UNIVERSITY OF NAIROBI SCHOOL OF BIOLOGICAL SCIENCES CHIROMO CAMPUS LABORATORY MANUAL APPLIED MYCOLOGY SBT 405 PROF. GEORGE M. SIBOE APRIL 2008 STUDENT NAME: ---—-.r GENERAL REQUIREMENTS FOR PRACTICAL SESSIONS IN MYCOLOGY You will be expected to have the following during laboratory sessions + A pencil HB & B types * A lab book * Pad of drawing paper + One new, single-edge safety razor blade + 2 mounted needles + Swann morton scalpel preferably handle no. 4 with blade no. 11 and 22A (or any other round edge blade (| * A pair of fine forceps 2. General mules for drawings of fungal structures: i. Always provide a bar scale (10m, 50 1m) ii. Use a single line for a fungal hyphal wall iii, Draw the mimimum to characterize the subject & to occupy half a page iv. Label fully v. Label in pencil vi. Use stippling not shading, but if shading is necessary use single line hatching vii. Use a dotted line for protoplasmic boundaries LABORATORY EXERCISE 1 Preparation of solid culture media for isolation of fungi. Introduction fungi can be grown outside their living hosts or natural substrates on prepared substances, which provide essential nutrients. These substances are called culture media. An organism maintained on such media is said to be ‘maintained in culture’. The base for most of the common culture media is agar, whtch is processed from kelp (brown iigae). The unique property of agar is that it is liquid at 100°C, but olidifies into a soft 40°C. This substance 1s ideally suited for »wth of many fungi and most bact amended w ents to stir em will cont carbon nitt organic forn a cultu’tassium, calcium ° im, P% small S as‘sodiu ¢ ee tare also necessary ee: i al er ule aired ey eocemucoeen rere. ene vitamins themselves. These some microbes can produce the vitamins NO th) and then usually added to water (also cri joGiens are supplied solidified using agar. If all these required Pater’ Th’ TT jerined or forms). Other metallic clement eae magnesium, iron, zinc, coppe quantities. Vitamins may @ nmeal a aa individually in pure form, we term the medlurn CMTE act, cor the medium i If raw materials such as y' I ee of these nutrients, a ned tn preven hers to grow. These are des can be used to render a to prevent the growth of growth of some organisms, while allowing ot termed selective media. Antibiotics or fungici medium selective. Antibiotics are added to media ree bacteria. These are especially used for first platirigs, and also to purify cultures contaminated with bacteria. Useful antibiotics are e.g. chloramphenicol, novobiocin, streptomycin, and tetracycline. They are used singly or in various combinations, usually at concentration cf Sml/l. Antibiotics are added to the medium with a syringe and needle only after sterilization, and when the agar has cooled down to about 45°C - 48°C Some media are poor in nutrients while others are rich. Nutrient poor media are generally recommended because they do not encourage abundant growth, so slower-growing fungi will not be overgrown by others. Also, the measurements (dimensions) of some fungi on nutrient- poor media are closer to those found in nature. Examples are water agar and potato carrot agar. First platings of material are usually made on nutrient-poor media. gredients in distilled water, adjusting Media are prepared by dissolving in the pH (fungi grow best on media which are slightly acidic, bacteria are favored by alkaline conditions), and autoclaving the medium for 20 minutes at 121.5°C at 15 psi pressure. The autoclave is like a large perature only if under pressure pressure cooker and can reach this tem, This temperature and time will generally sterilize most substances or is sterile, it can be poured into pieces of equipment. After the medium sterile Petri dishes and the medium will solidify as it cools. Conditions for the successful preparation of media The prevention of contamination is of utmost importance when preparing media (i.e. work aseptically) : hzed, cooled apparatus and media: z * Use r water when needed+ Always used ethyl alcohol (at least 70%) to wipe the surface on which you are going to work. * Do not work in a lab where there is a current of air (ie. ina draught): spores are carried in air currents. » Agar requiring sterilization should be placed in bottles with screw- on tops. Ehrlenmeyer flasks with cotton-wool stoppers are also suitable. * The type of medium should be clearly written on the containers with a felt-tipped pen, in permanent ink. * The container iri which the agar is sterilized must not be