WO2018013603A1 - Champignons blancs hybrides destinés à la production commerciale - Google Patents

Champignons blancs hybrides destinés à la production commerciale Download PDF

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WO2018013603A1
WO2018013603A1 PCT/US2017/041587 US2017041587W WO2018013603A1 WO 2018013603 A1 WO2018013603 A1 WO 2018013603A1 US 2017041587 W US2017041587 W US 2017041587W WO 2018013603 A1 WO2018013603 A1 WO 2018013603A1
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culture
hybrid
white
mushroom
strain
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PCT/US2017/041587
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Christopher William Robles
Stephen Christopher Lodder
Maykoe ABAUNZA
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Amycel, Inc.
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H15/00—Fungi; Lichens
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers

Definitions

  • the present invention relates to a novel class of hybrid cultures of the edible, cultivated mushroom fungus Agaricus bisporus (Lange) Imbach var, bisporus, and methods of producing and using the said hybrid culture. Specifically, to one or more newly developed cultures of white hybrid mushrooms produced by successively crossing progeny of wild strain AA-0096 to progeny of commercial white mushrooms. More particul arly, this invention relates to a newly developed hybrid strain designated 472x65 and to cultures that are descended, or otherwise derived, from Agaricus bisporus strain 472x65 crossed with commercial white mushroom strains including Ul derived commercial off-white hybrids to produce a new strain designated 9027, and descendants thereof, for commercial production. BACKGROUND OF THE INVENTION
  • the cultivated white button variety of Agaricus bispor s known as A. bisporus (Lange) Imbach (syn. A. brannescens Peck), is the predominant mushroom species in cultivation in the world today. Published data suggests that all the commercial white hybrid strains available for sale in the world are essentially derived varieties (EDV) of the Ul strain originally developed by Horst. For the last 30 years, mushroom breeders have been trying to improve this original hybrid by introducing a wild genomic parent material into commercial white mushroom hybrid (originally developed by a laboratory at Horst, the Netherlands) through a process known as crossing (Kerrigan et. al, US # 9,017,988 and A. Sormenberg et al, Fungal Genet Biol, 2016 June 8; 93; 93 35-45).
  • strains may have different ancestry, which will be reflected directly by the genotype and indirectly, in some cases, by the phenotypical characteristics.
  • strains may be differentiated on the basis of traits associated with the hybrid strains, such as, but not limited to, cap shape, surface texture, flesh thickness, color, shelf life, basidial spore number, sporelessness, resistance to infection by, symptoms of, or transmission of bacterial, viral or fungal diseases, amongst many other traits, to give some examples. Many of these traits directly contribute to factors that influence productivity, revenue capture, reduced costs, crop performance, and consumer and market demand.
  • bisporus spores derived from two-spored basidia preferentially contain nuclei of complementary mating type. These spores germinate to produce functionally diploid and self-fertile mycelium, known as heterokaryons. Heterokaryons contain two nuclei 'a' and 'b ⁇ These self-fertile heterokaryons can under correct environmental conditions, undergo several fruiting cycles commonly referred to as "breaks,"
  • viable non self-fertile spores are produced at the rate of 1 to 20%. These homokaiyotic spores arise from aberrant three-and four-spored basidia.
  • the homokaryotic mycelium so derived are used in controlled crossing that is the foundation of A. bisporus breeding. The homokaryons taken from the homokaryotic spores can be used in crosses to produce new strains of A. bisporus hybrids, with improved traits leading to increased consumer demand and revenue.
  • the present invention addresses the need for an improved white variety of Agaricus bisporus mushrooms with superior commercial characteristics relative to other commercial white mushrooms.
  • this invention advantageously provides white hybrid mushrooms for commercial production that have one or more (most preferably all) of the following beneficial characteristics:
  • Another aspect of the current invention is to advantageously provide a new and distinct A. bisporus mushroom pedigree, derived from an intermediate brown hybrid (a bridging cross strain), and its progenies, wherein the intermediate hybrid is obtained from crossing a wild strain AA-0096 or a progeny thereof!, to either protoplasts or single spore homokaryons obtained from commercial white mushrooms, industry referred to as Ul derived off-white hybrids,
  • a further aspect of the current invention is directed to an embodiment that provides a novel pedigree of hybrid strains and progenies thereof, derived from the newly developed intermediate hybrid strain designated 472x65, which is then crossed to compatible white homokaryons (single spore derived homokaryons and protoplasts), collected from commercial white strains including Ul derived commercial off-white hybrids to produce a new viable strain of white mushrooms designated as 9027, and descendants thereof, for commercial production.
  • white homokaryons single spore derived homokaryons and protoplasts
  • Another feature of the present invention includes a new white hybrid mushroom having improved agronomic traits leading favorably to increased consumer and market demand, productivity, crop performance, and revenue,
  • a further feature of the present invention is a novel breeding pedigree having beneficial characteristics unique to the new hybrid strain differentiating it from other commercial white strains.
  • FIG. 1 is directed to an illustration of an example of a breeding program.
