Lecture 3 Effect Of Genetic Factors, Temperature, Light, Humidity .
Lecture 3Effect of genetic factors, temperature, light, humidity, mediumon cultivation of mushrooms. Techniques of Commercialcultivation of some important mushrooms, Single sporeisolation/pure culture and spawn production techniques,Present situation and prospect of mushroom cultivation inNepal.Mitesh Shrestha
Genetic Factors For breeding work in edible species it is desirable toknow how fruiting is controlled genetically as wellas nutritionally and physiologically.
Genetic Factors
Temperature The conditions that are favorable for reproduction are always lessfavorable for growth. An increase in temperature generally increases enzymatic activity. Inthat part of the growth curve that is linear, the growth ratesapproximately double for each 100C increase in temperature (i.e., theQ10 value is 2). High temperatures inactivate enzymes with a resulting effect onmetabolism and, consequently, on growth. The failure of a fungus togrow at high temperature may be the result of inability to synthesizea needed vitamin, for example.
Effect of Temperature, Light and HumidityTemperature (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 rangeof 5 to 33 C; whereas fruiting occurred only from 13to 24 C. The optimal temperature for mycelialgrowth was 27 C and for fruiting body formation 18to 21 C.
Light The growth of most fungi is not sensitive to light, although strong light mayinhibit 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 offruiting body development in which short cylindrical stipes with terminalapical pits are formed. It has been reported that this inhibition by strong light may be overcome bythe addition of natural materials, such as yeast extract, to the medium. Apossible interpretation for this phenomenon is that the light may havedestroyed certain vitamins, which are then replaced by those present in thenatural material. Because light is actually inhibitory to the development of primordia andaffects stipe elongation and pileus expansion of the button mushroom(Agaricus bisporus), this mushroom is grown in caves, tunnels, ormushroom houses in the dark.
Light (sun light / electrical light / no light ) Intense lightSemi lightDarkOpen (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 substratebetween 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 at0% relative humidity. Another exception is the dry rot fungus, Serpula lacrymans, whose mycelialstrands can grow through substrates lacking moisture as a result oftranslocation of nutrients farther back in the hyphae and by the formationof “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 areoxygen and carbon dioxide. Most fungi are obligate aerobes, but many willalso grow in reduced levels of oxygen. The mushrooms of Basidiomycetes may be malformed in the presence oftoo much respiratory carbon dioxide - a fact that emphasizes the need forproper ventilation in mushroom growing houses. The effect of high concentrations of carbon dioxide on the development offruiting 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 pileusformation as well as extensive stipe elongation. The vegetative growth of A. bisporus has been shown to require low levelsof carbon dioxide with an optimum concentration in the range of 0.1 to0.5% reported by San Antonio and Thomas
pH It is well known from experimental studies that theoptimal pH values for fruiting may differ from those forgrowth. It is also known that species differ in theiroptimal pH values for fruiting. During the course of an experiment the pH value of themedium may change because the fungus has producedmetabolites, e.g., organic acids, that affect thehydrogen ion concentration. Media that are strongly buffered or that areperiodically neutralized by the addition of base or acidmay not fruit because the pH value required for themetabolic reactions necessary for fruiting is notreached.
pH
Media Carbon– Carbon sources provide for both the structural and energy requirements of thefungal 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 naturalproducts 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 CompostFinishing the CompostSpawningCasingPinningCropping
Materials necessary for cultivationWater for cleaning or washingAutoclave, incubators, Inoculatingchamber, Laminar Air flowsSpirit lamp. Rectified spirit, lighterCotton non absorbentInoculating needle BladesTest tubes petri dishes.Paddy strwa, Wood logs, Eledtric boarerWaxWheat, ChemicalsPlastic bags, wooden trays, Rubberbands
Chemicals and materials necessaryPDA (for slants) Boil and pour into test tubes and Strelize1. Potato raw200 g.2. Dextrose sugar15 g.3. Agar Agar20g.Or25 grms of PDA in 1000 ml of waterFor making spawn (growing mycelium on wheat seeds)1. Wheat seeds20 kg.2. Lime30 – 25 gr.Wash and soak wheat grains for one day, mix lime, fill the bags, plugthem, sterelixe
Preparation of PDA medium for mushrooms
Making spawn for cultivationCulture tubes of mushrooms
Making spawn for cultivation
Substrate for growing Agaricus bisporusCow dung . Horse manure, droppingsPaddy straw, Chopped strawsChemicals –Ammonium sulphate, Potassium, UreaCalcium carbonateWaterTemperature thermometerWooden wracks, trays
ProcessFor paddy straw mushrooms1. Select good straw2. Chop upper and lower parts and throw3. Cop whole part in to 3- 4 “4. Wash throughly by water5. Soak in water for one day6. Wash throughly by water7. Keep in shady place for 2 – 3 hours, drain water8. Pack up straw in plastic bags with grains of mushroom spawn9. Seal with rubber bads