filled completely, because the contents will boil over (the volume of agar increases when the medium is heated in an autoclave or pressure cooker). * Unscrew all bottle tops a quarter of the way before putting them in an autoclave. Close them properly after sterilization, before cooling, * Petri dishes, test-tubes, and McCartney bottles should also be clearly marked with the name of the medium they contain (e.g. WA, PCA, etc.) « Pouring agar into different containers 1. Petri dishes: Lift one side of the lid carefully, just enough to be able to put the agar into the plate. Do not remove the lid completely, and do not place it on the work surface bought sterile and can only be re-used when sterilized by radiation If this facility W& not available they use. Glass Petri dishes must be steril f them: they must be placed in suitable cl foil and then sterilize preferable because tt emain sterile long McCartney bottleshe agar and water suspension is boiled unt ] the agar is proper! Bi Pp Bi : i ie 2 dissolved. Alternatively, it can be placed in the autoclave for one he # 5 after it has cooled down a litt It is then carefully poured into bottle ni stirring the mixture each time before pouring It. to che aucec sved on properly. The bottles are then positioned at a caps are slight angle to make agar slants and allowed to cool down. 3. Test-tubes Test-tubes are filled in the same way as McCartney bottles. Non- absorbent cotton wool stoppers are preferable. The test tubes should be positioned upright in wire baskets with their stoppers in place. The top of the basket is covered with aluminium foil or a paper bag. After sterilization, the test-tubes are positioned at an angle and then allowed to cool down. Note: Be careful when using flammable liquids, hot equipment and hot agar or other potentially dangerous materials needed for culture work. Sterilized agar should be allowed to cool down to about 45°C - 48°C before being poured. Antibiotics should be added just before the agar is poured. Recipes for some common media Note: PDA, OMA, and MEA are nutrient-rich media; PCA and WA ar nutrient-poor media. Depending on the grade of agar, 10 ~ 20g aga per litre can be used. The more agar used, the stiffer the media i, Water agar (WA): o Agar - 15g o Water - tol.Ohtre= Method: 1. Dissolve the agar in water 2. Sterilize forl4 min. at 121°C, and 15 p.s.i ii, Malt Bxtract Agar (MEA): 2 Malt Extract (e.g. Difco, etc) - 20g o Agar - 20g o Water — tol.Olitre * Method: 1. Boil the malt extract in water until dissolved 2. Add agar ’ 3. Autoclave for 20 mins at 121°C, and 15 p.s.i iii. Oatmeal agar (OMA) Oatmeal - 100g Water - 600-700ml After cooking for 30min. put through a wire sieve. Agar - 15g Water - tol.Olitre iv. Potato dextrose agar (PDA) o Potato -200g =x o Dextrose -20g ~ o Agar - 20g o Water - tol.Olitre oo000 = Method: 1. Scrub potatoes and cut into cubes; do not peal . Weigh, and boil in 1.01 water until soft . Decant the broth Add agar to broth and boil until dissolved . Add dextrose and stir until dissolved . Make up to 1l For Fusarium, use sucrose (i.e. PSA) instead of dextrose, mix and adjust the pH to 6.5 ANswWND v. Potato-carrot agar (PCA) o Grated or chopped potato - 20g o Grated or chopped carrot - 20g o Agar - 20g o Water - tol.Olitre * Method 1. Boil the vegetables in water for | hour 2. Press through a sieve and add agar3, Boil until the agar 1s dissolved 4, Sterilize as above OBJECTIVES you to become familiar with the The objectives of this lab are to enable ae ees of sterile technique and with the are ee Bae ae a complex culture medium. Today you will prepare and po Pp an Potato dextrose agar (PDA). To limit contamination by bacteria, } i ac jaa anc otic as described abi MATERIALS 1. Alcohol for rubbing down tables 2. Cotton wool 3. PDA recipe components as listed above 4. A given number of Petri dishes per group Petri METHOD a) Follow PDA preparation recipe above b) Swab down your working area with 95% alcohol Remove the cap of a bottle containing PDA. Very slightly open the lid of a Petri dish and pour the molten agar into the dish, about 25ml. Each person in your group should pour a plate d) Each plate with your initials €) Allow the agar to solidify When the agar has solidified (15-30 min) place all plates from your b) group in a plastic bag, label with your group designation and names of group members, and hand them over to the technologist in-charge. The plates of media will be used in future labs ¢) (refer QUESTIONS FOR THOUGHT “1. How could you describe the media used to pour the plates to the introduction)? Comment on the truth of the statement that “In preparing a sterile culture medium, one should use caution to prevent the introduction of contaminant microorganisms such as bacteria fungi and viruses.” 