How to Collect Spores

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A culture arising l'rom cloning is fundamentally di Jerent than a culture originating from spores. When spores are germ' ated, many different strains are created, some incompatible with one another. A cultivator will not know what features will be expressed until each and every strain is grown out to the final stage, that of mushroom production.T1 is form of genetic roulette results in very diverse strains, some more desirable than others.

Mushroom spores are collected by taking a spore print. Spore prints are made by simply severing the cap from the stem, and placing the cap, gills down, upon a pece of typing paper, or glass. Covering the mushroom cap with a bowl or plate lessens evaporation and distur bance from air currents. Within 24 hours spores fall in a Beautiful pattern according to the radi-

Figure 75. The spore print can be folded and rubbed together so that spores drop onto nutrient agai' media. This method is not the best as concentrated populations of spores are grouped together.

Figures 76-79. An inoculation loop is sterilized, in this case with a BactiCinerator and then :o< e into the "receiving" dish. The spores are picked up by touching the inoculation loop to the spore The sporeladen inoculation loop is streaked across the surface of the sterilized, nutrient filled medium in an "S pattern.

Figures 76-79. An inoculation loop is sterilized, in this case with a BactiCinerator and then :o< e into the "receiving" dish. The spores are picked up by touching the inoculation loop to the spore The sporeladen inoculation loop is streaked across the surface of the sterilized, nutrient filled medium in an "S pattern.

What Nutrient Found Drug
Figures 80. Spores germinate according to the streaking pattern. A small portion is excised and transferred to a new, nutrient agar-filled petri dish

at:ng symmetry of the gills. (See Figure 14.) A single mushroom can produce from tens of thousands to a hundred million spores!

I prefer to collect spores on plates of glass, approximately 6x8 inches.The glass is washed wi h soapy water, wiped dry, and then cleaned vi h rubbing alcohol (isopropanol). The two pieces of glass are then joined together with a length of duct tape to create 'n effect, a binding The mushrooms are then laid on the cleaned, open surface for spore collection Af ter 12-24 hours, the cont:' Duting mushroom is removed dried, and stored for reference purposes (See Figure 15.)The remaining edges of the glass are then taped. The result ;s a glass enclosed "Spore Booklet" which can be stored at room temperature for years. Spores are easily removed from the smooth glass surface for future use. And, spores can be eas!,y observed without increasing the likelihood of contami-

Bacteria Spore And Without Spore
Figure 81. Scanning electron micrograph of spores germinating
Figure 82. Scanning electron micrograph 01 spores infected frith bacteria

nation.

Spores have several advantages. Once a spore print has been obtained, i: can be sealed and stored, even sent through the mail, with little ill effect. For the traveller, spore prints are an easy way to send back potential new strains to the home laboratory. Spores offer the most diverse source of genetic characteristics, far more than the phenotypic clone If you want the greatest number of strains, collect the spores. If you want to capture the characteristics of the mushroom vou have found, then clone the mushroom by cutting out a piece of living tissue.

To germinate spores, an inoculation loop, a sterilized needle, or scalpel is brought into contact with the spore print. (I prefer an inoculation loop.) I recommend flame sterilizing an inocu lation loop until red hot and immeuiately cooling it in a petri dish filled ivith a sterilized nutrient medium The tip immediately sizzles as it cools.The tip can now touch the spore print without harm, picking up hundreds of spores in the process. (By touching the tip to the meoium-filled petri dish first, not only is it cooled, but the tip becomes covered with a moist, adhesive layer of media to which spores easily attach.) The tip can now be stieaked :n an "S" pattern across the surface of another media dish, VBth heavy spore prints, the"S" streaking techr:que may not sufficiently disperse the spores In this case, the scalpel or inoculation loop should be immersed into a sterile vial holding 10 cc. (ml.) of water. After shaking thoroughly, one drop (or l/10th of 1 cc.) is placed onto the surface of the nutrient medium in each petri dish. Each dish should be tilted back and forth so that the spore enriched droplet streaks across the surface, leaving a trail of dispersed spores. In either case, spores will be spread apart from one another, so that individual germinations can occur

Five days later, spores may be seen germinating according to the streaking pattern. Colonies of germinating spores are subcultured Ho more petri dishes. (See Figure 80.) After the mycelium has grown away from the subculture site, a small fragment of pure mycelium is again sub -cultured into more petri dishes. If these cultures do not sector, then back-ups are made for storage and future use. This last transfer usually results in individual dik^yotic strains which are labelled. Each labelled strain is then tested for productivity. Mini-culture experiments must be conducted prior to commercial-level production.

