Mycologia

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Coprinus comatus: A basidiomycete fungus forms

novel spiny structures and infects nematode

Hong Luo, Minghe Mo, Xiaowei Huang, Xuan Li & Keqin Zhang

To cite this article: Hong Luo, Minghe Mo, Xiaowei Huang, Xuan Li & Keqin Zhang (2004)
Coprinus comatus: A basidiomycete fungus forms novel spiny structures and infects nematode,
Mycologia, 96:6, 1218-1224, DOI: 10.1080/15572536.2005.11832870

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Published online: 30 Jan 2017.

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Mycologia, 96(6), 2004, pp. 1218–1225.
q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897

Coprinus comatus: A basidiomycete fungus forms novel spiny structures and

infects nematode

Hong Luo them are wood-decay mushrooms. Our knowledge in

Minghe Mo this area begins with the discovery of Nematoctonus
Xiaowei Huang (Drechsler 1941), with its teleomorph in the genus
Xuan Li Hohenbuehelia (Barron and Dierkes 1977). This is an
Keqin Zhang1 unfamiliar aspect of the biology of these saprophytic
Laboratory for Conservation and Utilization of Bio- fungi. It was suggested that the wood-decay fungi ob-
resources, Yunnan University, Kunming 650091, tain nitrogen supplement from prey, including nem-
Yunnan, P.R. China atodes, to survive in such nitrogen-restricted habitat as
rotting wood and forest soil (Thorn and Barron 1984,
1986). Along the hyphae of these nematophagous ba-
Abstract: Nematophagous basidiomycete fungi kill sidiomycete fungi, some appendages were found to be
nematodes by trapping, endoparasitizing and pro- attack or defense weapons. Nematoctonus is character-
ducing toxin. In our studies Coprinus comatus ized by the ability to capture nematodes by sticking to
(O.F.Müll. : Fr.) Pers. is found to be a nematode-de- the nematode cuticle with hourglass-shaped adhesive
stroying fungus; this fungus immobilizes, kills and knobs that are enveloped in a thick mucus sheath
uses free-living nematode Panagrellus redivivus Good- (Drechsler 1943, 1946, 1949, 1954). Pleurotus paralyzes
ey and root-knot nematode Meloidogyne arenaria nematodes with toxin droplets produced by tiny secre-
Neal. C. comatus produces an unusual structure des- tory cells on vegetative hyphae (Barron and Thorn
ignated spiny ball. Set on a sporophore-like branch, 1987). Similar secretory appendages were found on
the spiny ball is a burr-like structure assembled with lawn mushroom Conocybe lacteal, in which they are
a large number of tiny tubes. Purified spiny balls ex- more likely to be defense apparatus. After paralyzing
hibit moderate nematicidal activity. Experiments and killing nematodes, C. lacteal does not use them
show that spiny balls are not chlamydospores because as food (Hutchison et al 1995). Some species of Hy-
of the absence of nuclei in the structures and quick phoderma produce stephanocysts that once were con-
formation within 3 d in a young colony. Nematodes sidered asexual spores, dispersal propagules (Burdsall
added to C. comatus cultures on potato-dextrose agar 1969), chlamydospores (Kendrick 1979) or beneficial
(PDA) and cornmeal agar (CMA) become inactive in nutritional structures (Hallenberg 1990), but Tzean
hours. Infection of nematodes by the fungus occurs and Liou showed that they were trapping devices
only after the nematodes are immobilized (feeble or (Tzean and Liou 1993). In this article, a basidiomycete
dead), probably by a toxin. Electron micrographs il- fungus, Coprinus comatus, is shown to be a nematode-
lustrate that C. comatus infect P. redivivus by produc- destroying fungus, producing a new structure desig-
ing penetration pegs with which hyphae colonize nated spiny ball. The infection process of P. redivivus
nematode bodies. An infected nematode is digested by the fungus is studied with SEM and TEM. A simple
and consumed within days and hyphae grow out of method to purify spiny balls is suggested. The con-
the nematode. nection between the spiny balls and killing of nema-
Key words: Coprinus comatus, Nematophagous tode is discussed here.
fungi, spiny ball