3. What are the possible sources of contamination wher a sterile culture medium? What procedures have y eliminate or exclude contaminants from each arce? 2 preparing useLABORATORY EXERCISE 2 Isolation and microscopic study of fungi Introduction: A wide variety of microorganisms can be isolated from various substrate environments and cultivated (grown) on media in the laboratory. Different media will encourage the growth of different types of microbes through the use of inhibitors and specialized growth substrates. We will use PDA medium to isolate fungi from soil, diseased plant materials, and aquatic environments. We will count the numbers of fungi that are capable of growing on: PDA medium. These counts are referred to as colony forming units (CFUs). Our knowledge of soil microbes is derived primarily from dilution and plating techniques. These methods are biased in favour of rapidly growing and sporulating organisms. Dilution plate techniques measure only a small portion of the total soil community but, nevertheless, are a useful tool for studying the relative abundance of culturable populations and the changes in population density which occurs according to the medium used or the proximity to plant roots. Objectives; . To learn techniques for isolating fungi from different substrates 2 To learn how to make a basic microscope preparatio 3. To learn how to measure the density of 4. To learn techniques to identify unkno' Materials MicroscopesSterile pipettes Tube racks Spreaders for spread-plating (make from glass pipette) Balances 70% ethanol (in bottles and dishes) Beakers Bunsen bumers and strikers Inoculating loops (for transferring colonies) Ruier (for measuriz Bic: waste container Methods 1. Solid culture media for isolation of fungi Week 2 you made last week from plastic bags. Be careful to a) Remove the plates lids closed. Check plates for contarnination. keep the Petri dish Bacterial colonies usually appear as viscid (slimy) droplets on the agar surface. They may be a variety of colors and shapes. Fungal colonies will usually appear as filamentous or fluffy growth on the agar surface. If contaminants are present they should be removed as follows sterilize a dissecting needle/scalpel by heating to readiness in an alcohol lamp frame. Slightly open the lid of the dish, cut well around the margin of the contaminant colony with the dissecting needle /scalpel. Stab the contaminated section of agar, lift it out of the dish and discard it. Revisit the questions for thought in last week’s b lab exercise. Isolation and microscopic study of filamentous fungi from various substrates No fungi can be scientifically and correctly identified without proper microscopic examination. This entails the making of microsc ‘opic preparations. Making a basic microscopic preparation: nicroscop Place the microscope slide next to your dissecting place a small drop of stain or mov 14 the material is dark, use colourless lactophenol; if it is colourless use a Stain e.g. lactophenol in cotton blue). 2. Remove some of the hyphae/sporulating structures using a fine needle or sharp pointed scalpel (No. 11) and transfer them to the liquid 3. Take a clean cover slip and hold it on one side, then let it slip onto the liquid from one side without trapping air bubbles underneath. If there are air bubbles, first place a few drops of 70% alcohol on the slide, then the material to be studied and then add the stain or mountant 4. Press the cover slip down gently. There must be just enough mountant or stain but not too much to leak out of the cover slip. Record all the observable characteristics and identify the fungi at least up to generic level. Illustrate typical characteristics. Hint: Always aim to make the preparation as thin as possible: this ensures a better depth of field and the preparation will seal properly Isolation of fungi by Direct transfer method Pick a leaf of spinach (Spinacia oleracea) with leaf spots, probably caused by Cercospora beticola. Carefully examine the lesions on the leaf (both the lower and upper surfaces) using a hand lens, and a dissecting microscope. Locate the fruiting structures (sporophores): conidiophores and conidia, which may be present in the necrotic lesion. NB. Conidiophores and conidia may appear as