Effect of Nematode-Trapping Fungi on an Entomopathogenic Nematode Originating from the Same Field Site in California

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

68, 246–252 (1996)

0092

Effect of Nematode-Trapping Fungi on an Entomopathogenic Nematode Originating from the Same Field Site in California ALBRECHT M. KOPPENHO¨ FER, BRUCE A. JAFFEE, ANN E. MULDOON, DONALD R. STRONG,*

AND

HARRY K. KAYA

Department of Nematology, University of California, Davis, California 95616-8668; and *Bodega Marine Laboratory, University of California, Bodega Bay, California 94923 Received November 13, 1995; accepted July 25, 1996

INTRODUCTION

We determined whether nematode-trapping fungi may influence the dynamics of a coastal shrub community. The food chain interactions in the shrub community involve the dominant plant species, its major insect herbivore, and an entomopathogenic nematode, Heterorhabditis hepialus. Of the 12 nematode-trapping fungi previously isolated from soils at the study site, 5 were selected for this study. Arthrobotrys oligospora, Geniculifera paucispora, Monacrosporium eudermatum, and Monacrosporium cionopagum efficiently trapped and colonized H. hepialus on agar; in contrast Nematoctonus concurrens trapped but did not infect or colonize the nematode on agar. To determine whether these fungi can suppress H. hepialus in soil, we added the fungi in the form of fungal-colonized nematodes to pasteurized (2 hr at 62°C) and raw (nontreated) soil from the study site. Suppression was measured by comparing nematode invasion into a wax moth larva in fungus-treated and untreated soil in vials at 20°C. Fungal population density in soil was estimated using dilution plating and most probable number procedures. All fungi suppressed H. hepialus if the wax moth larvae were added 4 days after the nematodes. Suppression ranged between 37 and 54% and did not differ among fungi. Suppression was usually greater in raw than in pasteurized soil. Raw soil contained a constant background of nematode-trapping fungi, and A. oligospora was the most common among these; no background was detected in pasteurized soil. The presence of background fungi in raw soil may explain the higher suppression in raw than in pasteurized soil. Fungal propagule densities in our laboratory experiments were similar to those observed in the field, suggesting that nematode-trapping fungi may influence the dynamics of the plant, insect herbivore, and entomopathogenic nematode in the coastal ecosystem. r 1996 Academic Press, Inc.

KEY WORDS: Heterorhabditis hepialus; Monacrosporium eudermatum; Monacrosporium cionopagum; Nematoctonus concurrens; Arthrobotrys oligospora; Geniculifera paucispora; soil ecology; most probable number.

0022-2011/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

Entomopathogenic nematodes in the genera Steinernema and Heterorhabditis occur in soils throughout the world (Amarasinghe et al., 1994; Choo et al., 1995 and references therein). They are obligate pathogens of insects in nature and are used as ‘‘biological insecticides’’ against many soil-dwelling insect pests (Kaya and Gaugler, 1993). The third-stage infective juvenile (IJ) is the only stage that occurs outside the host in the soil, where it persists until a new host is found. The survival of IJs in natural soils is limited by abiotic (Kaya, 1990) and biotic factors (Kaya and Koppenho¨fer, 1996, and references therein). Among the potential natural enemies are those fungi that produce special mycelial, nematode-trapping structures (Barron, 1977; Gray, 1987). Observations on agar indicate that Steinernema spp. and Heterorhabditis spp. are highly susceptible to these fungi (Poinar and Jansson, 1986; Van Sloun et al., 1990; Jaffee et al., 1992). Observations in soil are limited but indicate suppression of entomopathogenic nematodes by trapping fungi and other nematophagous fungi (Van Sloun et al., 1990; Timper et al., 1991). Like entomopathogenic nematodes, nematode-trapping fungi are common in a wide range of soil habitats throughout the world (Gray, 1987). In the coastal shrubland at the Bodega Marine Reserve (BMR) (Bodega Bay, Sonoma County, California), Jaffee et al. (1996) found 12 species of nematode-trapping fungi; these fungi are of special interest because of their possible involvement in food chain interactions. The food chain consists of the dominant plant at BMR, the bush lupine, Lupinus arboreus, which is subjected to heavy subterranean herbivory by the ghost moth caterpillar, Hepialus californicus (Lepidoptera: Hepialidae), causing lupine die-off in patches (Strong et al., 1995). The caterpillars, in turn, are parasitized and killed by an entomopathogenic nematode, Heterorhabditis hepialus, in some lupine patches. The absence of the nematode in other patches correlates with the die-off of lupine.