Chemicals necessaryFor composting ( For One ton farm yard manure / organicmanure/ Dungs Paddy straw mix)1. Ammonium sulphate5.6 kg2. Super phosphate3. Lime4. Gypsum5.6 kg - At first and second20.0 kg – At first and second16 kg - At first and second5. Copper sulphate2.0 Oz. – At firstProcessMix in the stacks / heaps / debris breaking in definite ratio in eachturnover after one weekTurn over the heaps sprinkling the water after each three days
ProcessFor compost straw mushrooms1. Make heaps of dungs (one ton each)2. Make dust of dung / coarse garined dungs3. Mix with small pices of straw 2-3 “ (clean)4. Make heaps / stacks5. Sprinkle water regularly by turning6. Add chemical fertilizers7. Keep in shady place and turn the compost after 2 – 3 days8. Do not let exceed the temperature over 110 –120c
Spawning and Casing the trays1. Fill trays with well made manure2. Make ½” hole with glass rods3. insert spawn grains4. Cover slightly with manure5. Cover the trays with moist papers6. See development of mycelium after 15 – 20 days7. Remove the paper8. Case the trays with sterilized soli
Stages inGrowingAgaricusbisporus
Pleurotus eryngii
Flammulina velutipes
Materials necessary for Pleurotus cultivation:sGo] Rofp v]tLPaddy straw k/fn Chopping tool e':;f sf6\g] s}FrL Space, Drum, vessel, 8 «\d jf kmf]l; / kf]6f;L Plastic bag Knfl:6s y}nf -!@ ! OGr Plastic sheet Knfl:6s l;6 Plastic vessel Knfl:6s af6f bfp/f M !) lsnf] Spawn / seed of mushroom Rofpsf] lapm Rubber band / Rope 8f]/L MMMMMM% lsnf]! Uff]6f! Uf]f6f% j6f! ld6/! Uf]f6fM ! af]tnM 6's f
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' lgoGq0fsfnflu k/fn lr:ofpg] M % b]lv 306f
Mushroom huts for cultivation
Substrate for growingPleurotus ostreatus & Lentinus sajor-cajuPaddy straw, Chopped strawsChemicals –Ammonium sulphate, Potassium, UreaCalcium carbonateWaterTemperature thermometerPlastic bags or other containers
Agaricus bisporus In Phase I of the process (outdoor composting), locally available raw materials arearranged into piles which are periodically turned and watered. The initialbreakdown of the raw ingredients by microorganisms takes place in Phase I. Thisphase is usually complete within 9-12 days, when the materials have becomepliable, dark brown in colour and capable of olding water. There is normally astrong smell of ammonia. Phase II (indoor fermentation) is pasteurisation, when undesirable organisms areremoved from the compost. This is carried out in a steaming room where the airtemperature is held at 60 C for at least 4 hours. The temperature is then loweredto 50 C for 8 to 72 hours depending upon the nature of the compost. CO2 ismaintained 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 andto 25 C for A. bisporus for spawning.
Agaricus bisporus Production of Phase III or Phase IV composts for growing Agaricusmushrooms has been an advanced technological development in recentyears in Western countries. The production of Phase III compost is Phase II compost spawn run in a bulktunnel, and ready for casing when delivered to the grower. If the Phase III compost is then cased and spawn developed into casing layerbefore dispatching to the growing unit or delivering to growers, it is namedas Phase IV compost. The successes of bulk Phase III and Phase IV depend alot 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 (Dewhurst2002, Lemmers 2003).
Lentinula edodes Culture media and preparation:– The mushroom can grow on a variety of culture media and on different agarformulations, both natural and synthetic, depending on the purpose of thecultivation. Synthetic media are often expensive and time-consuming inpreparation; hence they are not commonly used for routine purposes.– The potato dextrose agar, or PDA, is the simplest and the most popular mediumfor growing the mycelium of the mushroom.
Lentinula edodes Examples of the different formulas for spawn substrates aredescribed below.– Mother grain spawn:1.2.3.Wheat/rye grain 1.5% gypsum or slaked lime.Cotton seed hull 40%, sawdust 38%, wheat bran 20%, sugar 1% andgypsum 1%.Sugar cane bagasse 40%, sawdust 38%, wheat bran 20%, sugar 1% andgypsum 1%.– Planting spawn: A number of materials, mostly agricultural and forest wastes can be used toprepare mushroom planting spawn. Three of them are given here asexamples:1.2.3.Sawdust 78%, rice/wheat bran 16%, sugar 1.5%, corn flour 1.7%, ammoniumsulphate 0.3%, Calcium superphosphate 0.5% and gypsum 2%;Sawdust 64%, wheat bran 15%, spent coffee grounds 20% and gypsum/lime 1%;andSawdust 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 fromplace to place and country to country depending upon the raw materials available and local climaticconditions.– In general, after mixing the dry ingredients by hand or with a mechanical mixer, water is added tothe mixture so that the final moisture content of the substrate is between 55 and 60%, dependingon the capacity of the sawdust to absorb water. The ingredients are then packed into autoclavablepolypropylene or high-density polyethylene bags. Although they are more expensive,polypropylene bags are the most popular since polypropylene provides greater clarity thanpolyethylene. After the bags have been filled with the substrate (1.5 to 4 kg wet weight, w/w), theend 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 givenhere as reference.1. Sawdust 82%, wheat bran 16%, gypsum 1.4%, Potassium phosphate, dibasic 0.2%, and lime0.4%.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% andgypsum 1%.4. Sawdust 76%, wheat bran 18%, corn powder 2%, gypsum 2%, sugar1.2% Calcium superphosphate 0.5% and urea 0.3%.