MATERIALS AND METHODS

INTRODUCTION Culture of C. comatus.—One isolate designated LHA-7 was

obtained by tissue isolation. This strain was grown on PDA
Nematophagous fungi draw much attention with their in 9 cm diam Petri dishes. When the colonies reached 2 cm
biological control potential of capturing, killing and diam, 5 mm agar plugs from the leading edge of the indi-
digesting nematodes. Nematophagous basidiomycete vidual colonies were transferred to new PDA and CMA. The
fungi were investigated about 63 y ago and many of colonies occupied the whole agar surface in 9–12 d at 24 C,
and these first-transferred Petri plates were used in bioassays.
Accepted for publication May 13, 2004. Nematode culture.—The free-living nematode P. redivivus
1 Corresponding author. E-mail: [email protected] was grown axenically in a semiliquid oat medium (10 g of

1218
 LUO ET AL: SPINY BALL STRUCTURE 1219

TABLE I. Immobilization of nematodes by the isolate LHA-7

Immobilized
No. of nematodes Control Statisticsa
Incubation nematodes
Plates Nematodes time Mobile Immobile (%) Mobile Immobile x2 P
PDA P. redivivus 4 h 129 122 48.6 318 6 176.0 ,0.005
PDA P. redivivus 8 h 12 218 94.8 355 11 499.0 ,0.005
CMA P. redivivus 4 h 132 109 45.2 135 2 78.6 ,0.005
CMA P. redivivus 8 h 74 194 72.4 187 2 227.3 ,0.005
PDA M. arenaria 4 h 51 204 80.0 213 66 167.0 ,0.005
PDA M. arenaria 8 h 11 252 95.8 170 84 220.8 ,0.005
CMA M. arenaria 4 h 73 115 61.2 201 35 96.3 ,0.005
CMA M. arenaria 8 h 17 146 89.6 185 24 221.9 ,0.005
a
x2 tests (df 5 1) comparing frequencies of mobile and immobile nematodes in treated versus control samples.

oat, 6 mL of distilled water) at 28 C for 4–6 d, stored at 4 Bioassays.—Bioassay with fungal cultures were conducted
C and used within 15 d. The root-knot nematode M. aren- on first-transferred PDA and CMA Petri plates. The nema-
aria was cultured on tomatoes in greenhouse conditions todes were washed thoroughly with sterile distilled water
and second-stage juveniles were extracted and stored ac- and the concentration was adjusted to 10 000 nematodes
cording to Kerry’s methods (Kerry and Bourne 2002). per mL. Each Petri plate received 0.5 mL of the nematode
Purification of spiny balls.—LHA-7 was inoculated on PDA suspension that then was scattered with a glass spreader.
in 9 cm diam Petri dishes and incubated at 24 C for 9 d. The plates were incubated at 24 C and mobile and immo-
Lens paper in 12 layers was folded to fit a glass funnel, bile nematodes in five random visual fields were counted
moistened in the funnel, and the assembly was sterilized at after 4 and 8 h. This experiment was carried out with three
121 C for 20 min. Seven mL of sterile water were added to replicates and conducted twice. Nematode-inoculated PDA
each culture plate. The surface of the culture was scraped and CMA plates without fungal cultures served as controls.
gently for 2 min with a glass sterile spreader. The suspen- The bioassay with purified spiny balls of the isolate LHA-
sion was transferred to the sterile funnel and the filtrate 7 was conducted with 96 well cell culture clusters (Costar).
was collected in sterile 1.5 mL Eppendorf tubes and cen- Each test well received 30 mL of purified ball precipitate
trifuged at 12 000 rpm for 5 min. After transferring the and ca 100 nematodes in 20 mL of suspension. Treatment
supernatant to other Eppendorf tubes, the precipitate was control wells contained 30 mL of the supernatant obtained
washed by resuspending in sterile distilled water, and the during the purification of spiny balls and 20 mL of nema-
suspension was centrifuged at 10 000 rpm for 3 min. The tode suspension. To maintain humidity, 100 mL per well of
precipitate was stored at 4 C. sterile water was added to the remaining wells. The plates