  • FIG. 2 shows that the whiteness of novel hybrids is maintained for a longer period than the Ul derived commercial off-white hybrids.
  • a method of producing a hybrid mushroom culture includes the steps of providing a first culture of homokaryons selected from a wild strain, designated AA-0096, or a progeny thereof, wherein said wild strain AA- 0096 is available from ATCC under Accession No.
  • PTA-6903 providing a second culture of homokaryons selected from a commercial white strain, which is compatible with said first culture; and crossing said first culture with said second culture to form at least one heterokaryotic hybrid mushroom culture being capable of producing hybrid mushrooms, and their descendants via further crosses or self-fertile spores, wherein cells from said hybrid mushroom culture, said homokaryons and said hybrid mushrooms have at least one preferred phenotype and at least one marker inherited from said wild strain AA-0096.
  • An optional embodiment provides a method of producing a first hybrid mushroom culture of Agaricus bisporus. This method is comprised of the steps of: providing a first culture of homokaryons selected from a wild strain, designated AA-0096, or a progeny thereof, the homokaryons selected from the wild strain AA-0096 are single homokaryotic spores designated AA-0096ssi#472, wherein said wild strain AA-0096 is available from ATCC under Accession No.
  • PTA-6903 providing a second culture of homokaryons selected from a first commercial white strain, designated Amycel XXXp#65, which is compatible with the culture of homokaryons selected from the wild strain; and crossing the first culture with the second culture to form at least one heterokaryotic hybrid mushroom culture being capable of producing the first hybrid mushroom,, designated 472x65, a culture of which has been deposited under N RL Accession No. 67367, wherein cells from the first hybrid mushroom culture and said homokaryons have at least one preferred phenotype and at least one marker inherited from said wild strain AA-0096.
  • Another embodiment of the present invention further includes the steps of producing a second white hybrid mushroom culture, this method further comprising the steps of: selecting a homokaryotic line, designated 472x65 ss#29, a culture of which has been deposited under NRRL Accession No.
  • 673687 from the first hybrid mushroom; providing a fourth culture of homokaryons selected from a second commercial white strain, the fourth culture being compatible with the homokaryotic line 472x65 ss#29; and crossing the homokaryotic line 472x65 ss#29 with the fourth culture to form at least one heterokaryotic white hybrid mushroom culture, designated 9027, being capable of producing the second hybrid mushroom for commercial production, and wherein a representative culture of 9027 is available from NRRL under Accession No. 67366.
  • a further embodiment incudes a hybrid mushroom culture of Agaricus hisporus designated as strain 9027 or a homokaryon derived thereof, wherein a representative culture of said strain 9027 is available from NRLL under Accession No, 67366.
  • An optional embodiment includes a method of producing commercial white mushrooms comprised of the steps of: inoculating a mushroom growth medium with hybrid mushroom strain 9027, a culture of which has been deposited under NRRL Accession No. 67366: maintaining said inoculated growth medium under conditions conducive to mushroom fruiting; and collecting mushrooms from said growth medium for producing the commercial white hybrid mushrooms.
  • the present invention also includes a method of producing a white hybrid mushroom culture comprising mating a homokaryotic Sine, designated 472x65ssi#29, a culture of which has been deposited under NRRL Accession No. 67368, with a homokaryotic line obtained from an off-white commercial white strain, designated Phoenixx ssi# 157.
  • the present invention further includes amethod of producing white mushrooms for commercial production comprised of the steps of: selecting a homokaryotic line, designated 472x65 ss#29, a culture of which has been deposited under NRRL Accession No. 67368, from a heterokaryotic culture of hybrid mushroom, designated 472x65, a culture of which has been deposited under NRRL Accession No.
  • homokaryotic line designated 472x65 ssi#29
  • a culture of homokaryons selected from a commercial white strain from Ul derived off-white hybrids, designated Phoenixx ssi#157
  • 9027 being capable of producing the white mushrooms for commercial production, and their descendants via further crosses or self-fertile spores, wherein a representative culture of 9027 is available from NRRL under Accession No. 67366.
  • An alternative embodiment of the present invention advantageously provides a hybrid mushroom culture of Agaricus bisporus designated as strain 472x65 or a homokaryon derived thereof, wherein a representative culture of said strain 472x65 is available from NRLL under Accession No. 67367. .
  • a hybrid mushroom culture of Agaricus bisporus designated as strain 472x65 or a homokaryon derived thereof, wherein a representative culture of said strain 472x65 is available from NRLL under Accession No. 67367.
  • the present invention of hybrid mushrooms arose from a breeding program that crossed compatible hornokaryons collected from an intermediate brown hybrid strain and a commercial white mushroom strain.
  • the intermediate brown hybrid strain also referred to as a bridging cross strain, was produced by crossing compatible hornokaryons from a wild strain of Agaricus bisporus and a commercial white mushroom strain.
  • subsequent hybrid strains can be produced by crossing cultures of compatible hornokaryons selected from these novel bridging cross hybrid strains and commercial white mushroom strains, to induce and/or eliminate certain parental characteristics in the hybrid strains.
