Experiments in Applied Biotechnology II

Experiments in Appl Biotech II: Bioremediation & RCA-Based Diagnosis of Geminiviruses – MSSHTAYEH - 1/27/2013

Experiments in Applied Biotechnology II

Mohammed S. Ali Shtayeh Professor of Biology & Biotechnology Biodiversity and Environmental Research Center, BERC Til, Nablus

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Experiments in Applied Biotechnology, Prof. Mohammed S. Ali Shtayeh

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TABLE OF CONTENTS Part A. Fungi as bioremediation agents Experiment 1. INTRODUCING FUNGI Exercise 1a: Preparation of media, pouring of plates, and preparation of agar slants Exercise 1b: Cultivation and microscopic examination of fungi Exercise 1c: Morphology of fungi Experiment 2. Keratinolytic activity of fungi Part B. Use of white rot fungi in soil bioremediation. Introduction Experiment 3. Bioassay of benomyl persistence in soil. Experiment 4. Selective isolation of Phanerochaete chrysosporium from soil. Experiment 5. Use of Phanerochaete chrysosporium in the bioremediation of soils Part C. RCA-based diagnosis of plant viruses: Rolling Circle Amplification (RCA) Introduction Experiment 6. Sample collection and DNA isolation from symptomatic plants- ‘Fast Eddie’ Experiment 7. DNA Extraction check Experiment 8. RCA-based diagnosis of plant viruses: Rolling Circle Amplification (RCA) Experiment 9. Check RCA product Experiment 10. Rolling Circle Amplification (RCA)/ RFLP Experiment 11. Detection of Squash leaf curl virus (SLCV) in Palestine by polymerase chain reaction using degenerated primers and isolate-specific primers References

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PART A. Fungi as bioremediation agents Experiment 1. Introducing fungi Exercise 1a: Preparation of media, pouring of plates, and preparation of agar slants Materials needed: Culture media and chemicals: Corn Meal Agar (CMA); Potato Dextrose Agar (PDA); Malt Extract Agar (MEA); Sabouraud Dextrose Agar (SDA); 95% Ethyl alcohol. Antibiotics: Penicillin; Rifampicin; Ampicillin, Chloramphenicol, Cycloheximide, Cycloheximide, Pimaricin. Procedure: 1. Prepare 500-ml of each of the following media: CMA; PDA; SDA; MEA. 1. Weigh the designated quantities of ingredients. 2. Dissolve in the required amount of distilled water in a flask. 3. Plug the flask with cotton and sterilize by placing the media in an autoclave for 15-20 minutes at 120 oC and 15-psi pressure. What a complete medium should provide a fungus with? ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… …………………………………………………………………………………… When preparing a medium its necessary to adjust its pH. Why? ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………….……… 2. Prepare agar slants (slopes) in screw-capped bottles or in tubes as follows: 1. Sterilize the medium in the bottles or tubes (15 ml per bottle). 2. While the agar is still hot and molten lay the tubes on a bench with the plugged ends supported (in a slanted position) so that the medium forms a layer of decreasing thickness from the bottom of the bottle to within about 2 cm of the plug. Sloping the agar obviously provides the growing fungus a relatively large surface. Agar slants are sometimes superior over agar plates for growing fungi. Explain. ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………… 3. Pour the media into 9-cm plates as follows: 1. Clean the working area of the bench with a detergent and disinfectant as mentioned before. [