grey-white fine hairs at the centre of the necrotic spot. To pick a few fruiting bodies (conidiophores and conidia) gently and carefully remove by brushing a sterile and moistened pointed tip of a Swann Morton no. 1! scalpél blade mounted on no.3 blade holder across the lesion, without removing the plant tissue, thus causing the spores to adhere to the moistened tip.3) Transfer the trapped sporophores into a drop of lactophenol in cotton blue on a glass slide. Put a cover slip and examine at x10 and x40. Illustrate the fruiting bodies. b) Identify the fungus. Use the following reference books i Host list of Kenya fungi and bacteria u. Illustrated Genera of Imperfect Fungi Isolation of fungi by direct plating: Aseptic isolation of fungi from plant Tissue for study oe 1 Diseased Leaves of Spinacea oleracea: =—sseased Leaves of Spinacea oleracea: * Choose leaves with young lesions (Fig. 1) ( + Aseptically cut out tissues from edge of lesion (ca. 5 mm2) * Surface sterilize for 1-3min in 10% NaOCl and wash in sterile water * Using sterile forceps place leaf pieces onto the agar surface (Fig. 3)Fig. | Isolation from leave lesions ‘small immature diseased and heathy tissue, wee Fg verand's: invading nee tisues 2. Diseased stem of Citrus Split longitudinally from healthy to diseased area (Fig 2) Remove tissue from leading edge of lesion on newly exposed internal surface Transfer 3 -5 mm tissue onto agar surface For thin stems, small pieces of diseased tissue are excised from lesion margin, sterilized for 1 - 3 min in 10% NaOCl & washed in sterile water and plated onto agar surface (Fig. 3) Fig. 2 Isolation from stem lesionsFig. 3 Multi and single point inoculation ‘nult-coint Inocuiation Isolation of fungi by the Dilution plate method Specimens Three types of soil: 1. Loam soil ~ 2. Sandy soil 3. Clay soil — 4. Saw dust First stage Spread plating procedure Prepare soil dilutions. Prepare a 1/10 dilution of soil/other materials by suspending 10 g of soil in 96ml of sterile water. Shake well and immediately transfer 1 ml of the suspension to 99 ml sterile water and continue this procedure until you have a 10-fold dilution series ranging 1 1/10 to 1/100,000. Throughout the whole procedure, use sterilesfer 5 mL of the 1/100, 1/1000, and the 1/10,00t test tubes. Proceed to plate these dilutions on PDAaee Agar plates are needed: PDA Label plates with the type of agar and the follo triplicate plates for each dilution (1/100, 1/1000, and 11 the plate indicates the bottle from which yo ul) aliquot to spread on the plate. Note be 10X of that labeled since you are Spreading procedure. Pipet .10 ml (11 onto the agar a surface (be sure to shake before (bent glass rod) which as been the surface as evenly as possi spreading). Flame sterilize the’ media or if it comes into contat factors are spread in order of d 1/100 etc, spreaders need media.) Allow the moistu incubation. After the c lowest dilution dilution still counts are CO! + After making total col bacteria and fungi an bacteria teria) and € s in your write:tentify the microbes the best you can. Make representative Pid drawings. In the Mentiiceten ‘conesae ate bacteria Saat to isolate in pure culture. i you are attemptir port Its Section: ar CEUs s of Isolates plate count data and one sample calculation of CF 8) & Conch. 1. Discuss and compare the results obtained from of soil and saw dust 2. What is your conclusion? 4 3. Briefly discuss the pros and cons of the plate 6 enumerating soil fungi 4 Compare the species diversity and abunda 'ypes of soil and saw dust. Explain your obs Explain any real or potential economic si that you have isolated from the different sub assess the economic potential of the - known uses of the members of the genus, oF th ; of the genus that you have identified during the e Isolation of fungi by Baiting method a) Isolation from waters a Float sterile seeds of Sesamum in pond water in a Pet mycelia develop on the bait material, transfer it to fresh sterile bait to assist purification. Examine re when ready to prepare a slide for microscopi b) Isolation from soil (f Disinfect the surface of the green cock borer to bore and remove tisst with a sterile scalpel. Transfer < and place into the hole made | replace the tissue that had b When a lesion develops, portion from the edge o}inocul seful for solating Pi fung. ig. 4 Apple as bait for selective isolation of some soil fungi. sfe s iece to petri dish containing Potato eee ea the petri dish lid carefully to allow only the scalpel and tissue piece in. Place