims 1 to 4 or a mushroom culture prepared by the method described in claim 15.

20. Use of a hybrid mushroom culture according to any one of claims 1 to 4 or a hybrid mushroom culture prepared by the method described in claim 15.

21. Use of a hybrid mushroom culture according to any one of claims 1 to 4 for producing an isonuclear symbiont, EDV, strain, mushroom, or spore.