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2. Melt the agar medium in the bottles or flasks and then allow the medium to cool to about 60 oC. Antibacterial agents (e.g., penicillin 50 mg/L, rifampicin 10 mg/L, ampicillin 250 mg/L) may be incorporated in the medium before pouring in plates. 3. Place the Petri dishes on the bench. 4. Light a Bunsen burner and proceed as follows. 5. Remove the cotton or plastic plug and flame the mouth of the bottle or flask gently. 6. Pour the medium into the sterile plastic or glass Petri dishes and rotate each dish so as to spread the agar evenly over its bottom. The lids of the dish is raised just enough to insert the mouth of the bottle or flask, thereby reducing the danger of airborne contamination. The dishes are allowed to cool sufficiently for the medium to harden before using. `Store labeled agar plates and agar slants in the refrigerator at 10 oC until use. You should cool the medium to about 50 oC before pouring in plates. Why? ………………………………………………………………………………………………………… ……………………………………………………………………………………………… …………………………………………………………………………………………………… Agar plates are usually stored upside down. Why? ………………………………………………………………………………………………………… ………………………………………………………………………………………………

Exercise 1b: Cultivation and microscopic examination of fungi Materials needed: Water cultures of: Pythium aphanidermatum, P. ultimum, Phytophthora parasitica, and Saprolegnia sp. Agar cultures of: Fusarium, Penicillium, Microsporum canis, Trichophyton, and Rhizopus stolonifer. Culture media and chemicals: CMA; PDA; SDA; MEA; Sterile 20 % solution of glycerol; Lactophenol; Suitable mounting fluid; Nutrient broth; 7% Aqueous safranin; 0.5% Basic fuchsin; 25, 50, 75, 95, and 100% Ethanol; 100% Xylene; Fingernail varnish or commercial water-based PVA (polyvinyl acetate). Other materials: Ocular micrometer; Stage micrometer; Cavity slide. Procedure: A. Transferring of fungi to Petri dishes and slants Fungal cultures can be perpetuated in the laboratory as follows: 1. Using a sterile straight inoculating needle or a scalpel, transfer spores or fragments of the mycelium from one or more of the provided fungal cultures to sterile media (in plates or culture bottles) which you prepared in the last lab period. 2. Place the inoculated plates or slants in the incubator at 25 ºC until the next lab period. After 7 days of incubation examine the inoculated plates or culture tubes and record your observations by describing the growing colonies.

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………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………………………………… B. Slide culture technique 1. Prepare a suitable agar medium (e.g. MEA, SDA, CMA, and PDA), and pour into a plate to a depth of about 2-mm. 2. When it has set completely, cut out a small block, about 1-cm square using a sterile scalpel and transfer to the center of a sterile slide. 3. Inoculate the block with the fungus on all four edges. 4. Place a sterile coverslip on top of the block of agar. 5. Incubate the slide in a moist chamber (Figure 2.1). This may be made from a sterile Petri dish with filter paper in the bottom. Two sterile glass rods (or a bent glass rod) are placed on the filter paper to function as supports for the slide. About 10 ml of a sterile 20 % solution of glycerol is poured into the dish. This will keep the agar moist. The dish with the inoculated slide is incubated until the growth reaches a desired stage (until the mycelium formed from each edge of the block has reached the edge of the coverglass). The fungus usually spreads out from the agar block and tends to attach itself to the two glass surfaces. 6. When the desired stage of fungal growth has been reached, lift off the coverslip and carefully lower fungus side down onto a drop of mounting fluid such as lactophenol (with or without stain) on a clean slide. Carefully remove the agar block, add a drop of mountant (e.g., lactophenol) to the growth adhering to the slide, and apply a clean coverslip. Then seal the slides using nail varnish. 7. Examine your preparations under the microscope (10 X, 40 X).

Figure 2.1 Slide culture technique: A, culture plate; B, blotter paper; C, bent glass rod; D, glass slide; E, block of agar; F, inoculum; G, cover slip.