When a concentrated mass of spores is germinated, the likelihood of bacteria and weed fi ingi infesting the site is greatly increased. Bacteria replicate faster than mushroom spores can germinate. As a result, the germinatng spores become infected. (See Figure 81.) Mycelium arising from such germinations are frequently associated with a high contamination rate, unfortunately often not experienced until the mycelium is transferred to grain media. However, if the spore prints are made correctly, contamination is usually not a problem

Once inoculated, the petri dish cultures should be taped with an elastic film (such as Parafilm™) which protects the incubating mycelium from intrusive airborne contaminants. (See Figure 58.)

Many cultures originating from spores or tissue are associated with other micrc organisms. Several techniques are at one's disposal for cleaning up a culture. Depending upon the type and level of contamination, different measures are appropriate

One way of cleaning a bacterially infested culture is by sandwiching it between two layers of media containing an antibiotic such as gentamycin sulfate. The hyphae, the cells composing the mycelium, are arranged as long filaments.These filamentous cells push through the media while the bacteria are left behind. The mycelium arising on the top layer of media will carry a greatly reduced population of bacter a, if any at all. Should the culture not be purified the first time using this procedure, a second treatment is recommended, again subculturing from the newly emerged mycelium. Repeated attempts increase the chances of success.

If the culture is mixed with other molds, then the pH of the media can be adjusted to favor the mushroom mycelium. Generally speal.'ng, many of the contaminant fungi are strong acidophiles whereas Oyster mushrooms grow well in environments near to a neutral pH. If these mold fungi sporulate adjacent to the mycelia of mushrooms, isolation becomes difficult. * Remember, the advantage that molds have over mushroom mycelia is that their life cycles spin far faster, and thousands of mold spores are generated in only a few days. Once molds produce spores, any disturbance—including exposure to the clean air coming from the laminar flow hood—creates satellite colonies.

One rule is to immediately subculture all points of visible growth away from one another as soon as they become visible. This method disperses the colonies, good and bad, so they can be dealt with individually. Repeated subculturing and dispersal usually results in success. If not, then other alternative methods can be implemented.

Mycelia of all fungi grow at different rates and are acclimated to degrading different base materials. One method I have devised for separating mushroom mycelium from mold mycelium is by racing the mycelia through organic barriers. Glass tubes can filled with finely chopped, moistened straw, wood sawdust, even crushed corncobs (without kernels) and sterilized. The contaminated culture is introduced to one end of the tube. The polyculture of contaminants of mushroom mycelium races through the tube, and with luck, the mushroom mycelium is favorably selected, reaching the opposite end first. At this point, the cultivator simply transfers a sample of emerging mycelium from the end of the tube to newly poured media plates. The cultures are then labelled, sealed and ob* The spores of most mold fungi become distinctly pigmented at maturity. SomePenicillium molds are typically blue-green.As-pergillus species range in color from black to green to yellow. Neurospora can be pink. A few molds, such as Monilia or Verticillium, produce white colonies. For more information on these competitors, please consult The Mushroom Cultivator (1983) by Stamets & Chilton.

served for future verification. This technique relies on the fact that the mycelia of fungi grow at different rates through biodegradable materials. The semi-selectivity of the culture/host substrate controls the success of this method.

Every cultivator develops his own strategies for strain purification. Having to isolate a culture from a background of many contaminants is inherently difficult. Far easier it is to implement the necessary precautions when initially making a culture so that running away from contamination is unnecessary.