tissue pieces onto agar surface. Examine the inoculated petri dishes daily for growth. Identify the fungus/fungi associated with the off- colouration of the kernel tissue. uestion: Is the fungus you have isolated of any economic nce? Explain. eo) signif Specimens 2a & b:Wood decay: D “£nS 2a & b:Wood decay: Samples 2A & B are examples oficharacteristic White and Brown rots> carefully examine the samples and describe the characteristics of each type of decay and how they were produced. Is the white soft mass the wood pulp or fungal mycelia? What is the economic significance of each of these two types of rot? COMPOSTING Making a Compost Column You can make compost cohimns to Study the composting process on a small scale. Compost columns can be made casily with the following materials: * Three clear, plastic, 2-liter soda bottles with caps - cleaned, with the labels removed * Window screen - 2-inch by 2-inch (5-cm by 5-cm) square Piece (hardware stores may have scrap pieces this size) * Piece of wire - 6 inches (15 cm) long; picture frame wire or electrical wire works well (hardware stores may have scrap pieces available) + Small nail, skewer or dissecting needle + Clear, plastic packing tape + Coffee filter - basket type + Pair of pantyhose + Scissors + Newspaper + Soil + Trash to be compostedAssembling the compost column from pieces of 2-liter soda bottles (top left): Window screen is wrapped around Bottle 3 (top right); air holes are poked into Bottles 2 and 3 with a needle (bottom left, bottom right). To assemble the compost column, do the following: 1 Use the scissors to cut the three bottles as shown above. Cut Bottle 1 in half and trim the top portion just below the curve. Cut the top and bottom of Bottle 2 along the curved portions Cut the bottom off Bottle 3 along the curved portion. Take Bottle 3, wrap the window screen over the mouth of the bottle and fasten it below the neck with the wire, as shown above. With the nail, skewer or needle, poke many air holes in the sides of Bottle 2 and 3, as shown above. e Bottle 3 upside down in the bottom half that was cut Bottle 1, as shown below, and fasten it with tape. You a few pieces of tape that you can remove iodically to drain the water that will collect in the bottom Bottle 2 inside Bottle 3, as shown below, and fasten it th tape.¢ top piece fr \t with i andu tle 1, with its cap, Take the top piece from Bot " ae ver the open end of Bottle 2. This will be the cap of the low. finished compost column, as shown belo ee Stacking the pieces of the compost column (tape has been omitted for clarity) To load the compost column, do the following: 1. Place the coffee filter snugly in the bottom of Bottle 2 so that it sits just above the opening. The coffee filter acts as a pre- filter to keep the screen from becoming clogged with soil Particles aes © oe Coffee filter used as a pre-filter 2. Ona sheet of newspaper, mix the soil omposted. You can experiment with the trash used3. Fill the compost column with the soil/trash mixture 4. Cap the compost column. Fasten the cap with tape. 5. Add water to the column through the top-bottle cap. You can measure the amount of water if you wish, and see how much works best. Water will percolate down through the soil/trash mixture and collect in the bottom. You will have to drain the bottom periodically. 6. Cut one of the legs in the pantyhose, turn it upside down and cover the compost column with it. The pantyhose cover is effective in keeping fruit flies and gnats from moving in and out of the column. You will have to continually add water and drain the bottom, either daily yor every other day. You can turn the compost if you want to, but you can also leave it as is -- the air holes in the side should provide sufficient aeration. Here are some experiments that you can do with your column: Weigh the column daily and graph the change in weight as the compost develops. Monitor and graph the amount of water used by the column daily. Do this by subtracting the volume of water collected at * the bottom from the volume of water you've added to the top. Measure the temperature of the column with a soil thermometer daily and graph it. Take notes about the appearance of the trash in the column daily. How long does it take to decompose? Collect the drainage water and look at it under a microscope to see the microscopic organisms that live in the compost. Fungi with potential for Industrial products: S i. 3, 4. d5: a i ecimens an 2 ae ae 1. Examine the cultures with S Tae i. Naked eye ii. Dissecting microscope (DO NOT OPEN PETRI DISH). 