FIG. 1. Scanning electron micrograph of the spiny balls. A. The spiny balls on specific aerial hyphae. B. High-magnification
view of 5 d old spiny balls on a furcated branch. C. High-magnification view of 15 d old spiny balls. D. The nascent spiny
balls with less fuzz (arrows) compared with the mature ones. Bars: A 5 10 mm, B, C 5 1 mm, D 5 5 mm.
1220 MYCOLOGIA

FIG. 2. Nuclear staining of spiny balls. A. A light microscopic picture of the spiny balls. B. The fluorescence micrograph
of the same balls. The white spots (arrows) in the hyphae were nuclei. Bars: A, B 5 10 mm.

were incubated at 24 C and examined after 24 and 48 h. nematodes used in electron microscopic observation were
Mobile and immobile nematodes were counted with an in- prepared by adding ca 5000 nematodes (P. redivivus) in 0.5
verted light microscope. This experiment was performed mL of suspension at each center of the mycelia. The plates
with three replicates and conducted twice. then were incubated 12 h, 24 h, 48 h, 72 h, 5 d, 7 d or 9
d. Dense aerial hyphae covering the infected nematodes
Nuclear staining and germination assays.—Fluorescent dye
were removed with adhesive tape in SEM study. Samples for
Hoechst 33258 (Sigma) was used as the stain. To keep del-
both TEM and SEM studies underwent the same initial
icate structures intact, the slide culture technique was
steps: small blocks of agar (7 3 7 mm for SEM, 2 3 2 mm
adopted (Riddell 1950). Mycelia of LHA-7 with spiny balls
for TEM) containing spiny balls or infected nematodes were
expanded across the cover slips in 5 d, when the cover slips
cut out and fixed in 2.5% glutaraldehyde solution at 4 C
were used for fluorescence staining. Stock stain solution was
for 24 h. The agar pieces were rinsed in 0.2 M phosphate
prepared by dissolving 5 mg of Hoechst 33258 in 5 mL of
buffer (pH 7.2) three times and postfixed in 1% OsO4
distilled water and diluting the solution to 100 mL; working
made up in the same buffer for 1 h at room temperature.
solution contained 10% stock solution, 90% 0.2 M phos-
A graded acetone series was used in dehydration. For SEM
phate-citric acid buffer (pH 7.0). Fifteen percent glycerol
the agar blocks were exchanged in an isoamyl acetate series,
was added to the working solution before use. The cover
dried with a HCP-2 Critical Point Dryer (Hitachi) for 7 h,
slips with mycelia downward were laid on drops of working
mounted on copper stubs, coated with a gold/palladium
stain solution placed on slides beforehand. After 1 min the
mixture by an IB-3 ion coater (EIKO) and viewed with a
slides were observed in a Nikon Eclipse 800 microscope and
JSM-5600LV scanning electron microscope ( JEOL). For
photos were taken with Kodak Gold 400 film.
TEM samples were embedded in epoxy resin (Epon 812)
The germination experiment was conducted with puri-
after fixation and dehydration and cut with glass knives.
fied spiny balls. Approximately 10 mL of spiny-ball precipi-
Ultrathin sections were mounted on formvar film on 100
tate was added to 1 mL of sterile water, and the suspension
mesh copper grids and stained with uranyl acetate and lead
was spread on PDA and CMA plates with a glass spreader,
citrate. Observation was taken with a JEM-100 CX transmis-
200 mL of the suspension each. These plates then were in-
sion electronic microscope ( JEOL) operating at 60 kV.
cubated at 24 C and examined with a light microscope ev-
In the SEM study for purified spiny balls, 10 mL of spiny-
ery 24 h for 9 d.
ball precipitate was added to 1 mL of 2.5% glutaraldehyde
Scanning electron microscopy (SEM) and transmission electron solution at 4 C for 24 h. After centrifugation the precipitate
microscopy (TEM).—The isolate LHA-7 was grown on PDA was transferred to a cover slip and spread evenly with an
in 9 cm diam Petri dishes at 24 C for 5 d. Mycelia did not inoculating loop. The spiny balls on the cover slip were
cover all the agar surface and contained less dense aerial coated and viewed as described above without being post-
mycelia, which could aid TEM and SEM processes. Infected fixed and dehydrated.