CHAPTER

-j very sexually reproducing organism on this planet is limited in 'the number of its cell replications. Without further recombination of genes, cell lines decline in vigor and eventually die. The same | is true with mushrooms. When one considers the exponential expansion of mycelial mass, from two microscopic spores into tons of mycelium in a matter of weeks, mushroom mycelium cell division potential far exceeds that of most organisms. Nevertheless, strains die and, unless precautions have been taken, the cultures may never be retrieved.

Once a mushroom strain is taken into culture, whether from spores or tissue, the resultant strains can be preserved for decades under normal refrigeration, perhaps centuries under liquid nitrogen. In the field of mycology, cultures are typically stored in test tubes. Test tubes are filled with media, sterilized and laid at a 15-20 degree angle on a table to cool. (Refer to Chapter 12 for making sterilized media.) These are called test tube slants. Once inoculated, these are known as culture slants.

Culture slants are like "back ups" in the com puter industry. Since every mushroom strain is certain to die out, one is forced to return to the stock library for genetically younger copies. Good mushroom strains are hard to come by, compared to the number of poor performers isolated from nature. Hence, the Culture Library a.k.a. the Strain Bank, is the pivotal center of any mushroom cultivat'on enterprise.

One culture in a standari 100 x 15 mm. petri dish can inoculate 50-100 test tube slants measuring 100 x 20 mm. After incubation for 1-4 weeks, or until a luxurious myce!:um has been established, the test tube cultures are placed into cold storage, I seal the gap between the screw cap and the glass tube with a commercially available elastic, wax-like film. (Those test tube slants not sealed with this film are prone to contaminate with molds after several months of cold storage.) Culture banks in Asia commonly preserve cultures in straight test tubes whose ends are stuffed with a hydrophobic cotton or gauze The gauze is sometimes covered with plastic film and secured +ightly with a rubber band. Other libraries offer cultures in test tubes fitted with a press-on plastic lid especially designed for gas exchange. The need for gas exchange 'S minimal—provided the culture's growth is slowed down by timely placement into cold storage. Culture slants stored at room temperature have a maximum life of 6-12 months whereas cultures kept under refrigeration survive for 5 years or more. Multiple back-ups of each strain are strongly recommended as there is a natural attrition over time.

I prefer to seal test-tube slants in plastic zip-lock bags.Threeto four bags, each containing 4 slants,

Pleurotus Ostreatus Columbinus
Figure 33. Stock Cultures, in quadruplicate, sealed in a plastic bag, stored in a cedar box, and ref derated for years at 35° F. (1-2° C.) until needed.

are then stored in at least two locations remote from the main laboratory. This additional safety precaution prevents events lik: fires, electrical failure, misguided law enforcement officials, or other natural disasters from destroying your most valuable asset—'The Culture Library.

Household refrigerators, especially modern ones, suffice Those refrigerators having the greatest mass, with thermostatic controls limiting variation in temperature are best for culture storage. With temperature vari?t;on, condensation occurs within the culture tubes, spreading a contaminant, should it be present, throughout the culture. Therefore, limiting tem perature fluctuation to 2-3° F. (10 C.) is crucial for long term culture preservation. Furthermore, when mushroom cultures freeze and thaw repeatedly, they die

If one has ten or more rephcates, stock cultures of a single strain can be safely stored for 5 years by th:s method. As a precaution, however, one or two representative culture slants should be retrieved every year, brought to room temperature for 48 hours, and subcultured to newly filled media dishes Once revived, and determined to be free of contamination, the mycelium can once again be subcultured back into test tube slants, and returned to refrigeration. This circular path of culture rotat e n ascertains viability and prolongs storage with a minimum number of cell divisions. I can not over-emphasize the importance of maintaining cell lines closest to their genetic origins.

Cryogenic storage—the preservation of cultures by storage under liquid nitrogen—is the best way to preserve a strau. liquid p'trogen storage vessels commonly are held at -302° F.