2. Mount in lactophenol in cotton blue and examine at x10 and x40 ) Identify the Fungi in culture (Specimen 3, 4, and 5) using the descriptions given below. Illustrate characteristic structures Description 1:— n, or yellowish, sometimes bre , colored, often gree | Stroma none Sek ell eee partly oa mononematous xc, Mycelium partly cs macron®) eae psent. Conidiophor 2 straight or flexuous, ae eae ae eee smooth, swollen at the ef eae ich is covered 9. ir ‘nerical or clavate vesicle the surface of et SS beatae branches or in some species by Phialides; pes aches 1 inal ones in the si 9 DEAT Eea Seana: ae discrete, several arising cogether ee r the surface of the vesicle, mostly collarettes and Hyphopodia al colourless OF often with a foot ce part mid to dark brown, Conidiogenous cells monop. the ends of termina] branches or over de geniform, determinate, rarely percurrent, ampullitorm or lagen on ee sometimes present. Conidia catenate dry, semi-endoge! Be or a acrogenous, spherical, variously colored smooth, rugose, ee echinulate, sometimes with spines arranged spirally, O-sep' ‘ Descripti Mycelium partly immersed, partly superficial; hyphae colourless to very pale yellow, 2 ~ 4 um thick.Conidiophores erect, straight or flexuous, mostly arise directly from the substratum, smooth, septate or nonseptate, varying greatly in length and diameter, 200 - 400 x 7-10 um or several millimeters long and 201m in diameter. Conidial heads blackish-brown, purple-brown, in every shade to black, varying from small, almost columnar masses of a few conidial chains to the more common globose or radiate heads, up to 300, 500, or 100um in diameter. Phialides typically in two series, thickly covering the vesicle, primary varying greatly in length, secondary 6 ~ 10 x 2 -3um. Conidia globose, at first smooth, but later spinulose and pigmented, mostly 2.5 - 4m less trequently Sum. Globose, superficial sclerotia produced in some strains, but not common. Description 3: Vegetative hyphae creeping, septate, branched. Conidiophores erect usually unbranched, septate, at the apex with a verticil of erect primary branches, each with a verticil of secondary (Metulae = branch from which phialids arise) and sometimes tertiary branchlets or with a verticil of conidia-bearing cells (phialides) borne directly on the slightly inflated tpex of the conidiophores, sometimes with secondary conidiophores wince On the apes Bets GammeaaHiephcre. Conidia borne in chains Which typically, ferns BaasieiesHead, not enclosed in slice, ann erentiated (06% SAUMUR MNNREGE Gnidia globose, ovate ellipti oth or rough ee a,5) Descriptions 1 & 2 are more or less similar, what is the functional distinction between the two descriptions? ©) Do you think that the fungi are of any.economic importance? Explain. 4) Ifyou are sure that the fungus you are handling is Aspergillus niger, use it to produce some citric acid (Work in groups) using the surface culture technique as follows (Consult Mr. Tepeny): + Dispense 500ml diluted molasses into sterile conical flasks/beakers and sterilize. ‘ Aseptically inoculate the medium with spores of A. niger. Plug the flasks with cotton wool or aluminium and e incubate at room temperature for 5 - 7 days. * Remove the mycelia by filtration using double muslin cloth. * Add calcium oxide (lime) to precipitate calcium citrate, an insoluble salt that can be collected by filtration. The citric acid can be recovered from its calcium salt by adding sulphuric acid. (DO NOT TASTE!) 2) Ifyou did not succeed in obtaining calcium citrate, what could be the reason? b) If you succeeded in isolating calcium citrate, do you think that the A. niger isolate you have is good enough to be considered as a train with industrial potential? Why? c) What is the use of citric acid? Diagrams for guidanceSpecimen 6:The fungus in culture is suspected to be a Trichoderma sp., use the reference “Illustrated Genera of Imperfect Fungi” by Barnett to confirm this suspicion. Illustrate the features that enabled you to identify the fungus. Does this fungus have any economic value? Explain. Specimen 7 is a yeast culture: Identify the yeast using one of the following sets of characteristics. 1. No mycelia; sometimes pseudemycelium; bipolar budding. 2. Fermentative, oidia- sometimes mycelium, fission - no budding, round spores. 3. Fermentative lemon- shaped cells; Buds fission spores smooth. 