  • the specific wild mushroom strain of A. bisporus used to provide the desired genetic and phenotypical characteristics is known as AA-0096.
  • strain AA-0096 described in the scientific literature has unique genetic characteristics.
  • Strain AA-0096 also known as BP-1 and ARP-023, is available from the American Type Culture Collection (ATCC) under the accession number 76562 as a non-patent deposit.
  • ATCC American Type Culture Collection
  • This strain was re-deposited by the current inventors under the Budapest Treaty governing the deposit of organisms for patent purposes at the American Type Culture Collection, Rockville, MD, USA, under ATCC Accession No. PTA-6903, Deposits of the hybrid mushroom, strains and hornokaryons of this invention, as disclosed herein, are made with the Agricultural Research Services Culture Collection ( RRL) 1815 North University Street, Peoria, IL 61604, USA.
  • Hybrid strains obtained by crossing intermediate hybrid strains (or bridging cross strains) of A. bisporus and commercial white hybrid strains including Ul derived commercial off-white hybrids, and successive hybrids obtained by crossing such hybrids with other commercial white strains can be cultivated to have at least one (or preferably all) of the improved genetic and phenotypic characteristics described herein by beneficially using the procedures described herein.
  • Agaricus bisporus a nutritious edible fungus with a unique taste and having beneficial medical properties, is cultivated worldwide for commercial purposes.
  • A. bisporus has shown to introduce positive agronomic characteristics in hybrid progeny. These positive traits include rounder cap, denser cap tissue, improved shelf-life, better disease resistance, higher percentage of solids, etc.
  • A. bisporus exhibits arnpithallisrn, a reproductive syndrome with two distinct lifecycles, heteromixis and intramixis, operating concurrently. In heteromixis or more commonly outbreeding, the A. bisporus spores each receive a single haploid postmeiotic nucleus.
  • spores In intramixis or more commonly inbreeding, most spores receive two Non-Sister Nuclear Pairs (NSNPs) post-meiotic nuclei.
  • NNPs Non-Sister Nuclear Pairs
  • the haploid spores from heteromixis germinate and produce homokaryotic offspring that can mate with other compatible homokaryons to produce novel hybrid heterokaryons, competent of producing mushrooms.
  • the intramixis spores are referred to as self-fertile spores. Relatively, little chromosomal crossing-over is observed to have occurred in postmeiotic offspring of A. bisporus. Consequently, parental and offspring heterokaryotic genotypes and phenotypes tend to closely resemble each other (Kerrigan et. al. in Genetics, 133, 225-236, 1993, Sonnenberg et. al., Fungal Genet. Biol., 93 35-45, June 8, 2016).
  • a culture of selected homokaryons collected from a wild strain of A. bisporus is first crossed with compatible homokaryons from commercial white strains to form a first hybrid strain, also referred to as an intermediate or a bridging cross strain.
  • the homokaryons are usually collected from the natural process of spore formation, but they are also obtained through a technique called protoplasting.
  • Homokaryons collected from spores are called Single Spore Isolates (SSI or ssi), and those created from protoplasting are called Protoplasts (p).
  • SSI or ssi Single Spore Isolates
  • Protoplasts Protoplasts
  • the fundamental difference between these two types of homokaryons is that the SSIs have gone through a meiosis step, and protoplasts have not.
  • SSIs are collected from spore prints, and protoplasts which are created from the enzymatic removal of the mycelial fungal cell wall.
  • spores from wild mushroom strain designated AA- 0096 are used. Furthermore, a specific homokaryon designated as AA-0096ssi#472 is crossed to another homokaryon designated as XXXp#65, and this bridging cross strain or the intermediate hybrid is one or more embodiments of this invention.
  • the selection of AA- 0096 as one parent was not apparent until after the completion of the breeding program because in Agaricus bisporus, the brown color is dominant to white and it was therefore not apparent that a brown parent could lead to improved white hybrids.
  • the white button mushroom is the predominant mushroom species in cultivation. Since the release of Ul and U3 strains (developed by Horst), these strains have dominated the industry. Ul derived commercial off-white hybrids were used in this invention because of their accepted agronomic traits in the marketplace, these include but are not limited to, yield, speed, overall commercial performance, shelf-life, etc. Most white hybrid mushrooms are formed by the introduction, through crossing, of novel genomic material into a commercial white mushroom or derivative, in one or more embodiments, Ul derived commercial off-white hybrids were used. In one or more embodiments, protoplasts derived from Amycel Ul derived commercial off-white hybrids are used to produce bridging cross strains.
  • Amycel Ul derived commercial off-white hybrids were used as an example in one or more of the embodiments of this invention because of their availability. Likewise, any commercial white strain including Ul derived commercial off-white hybrids could be used replacing Amycel Ul derived commercial off-white hybrid strains in one or more eemmbbooddiimmeennttss ooff tthhiiss iinnvveennttiioonn ssiinnccee tthheeyy aarree a allll ggeenneettiiccaallllyy aanndd pphheennoottyyppiiccaallllyy ssiimmiillaarr..
