Lecture 3 Effect of genetic factors, temperature, light, humidity, medium on cultivation of mushrooms. Techniques of Commercial cultivation of some important mushrooms, Single spore isolation/pure culture and spawn production techniques, Present situation and prospect of mushroom cultivation in Nepal.

Mitesh Shrestha Genetic Factors • For breeding work in edible species it is desirable to know how fruiting is controlled genetically as well as nutritionally and physiologically.

Genetic Factors Temperature

• The conditions that are favorable for reproduction are always less favorable for growth. • An increase in temperature generally increases enzymatic activity. In that part of the growth curve that is linear, the growth rates approximately double for each 100C increase in temperature (i.e., the Q10 value is 2). • High temperatures inactivate enzymes with a resulting effect on metabolism and, consequently, on growth. The failure of a fungus to grow at high temperature may be the result of inability to synthesize a needed vitamin, for example. Effect of Temperature, Light and Humidity

Temperature (in oC) <0 = no growth >0 – 10 = Very very slow growth >10 – 15 = Slow growth .15 – 22 = Good growth >22 – 25 = very fast growth >25 = may case harm to mycelia and its growth

Pholiota adiposa

• Mycelial growth was found to occur over the range of 5 to 33°C; whereas fruiting occurred only from 13 to 24°C. The optimal temperature for mycelial growth was 27°C and for fruiting body formation 18 to 21°C.

Light

• The growth of most fungi is not sensitive to light, although strong light may inhibit or even kill (possibly a temperature effect). • One exception to this latter statement concerning S. commune is of interest. Bromberg and Schwalb have demonstrated that light is required in S. commune for the formation of primordia and also for the early stages of fruiting body development in which short cylindrical stipes with terminal apical pits are formed. • It has been reported that this inhibition by strong light may be overcome by the addition of natural materials, such as yeast extract, to the medium. A possible interpretation for this phenomenon is that the light may have destroyed certain vitamins, which are then replaced by those present in the natural material. • Because light is actually inhibitory to the development of primordia and affects stipe elongation and pileus expansion of the button mushroom (Agaricus bisporus), this mushroom is grown in caves, tunnels, or mushroom houses in the dark. Light (sun light / electrical light / no light ) • Intense light • Semi light • Dark • Open (without shade) – In forest – In land / field • Shady (in forest, Hut / house / covered area) – semi shady – Shady • Dark • Semi dark Humidity

• It is generally recognized that most fungi require high moisture levels. • A relative humidity of 95 to 100% and a moisture content of the substrate between 50 and 75% support maximum growth of most Basidiomycetes; but, of course, there are fungi adapted to lower moisture concentrations. • The extreme is the germination of powdery mildew spores that occurs at 0% relative humidity. • Another exception is the dry rot fungus, Serpula lacrymans, whose mycelial strands can grow through substrates lacking moisture as a result of translocation of nutrients farther back in the hyphae and by the formation of “metabolic water.”

Humidity

• <0 = no symptom of growth • >0 – 15 = very very slow growth • >15 – 30 = very slow growth • > 30 – 50 = medium growth • >50 – 70 = Good grwoth • > 70- 80 = Fast growth • >80 – 100 = very fast growth but may cause harm