4. Fermentative; round, oval to long cells; pseudomycelium at times; Spores round kidney- shaped. a) Illustrate all the observable features. Suggest any application(s) (with justification, and relevant to Kenya) that this fungus could be used for product development Inoculate sterilized 500ml diluted molasses (pH 4 - 5) in flasks with the yeast. Put on a shaker and incubate for 5 — 7 days. c) Harvest yeast cells by centrifugation. d) Describe and interpret your results. Specimen 8: Decomposing material (Composting process). Study the diversity of fungi on the decomposing and their value in composting, b} LABORATORY EXERCISE 4 Ul. MUSHROOM CULTIVATION: Objective: °o learn how to Produce a mother mushroom culture from a young mushroom b) Produce spawn using cereal grains as a substrateu Spawn g Inoculum and Mother 3 The Starting culture can be made from a fresh and health fruit body or obtained from a type collection. It is not possible to keep on transferring the cultures on agar forever. It is advisable not to transfer more than 8 times (Why?) Young and vigorous mycelium can be obtained from a fruiting body in the following way, using: Young mushroom Scalpel Alcohol Sterilized agar slants or bi Flame (non-smoking) Clean table to work on, or inoculation box. (1-3 days old) preferably in button stage, ottles with agar Preferably a laminar flow cabinet or Step: 1. Wash the mushroom thoroughly 2. Dip the scalpel in alcohol, cool for 10 seconds. 3. Break the mushroom lengthwise, Do not touch the inner surface, and then flame it until red-hot. Let it Which part to use in Agaricus, shiitake, Flammudina, oyster mushroom and monkey head mushroom (from lefe to right). Use the heated scalpel to take a small Piece (2x2mm) of the tissue out. ge . Open the test tube and heat the mouth (or Use Petri dish). . Using the scalpel put the tissue in the middle of the agar. Immediately replace the plug. 7. Inoculate at least 3 cultures. Within 3-4 days, mycelium will cover the tissue and branch out on the agar. The mycelium should be white, and grow from the tissue. Anything else is a fungal contaminant. (NB Work with the technologist all the time)Preparation of final spawn (Use a ready culture of Pleurotus sp. The Spawn Preparation of the Substrate Different grains can be used, e.g. wheat, millet, rice, or sorghu quality is very important: should contain few broken kernels, extraneous debris and be recently harvested » drain and cook for 10 -15 minutes, 1. Soak grain for 2hrs in water, and drain again to Mix the grain with vermiculite (this prevents the grain from getting Sticky), chalk or ground limestone (CaCO3 and gypsum (CasOs.2H20) (e.g, 4.5 kg wheat, 100g gypsum and 25g chalk 3. Fill the bottles and clean the inner part of the mouth to prevent Spores from germinating near the mouth of the bottle 4. Sterilize the bottles in the autoclave for 30 minutes at 121°C S. Shake bottles when taking them out of autoclave 6. Inéculate after the temperature in the center of the container has dropped below the maximum mycelial growth temperature. 7. Use one (for 250ml bottles) or 2 (for bigger bottles) small squares of SxSmm from full-grown agar from the mother culture for each bottle. 8. All equipment must be sterilized before use 9. Incubate the bottles until the mycelia have grown all over the substrate, Shake once (after 8 days) or twice during incubation (or every 3-4 days) to distribute the mycelium evenly and to prevent kernels from sticking together. It takes about two weeks fey most species to colonize the substrate 10 Keep the spawn in the refrigerator and only take it out wh needed. Grain spawn can spoil in one night at temperatures above 2590 cMushroom substrate preparation: Pasteurization: 1. Chop the straw/maize waste/banana leaves etc into bits of 3-S5cm in length and pack in bags 2. Boil water in'a drum/ appropriate container: When the water comes to boiling, place the substrate bag along with the substrate in the boiling water and boil for 15 — 20 minutes 3. Remove the bag from the hot water and leave for 8 — 10 hrs to drain excess water and also to allow the substrate to cool, Spawn ‘planting’ and incubation: 1. Thoroughly mix the spawn with the substrate in the plastic bag. 2. Punch holes (2mm diameter) on all sides of the bag for aeration. 