  • AAddddiittiioonnaall eemmbbooddiimmeennttss iinncclluuddee hhoommookkaarryyoonnss ffrroomm AAmmyycceell DDeellttaa,, AAmmyycceell XXXXX,, AAmmyycceell PPhhooeenniixxxx,, aanndd ootthheerr c coommmmeerrcciiaallllyy aavvaaiillaabbllee wwhhiittee ssttrraaiinnss,, iinncclluuddiinngg 1 U11l ddeerriivveeddd ccoommmmeerrcciiaall ooffff--wwhhiittee hhyybbrriidds
  • Homokaryotic lines are the preferred starting cultures for breeding as they have maximal ability to anastomose and achieve plasmogamy with other cultures.
  • Heterokaryotic hybrid strains of Agaricus bisporus are created by combining compatible homokaryons (cells that have multiple identical nuclei).
  • the homokaryotic isolates are collected from the natural processes of spore formation, or by the techniques of protoplasting.
  • Homokaryons collected from spores are called Single Spore Isolates (SSI or ssi), and those from protoplasting are called Protoplasts (p).
  • Homokaryons derived from single spore isolates go through the meiosis step.
  • homokaryotic spores are collected from spore prints to give an example, in another embodiment, spores may be isolated under a microscope using sterile microtools, such as needles. In some of the other embodiments, protoplasts are collected by removing the fungal cell wall through the action of enzymes.
  • Protoplasting techniques have been described in many publications, for example "A protoplasting technique with general applicability for molecular karyotyping of hymenomycetes," J. Gen. Microbio., 139, 1063- 1067, 1993, by S. Lodder et al. Protoplasts are considered homokaryons depending on the type of nuclei present in the cytoplasm.
  • viable non-self fertile spores are produced at the rate of 1 to 20%. These homokaryotic spores arise from aberrant three-and four-spored basidia.
  • the homokaryotic mycelium derived from these spores can be used for the controlled crossing for A, bisporus breeding.
  • homokaryotic lines are associated in predetermined pairwise combinations. In one method, homokaryotic pairs are placed in close proximity on a growth medium to achieve anastomosis.
  • the homokaryons crossed were AA-0096ssi#472 and Amycel XXXp#65 for the creation of a bridging cross strain, or intermediated brown hybrid, and the resultant hybrid, designated 472x65.
  • a good example of this embodiment is homokaryons 472x65ssi#29, A series of hybrids derived from crossing homokaryons 472x65 ssi#29 from bridging cross strain 472x65 with homokaryons from commercial white mushroom strains, including Ul derived commercial off-white hybrids, are some of the examples of white hybrid mushrooms.
  • a good example of a hybrid embodiment is a cross between 472x65ssi#29 and Amycel Phoenixx ssi#157, designated 9027.
  • Essentially Derived Variety is a variety that is predominantly derived from an initial variety or from an EDV of an initial variety, and retains the essential characteristics of the initial variety except where the differences that arise are solely from such a derivation.
  • ED s incorporate elements of (1) relatedness, (2) methods of derivation, (3) and empirical tests.
  • a strain or culture predominantly or entirely derived from a single initial strain or culture, or from the modification of an initial culture using methods including somatic selection, tissue culture selection, selling (mating among sibling lines) including intramixis (reproduction via single spores and multiple spores and mating of sibling offspring lines), back-mating to the initial variety, or mutagenesis and/or genetic transformation of the initial variety to produce a distinct culture in which the genotype of the resulting culture remains predominantly identical to the initial variety, having most or all, but at least 75% of its genome present in its initial strain or culture, is an EDV.
  • an Essentially Derived Variety of A. bisporus strain or intermediate strain or hybrid strain may be used. It is to be noted that the usage of the term “derived” in many techniques and processes listed herein is not in any way congruent with "Essentially Derived Variety.” The two processes are mutually exclusive and well understood in the art of mushroom breeding.
  • Unique markers help differentiate the white hybrid mushrooms of this breeding program, from Ul derived commercial off-white hybrid mushrooms.
  • New genotypical characteristics like incompatibility or non-anastomosis along with some of the phenotypical traits like cap shape, cap density, percent solids, shelf-life amongst other phenotypical traits also help distinguish these white hybrids from other commercial Ul derived off-white type mushrooms.
  • An example of one of the embodiments is 9027 that has one or more unique genetic markers differentiating it from commercial Ul derived off-white type mushrooms, as shown in Table 1. It is to be noted that the markers shown in Table 1 are just a few of the thousands of markers that can differentiate novel hybrids from Ul derived off-white commercial hybrids.
  • Table 1 One or more markers differentiating white hybrid mushrooms of this invention from commercial Ul derived off-white type mushrooms.
  • the novel strains of this invention are genetically different and incompatible with other commercial strains. That is, these novel strains are incompatible with all of the white mushrooms strains currently on the market and hence could act beneficially as a virus break.
