carnivorous fungus kit -...

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Carnivorous Fungus Kit Instructions Many species of flowering plants are known to be carnivorous, cap- turing and digesting an animal prey. Less well known are the carniv- orous fungi. Fungi that entrap and digest nematodes, or round- worms, are found in soil and in fresh and salt waters. Over 150 species of nematode-trapping fungi have been described. Nematodes are extremely abundant. Although 'Some are serious plant and animal pathogens, most play a vital role in soil aeration and organic decomposition. They are secretive animals with limited behavioral patterns. However, for their size, they are powerful and enormously active. When you consider the delicate nature of fungal hyphae. it is remarkable that the hyphae can trap and hold these worms until escape is impossible. MATERIALS The materials in the Carnivorous Fungus Kit are sufficient for 10 groups of 34 students. The materials are supplied for use with the exercise in this kit only. Carolina- Biological Supply Company dis- claims all responsibility fur any other uses of these materials. Included in the kit are: Dish Culture Arthrobotrys conoides (Carnivorous Fungus) Tube Culture Rhabditis (Nematode) Two Bottles Cornmeal Agar Inoculating Loop 10 Sterile Petri Dishes Other materials needed but not included are a bunsen burner. a water bath with thermometer, scalpels, a disinfectant such as 70% ethanol or phenol, and a compound microscope or stereomicroscope (40x magnification). If a water bath is not available, a container of boiling water can be substituted.

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Page 1: Carnivorous Fungus Kit - Winonacourse1.winona.edu/RRICHARDSON/242/documents/documents/scan0016.pdfCarnivorous Fungus Kit Instructions Many species offlowering plants are known to be

Carnivorous Fungus Kit

Instructions

Many species of flowering plants are known to be carnivorous, cap-turing and digesting an animal prey. Less well known are the carniv-orous fungi. Fungi that entrap and digest nematodes, or round-worms, are found in soil and in fresh and salt waters. Over 150species of nematode-trapping fungi have been described.Nematodes are extremely abundant. Although 'Some are serious

plant and animal pathogens, most play a vital role in soil aerationand organic decomposition. They are secretive animals with limitedbehavioral patterns. However, for their size, they are powerful andenormously active. When you consider the delicate nature of fungalhyphae. it is remarkable that the hyphae can trap and hold theseworms until escape is impossible.

MATERIALS

The materials in the Carnivorous Fungus Kit are sufficient for 10groups of 34 students. The materials are supplied for use with theexercise in this kit only. Carolina- Biological Supply Company dis-claims all responsibility fur any other uses of these materials.Included in the kit are:

Dish Culture Arthrobotrys conoides (Carnivorous Fungus)Tube Culture Rhabditis (Nematode)Two Bottles Cornmeal AgarInoculating Loop10 Sterile Petri Dishes

Other materials needed but not included are a bunsen burner. awater bath with thermometer, scalpels, a disinfectant such as 70%ethanol or phenol, and a compound microscope or stereomicroscope(40x magnification). If a water bath is not available, a container ofboiling water can be substituted.

Page 2: Carnivorous Fungus Kit - Winonacourse1.winona.edu/RRICHARDSON/242/documents/documents/scan0016.pdfCarnivorous Fungus Kit Instructions Many species offlowering plants are known to be

PROCEDURES

Laboratory Procedures

The agar dishes should be prepared at least two hours before the lab-oratory period. After the agar solidifies, it can be stored in the refrig-erator for up to one week.

1. Slightly loosen the caps and set the bottles of cornmeal agar in ?

boiling water bath to melt the agar. This melting requiresapproximately 20-40 minutes. Swirl the agar within the bottlesto be sure it. has completely melted.

2. Cool the medium in the bottles to about 45° C (should be com-fortably hot to the touch) by cooling the water bath to that tem-perature or by removing the bottles and letting them sit forseveral minutes at room temperature.

3. Wipe down work area with a disinfectant. Wash hands.4. Swirl the bottles. Remove the cap from a bottle, flame the

mouth over the bunsen burner for a few seconds, and distributethe contents among five petri dishes. Cover each dish immedi-ately after pouring to prevent contamination.

5. Repeat Step 4 with the other bottle.6. Allow the dishes to remain undisturbed until the agar has solidified.7. Set up an inoculating station at a convenient location. The sta-

tion should have the culture of A. conoides, a buns en burner,and a supply of scalpels.

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Figure 1Cornmeal agar dish

containing agar block ofArthrobotrys conoides.