(-150° C.). Test tubes slants filled with a specially designed cryoprotectant media help the myce;,'um survive the shock of sudden temperature change. (Such cryoprotectants involve the use of a 10% glycerol and dextrose media.) Wang and Jong (1990) discovered that a slow, controlled cooling rate of -1 degrees C. per minute resulted in a higher survival rate than sudden immersion into liquid r rogen. This slow reduction in temperature allowed the mycelium to d:scharge water extracellularly, thus protecting the cells from the severe damage crystals pose. Further, they found that strains were better preserved on grain media than on agar med' .. However, for those with limited iiqrid nitrogen storage space and large numbers of straii.s, preservafon on grair. media is not as practical as preservig strains in ampules or test tubes of liquid cryoprotectant

Figure 84. Examples of cultures originating from stock culture libraries from sources in United States, Canada, Thailand, and China. Most culture libraries do not send cultures in duplicate nor indicate how far the cultures have grown since inception.

media.

Of all the mushrooms discussed in this book, only strains of the Paddy Straw Mushroom, Volvariella volvacea, should not be chilled. V volvacea demonstrates poor recovery from cold storage—both from simple refrigeration at 34° F. (2° C.) or immersion in liquid nitrogen at -300° F. (-150° C.). When the mycelium of this tropical mushroom is exposed to temperatures below 45° F. (7.2° C.) drastic die-back occurs. Strains of this mushroom should be stored at no less than 50° F. (10° C.) and tested frequently for viability. When cultures are to be preserved for prolonged periods ai room temperature, many mycologists cover the mycelium with liquid paraffin. (For more information, consult Jinxia and Chang 1992).

When retrieving cultures from prolonged storage, the appearance of the cultures can immediately indicate potential viability or clear inviability. If the mycelium is not aerial but is flat, with a highly reflective sheen over its surface, then the culture has likely died. If the culture caps have not been sealed, contaminants, usually green molds, are often visible, giving the mycelium a specked appearance. These cultures make re-isolation most difficult Generally speaking, success is most often seen with cultures having aerial, cottony mycelium. Ultimately however, cultivators can not determine viability of stored cultures until they are subcultured into new media and incubated for one to fhree weeks.

The Stamets "P'? Value System for Age Determination of a Strain

The Stamets "P" value system is simply an arithmetic scale I have devised for measuring the expansion of mycelium through successive

Figure 35. AnEnokitake (Flammulina velutipes) culture is labelled to indicate genus, species, variety, "P" value, voucher collection number, and—if necessary—type of med'um

inoculations from one 100 x 15 mm. petri dish to the next.The number of cells divisions across a petri dish is affected by the range of cell wall lengths. Of the septate strains of fungi, some have cells as short as 20 p while others have cells 200 p and longer. The Stamets "P" Value (SPV) benefits cultivators by indicating how close to the origin their culture is at any point in time by simply recording the number of petri dishes the mycelium has grown across. When a culture has been isolated from contaminants, usually in one or two transfers, the first pure culture is designated as P,. When the mycelium has filled that dish, the next dish to receive the mycelium is called ?r Each culture is labelled with the date, species, collection number, strain code, P-Value and medium (if necessary). Thus, a typical example from one of my culture dishes reads:

11/16/92 C # 0825905

This means: the strain,FIammuIina velutipes isolated from Telluride, Colorado, has grown out over two petri dishes since its inception (in this case 8/90).The collection number refers to the date the mushrooms were^llected in the wild: it was the fifth group afon^firooms found that day. (Others sequei^flM ||t their collections, from 1 to infinity.)'iwcyjiure should be referenced to a dried voucher specimen from which the strain was generated. The dried specimens are either kept in your own private herbarium, or, better yet, deposited in an academically recognized herbarium which cross-indexes collections by date, species name, and collector. Keeping a voucher collection is critical so future researchers can commonly refer to the same physical standard.

The date "11/16/92" refers to the time the medium was inoculated. Spawn created from such young cultures, in contrast to one grown out twenty times as far, gives rise to more highly productive mycelium. The "P" value system is essentially a metric ruler for measuring relative numbers of cell divisions from the culture's birth. (Note that a square centimeter of mycelium is generally transferred from one culture dish to the next.) I have strains in my possession, from which I regularly regenerate cultures, which are ten years old and kept at a P2 or P . Having ten to twenty back-up culture slants greatly helps in this pursuit.