  • This invention due to its novel genetics, may also offer improved resistance to know r n and emerging mushroom pathogens (the importance of providing disease resistance is discussed in Kerrigan, R., Mushroom News Volume 53 No. 14-23, 2005). Worldwide, all of the white commercial white mushroom strains that are in the market are compatible with each other.
  • the novel strains of this invention are also incompatible with commercial brown strains.
  • 472x65 is one embodiment of the invention and, additionally, the homokaryon designated 472x65ssi#29 is an example of another embodiment of the present invention.
  • Ail crosses between homokaryons from 472x65 fruiting bodies and commercial white homokaryons that have at least one improved characteristics noted herein are representatives of this preferred class of crosses.
  • strains derived from this pedigree exhibit improved characteristics over the Ul derived off-white type strains. These improvements include cap roundness, improved shelf-life, percent solids, density and improved disease resistance (incompatibility). These improvements are a result of the introduction of the wild parent, AA-0096, and subsequent crosses and selection. These improvements are in addition to the many traits that have made Ul derived oft- white hybrids successful commercial mushroom strains. Mushrooms with improved cap roundness will attract a higher price than conventional Ul derived commercial off-white hybrids. [0041] The invented class is suitably configured to produce white hybrid mushrooms with thicker domed shape caps, giving them a distinct shape compared to commercial Ul derived off-white type hybrids.
  • the cap roundness is a big plus for the grower, because it can significantly improve crop profitability.
  • the round shape of this mushroom reduces the amount of open mushrooms that a grower cannot sell and thus minimizes loss.
  • These open mushrooms are referred to as "Number 2s" in the industry and are sent to a cannery or even thrown away.
  • Table 2 below shows fewer open, or "Number 2s", for the 9027 white hybrid mushroom, an example of this invention as compared to Amycel Delta, a commercial Ul derived off-white type white mushroom. In each trial, the mushrooms were grown under exactly the same conditions.
  • the caps of hybrid strains of this invention remained closed longer than the Ul derived off-white type hybrids, due to their rounder cap shape.
  • Cap roundness or cap shape is a ratio of the maximum distance between the top and bottom of the mushroom cap (CH) divided by the diameter of the cap (CD). A higher number indicates a rounder mushroom. Measurements are made after the mushroom has been bisected. Table 3 summarizes the result of experiments to show the improved rounder caps of hybrids. As an example, 150 muslirooms from each treatment were collected for first and second break. 100 mushrooms were collected for third break, which was harvested over a shorter period. The mushrooms were sliced in half and then cap height and width were measured. All mushrooms were from the same test crop, and treated in exactly the same manner.
  • This invention is also configured to produce mushrooms with higher percent solids on the first and second break.
  • the percent of mushroom that is solid is determined by drying a defined weight of sliced mushrooms. As an example, 150 mushrooms from each treatment were collected for first and second break. 100 mushrooms were collected for the third break, which was harvested over a shorter period. A clean Aluminum pan was placed on a tared balance scale. The weight of the pan is recorded to ⁇ 0.0 Ig. Mushrooms of similar size harvested at the same time were used for the measurement. The stems were removed and mushrooms were sliced to 5mm thickness using an egg sheer. The weight, of sliced mushrooms on the pan was recorded before drying. The pan of mushrooms was then placed in a drying oven at 80°C until no additional weight loss is recorded, where a typical process like this takes 2 days. The difference between fresh weight and final dry weight is the dry matter of the mushroom.
  • Table 5 refers to data comparing the percent solids of hybrid mushrooms of the current invention to the commercial Ul derived off-white type hybrids.
  • Table 5 Higher Percent Solid s of Novel White Hybrid s Non-anastomoses ' to Prevent .. Virus, . Transmission:
  • Electron microscopic examinations have shown the presence of mycoviruses generally in large amounts in the hyphal cytoplasm (Albouy and Lapierre, 1971; Dieleman-van Zaayen,
  • mycoviruses are readily transmitted from an infected strain to a healthy compatible strain.
  • the mycelia of compatible strains of mushrooms are readily able to anastomose (fuse together) and facilitate the transportation of nutrients and nuclear constituents along with mycoviruses.
  • One trait of biological and commercial interest is the heterokaryon incompatibility, where the virus transmission is inhibited. In non-compatible strains, mycelial fusion is diminished. The lack of anastomosis between strains has the potential of limiting the propagation of the viral infection.
  • a typical experiment to ascertain incompatibility is to grow the putative incompatible strains side by side by the standard methods using two inoculated substrates. The combination of strains will result in retarded growth and lower mushroom yields, demonstrating incompatibility. While the present invention achieves better commercial properties of cap roundness, higher percent solids etc., relative to the commercial white mushrooms, incompatibility is said to be present, rather than being "better.”
  • a white hybrid strain was tested with Amycel Delta, Trays of 9027 and Ul derived commercial off-white hybrid type Amycel Delta were grown under exact same conditions. Reduced yield indicates the incompatibility of the two hybrids.
  • Table 6 shows existence of incompatibility through reduced yield of the novel white hybrids for four experiments where different combinations were grown side by side.
  • Spawn strain refers to the strain that was inoculated into the mushroom compost.