Aeration

• The components of the air that are of greater importance to most fungi are oxygen and carbon dioxide. Most fungi are obligate aerobes, but many will also grow in reduced levels of oxygen. • The mushrooms of Basidiomycetes may be malformed in the presence of too much respiratory carbon dioxide - a fact that emphasizes the need for proper ventilation in mushroom growing houses. • The effect of high concentrations of carbon dioxide on the development of fruiting bodies was observed with elongation of the stipe. • The influence of carbon dioxide on fruiting body development of A. bisporus was extensively studied and reported as abnormal pileus formation as well as extensive stipe elongation. • The vegetative growth of A. bisporus has been shown to require low levels of carbon dioxide with an optimum concentration in the range of 0.1 to 0.5% reported by San Antonio and Thomas pH • It is well known from experimental studies that the optimal pH values for fruiting may differ from those for growth. It is also known that species differ in their optimal pH values for fruiting. • During the course of an experiment the pH value of the medium may change because the fungus has produced metabolites, e.g., organic acids, that affect the hydrogen ion concentration. • Media that are strongly buffered or that are periodically neutralized by the addition of base or acid may not fruit because the pH value required for the metabolic reactions necessary for fruiting is not reached. pH Media • Carbon – Carbon sources provide for both the structural and energy requirements of the fungal cell. The fungi are quite versatile in utilization of carbon compounds. There are fungal species that utilize various polysaccharides, monosaccharides, organic acids, amino acids, certain alcohols, polycyclic compounds, and natural products such as lignin and cellulose as carbon sources. • Nitrogen – Chitin, a polysaccharide of common occurrence in the cell walls of many fungi, also contains nitrogen. • Minerals – Phosphorous, Potassium, Magensium etc. • Vitamins – Thiamine, Biotin, Nicotinic Acid, Pantothenic Acid etc.

Six steps for growing Mushroom

• Making Mushroom Compost • Finishing the Compost • Spawning • Casing • Pinning • Cropping

Materials necessary for cultivation Water for cleaning or washing Autoclave, incubators, Inoculating chamber, Laminar Air flows Spirit lamp. Rectified spirit, lighter Cotton non absorbent Inoculating needle Blades Test tubes petri dishes. Paddy strwa, Wood logs, Eledtric boarer Wax Wheat, Chemicals Plastic bags, wooden trays, Rubber bands Chemicals and materials necessary PDA (for slants) Boil and pour into test tubes and Strelize 1. Potato raw 200 g. 2. Dextrose sugar 15 g. 3. Agar Agar 20g. Or 25 grms of PDA in 1000 ml of water

For making spawn (growing mycelium on wheat seeds) 1. Wheat seeds 20 kg. 2. Lime 30 – 25 gr. Wash and soak wheat grains for one day, mix lime, fill the bags, plug them, sterelixe Preparation of PDA medium for mushrooms Making spawn for cultivation Culture tubes of mushrooms

Making spawn for cultivation

Substrate for growing Agaricus bisporus

Cow dung . Horse manure, droppings Paddy straw, Chopped straws Chemicals – Ammonium sulphate, Potassium, Urea Calcium carbonate Water Temperature thermometer Wooden wracks, trays Process For paddy straw mushrooms 1. Select good straw 2. Chop upper and lower parts and throw 3. Cop whole part in to 3- 4 “ 4. Wash throughly by water 5. Soak in water for one day 6. Wash throughly by water 7. Keep in shady place for 2 – 3 hours, drain water 8. Pack up straw in plastic bags with grains of mushroom spawn 9. Seal with rubber bads Chemicals necessary For composting ( For One ton farm yard manure / organic manure/ Dungs + Paddy straw mix) 1. Ammonium sulphate 5.6 kg 2. Super phosphate 5.6 kg - At first and second 3. Lime 20.0 kg – At first and second 4. Gypsum 16 kg - At first and second 5. Copper sulphate 2.0 Oz. – At first

Process Mix in the stacks / heaps / debris breaking in definite ratio in each turnover after one week Turn over the heaps sprinkling the water after each three days Process For compost + straw mushrooms 1. Make heaps of dungs (one ton each) 2. Make dust of dung / coarse garined dungs 3. Mix with small pices of straw 2-3 “ (clean) 4. Make heaps / stacks 5. Sprinkle water regularly by turning 6. Add chemical fertilizers 7. Keep in shady place and turn the compost after 2 – 3 days 8. Do not let exceed the temperature over 110 –120c

Spawning and Casing the trays

1. Fill trays with well made manure 2. Make ½” hole with glass rods 3. insert spawn grains 4. Cover slightly with manure 5. Cover the trays with moist papers 6. See development of mycelium after 15 – 20 days 7. Remove the paper 8. Case the trays with sterilized soli