Record and illustrate your observations. ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… [


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………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… C. Single spore cultures Several methods for preparing single spore isolations are available. The following is a quick method that does not involve the usage of micromanipulators. 1. Using a sterile looped wire needle place a few drops of sterile water on a sterilized slide. 2. Transfer a small amount of the fungus spores to the water drop using a sterile inoculating needle and mix to form a suspension. 3. Using a sterilized wire needle, pick up a loopfull of the suspension, and streak it onto a nutrient agar plates using 4 rapid strokes. 4. After incubation for e.g. 17 hr, study the plate with a dissecting microscope when colonies just starting to develop from single spores. Pick off an isolated colony with an inoculating needle and transfer to fresh petri dish. Look for isolated colonies at the end of the fourth streak where the suspension is the finest. Record and illustrate your observations. ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………… D. Hanging drop Hanging drop cultures can be prepared as follows: 1. Place a few spores in water or nutrient broth on a coverslip. 2. Invert the coverslip over the cavity in a cavity slide or a suitable glass or plastic ring. 3. Place the slide in a moist chamber overnight. Record and illustrate your observations.

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………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………………………………………………………………… E. Microscopic preparations a. Temporary mounts: 1. Place a small drop of mounting fluid in the center of a clean glass slide. 2. Pick off a very small portion of the fungal material from the culture, with a sterile needle, place it in the drop of fluid, and tease it out with a pair of needles. 3. Place a cover glass over the preparation in a way as to avoid air bubbles. Placing the object directly in the mounting fluid, particularly if this be lactophenol, may result in the inclusion of air bubbles in the preparation. To avoid such a problem the fungal material is placed on a slide and moistened with a drop of alcohol. Most of the alcohol is allowed to evaporate before being replaced by the mounting fluid. 4. Seal the coverslip on the slide using fingernail varnish or commercial water-based PVA (polyvinyl acetate). Apply two coats of nail varnish and when the nail varnish seal has dried, apply a thick layer of PVA diluted with water (1:1) over the nail varnish. Examine the preparation under the microscope. Record and illustrate your observations. ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… …………………………………………………….………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………………………………….………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… …………………………………………………………………………………… b. Permanent mounts: 1. Using a sterile needle pick off a very small portion of the fungal material from an agar culture of Penicillium or Fusarium species and place it in a drop of water, and tease it out with a pair of needles. 2. By holding the slide with the fungus side upwards, pass the slide rapidly 10-cm above an open flame a few times. 3. Add a few drops of 7% aqueous safranin and leave for 1 min or for 5 to 20 sec in 0.5% basic fuchsin. 4. Wash the stained mount in distilled water. 5. Dehydrate the fungus material by dipping the slide for 30-60 sec through a 25, 50, 75, 95, and [


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100% ethanol series, then through a 100% xylene and finally in 3:1 and 1:1 mixtures of xylene and mounting fluid before mounting in the mounting fluid. Examine the preparation under the microscope. Record and illustrate your observation. ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… c. Measurement of microscopic fungal structures: Determine the value for each of the lines in the ocular micrometer by matching them with lines on the stage micrometer. This value is fixed for each objective lens. Record your results in a tabulated form. The ocular micrometer can now function as a ruler in the microscope. ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………… Using the method described above, measure the dimensions of conidia and hyphae in Penicillium and Fusarium. Record your results. ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ……………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… [


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………………………………………………………………………………………………………… …………………………………………………………………………………… Cultures prepared in this lab period may be used for the study of morphology of fungi in the next lab. Questions: 1. What conditions of nutrition and environment favor the growth of most fungi in the laboratory? 2. What is meant by absorptive nutrition? 3. How are fungi commonly propagated in the laboratory? 4. Briefly discuss the role of light in the growth and reproduction of fungi.

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Exercise 1c: Morphology of fungi Materials needed: Fungal cultures: Four-day-old water cultures of Pythium aphanidermatum, P. ultimum, Phytophthora parasitica, and Saprolegnia sp.; 1-2 week old agar cultures of Fusarium, Penicillium, Microsporum canis, Trichophyton, and Rhizopus stolonifer; Cultures prepared in the previous lab period. Prepared slides: Claviceps purpurea (l.s., stroma; c.s., sclerotium), Puccinia graminis (teliospores), Rhizopus stolonifer (sexual stage). Procedure: 1- Mycelium a.Coenocytic mycelium: Examine a culture of one or more of the following fungi: Saprolegnia, Pythium, and Phytophthora, growing on corn kernels in water, by directly putting the Petri dish on the stage of the microscope and examining it with 10 X objective. Prepare a wet mount using a small excised portion of the mycelium, and examine under 10 X and 40 X objectives and note the distribution of protoplast in the hyphae. Look for any septa. Are there any? If yes, at what locations? ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… …………………………………………………………………………………… Can you see any cytoplasmic movement? …………………………………………………………………………………………………… Illustrate.