  • Case strain refers to the strain inoculated into the commercial casing soil.
  • This invention is also suitably configured to produce mushrooms with longer shelf- life.
  • This particular parameter is measured by the whiteness of the white hybrids as compared to commercial Ul derived off-white hybrids. Error! Reference souree not found, shows that the whiteness of novel hybrids is maintained for a longer period than Ul derived commercial off-white hybrids. Mushroom surface color data was evaluated with a chroma meter ( onica Minolta BC-10, Osaka, Japan), by measuring the L* parameter. L* is a brightness variable and extends from 0 (black) to 100 (white). Whiteness indicative of longer shelf-life of novel white hybrids (bars represent standard error as shown in Figure 2).
  • Mushrooms of similar cap diameters were collected from both 9027 and Amycel Delta at the same crop stage, and mushrooms were placed into mushroom storage tills as per normal commercial practice and held at 36 0 -38° F for shelf-life evaluation. Between 25 -60 muslirooms were analyzed for L* value at each time point of the shelf life trial (day 0, 1 , 3, 6, 8, 10, 13, 15, and 17). Measurements were taken on the tops of the caps at random. The 9027 cap surface remained whiter over the period of the 17 ⁇ day shelf-life trial. The shelf-life improvements became apparent after the completion of larger test in the test facility. It was apparent that new hybrids from 472x65 pedigree offer a distinct advantage over the Ul derived hybrids that are sold commercially all over the world.
  • a "genetic characteristic” is any property of the genetic material of the mushroom strain (usually a gene sequence) that is measurable by a standard analytical technique. Mushrooms within the scope of the present invention are characterized by having at least one or more genetic characteristics of the first wild parent strain, AA-0096ssi#472, that is not a corresponding genetic characteristic of the second parent strain. Table 7 below shows one or more of the genotype differences between AA-0096ssi#472 and the Ul parent.
  • Table 7 Table showing a partial genetic marker profile of AA ⁇ QG96ssi#472 based on SNP
  • progeny strain genetically derived from crossing an AA-0096 with a commercial white mushroom strain including Ul derived commercial off- white hybrid that retains a commercially desirable characteristic remains within the scope of this invention.
  • Progeny strains can be selected by the aforementioned characteristics of interest. In Agaricus bisporus, brown color is dominant to white, and to create a white hybrid using a brown mushroom parent AA-0096, AA-0096 parent is first crossed with a white mushroom. Ul derived commercial off-white hybrids were selected, as they are the most widely grown commercial mushroom variety in the US. The first step in the process of choosing progeny is to determine mating type and color.
  • homokaryons were determined by using techniques described using the MAT marker (Xu J et. al,, 1993; Localization of the mating type gene in Agaricus bisporus. Appl Environm. Microbiol 59:3044-3049), and by doing tests of fruiting crosses. Homokaryons derived from 472x65 were screened for color with a marker based on the work described in prior publications (Loftus, et. al., 2000 and Science and Cultivation of Edible Fungi: 201 -202).
  • homokaryons have been collected from the 472x65 (or other AA-0096 homokaryons crossed to commercial white homokaryons, including those from Ul derived commercial off-white hybrids, or other intermediate brown hybrids, or other bridging cross strains) they are crossed to an initial pool of homokaryons collected from the Ul derived commercial hybrids. Homokaryons like 472x65ssi#29 are then identified by the promising phenotypical characteristics that they exhibit. These are then crossed to a larger pool of white homokaryons. Progeny that have commercially desirable characteristics are determined through observation and comparison with Ul derived commercial off-white hybrids as controls. In the case of 9027, and other related crosses involving progeny of 472x65, rounder caps were observed. Measurements were then taken to compare these caps to caps of the control mushrooms and hence resulted an example of one embodiment of this invention.
  • DNA fingerprinting of novel strains was determined by polymerase chain reaction (PGR) analysis using sequence-characterized amplified region markers (SCAR) based on SNP data.
  • PGR polymerase chain reaction
  • SCAR sequence-characterized amplified region markers
  • DNA fingerprinting techniques were adapted from those described in Khush, R.S., Becker, E. & M. Wach (1992); DNA Amplification Polymorphisms of the cultivated mushroom Agaricus bisporus; Appl Environmental Microbiol 58(9):2971 ⁇ 2977, and Williams, J.A., Kubeliki, K.5 Livat, K., Rafalski, J. & S. Tingey. (1991); DNA polymorphisms amplified by arbitrary primers are useful as genetic markers; Nuclei Acids Research. 22:6531 -6525.
  • DNA isolation cultures were grown either in MPYFE liquid medium (Castle et al., 1987) or on cellophane over CL Agar. Harvested tissue was frozen at -70° C. and was freeze dried prior to DNA isolation. DNA was prepared from freeze-dried mycelium. First, freeze dried tissue was ground with a glass rod, and 0.6 ml of 65° C. DNA extraction buffer (0.7 M sodium chloride/0.1 M sodium sulphite/0.1 M Tris-HCl, pH 7.5/0.05 M EDTA/1% SDS) was added. Tubes were mixed and placed at 65° C. for 30 minutes.