Stages in Growing Agaricus bisporus

Pleurotus eryngii Flammulina velutipes Materials necessary for Pleurotus cultivation : sGo] Rofp v]tL k/fn M % lsnf]  Paddy straw e':;f sf6\g] s}FrL M ! Uff]6f  Chopping tool 8|||«|||\|d jf kmf]l; / kf]6f;L M ! Uf]f6f  Space, Drum, vessel, Knfl:6s y}nf -!@ !^ OGr_ M % j6f  Plastic bag × Knfl:6s l;6 M ! ld6/  Plastic sheet Knfl:6s af6f M ! Uf]f6f  Plastic vessel bfp/f M !) lsnf]  Rofpsf] lapm M ! af]tn  Spawn / seed of mushroom 8f]/L M $ 6's|f  Rubber band / Rope k|ljlw k/fnsf] u'0f:t/ M ;kmf . xl/of]kg gePsf] .  k/fn 6'qmf kfg]{ M @ b]lv # OGr .  k/fnsf] 6'qmf tf}ng] M % s]hL k|lt af]tn -@%) u|f aLpm _  k/fn lehfpg] M @ b]lv # 306f .  k/fn w'g] M # b]lv $ k6s .  kfgL tsf{pg] M % b]lv ^ 306f jf ! /ft  k/fn akmfpg] M ! b]lv !=% 306f - /f]u, ls/f zq' lgoGq0fsf  nflu_ k/fn lr:ofpg] M % b]lv ^ 306f  Mushroom huts for cultivation Substrate for growing Pleurotus ostreatus & Lentinus sajor-caju

Paddy straw, Chopped straws Chemicals – Ammonium sulphate, Potassium, Urea Calcium carbonate Water Temperature thermometer Plastic bags or other containers Agaricus bisporus

• In Phase I of the process (outdoor composting), locally available raw materials are arranged into piles which are periodically turned and watered. The initial breakdown of the raw ingredients by microorganisms takes place in Phase I. This phase is usually complete within 9-12 days, when the materials have become pliable, dark brown in colour and capable of olding water. There is normally a strong smell of ammonia.

• Phase II (indoor fermentation) is pasteurisation, when undesirable organisms are removed from the compost. This is carried out in a steaming room where the air temperature is held at 60°C for at least 4 hours. The temperature is then lowered

to 50° C for 8 to 72 hours depending upon the nature of the compost. CO2 is maintained at 1.5 to 2% and the ammonia level drops below 10 PPM.

• Following Phase II composting, the substrate is cooled to 30° C for A. bitorquis and to 25° C for A. bisporus for spawning.

Agaricus bisporus

• Production of Phase III or Phase IV composts for growing Agaricus mushrooms has been an advanced technological development in recent years in Western countries.

• The production of Phase III compost is Phase II compost spawn run in a bulk tunnel, and ready for casing when delivered to the grower.

• If the Phase III compost is then cased and spawn developed into casing layer before dispatching to the growing unit or delivering to growers, it is named as Phase IV compost. The successes of bulk Phase III and Phase IV depend a lot on the quality of Phase I and Phase II processes.

• Phase II on shelves produce an average of 4.1 crops per year. Since 1999, growers using Phase III production enjoyed an average of 7.1 crops per year. In recent years, Phase IV can generate 10-12 crops per year (Dewhurst 2002, Lemmers 2003).

Lentinula edodes

• Culture media and preparation: – The mushroom can grow on a variety of culture media and on different agar formulations, both natural and synthetic, depending on the purpose of the cultivation. Synthetic media are often expensive and time-consuming in preparation; hence they are not commonly used for routine purposes. – The potato dextrose agar, or PDA, is the simplest and the most popular medium for growing the mycelium of the mushroom.