For purposes of commercial production, I try to maintain cell lines within P , that is, within 10 successive transfers to medium-filled petri dishes. Many strains of Morels. Shiitake and King Stropharia express mutations when trans ferred on media for more than 10 petri dishes. Morels seem particularly susceptible to degeneration. Morchella angusticeps loses its ability to form micro-sclerotia in as few as 6 or 7 plate transfers from the original tissue culture.

The slowing of mycelium may also be partly due to media specificity, i. e. the agar formula selectively influences the type of mycelial growth.To ameliorate degenerative effects, the addition of extracted end-substrates (sawdust, straw, etc.) favors the normal development of mycelium. The introduction of the end-substrate acquaints the mushroom mycelium with its destined fruiting habitat, challenging the mycelium and selectively activating its enzymatic systems. This familiarity with the end-substrate greatly improves performance later on. Parent cells retain a"genetic memory" passed downstream through the mycelial networks. Mycelia grown in this fashion are far better prepared than mycelia not exposed to such cultural conditions. Not only is the speed of colonization accelerated, but the time to fruiting is shortened. Only 1-3 grams of substrate is recommended per liter of nutrient medium. Substrates high in endospores (such as manures or soils) should be treated by first boiling an aqueous concoction for at least an hour. After boiling, sugar, agar and other supplements are added, and the media is sterilized using standard procedures described in Chapter 12.

By observing the cultures daily, the changeover of characteristics defines what is healthy mycelium and what is not. This book strives to show the mycelium of each species and its transformations leading to fruiting. Variations from the norm should alert the cultivator that the strain is in an active state of mutation. Rarely do mutations in the mycelium result in a stronger strain.

Subculture Paddy Straw Mushrooms
Figi-re 86. Ciassic forms of mushroom myceli«.

Each mushroom species produces a recognizable type of mycelium whose variations fall within a range of expressions Within a species, multitudes of strains can differ dramatically in their appearance. In culture, mushroom strains reveal much about the portion of the mushroom life cycle which is invisible to the mere forager for wild mushrooms This range of characteristics—changes in form and color, rate of growth, flagrance, even volunteer fruitings of mushrooms in miniature—reveals a wealth of information to the cultivator, defining the strain's "personality".

Form. Mycelia can be categorized into several different, classic forms. For ease of explanation- these forms are delineated on the basis of their macroscopic appearance on the two dimensional plane of a nutrient-filled petri dish. As the mycelium undergoes changes in its appearance over time this progression of transformations defines what is normal and what is abnormal.The standard media I use is MaltYeast Agar (MYA) often fortified with peptone (MYPA).

1. Linear: Linear mycelium is arranged as diverging, longitudinal strands. Typically, the mycelium emanates from the center of the petri dish as a homogeneously forming mat Shiitake (Lentinula edodes) and initially Oyster (Pleurotus ostreatus) mycelia fall in this category. Morels produce a rapidly growing, finely linear mycelium, which thickens in time. In fact, Morel mycelium is so fine that during the first few days of growth, the mycelium is nearly invisible, detected only by tilting the petri dish back and forth so that the fine strands can be seen on the reflective sheen of the agar

Spore Use Agar Mycelium Growing

Figure 87. Riiizoniorphic mycelium diverging from cottcnv mycelium soon after spore germination.

Figure 80. Classie rhizomorphic mycelium.

Figure 87. Riiizoniorphic mycelium diverging from cottcnv mycelium soon after spore germination.

medium's surface.