  • DNA extraction buffer 0.7 M sodium chloride/0.1 M sodium sulphite/0.1 M Tris-HCl, pH 7.5/0.05 M EDTA/1% SDS
  • Homokaryons used in the invention were derived from spontaneous hornokaryons identified from single spore isolates (SSI), and from protoplasting (p). Spores were collected from mushrooms, and the spores were diluted in H 2 0 containing 1% Tween 80. Spore density was calculated on a hemocytometer slide, and spore dilutions were plated out on PDA. Individual spores were isolated, and then tested to determine if they were homokaryons or heterokaryons. Slow growing protoplast homokaryons were isolated from the mycelia of Ul and AA-0096 that were protoplasted as described in Khush, R. S., Becker, E. & Wach, M. W. (Applied and Environmental Microbiology 58 2971- 2977(1992)). DNA was isolated as described in the same Khush et al. publication. A brief description of the techniques are provided in the following section.
  • Protoplast density was measured with a hemocytometer slide, and protoplast concentration was adjusted through centrifugaiion and resuspension in protoplast buffer.
  • Protoplasts were regenerated on Regeneration Medium (RM: 200 ml compost extract/5 g Peptone (Bacto)/205.4 g sucrose/15 g agar per liter).
  • Protoplasts were spread on plates of fresh KM with a sterile glass rod. Regenerants appeared after 7 days, and were selected for the slow growing types.
  • Deposits of parental line 472x65 and homokaryotic line 472x65ssi#29 are made with the Agricultural Research Services Culture Collection (NRRL) 1815 North University Street, Peoria IL 61604, USA. Specifically, a representative culture of 472x65 is available from the NRRL under Accession No. 67367, and a representative culture of 472x65ssi#29 is available from the NRRL under Accession No. 67368. Further, a representative culture of 9027, the commercially viable white hybrid mushrooms for commercial production, is available from the NRRL under Accession No. 67366. The date of deposits was made on or about Feb. 8, 2017. All restrictions upon the deposit have been removed, and the deposit is intended to meet all deposit requirements of the U.S.
  • Patent and Trademark Office including 37 C.F.R. Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty.
  • the deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.
  • the cultures are irrevocably and without restriction or condition, released to the public upon the filing of the priority application or upon the issuance of a patent on this strain according to the patent laws.
  • the hybrid mushrooms of this invention remain Agaricus bisporus mushroom strains, they can be grown using standard commercial mushroom growing processes that have been developed for Agaricus bisporus. Such processes are well known in the industry, also available from technical publications.
  • the process comprises inoculating a mushroom growth medium with a hybrid Agaricus bisporus mushroom strain of this invention, maintaining the inoculated growth medium under conditions conducive to mushroom fruiting, and collecting mushrooms from the growth medium after they have reached the desired maturity level.
  • Commercial mushroom spawn production is a controlled process that involves the long term storage of mushroom strains, and the subsequent bulking up of inoculum for commercial production. All strains of this invention will be maintained in long term storage, and go through the regular commercial production process.
  • the methods for storage and mushroom spawn production differ from spawn producer to spawn producer and are largely proprietary. This is based on typical commercial growing parameters.
  • heterokaryons were fruited on standard phase II mushroom compost.
  • the compost was colonized with inoculated millet spawn for thirteen to fourteen days, with bed temperatures in the range 21°-27° C, and CO2 between 5000 and 10000 parts per million (pprn).
  • Beds were then covered with a 5 ⁇ cm layer of casing formulation (approximately 75% peat/25% CaCCte), and the cased beds were scratched after five days to encourage mycelial growth into the casing layer.
  • Two days after scratching the beds were flushed, with the air temperature dropping to 16° C and the CO2 dropping to 1000 to 1500 ppm. Mushrooms appeared approximately two weeks after flushing, and during first break bed temperatures were held at 18° to 21° C. For every crop, yield was assessed using three breaks of production.
  • Mushrooms were harvested over a three-week period, and comprehensive yield data was collected using a system designed specifically for this purpose.
  • the mean yield of the two strains, expressed in pounds of mushrooms produced per square foot data is as shown in Table 5 .
  • Any intermediate hybrid produced by crossing a wild genomic parent, such as AA- 0096, to a commercial Ul derived off-white parent and having the potential of introducing improved characteristics of commercial appeal when crossed to a second commercial U 1 derived off-white parent remains within the scope of this invention.
  • An example of such an intermediate brown hybrid is 472x65. Homokaryons were collected from a spore print of AA-0096 using standard techniques. AA-0096ssi#472 was crossed to a protoplast derived homokaryon, XXX p#65, and the resulting hybrid was fruited in a small test pot and spores were collected. Homokaryons from this spore print were collected and analyzed for color and mating type.
  • Amycel XXX is a Ul derived white mushroom strain sold commercially in the US and E urope.
  • XXX p#65 is a homokaryon collected from protoplasting mycelium of XXX following standard methods. This protoplast was chosen to use to cross to AA-0096ssi#472 because of its mating compatibility, and would introduce white color and other desired characteristics into the breeding pedigree.