3. Place the bags in an incubation room at about 25°C. 4. It takes 10-15 days for the spawn to spread throughout the straw. Spawn running is complete when the entire block is completely white. After spawn running open the bags. S. From this stage onwards, the relative humidity of the room should not be Iess than 85%. Maintain this by periodically spraying water on the walls and floor of the room. If it is a cemented floor, pour water on the floor (or trenches around the room & fill with water), so that water always remains on the floor. If there are signs of drying, spraying can be done with the help of a sprayer. Fruiting: (Within 7 — 10 days, tiny mushroom pinheads appear on the surface of the block and these grow into full-size mushrooms within 1 - 2 days When fruit bodies start forming, the requirement of air is increased; therefore ensure exchange of fresh air 6 - 12 hrs by opening the ventilators provided at the front and back sides of the room. LABORATORY EXERCISE 5 Diagnosis of Fungal Plant Pathogens: Objectives: To learn how to recognize and describe symptoms and signs of a diseased plant issue To learn how to detect fungal pathogens from diseased plant tissueHow Do You Diagnose the Cause of a Plant Disease? Symptoms Signs Know the Plant Species Reference Books Examine Symptoms in Detail Explore Beyond the Obvious Pare Examining Symptoms and Searching for Pathogens 7. Leek on Surface 2. Look Inside Plants What Do You Look With? 3. Naked Eye or Magnifying Lens 10. Microscope E 11. More Sophisticated Technology Where Do You Look? 12. Affected Area (e.g. necrotic lesions) 13. Base of Plant 14, Vascular Tissues 15, On or In Roots Diagnosing Cause of a Disease 1. Fungi a} Identify Species With Microscope 2) 7 Fruiting Structures Not Present, Try to Induce Them eipte neal Identification Requires Presence of Spores ee Fruiting Structures 2, Multiple Pathogens a) Recognize Additional Pathogens ») Use Above Procedures to ID Pathogens and Diagnose Diseases All Plant Pathogens Can Be Diagnosed by a) Immunodiagnostic Techniques b) Molecular Techniques 3. Noninfectious (Abiotic) Diseases a) Usually the Cause of the Disease Transmitted bir Usually Interfere with Normal Physiological Processes ©) Usually Difficult to Diagnose d) Causes of Abiotic Diseases i. Toxic Substance in Soil or Air i, Lack of Essential Substance ui, Climate Extreme itNo Pathogen Can Be Found, Cultured or - What Resources Do You Use to Diagnose a Disease? 16. Plant ID Books 17. Textbook 18. Extension Publications 19 Online ResourcesProcedure: 1. Examine the following specimens for fungal infections: } Spinach (Spinacia oleracea) probably infected with Cercospora beticola i. Celery (Apium graveolens) probably infected with Septoria api or Cercospora apii (Confirm) : Fe Goses (Rosa sp) probably infected with Sphaerotheca pannosa (Brysiphe) W. Carica papaya frnit probably infected with Rhizopus sp ‘\_ Banania fingers with cigar end rot infection probably caused by a) Verticillium theobromae ST ry i acre breligesa > Ciga cd of i. Coffea arabica (coffee) berries wit dniblte e [shies boy dizease (CBD}]causes by Colletotrichum coffeanum “st Bean pods showing anthracnose infection caused by Colletotrichum lindemuthianum uiii, Citrus fruits with scab catised by Blsinoe fawcetti V~ a ix. Coffee leaves with rust caused by Hemilea vastatrix,~ = * Maize with Smut disease caused by Ustilago maydis,~ xi xi Irish potato “Citrus twigs showing dieback infection probably caused by Diplodia sp., y (Confirm.) a) Describe the symptoms and signs of infection on diseased plant materials, 2. Carefully examine the lesions on each plant material (both the lower and upper Surfaces if it's a leaf) using a hand lens, and a dissecting microscope. Locate the Guiting structures (Sporophores): conidiophores and conidia, pycnidia, perithecia, etc, which may be present in the necrotic lesion. NB. Conidiophores and conidia may appear as fine hairs, pycnidia/perithecia appear as pinheads in necrotic lesions, pick a few fruiting bodies: If conidiophores and conidia, gently and carefully remove by brushing a sterile and moistened pointed tip of a Swann Morton no. 11 scalpel plade mounted on no. 3 blade holder across the lesion, without removing the plant Ussue, thus causing the spores to adhere to the moistened tip; or [fit is wood or Haut Peel off a thin portion of the “skin” and cut thin sections from parts of the plant tissue showing pycnidia, perithecia, or stroma, ) ‘Transfer the trapped sporophores into a drop of lactophenol in cotton f blue on a glass slide. Put a cover slip and examine at x10 and a0, lustrate the fruiting bodies. a ©) Identify the causal agents of the diseases of each plant. Use the Loud { Ae following referee aaa at’ Ded 1 Westoot's plant disease handbook, pul Host lists of Kenya Fungi and Bactena Nlustrated Genera of Imperfect Fungi 4