FIG. 3. Transmission electron micrograph of spiny balls. A. Longitudinal section of a spiny ball. The ball was composed
of an electron-dense shaft and many irregular tubes (TB). B. Longitudinal section of a spiny ball on a branch (TG). The
shaft (SH) was electron-dense except the slightly inflated end. Bars: A, B 5 0.5 mm.
 LUO ET AL: SPINY BALL STRUCTURE 1221

FIG. 4. Scanning electron micrograph of immobilized and infected nematodes. A. An inactive nematode (treated 12 h).
Two hyphae wound the nematode. B. A nematode treated 7 d was wrapped in hyphae. C. Hyphae protruded from infected
nematodes (treated 5 d). D. Pores (arrows) in nematode cuticle left by pulling out hyphae with adhesive tape. Bars: A 5 10
mm, B 5 50 mm, C 5 5 mm, D 5 2 mm.

FIG. 5. Transmission electron micrograph of infected nematodes. A. A hypha grew toward and pressed the cuticle (CU)
of a nematode. B. A penetration peg (PG) was forming. C. A hypha penetrated nematode cuticle (CU) with a penetration
peg (PG). D. High-magnification view of the penetration peg (PG). E. A hypha was about to grow out from an infected
nematode. F. A hypha protruded from the nematode. Bars: A, B, C 5 1 mm, D 5 0.5 mm, E 5 0.25 mm, F 5 2 mm.
1222 MYCOLOGIA

FIG. 6. Colonization and digestion of P. redivivus by LHA-7. A. A nematode was digested partially 3 d after adding
nematodes. B. An infected nematode was digested fully and occupied by the hyphal body 7 d after adding nematodes. Bars:
A 5 5 mm, B 5 1 mm.

Culture of basidiocarps.—To verify the identity of the cul- es were measured 0.5–8 mm in length, the lower parts
tures we isolated, solid-state cultivation was done to obtain were somewhat wider (0.5–1.3 mm), and the upper
basidiocarps. In 250 mL flasks, mycelia of LHA-7 were parts were slender (0.3–0.6 mm). In high-magnifica-
grown in potato dextrose broth (PDB) medium at 24 C for
tion TEM, the spiny balls were assembled by many
7 d by shaking at 150 rpm. Mushroom culture bags were
tiny tubes (ca 0.2 mm wide) with slightly narrowed
prepared with the medium containing 90% cottonseed hull,
4.5% bran, 4.5% cornmeal and 1% lime. The bags were apices (FIG. 1B, C). The development of the spiny
sterilized at 121 C for 4 h and inoculated with the liquid balls differed from that of spores: Specific aerial hy-
culture. The bags were incubated at 24 C in a greenhouse phae branched to form sporophore-like branches,
for 40 d until all bags were colonized fully. The cores were many fibers grew out from tips of the branches and
transferred to barrels and covered with forest soil contain- the fibers elongated themselves and surrounded the
ing abundant organic debris. Relative humidity was adjusted ends of the branches to form spiny balls (FIG. 1D).
to 85% and the containers were placed in the greenhouse Nuclear staining illustrated the absence of nuclei in
for induction of basidiocarps at 20 C.
the structures and in the sporophore-like branches
and that the aerial hyphae bearing these balls were
RESULTS dikaryotic (FIG. 2). Purified spiny balls did not ger-
minate on PDA and CMA. After 9 d no obvious
Immobilization of nematodes.—On PDA and CMA
change of the balls was found on either medium. In
plates, LHA-7 immobilized nematodes with high ef-
TEM the core of a spiny ball was an electron-dense
ficiency (TABLE I). At same intervals, LHA-7 immo-
shaft that had a slightly inflated end with some sac-
bilized more nematodes on PDA than that on CMA.
cular organelles and the shaft was surrounded by
Spiny balls.—LHA-7 produced abundant spiny balls many tubes (FIG. 3A). The shaft sprouted from the
on PDA, and spiny balls were formed quickly in cul- top of a sporophore-like branch and was the base of
ture, usually within 3 d of inoculation. On an aerial a spiny ball (FIG. 3B). Consistent with the result of
hypha, a spiny ball was a ‘‘fluffy’’ structure having a nuclear staining study, no nuclei were found by using
diameter of about 2.5 mm. The spiny balls and the TEM technique.
support branches, respectively, resembled spores and
sporophores and the sporophore-like branches ar- Infection of nematodes.—Most of the P. redivivus add-
ranged in a sympodial pattern (FIG. 1A). The branch- ed to the cultures on PDA and CMA were immobi-