  • any progeny strain genetically derived by first crossing AA-0096 and commercial white strain, including Ul derived commercial off-white hybrid strain, and then crossing homokaryons from this intermediate hybrid with white commercial homokaryons, collected from commercial white strains (including Ul derived commercial off-white hybrids), that retain commercially desirable characteristics of the strains of the invention remains within the scope of the invention.
  • white progeny strains can readily be selected by shape of the cap, density, percent solids, antagonism with the commercial Ul derived off-white hybrids, as well as the identification of genetic characteristics inherited from the wild AA-0096 parent.
  • the commercially desirable characteristics like non-anastomosis Incompatibility, cap shape, percent solids, shelf-life amongst other desirable characteristics can be measured quantitatively using the general techniques described earlier.
  • the current invention pertains to white hybrid mushrooms for commercial production, which may be of value or importance to various industries such as, but not limited to, growers, processors and marketers of mushrooms, farming industry, produce industry and/or the agricultural industry.

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Abstract

Cette invention concerne une nouvelle classe de cultures hybrides du champignon cultivé, comestible, Agaricus bisporus (Lange) Imbach var. bisporus, ainsi que des procédés de production et d'utilisation de ladite culture hybride. L'invention concerne plus particulièrement des cultures nouvellement développées de champignons blancs hybrides produits par croisement successif de la descendance de la souche sauvage AA-0096 à la descendance de champignons blancs du commerce. En particulier, l'invention concerne des homocaryons provenant d'un hybride intermédiaire nouvellement développé désigné 472x65 et des cultures qui descendent de ces dernières, ou sont autrement dérivées de ces dernières, croisés à des homocaryons provenant de souches commerciales de champignons blancs. Les procédés de cette invention produisent des champignons blancs hybrides présentant des caractéristiques commerciales supérieures par rapport à d'autres champignons blancs du commerce. De manière spécifique, cette invention fournit avantageusement des champignons blancs hybrides, désignés 9027, qui présentent une ou plusieurs des caractéristiques avantageuses du point de vue commercial telles que des matières solides de champignon à un pourcentage plus élevé, un chapeau plus arrondi, une durée de conservation plus longue, un chapeau plus dense, une meilleure barrière contre les maladies génétiques, d'autres qualités esthétiques et similaires.
PCT/US2017/041587 2016-07-12 2017-07-11 Champignons blancs hybrides destinés à la production commerciale WO2018013603A1 (fr)

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CN108531641A (zh) * 2018-05-25 2018-09-14 上海市农业科学院 基于snp位点差异的鉴定双孢蘑菇同核不育单孢菌株及其交配型的方法及引物
CN109207625A (zh) * 2018-10-29 2019-01-15 福建省农业科学院食用菌研究所（福建省蘑菇菌种研究推广站） 一种区分双孢蘑菇4个主栽品种的ssr-pcr鉴定方法
CN111394491A (zh) * 2019-01-03 2020-07-10 中国科学院微生物研究所 蘑菇交配型分子标记及其在鉴定蘑菇交配型中的应用
WO2022023290A1 (fr) * 2020-07-27 2022-02-03 Somycel Lignée de champignon n-s34, incorporée dans la souche de champignon hybride la3782, et ses dérivés
CN114250151A (zh) * 2021-11-22 2022-03-29 海南省林业科学研究院(海南省红树林研究院) 一种金福菇的杂交育种方法

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
CN108531641A (zh) * 2018-05-25 2018-09-14 上海市农业科学院 基于snp位点差异的鉴定双孢蘑菇同核不育单孢菌株及其交配型的方法及引物
CN108531641B (zh) * 2018-05-25 2022-03-25 上海市农业科学院 基于snp位点差异的鉴定双孢蘑菇同核不育单孢菌株及其交配型的方法及引物
CN109207625A (zh) * 2018-10-29 2019-01-15 福建省农业科学院食用菌研究所（福建省蘑菇菌种研究推广站） 一种区分双孢蘑菇4个主栽品种的ssr-pcr鉴定方法
CN109207625B (zh) * 2018-10-29 2022-01-04 福建省农业科学院食用菌研究所（福建省蘑菇菌种研究推广站） 一种区分双孢蘑菇4个主栽品种的ssr-pcr鉴定方法
CN111394491A (zh) * 2019-01-03 2020-07-10 中国科学院微生物研究所 蘑菇交配型分子标记及其在鉴定蘑菇交配型中的应用
WO2022023290A1 (fr) * 2020-07-27 2022-02-03 Somycel Lignée de champignon n-s34, incorporée dans la souche de champignon hybride la3782, et ses dérivés
CN116456825A (zh) * 2020-07-27 2023-07-18 索米赛尔公司 掺入杂交蘑菇菌株LA3782的蘑菇品系N-s34及其衍生物
CN114250151A (zh) * 2021-11-22 2022-03-29 海南省林业科学研究院(海南省红树林研究院) 一种金福菇的杂交育种方法

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