Coenocytic mycelium b. Septate mycelium: Examine microscopic preparations of excised portions of mycelium from an agar culture of one or more of the following fungi: Fusarium, Penicillium, Microsporum, and Trichophyton. Note the distribution of protoplast in the hyphae. Look for septa (partitions) that divide hyphae into compartments. Note the pattern of branching in the mycelium. Can you distinguish any differences in the branching of hyphae between different fungi? Note the differences in the morphology of the colonies of different fungi that you examined. Illustrate. [


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Septate mycelium 2- Fungal tissues (plectenchyma) a. Prosenchyma: Examine the stroma (longitudinal section, l.s.) of Claviceps purpurea and observe its structure with reference to the organization of hyphae within it. Illustrate.

L.s. in the stroma of Claviceps purpurea showing prosenchyma. b. Pseudoparenchyma: Examine a cross section (c.s.) of a sclerotium of Claviceps purpurea. Note its organization with reference to the relationships between hyphae in this structure. What is the role of such a structure relative to the fungus survival? ………………………………………………………………………………………………………… …………………………………………………………………………………………………………

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………………………………………………………………………………………………………… …………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… ………………………………………………………………………………………… Illustrate.

C.S. of a sclerotium of Claviceps purpurea showing Pseudoparenchyma. 3- Main reproductive structures 1- Asexual structures a. Chlamydospores: Examine under the microscope (45 X) an excised part of mycelium of Trichophyton. Observe the thick-walled portions, usually intercalary, of the mycelium. Illustrate.

A

B Chlamydospores (Pythium undulatum, A. Sporangia, B. Chlamydospore) [


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b. Buds: These can be seen in actively growing yeast. Illustrate.

Yeast buds in actively growing yeast

c- Sporangiospores (formed inside sporangia) (i) Aplanospores (non-motile): Examine a culture of Rhizopus and observe the sporangia with the nonmotile spores (sporangiospores) within them. Illustrate.

Sporangia and sporangiospores (aplanospores, non-motile spores) of Rhizopus

How do you think these spores disseminate? ………………………………………………………………………………………………………… ………………………………………………………………………………………………………… …………………………………………………………………………………………

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(ii)

Planospores (motile): Examine under the low power of a microscope chilled

undisturbed water culture of Pythium aphanidermatum, Saprolegnia, or Phytophthora parasitica and observe the biflagellate moving zoospores. Pick a sporangium that is about to sporulate and watch it for a few minutes until zoospore discharge takes place. Describe and illustrate the process.

Sporangia, zoospores (planospores, motile spores), and zoospores discharge of … …. …………

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d- Conidia (formed at tips of hyphae): Examine under the high power of a microscope a small portion of mycelium of each of the following fungi: Microsporum canis, Epidermophyton floccosum, and Fusarium sp. Observe the conidiophores and the various types of conidia. Illustrate.

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a. Oospores (result of gametangial contact): Examine under the low powers of a microscope a water culture of Pythium ultimum (or a similar fungus). Prepare a wet mount of the fungus and examine under the high power of the microscope. Observe the sexual structures present (especially oogonia, antheridia, and oospores). Illustrate.

Oospores of Pythium ultimum

b. Zygospores (result of gametangial copulation): Examine under the high powers of a microscope the permanent slide of the zygospores of Rhizopus stolonifer. Observe the various steps of the sexual process that ends up with the formation of zygospores. Illustrate.