246

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TRAPPING FUNGI/ENTOMOPATHOGENIC NEMATODE

The purpose of the present study was to further our understanding of the food chain interactions in the ecosystem at BMR. Our objectives were to determine the susceptibility of H. hepialus to the most common nematode-trapping fungi isolated from BMR, to determine whether the fungi can suppress H. hepialus populations in soil, and to relate experimental fungal population densities to natural densities in the field (Jaffee et al., 1996). MATERIAL AND METHODS

TABLE 1 Nematode-Trapping Fungi Used in Study Fungus Arthrobotrys oligospora Geniculifera paucispora Monacrosporium eudermatum Monacrosporium cionopagum Nematoctonus concurrens

Infective structures

ARSEF ATCC No. No.

Adhesive networks Adhesive networks Adhesive networks

4939 4941 4945

96709 96704 96679

Adhesive branches

4943

96677

Adhesive, glandular cells

4947

96681

General Procedures A sandy soil (89% sand, 9% silt, 2% clay; 4.3% organic matter; pH 5.3) was collected from BMR. The sampling site has exhibited frequent bush lupine die-offs (Strong et al., 1995) and was designated ‘‘Upper Draw high.’’ After sieving (2-mm aperture) to remove larger particles and plant parts, the soil was kept (4–10 weeks) at room temperature (23 6 2°C) and 7% soil moisture (7 g water per 100 g dry soil) until used in experiments. Based on Baermann funnel extraction, the soil contained small numbers (,1/cm3 of soil) of plant-parasitic and microbivorous nematodes after storage for 4 weeks. A portion of the soil was heated to 62°C for 2 hr (pasteurized) to kill any nematodes and nematophagous fungi naturally present. Thereafter, the pasteurized soil was air-dried to 2% soil moisture and kept at room temperature. Both soils were moistened to 11.5% 48 hr before use in experiments. H. hepialus was cultured at 20°C in last instar larvae of the greater wax moth, Galleria mellonella. The emerging IJs were harvested from White traps and stored in sterilized distilled water at 10°C (Woodring and Kaya, 1988) for 7–21 days. Before use in experiments, the IJs were kept at 20°C for at least 12 hr. Steinernema glaseri NC strain IJs were obtained in the same way, except that they were cultured at room temperature (23 6 2°C) and stored for 7–40 days. The cyst nematode, Heterodera schachtii, was cultured on sugarbeet, Beta vulgaris, in the greenhouse. To obtain second-stage juveniles (J2) of H. schachtii, cysts were placed on Baermann funnels, and J2 were collected every 30–120 min and stored at 10°C with aeration for less than 5 hr. The fungi selected for this study are listed in Table 1. The first three fungi were the most common among the 12 nematode-trapping fungi detected at BMR (Jaffee et al., 1996); the highest densities detected of Arthrobotrys oligospora, Geniculifera paucispora, and Monacrosporium eudermatum were 686, 32, and 15 propagules per gram of soil, respectively. Monacrosporium cionopagum was selected because of its potential importance as a biological control agent of plant-parasitic nematodes (Jaffee and Muldoon, 1995a), and Nematoctonus concurrens was selected because it differs substantially

from the other fungi. First, N. concurrens is a basidiomycete rather than a hyphomycete. Second, while it can be classified as a nematode-trapping fungus based on the production of traps on hyphae, it also can be classified as an ‘‘endoparasitic’’ fungus based on its production of traps on conidia (Barron and Dierkes, 1977). Fungal-colonized S. glaseri IJs (approximately 1130 µm long and 43 µm wide) were used as inocula. To obtain fungal-colonized nematodes, about 1000 healthy IJs were added to fungal colonies growing on quarterstrength corn meal agar (CMA/4) in 10-cm petri plates (Jaffee et al., 1992); after 24 hr, the colonized nematodes were transferred individually from the agar surface to soil using a needle. Trapping on Agar Although observation of trapping of nematodes on agar may be unrelated to trapping of nematodes in soil (Galper et al., 1995), it is a simple and fast way to qualitatively determine physiological susceptibility of nematode species to different species of fungi (Jaffee and Muldoon, 1995a). We studied the susceptibility of H. hepialus to the trapping fungi on agar and included another species of entomopathogenic nematode, S. glaseri, known to be very susceptible to many nematophagous fungi (Jaffee et al., 1992). We also included two temperature treatments (15 and 20°C). At BMR, cool soil temperatures (range approximately 9 to 17°C) prevail. If differences between 15 and 20°C were not important, however, running the soil experiments at the higher temperature would allow shorter incubation periods and direct comparison with other data generated at 20°C (e.g., Jaffee and Muldoon, 1995a,b). Five plugs (0.25 cm3) from cultures of the fungi growing on CMA/4 were added to sterile CMA/4 in 10-cm petri plates. To ensure that the fungus colonies had an average diameter of 2.5 cm when nematodes were added to the plates, the plugs were added at different times, depending on the fungus growth rates. At 15°C, the plugs were added 12 days before nematodes for N. concurrens and M. cionopagum, 6.5 days for