Commercial scale cultivation In general, the operation can use oak or other hard woodsawdust medium to grow the mushroom. The basic steps are1. 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 colonisationof the sawdust mixture, in other words, to allow themycelium to be established for ready fructification;6. fruit the colonised and established sawdust logs/bags/blocks6 times using a 21 days cycle at 16 to 18o C; and7. harvest, clip steps, grade, box, and cold store for freshmarket, or harvest, dry, cut steps, grade and dry again beforebox for dry market.
Lentinula edodes
Ganoderma lucidum Artificial cultivation of this valuable mushroom wassuccessfully achieved in the early 1970s and, since1980 and particularly in China, production of G.lucidum has developed rapidly. Currently, the methods most widely adopted forcommercial production are the wood log, shortwood segment, tree stump, sawdust bag and bottleprocedures.
Ganoderma lucidum Log cultivation methods include the use of natural logs and treestumps which are inoculated with spawn directly under naturalconditions. The third alternative technique involves the use of sterilized shortlogs about 12cm in diameter and approximately 15 cm long whichallow for good mycelia running. This method provides for a shortgrowing cycle, higher biological efficiency, good quality of fruitingbodies, and, consequently, superior economical benefit. However,this production procedure is more complex and the production costsmuch higher, than natural log and tree stump methods. For thisproduction procedure, the wood logs should be prepared frombroad-leaf trees, preferably from oak. Felling of the trees is usuallycarried out during the dormant period, which is after defoliation inautumn and prior to the emergence of new leaves the followingspring. 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 tomaturity---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 insoil inside a greenhouse or plastic shed. The soil should allow optimumconditions of drainage, air permeability and water retention, but excessivehumidity should be avoided.
Ganoderma lucidum Examples of cultivation substrates, using plastic bags or bottles ascontainers, include the followings (please note that these examples are forreference purposes only and can be modifiedaccording to the strains selected and the materials available in differentlocalities):1. Sawdust 78%, wheat bran 20%, gypsum 1% and soybean powder 1%;2. bagasse 75%, wheat bran 22%, cane sugar 1%, gypsum 1% and soybeanpowder 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 ash1%. After sterilisation, the plastic bags can be laid horizontally on beds or theground for fruiting.
Spawn production
Pure culture (Nucleus seed)1.2.3.4.5.6.7.8.The culture should be genetically pure and true to type.Culture should be obtained from Research organization or authenticsource.Free from any kind of fungal and viral contamination.Culture should be maintained on compost extract agar medium.Culture should indicate specific growth rate on defined medium and atdefined temperature.Visually the culture should be strandy and off white in colour in Agaricus,pure white and thick fluffy growth in Pleurotus, cottony fluffy with brownsclerotia (after 12-15 days) in Volvariella, pure white, dense, thick andfluffy growth in Calocybe indica and pure white later on turning to lightbrown pigmentation in Lentinula edodes.Culture should be stored at 4-60C for Agaricus, Pleurotus and Lentinulaand between 18-220C in Volvariella and Calocybe indica.The incubation temperature should be between 32 2 C for Volvariella andCalocybe 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 barleygrains.4. Breeder seed should be incubated at 25 20C for Agaricus,Pleurotus, Lentinula and 32 20C for Calocybe indica andVolvariella.5. The master spawn should be stored at 4-60C for 40-45 daysin Agaricus, Pleurotus, Lentinula and 18-200C days inCalocybe indica and Volvariella for maximum 30-40 days.6. It should be produced in autoclavable transparent glassbottles
Commercial spawn (Foundationseed/Certified spawn)1.The incubation temperature should be 25 20C for Agaricus, Pluerotus, Lentinula and32 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 and30-40 days in Calocybe indica and Volvariella.6.Certified spawn should be stored at 4-60C in Agaricus, Pleurotus and Lentinula and 18200C in Calocybe indica and Volvariella.7.The bag should indicate lot no., date of inoculation, variety/strain and quantity. h For everynew lot of commercial seed (foundation seed), fresh master spawn (breeder seed) shouldbe used. Commercial spawn may not be used for further multiplication of seeds as it maylead to higher contamination and decline in yield.
Preparation of Mother Spawn
Preparation of Commercial Spawn
Spawn production
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 .
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