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Stages of Zygosporangium and zygospores formation in Rhizopus stolonifer zygospores.

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c. Ascospores (spermatization): Examine the ascocarps of Claviceps purpurea. Observe the asci and the ascospores. The ascospores (perithecia) are embedded in the stroma. How many ascospores can you see in the ascus? Illustrate.

Ascospores, asci and ascocarps (embedded in the stroma) in Claviceps purpurea

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d. Basidiospores: Examine a permanent slide of Puccinia graminis. Observe the teliospores which give rise to basidia and basidiospores. Illustrate.

Teliospores in Puccinia graminis.

Questions: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Define the fungi by listing their characteristic features. Differentiate between prosenchyma and psedoprosenchyma. How can parasitic fungi obtain their nutrients? Define heterokaryosis and explain how this phenomenon occurs in fungal thallus. Define or explain: planogametes, hypha, mycelium, septum, mitospore, meiospore. What is the distinguishing chemical component of the cell wall of most fungi? How does the fungal cell wall differ in chemical composition from that of plants? How does mitosis differ from that in plants? Some fungal chromosomes are very minute and difficult to count accurately, what other criterion is used to determine the occurrence of meiosis in fungi? 10. Where does growth take place in a hypha?

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MORPHOLOGY OF FUNGI

Claviceps purpurea -mature stroma ergot

Puccinia graminis (teliospores)

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KINGDOM FUNGI: PHYLUM ZYGOMYCOTA

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KINGDOM FUNGI: PHYLUM ASCOMYCOTA

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KINGDOM FUNGI: PHYLUM BASIDIOMYCOTA

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KINGDOM FUNGI: DEUTEROMYCETES: ASEXUAL ASCOMYCETES AND OTHER ASEXUAL FUNGI

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KINGDOM STRAMENOPILA: PHYLUM OOMYCOTA

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EXERCISE 2. Keratinolytic activity of fungi Materials needed: Samples representing varied polluted and non-polluted soils (e.g., roadside, home garden, irrigated vegetables field, rain-fed field, forest, farmyard, factory yard, landfill vicinity, olive mill vicinity, …etc. Other materials: Sterile sand; Hairs from a blond-child, 1 cm long; Lactophenol cotton blue. Procedure: Keratinolysis test. The hair-soil method of English (1976) as modified by Filipello Marchisio (1986) will be used to detect the keratinolytic activity. Wash sand under tap water, dry and autoclave. 1.

Place 25-ml soil in Petri dishes (9-cm diameter), and moisten with 10-ml sterile distilled water.

2.

Scatter hairs from a blond-child, 1 cm long, on the surface of the soil.

3.

Incubate the cultures at room temperature for 20 days, and moisten with sterile distilled water when necessary. Employ three replicate plates for each soil.

4.

Mount the inoculated hairs in lactophenol cotton blue for microscopic examination (10 X, 40 X, and 100 X).

5.

Explain results (based on the examination of 10 hairs, 5 from each replicate plate for each isolate) in light of model proposed by Filipello Marchisio (1986) (Figure 2.5). The term surface erosion (SE) is used to indicate progressive destruction of the hair from the exterior or inwards; this may occur either uniformly (U) along the length of the hair, or in localized areas forming more or less extensive pockets (P). Random attack on the hair by more or less specialized hyphae that penetrate the hair at right angles to the surface is termed radial penetration (Rp). A boring hypha is used to describe fine hypha about 1 μm diam. which penetrates the substratum at right angle to the layer of keratinized cells, [


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while a perforating organ is used to describe single column of up to 10 short hyphal cells, usually 3-4 times wider than normal mycelial cells, that penetrates radially into the hair cortex, passing straight through its keratinized cells irrespective of their boundaries. Wider boring hyphae (wbh) indicate structure intermediate in diameter between boring hyphae and perforating organs. Swollen boring hyphae (sbh) are structures that are similar to boring hyphae when penetrating the outer cortex, but dilated in a series of balloons on reaching what are probably less compact regions of the hair. 6.