Lentinula edodes

• Examples of the different formulas for spawn substrates are described below. – Mother grain spawn: 1. Wheat/rye grain + 1.5% gypsum or slaked lime. 2. Cotton seed hull 40%, sawdust 38%, wheat bran 20%, sugar 1% and gypsum 1%. 3. Sugar cane bagasse 40%, sawdust 38%, wheat bran 20%, sugar 1% and gypsum 1%. – Planting spawn: • A number of materials, mostly agricultural and forest wastes can be used to prepare mushroom planting spawn. Three of them are given here as examples: 1. Sawdust 78%, rice/wheat bran 16%, sugar 1.5%, corn flour 1.7%, ammonium sulphate 0.3%, Calcium superphosphate 0.5% and gypsum 2%; 2. Sawdust 64%, wheat bran 15%, spent coffee grounds 20% and gypsum/lime 1%; and 3. Sawdust 78%, sucrose 1%, wheat bran 20% and Calcium carbonate 1%.

Lentinula edodes

• Lentinula edodes mushroom is produced both on a cottage and a commercial scale. • Cottage scale cultivation: – There are many formulas for the composition of the substrate. The ingredients can be variable from place to place and country to country depending upon the raw materials available and local climatic conditions. – In general, after mixing the dry ingredients by hand or with a mechanical mixer, water is added to the mixture so that the final moisture content of the substrate is between 55 and 60%, depending on the capacity of the sawdust to absorb water. The ingredients are then packed into autoclavable polypropylene or high-density polyethylene bags. Although they are more expensive, polypropylene bags are the most popular since polypropylene provides greater clarity than polyethylene. After the bags have been filled with the substrate (1.5 to 4 kg wet weight, w/w), the end of the bag can be closed either by strings or plugged with cotton wool stopper. – Four formulas in the preparation of the substrate for the cultivation of the mushroom are given here as reference. 1. Sawdust 82%, wheat bran 16%, gypsum 1.4%, Potassium phosphate, dibasic 0.2%, and lime 0.4%. 2. Sawdust 54%, spent coffee grounds 30%, wheat bran 15%, and gypsum 1%. 3. Sawdust 63%, corncob powder 20%, wheat bran 15%, Calcium superphosphate 1% and gypsum 1%. 4. Sawdust 76%, wheat bran 18%, corn powder 2%, gypsum 2%, sugar 1.2% Calcium superphosphate 0.5% and urea 0.3%.

Commercial scale cultivation • In general, the operation can use oak or other hard wood sawdust medium to grow the mushroom. The basic steps are 1. to mix the sawdust,supplements, and water; 2. bag the mixture; 3. autoclave the bags to 121oC and cool the bags; 4. inoculate and seal the bags; 5. Incubate (18 – 25 0C) for 90 days to achieve full colonisation of the sawdust mixture, in other words, to allow the mycelium to be established for ready fructification; 6. fruit the colonised and established sawdust logs/bags/blocks 6 times using a 21 days cycle at 16 to 18o C; and 7. harvest, clip steps, grade, box, and cold store for fresh market, or harvest, dry, cut steps, grade and dry again before box for dry market.

Lentinula edodes Ganoderma lucidum

• Artificial cultivation of this valuable mushroom was successfully achieved in the early 1970s and, since 1980 and particularly in China, production of G. lucidum has developed rapidly. • Currently, the methods most widely adopted for commercial production are the wood log, short wood segment, tree stump, sawdust bag and bottle procedures.

Ganoderma lucidum

• Log cultivation methods include the use of natural logs and tree stumps which are inoculated with spawn directly under natural conditions. • The third alternative technique involves the use of sterilized short logs about 12cm in diameter and approximately 15 cm long which allow for good mycelia running. This method provides for a short growing cycle, higher biological efficiency, good quality of fruiting bodies, and, consequently, superior economical benefit. However, this production procedure is more complex and the production costs much higher, than natural log and tree stump methods. For this production procedure, the wood logs should be prepared from broad-leaf trees, preferably from oak. Felling of the trees is usually carried out during the dormant period, which is after defoliation in autumn and prior to the emergence of new leaves the following spring. The optimum moisture content of the log is about 45-55% . Ganoderma lucidum

• The flow-chart for the short-log cultivation method is as follows : --- selection and felling of the tree ---sawing/cutting the log into short segments ---transfer segments to plastic bags ---sterilization --- inoculation ---spawn running --- burial of the log in soil ---tending the fruiting bodies during development from the pinhead stage to maturity ---harvesting of the fruiting bodies ---drying of the fruiting bodies by electrical driers--packaging. • It should be noted that the prepared logs/segments are usually buried in soil inside a greenhouse or plastic shed. The soil should allow optimum conditions of drainage, air permeability and water retention, but excessive humidity should be avoided.