FIG. 7. Scanning electron micrograph of purified spiny balls. A. Low-magnification view of abundant purified spiny balls.
B. High-magnification view of purified balls. The pores (arrows) were the sites where shafts fixed in. Bars: A 5 20 mm, B 5
2 mm.
 LUO ET AL: SPINY BALL STRUCTURE 1223

TABLE II. Immobilization of nematodes by the purified spiny balls

Immobilized
No. of nematodes Control Statisticsa
Incubation nematodes
Nematode time Mobile Immobile (%) Mobile Immobile x2 P
P. redivivus 24 h 108 77 41.6 174 37 26.7 ,0.005
P. redivivus 48 h 40 149 78.8 155 51 113.2 ,0.005
a
x2 tests (df 5 1) comparing frequencies of mobile and immobile nematodes in treated versus control samples.

lized within 8 h. Many of the nematodes transferred Immobilization of nematodes by excised spiny balls.—
to a drop of water by hand moved feebly, but one The SEM micrograph showed a great number of pu-
hardly could recover or survive. When the nematodes rified spiny balls could be obtained in a simple way
were immobilized (i.e. they were very weak or dead) (FIG. 7A). A pore in each ball showed the site where
the hyphae of LHA-7 grew toward and wounded the a shaft fixed in (FIG. 7B). The purified balls showed
nematodes (FIG. 4A). An infected nematode envel- a moderate activity in immobilizing nematodes (TA-
oped by a layer of hyphae still could be recognized BLE II). The same situation was found when spiny
easily 15 d after adding nematodes (FIG. 4B). The balls were ground with liquid nitrogen.
nematode body was colonized fully by hyphal body,
and from the third day on hyphae protruded from Basidiocarps of C. comatus.—Basidiocarp primordia
the nematode (FIG. 4C). Adhesive tape was used to formed after 15 d and mushrooms emerged 30 d af-
prepare samples for SEM study so that pores perfo- ter placing the cores into soil (FIG. 8). Identification
rated by hyphae were exposed because some hyphae with well developed basidiocarps reconfirmed the
were pulled out (FIG. 4D). Transmission electron mi- identity of the cultures as C. comatus. Furthermore,
crographs revealed a detailed infection process, con- the isolates obtained from second-time tissue isola-
cerning how a nematode was colonized by C. comatus tion also possessed spiny balls and nematicidal activ-
and how hyphae grew from an infected nematode. ity.
In the first stage hyphae grew toward and pressed
onto the cuticle of the nematode (FIG. 5A); then a DISCUSSION
penetration peg was formed (FIG. 5B); in the third
stage the hyphae penetrated nematode cuticle with C. comatus was considered a saprophytic fungus be-
the peg and rapidly colonized the body (FIG. 5C, D); fore, but in this report it is shown to infect and kill
finally, hyphae protruded from the infected nema- the free-living nematode P. redivivus and the plant-
tode (FIG. 5E, F). The inward and outward defor- parasitic nematode M. arenaria. Our results support
mation of cuticle at the sites where hyphae invaded the view that some mushrooms develop the function
and emerged from nematodes indicated that me- of feeding on microfauna, enabling them to survive
chanical force was involved in penetration and out- or thrive in nitrogen-restricted niches (Thorn and
growth. After penetration of hyphae, the content of Barron 1984, 1986).
a nematode was digested in days and the nematode Many gilled fungi produce chlamydospores in cul-
was occupied fully by the hyphal body (FIG. 6). ture, and some of them are ornamented strongly.