Repeat step 4 every other fortnight twice. Record your observations. Illustrate.

7.

Construct a table showing the intensity and progress of keratinolytic activity in the different soils. Find out whether there was an association between soil pollution level and the soil-inhabiting keratinophilic fungi and keratinolytic activity.

8.

Discuss your results.

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Experiments in Appl Biotech II: Bioremediation & RCA-Based Diagnosis of Geminiviruses – MSSHTAYEH - 1/27/2013 2 weeks post inoculation Soil

USE

USE ELA BH PO WBH SBH

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ELA

BH

PO

4 wks PI WBH

SBH

USE

ELA

6wks PI BH

PO

WBH

SBH

USE

ELA

BH

PO

WBH

uniform surface erosion erosion in localized areas boring hyphae perforating organ wide perforating hyphae swollen perforating hyphae


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SBH


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Part B. Use of white rot fungi in soil bioremediation. Introduction Fungicide bioassays are based on the diffusion of fungicide from the sample through agar medium inoculated with spores of the test fungus. The fungicide concentration is then determined from the inhibition of fungal growth. A standard dosage-response curve is calculated from inhibition zones with known concentrations of fungicide, which permits estimation of concentration in samples (Munnecke, 1958). Although benomyl is not a particularly hazardous compound, it can be readily bioassayed and so used to determine the factors that influence the effectiveness of Phanerochaete chrysosporium (Figure 1-3) as a bioremedial agent in soils.

Figure 1. Zeiss drawing tube drawings of microscopic characters of Phanerochaete chrysoporium, HHB 6251. 1. cystidia. 2. basidia. 3. basidiospores. 4. subicular hyphae Author(s): Burdsall & Eslyn. Original in: Burdsall, H.H. Jr. & Eslyn, W.E. 1974, Mycotaxon 1(2): 123-133

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Figure 2. Description: Zeiss drawing tube drawings of microscopic characters of cultures of Phanerochaete chrysoporium, HHB 6251. 8. aerialhyphae. 9. submerged hyphae.. 10. aleuriospores and conidiophores. 11. arthrospores. 12 chlamydospores. Author(s): Burdsall & Eslyn. Original in: Burdsall, H.H. Jr. & Eslyn, W.E. 1974, Mycotaxon 1(2): 123-133 [


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A

B

Figure 3. A: Phanerochaete chrysosporium growth characteristics on malt extract agar. B. Description: Phanerochaete chrysosporium (holotype), a. subicular hyphae, b. cystidia, c. basidia, and d. basidiospores. Author(s): H. Burdsall. Original in: Burdsall, H.H. 1985, Mycologia Mem. 10: 1-165

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Experiment 3. Bioassay of benomyl persistence in soil. The bioassay technique used was originally used to determine the breakdown of nonvolatile fungicides in soil. Known concentrations of the fungicide under study are mixed in the soil and then soil plugs are prepared. These are then transferred to a medium seeded with spores of a fungus that is susceptible to the fungicide. The fungicide diffuses out of the soil plug and inhibits spore germination, producing an inhibition zone whose size is related to fungicide concentration. A standard curve is made relating fungicide concentration to the size of the inhibition zone. This is then used to determine an unknown concentration of the fungicide in soil. Objective of the experiment: Our aim is to detect the adsorption, persistence and amount of biologically-available benomyl residues in soil, using a bioassay technique. MATERIALS AND METHODS Chemicals. Benomyl, Methanol, Acetone, 70 % ethyl alcohol. Media. Potato dextrose agar, agar. Other items: Haemocytometer, plastic rings (12.7 mm diam, 10 mm height) Bioassay fungus and soils. Penicillium expansum, the bioassay organism, is obtained from the Department of Biology, An-Najah University. The soils to be used are typical local clay loam soils. The soils are air-dried and sieved (