2. Rhuomorphic: Often similar to linear mycelium, rhizomorphic mycelium is often called"iopey". In fact, rhizomorphic mycelium is composed of braided, twisted strands, often of varying diameters. Rnizomorphij mycelium supports primordia. Its presence is encouraged by selecting these zones for further transfer. The disappearance of rhizomorphs is an indication of loss of vigor. Lion's Mane (Hericium erinaceus), the King Stropharia (Stropharia rugoso-annulata), the Button Mushrooms (Agaricus brunnescens, Agaricus bitorquis), the Magic Mushrooms (Psilocybe cvbensir and Puilocybe cyanescens), and the Clustered Woodlovers (Hypholoma capnoides and H. sublateritium) are examples of mushrooms producing classically rhizomorphic mycel: . Some types of rh; omorphic mycelia take on a reflec tive quality, resembling the surface of silk

3 Cottony: This type of myceli im is common with strains of Oyster Mushrooms (Pleurotus species), Shaggy Manes (Coprinus comatus), and Hen-of-the-Woods (Grifola frondosa). Looking 'ike tufts of cotton, the mycelium is nearly aerial in: :s growth. Cottony mycelium is commonly called tomentose by mycologists. When a rhizomorphic mycelium degenerates with age, tomentose formations typically take over.

4. Zonate: Cottony mycelium often shows concentric circles of dense and light growth, or zones. Zonate myceliun is often characteristic of natural changes in the age of the mycelium. The newest mycelium, on the periphery of the culture, is usually light in color. The more-aged myce!:um, towards the center of the culture becomes strongly pigmented.

Mycelium Cottony Ryzo
Figure 89. Quasi-rhizomorphic and cottony mycelia.

Zonations can also be a function of the natural, circadian cycles, even when cultures are incubated in laboratories where the temperatures are kept constant. Growth occurs in spurts, creating rings of growth. This feature is commonly seen in species of Ganoderma, Hypholoma and Hypsizygus.

5. Matted orappressed: This type of mycelium is typical of Reishi (Ganoderma luc.dum) after two weeks of growth on 2% malt-extract agar media. So dense is ¿his mycelial type that a factory-sharpened surgical blade can't cut through it. The mycelium tears off in ragged sheaths as the scalpel blade is dragged across the surface of the agar medium. Many species develop matted mycelia over time, especially the wood rotters. Cultures that mysteriously die often have mycelium which appears matted but whose surface is flat and highly reflective.

6. Powdered: This form of myceliun is best

Mycelium Cottony Ryzo
Figure 90. Zonate mycelium

THE STOCK CULTURE LIBRARY

Lentinula Edodes Mycelium Growth

Figure 91. Hyplial aggregates of the Fairy Ring musnroom (Marasmius oreudes) and Shiitake (Lentinula edodes), exemplified by Laetiporus sulphureus (Polyporus sulphureus), a.k.a. Chicken of the Woods. The mycelium breaks apart with the least disturbance. In front of a lair'nar flow bench, the sterile wii.d can cause chains of mycelium (hyphae) to become a'rborne. Free-flying hyphae can cause considerable cross-contamination problems wLnin the laboratory.

7. Unique Formations: Upon the surface of the mycelial mat, unique forma ions occur which can be distinguished from the background mycelium. They are various in forms Common forms are hyphal aggregates, cottony ball-like or shelf-like structures. I view h) phal aggregates as favorable formations when selecting out rapidly fruiring strains of Sf "'take. Hyphal aggregates often evolve into primordia, the youngest visible stages of mushroom formation. Marasmi'-s oreades, the Fairy Ring

Mushroom, produces shelf-like forms that define the character of its mycelium. Stropharia rugoso-annulata, the King Stropharia, has uniquely flattened, plate-like zones of dense and li^ht growth, upon which hyphal aggre gates often form Morel mycelium produces dense, spherical formations called sclerotia These sclerotia can be brightly colored, and abundant, as is typical of many strains of Morchella angushceps, or dull colored, and spars, 1'ke those of Morchella esculenta and Morchella crassipes

The mycelia of some mushrooms generate asexual structures called coreima (broom-like bundles of spores) which resemble many of the black mold contaminants. Some of these peculiar formations typify Pleurotus cystidiosus, Pleurotus abalonus, and Pleurotus smiihii. I know of one Ph.D. mycologist who, not knowing that some Oyster mushrooms go through an

How Collect Oyster Mushroom Spores
Figure 92. A strain of the Abalone Oyster Mushroom, Pleurotos cystidiosus. This mushroom species is dimorphic—having an alternative life cycle path (see Figure 41). The black droplets are resplendent with spores and are not contanCiants.

asexual stage, promptly c^scarded the cultures I gave her because they were "contaminated". (See Figure 92.)