FIG. 8. Basidiocarps of the isolate LHA-7. A. Newly formed basidiocarps were emerging from soil. B. Mature basidiocarps.
Bars: A 5 1 cm, B 5 4 cm.
1224 MYCOLOGIA

The spiny balls first were thought to be terminal chla- ACKNOWLEDGMENTS

mydospores, but the evidence—lack of nuclei and
This research was financially supported by the National Nat-
quick formation—contradicted this conclusion. Con-
ural Science Foundation Program of China (No. 30470067,
sidering that the purified spiny balls showed moder- 30070006), the 973 earlier-stage program (No.
ate nematicidal activity, these unusual structures 2002CCC02800), National Key Technologies R&D Program
might be devices or appendages relevant to killing (No. 2002BA901A21), Key Applied Foundation Program of
nematode. Purified spiny balls showed a lesser ability Yunnan Province (No. 1999C0001Z) and National Key Foun-
to immobilize nematodes than the cultures, possibly dation Program (No. 2001DEA10009-10). We are grateful to
because they need proper physiological conditions. Dr Xuefeng Liu for his helpful suggestions on culture of the
So far the devices and the appendages found on ba- basidiocarps.
sidiomycetes are specialized cells producing toxins or
adhesive substances. On Pleurotus and C. lacteal toxin
LITERATURE CITED
droplets flow from secretory cells (Barron and Thorn
1987, Hutchison et al 1995). Stephanosysts and hour- Barron GL, Dierkes Y. 1977. Nematophagous fungi: Hoh-
glass-shaped adhesive knobs produce infection tubes enbuehelia, the perfect state of Nematoctonus. Can J Bot
or pegs (Drechsler 1946, 1949, 1954; Tzean and Liou 55:3054–3062.
1993). The spiny balls seem to be incomplete cells , Thorn RG. 1987. Destruction of nematodes by spe-
and do not resemble any of them. The assumption cies of Pleurotus. Can J Bot 65:774–778.
Burdsall HH, Jr. 1969. Stephanocysts: unique structures in
that the spiny ball might be a nematode-killing ap-
the Basidiomycetes. Mycologia 61:915–923.
pendage needs further investigation.
Drechsler C. 1941. Some hyphomycetes parasitic on free-
Although we cannot draw a conclusion about what living terricolous nematodes. Phytopathology 31:773–
the spiny ball is, experiments showed some evidence 801.
relevant to the nematode-killing capability of the fun- . 1943. Two new basidiomycetous fungi parasitic on
gus. We obtained 31 isolates after tissue isolation, and nematodes. J Wash Acad Sci 33:183–189.
they all produced the spiny balls and immobilized . 1946. A clamp-bearing fungus parasitic and preda-
nematodes with varying efficiency when grown on cious on nematodes. Mycologia 38:1–23.
PDA and CMA. Among them isolates with higher . 1949. A nematode-capturing fungus with anasto-
nematode-killing capability produced more spiny mosing clamp-bearing hyphae. Mycologia 41:369–387.
. 1954. A nematode-capturing fungus with clamp-
balls than those with lower nematode-killing capabil-
connections and curved conidia. J Wash Acad Sci 44:
ity. In the bioassays nematodes remained alive and 82–85.
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induced by the presence of nematodes, and no no- lactea. Can J Bot 74:431–434.
Kendrick B, Watling R. 1979. Mitospores in Basidiomycetes.
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a drop of water, many of them made feeble move- agent for root-knot nematodes. Merckstr, Germany:
ments suggesting they were alive. A similar occur- Druckform GmbH. 171 p.
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todes and a toxin-producing nematophagous fungus nematicidal toxin from Pleurotus ostreatus NRRL 3526.
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Organisms are energy-efficient systems and tend , . 1986. Nematoctonus and the tribe Resupi-
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