Figure 2Typical zig-zag streakingpattern for inoculatingdishes with Rhabditis.

Page 3: Carnivorous Fungus Kit - Winonacourse1.winona.edu/RRICHARDSON/242/documents/documents/scan0016.pdfCarnivorous Fungus Kit Instructions Many species offlowering plants are known to be

Inoculation of dishes with A. conoides (Day 1)

1. Wipe all work areas with a disinfectant. Wash hands.2. Distribute a cornmeal agar dish to each team.3. Flame-sterilize a scalpel and cut a block (about 1 em square) of

the A. conoides culture. Transfer the culture using the samescalpel to the dish of cornmeal agar. Invert the culture onto theagar (Fig. 1). Lift the cover of the dish only long enough toinsert the culture. Care should be taken to avoid touching thecutting surface of the sterilized scalpel or the inside surfaces ofthe petri dishes.

4. Flame the scalpel.5. Label the dishes with the students' names, the date, and the

name of the fungus.6. Incubate at room temperature (680 to 78° F).7. Observe the dishes daily as the hyphae spreads across andunder the surface of the agar.

Inoculation of A. conoides dishes with Rhabditis (Day 5-7)

1. When growth has reached the edge of the dish (5 to 7 days),inoculate the dishes with Rhabditis. Again wipe the work areawith a disinfectant. Wash hands. Hold the tube culture ofRhabditis in one hand. Flame the inoculating loop held in theother hand and then remove the top of the Rhabditis tube.Flame the mouth of the tube. Insert the cooled loop into thetube and remove a small amount of the Rhabditis culture (aboutthe size of match head). Replace the tube top. Raise the cover ofthe dish just enough to insert the loop. Streak the dish in a zig-zag pattern (Fig. 2). Replace the cover of the dish. Flame theloop.

2. Observe the petri dish under a microscope to be sure that theRhabditis nematodes were transferred to the dish. The studentsmay need to repeat step #1 if the nematodes are not observedafter the first transfer.

3. Note the thrashing movement and the amount of activity of theRhabditis. Also, note whether any traps or lassos are visiblealong the fungal hyphae.

4. Observe the plates at 24, 48, 72, and 96 hours and record obser-vations.

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Page 4: Carnivorous Fungus Kit - Winonacourse1.winona.edu/RRICHARDSON/242/documents/documents/scan0016.pdfCarnivorous Fungus Kit Instructions Many species offlowering plants are known to be

DISCUSSION

Arthrobotrys cono ides is a member of the Deuteromycetes, orImperfect Fungi, those fungi that have no known or a poorly under-stood sexual stage in their life cycles. Found in soil, A. conoides is arather innocuous-looking fungus that produces colorless hyphae. Itcan be best observed by holding the petri dish against a light source.When the hyphae have reached the edge of the dish, sporulationoccurs with the formation of conidia (asexual spores). A. conoidesgrows and reproduces in this characteristic manner until exposed tonematodes.When exposed to nematodes, the fungus produces many hyphal

traps or lassos. Evidently, the change in fungal structure is stimulatedby substances collectively called nemin that are produced by thenematodes. The fungal lassos are adhesive, and when a nematodecomes into contact with the adhesive lassos, it becomes trapped.The nematode may struggle for a time after its capture, but it dies

within a short period of time. Once a nematode is trapped, A.conoides produces additional hyphae that penetrate the worm's bodythen digest and absorb its contents. All fungi exhibit this absorptivenutrition.Within 24 hours after inoculation with Rhabditis, lassos are evi-

dent on the hyphae. During .the next 24 hours, Rhabditis becomeentrapped in these lassos. From 48 to 96 hours, the entrapped nema-todes cease movement and show signs of deterioration. By the end of96 hours, almost all traces ·of Rhabditis have disappeared. A.conoides then exhibits increased sporulation, especially at the siteswhere the nematodes were digested and absorbed. Evidently,feeding triggers the increased amount of sporulation.

FURTHER READING

Alexopoulos, J. and Mims, C.W. 1979. Introductory Mycology. JohnWiley and Sons, New York.

Barnett, H.L. and Hunter, Barry B. 1972. Illustrated Genera ofImperfect Fungi. Burgess Publishing Co., Minneapolis.

Barron, G.L. 1977. The Nematode-Destroying Fungi. CanadianBiological Publications, Ltd., Guelph, Ontario.

Lutz, P.E. 1985. Invertebrate Zoology. Addison-Wesley PublishingCo., Reading, MA.

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