Mushroom strains, once characterized by rhizomorphic mycelia, often degenerate after many transfers. Usually the decline in vigor fol lows this pattern: A healthy strain is first rhizomorphic in appearance, and then af'er months of transfers the culture sectors, form ing diverging "fans" of linear, cottony and appressed mycelium Often an unstable strain develops mycelium with aerial tufts of cottonlike growth. The mycelium at the center of the petri dish, giving birth to these fans of disparate growth, is genetically unstable, and being in an active state of decline, sends forth mutation-ridden chains of cells. Often times, the ability to give rise to volunteer primordia on nutrified agar media, once characteristic of a strain, declines or disappears entirely. Speed of growth decelerates. If not entirely dying out, the strain is reduced to an anemic state of slow growth, eventually incapable of fruiting. Prone to disease attack, especially by parasitic bacteria,, the mushroom strain usually dies.

Color: Most mushroom speciei produce mycelia that undergo mesmei zing transformations in pigmentation as they age, from the youngest stages of growth to the oldest. One must learn the natural progression of colorations for each species' mycelium Since the cultivator is ever watchful for the occurrence of certain colors which can forebode contamination, knowing these changes is critical Universally, the color green I s bad in mushroom culture, usually indicating the presence of

Button Mushroom Cultivation Pda
Figure 93. Miniature mushroom (Gymnopilus luteofolius) forming on malt agar media Note pro portion of mushroom relative to mycelial mat.

molds belonging to Pénicillium. Aspergillus or Trichoderma.

1. White: The color shared by the largest population of saprophytic mushrooms is white. Oyster (Pleurotus spp.), Shiitake (Lentinula edodes), Hen-of-the-Woods (Grifolafrondosa), the King Stropharia (Stwpharia rugoso-amulata) and most Magic Mushrooms (Psilocybes) all have whitish colored mycelium. Some imperfect fungi, like Monilia, however, also produce a whitish mycelium. (See The Mushroom Cultivator, Stamets and Chilton 1983.)

2 Yellow/Orange/Pink: Nameko (Pholiota nameko) produces a white mycelial mat which soon yellows. Oyster mushrooms, particularly Pleurotus ostreatus, exude a yellowish to orangish metabolite over time. These metabolites are sometimes seen as droplets on the surface of the mycelium or as excessive liquid collecting at the bottom of the spawn containers, Strains of Reishi Ganoderma lucidum, vary considerably in their appearance, most often projecting a white mycelium which as it matures becomes yellow as the agar medium is colonized. A pink Oyster mushroom, Pleurotus djamor, and Lion's Mane, Hericium erinaceus, both have mycelium that is initially white and, as the cultures age, develop strong pinkish tones. Chicken-of-the-woods (.Polyporus orLaetiporus sulphureus) has an overall orangish mycelium. Kuritake (.Hypholoma sublateritium) has mycelium that is white at first and in age can become dingy yellow-brown.

3. Brown: Some mushroom species, especially Shiitake, becomes brown over time. It would be abnormal for Shiitake mycelium not to brown in age or when damaged. Similarly, Agrocybe aegerita produces an initially white mycelium that browns with maturity. Morel mycelium is typically brown after a week of growth.

4. Blue: Lepista nuda, theWoodBlewit, pro duces a blue, cottony mycelium. Many species not yet cultivated are likely to produce blue mycelia. Although the number of species generating blue myce ium is few most of the psilocybian mushrooms are characterized by mycelium w__ich bruises bluish when damaged. Beyond these examples, blue tones are highly unusual and warrant examination through a microscope to ascertain the absence of competitor organisms, particularly the blue-greenPem'd/-lium molds. Although unusual, I have seen cultures of an Oyster mushroom, P. ostreatus var. columbinus, which produces whitish mycelium streaked with bluish tones.