Ganoderma lucidum

• Examples of cultivation substrates, using plastic bags or bottles as containers, include the followings (please note that these examples are for reference purposes only and can be modified according to the strains selected and the materials available in different localities): 1. Sawdust 78%, wheat bran 20%, gypsum 1% and soybean powder 1%; 2. bagasse 75%, wheat bran 22%, cane sugar 1%, gypsum 1% and soybean powder 1%; 3. cotton seed hull 88%, wheat bran 10%, cane sugar 1% and gypsum 1%; 4. sawdust 70%, corn cob powder 14%, wheat bran 14%, gypsum 1% and cereal straw ash 1%; 5. corn cob powder 78%, wheat/rice bran 20%, gypsum 1% and straw ash 1%. • After sterilisation, the plastic bags can be laid horizontally on beds or the ground for fruiting.

Spawn production Pure culture (Nucleus seed)

1. The culture should be genetically pure and true to type. 2. Culture should be obtained from Research organization or authentic source. 3. Free from any kind of fungal and viral contamination. 4. Culture should be maintained on compost extract agar medium. 5. Culture should indicate specific growth rate on defined medium and at defined temperature. 6. Visually the culture should be strandy and off white in colour in Agaricus, pure white and thick fluffy growth in Pleurotus, cottony fluffy with brown sclerotia (after 12-15 days) in Volvariella, pure white, dense, thick and fluffy growth in Calocybe indica and pure white later on turning to light brown pigmentation in Lentinula edodes. 7. Culture should be stored at 4-60C for Agaricus, Pleurotus and Lentinula and between 18-220C in Volvariella and Calocybe indica. 8. The incubation temperature should be between 32±2°C for Volvariella and Calocybe indica and 250C for Agaricus, Pleurotus and Lentinula

Master spawn (Breeder seed)

1. Breeder seed should always be prepared from pure culture. 2. Free from any kind of contamination. 3. It should be multiplied on wheat, jowar, bajra or barley grains. 4. Breeder seed should be incubated at 25±20C for Agaricus, Pleurotus, Lentinula and 32±20C for Calocybe indica and Volvariella. 5. The master spawn should be stored at 4-60C for 40-45 days in Agaricus, Pleurotus, Lentinula and 18-200C days in Calocybe indica and Volvariella for maximum 30-40 days. 6. It should be produced in autoclavable transparent glass bottles

Commercial spawn (Foundation seed/Certified spawn) 1. The incubation temperature should be 25±20C for Agaricus, Pluerotus, Lentinula and 32±20C for Volvariella and Calocybe indica.

2. Spawn should always be prepared from master spawn (Breeder seed).

3. Free from any kind of contamination.

4. It should be multiplied on wheat, jowar, bajra or barley grains.

5. Spawn should not be older than 60 days in Agaricus, 30-45 days in Pleurotus, Lentinula and 30-40 days in Calocybe indica and Volvariella.

6. Certified spawn should be stored at 4-60C in Agaricus, Pleurotus and Lentinula and 18- 200C in Calocybe indica and Volvariella.

7. The bag should indicate lot no., date of inoculation, variety/strain and quantity. h For every new lot of commercial seed (foundation seed), fresh master spawn (breeder seed) should be used. Commercial spawn may not be used for further multiplication of seeds as it may lead to higher contamination and decline in yield.

Preparation of Mother Spawn Preparation of Commercial Spawn Spawn production