5. Black: Few mushrooms produce black mycelium. Some Morel strains cause the malt extract medium to blacken, especially when the petri d ch culture is viewed from underneath. The parasitic Honey Mushroom, Armillaria mellea, forms uniquely black rhizomorphs. A pan-tropical Oyster mushroom, called Pleurotus cystidiosus, and its close relatives P. abalonus and P. smithii, have white mycelia that become speckled with black droplets. (See Figure 92.).

6. Multicolored: Myceliacan be zonate, with multicolored tones in concentric circles around the zone of transfer. The concentric circles of growth are usually diurnal, reflecting rates of growth dictated by the passage of day to night. All of the species described in the past 5 categories undergo unique color changea This sequence of color transformation defines the unique "personality" of each strain. I have yet to see a mycelium of greater beauty than that of the extraordinary Psilocybe mexicana, the sac-ramentalTeonanacatl of Mexico. Its mycelium is initially white, then yellow, golden, brown and sometimes streaked through with bluish tones. (See Color Plate 2, opposite page 176 in The Mushroom Cultivator by Stamets and

Mycelium Rhizomorphic Pleurotus
Figure 94. Mushroom primordia on malt agar media. Upper left: Ganoderma lucidum Reishi Upper right: Agrocybe aege-ü he F'.ack Poplar Mus . oom. Lowei left: Pleurotus djamor, the Pink Oyster Complex. Lower right: Hericium erinaceus, the Lion's Mane mushroom.

Chilton, 1983.)

Fragrance: The sensation most difficult to describe and yet so indispensable to the experienced spawn producer is that of fragrance. The mycelium of each species out-gasses volatile wastes as it decomposes a substrate, whether that substrate is nutrified agar media, grain, straw, sawdust, or compost. The com plexity of these odors can be differentiated by the human olfactory senses. In fact, each species can be known by a fragrance signature. As the mass of mycelium is increased, these odors become more pronounced. Although odor is generally not detectable at the per iish culture, it is distinctly noticed when a red-hot scalpel blade touches living mycelium The sudden burst of burned mycelium emits a fragrance that is specific to each species. More useful to cultivators is the fragrance signature emanating from grain spawn. Odors can constantly be used to check spawn quality and even species identification.

On rye grain, Oyster mycelium emits a sweet, pleasant, and slighdy anise odor. Shiitake mycelium has an odor reminiscent of fresh, crushed Shiitake mushrooms. Chicken-of-the-Woods (Laetiporus (Polyporus) sulphureus) is most unusual in its fragrance sig nature: grain spawn has the distinct scent of butterscotch combined with a hint of maple syrup! King Stropharia (Stropharia rugoso-annulata) has a musty, phenolic smell on grain but a rich, appealing woodsy odor on sawdust Maitake (Grifolafrondosa) mycelium on grain reminds me of day-old, cold corn tortillas! Worse of all is Enokitake—it smells like week-old dirty socks. Mycologists have long been amazed by the fact that certain mushrooms pro

THE STOCK CULTURE LIBRARY 115

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duce odors which humans can recognize elsewhere in our life experiences. Some mushrooms smell like radishes, some like apricots, and even some like bubble gum! Is there any significance to these odors? Or is it just a fluke of nature?

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  • mewael
    How to see rhizomorphic mycelium oyster?
    3 years ago
  • arja
    Do lentinula edods make rhizomorphic mycelium?
    3 years ago
  • abela
    How to collect lion's mane spores?
    3 years ago
  • donnamira
    How to collect oyster mushroom spores?
    3 years ago
  • Willow
    Why is my mycelium tissue culture partly yellow?
    2 years ago
  • bella
    How are mushroom spores collected?
    2 years ago
  • zane
    How to collect more spores from mushrooms?
    9 months ago
  • arrigo
    Is collection of abcteria easier than mold when subculturing from liquid media?
    6 months ago
  • aira per
    Which species of fungi has a bright white and cottony surface on SDA medium?
    5 months ago
  • kirsti
    How to collect spores sims?
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  • gregorio
    How to collect liquid spores?
    2 months ago
  • SARAH SYLVESTRE
    How to collect maitake spores?
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