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¨ FER ET AL. KOPPENHO

G. paucispora, 5 days for M. eudermatum, and 4 days for A. oligospora. At 20°C, the plugs were added 9 days before nematodes for N. concurrens and M. cionopagum, 5 days for G. paucispora, 3.5 days for M. eudermatum, and 3 days for A. oligospora. The experiment was started by adding healthy IJs (1000 H. hepialus or 500 of the larger S. glaseri) in 0.1 ml sterilized distilled water to each plate. Plates without fungi served as controls. After excess water had evaporated, the plates were covered and kept at 15 or 20°C. The plates were examined after 36, 72, and 108 hr with a dissecting microscope at 20–603 to determine the proportion of nematodes trapped or colonized (filled with hyphae). To select nematodes randomly, a plastic template with 11 1-cm-diameter circles was randomly placed beneath each plate, and all nematodes within each circle were scored until 50 specimen per plate were observed. The experiment was performed twice with three replicate plates per trial. Soil Procedures On Day -1, 20.3 g of soil (dry weight equivalent) and 100 H. schachtii J2 in 0.1 ml distilled water were placed in 250-ml plastic cups. Addition of H. schachtii ensured that both pasteurized and raw soil would have similar populations of ‘‘background’’ nematodes. At this point, the soil moisture of both pasteurized and raw soil was 12% or 218 kPa water potential based on the soil moisture release curve (Jaffee et al., 1996). On Day 0, 40 S. glaseri colonized by the respective fungus were added to the soil. Controls received no S. glaseri. Preliminary experiments had shown that the addition of dead, noncolonized S. glaseri IJs (killed by exposure to 62°C water for 10 min) to the soil had no effect on H. hepialus penetration into wax moth larvae. The soil was packed into vials (47 mm height 3 26 mm diameter), each of which had a 2-mm-diameter inoculation hole in the side. The soil column was 28 mm high (volume 15 cm3, bulk density 1.35), and the inoculation hole was 1 mm from the bottom. The vials were covered with a lid and stored in clear plastic boxes with moist paper towels (moisture chambers) at 20°C in the dark. On Day 14, each of eight vials per treatment received 200 healthy IJs of H. hepialus in 0.1 ml sterilized distilled water through the inoculation hole. After 2 or 4 days, depending on the trial, one wax moth larva weighing between 225 and 275 mg was added to the soil surface of each vial and covered with pasteurized soil. The larva was recovered after 2 days and incubated for 3 additional days in a petri dish at 100% RH. Dead larvae were dissected individually in a petri plate in a 0.5% Pepsin solution and incubated for 2 hr at 37°C to digest the insect’s tissues (Mauleon et al., 1993). Thereafter, the number of nematodes that had established in the larva was determined. To estimate fungal population density in soil, we used

dilution plating and most probable number procedures (Eren and Pramer, 1965; Alexander, 1982) as modified by Jaffee and Muldoon (1995b). For each combination of soil and fungus treatment (including controls), soil from four replicate vials was washed into a 250-ml flask on Day 14. Sterile distilled water was added to make the total volume 200 ml, and the flask was vigorously mixed on a wrist action shaker for 6 min followed by 1 min shaking by hand. A 10-fold dilution series (0.324, 0.0324, and 0.00324 g soil/ml) was prepared. Aliquots (0.1 ml) from each dilution were added to five replicate 10-cm-diameter petri plates (CMA/4) as a line of five drops across the dish. Each dish then received in 0.1 ml distilled water approximately 1000 healthy S. glaseri as a ‘‘bait nematode’’ (Eren and Pramer, 1965). After the excess water was absorbed or had evaporated, the plates were covered and left for 3 weeks at room temperature. Thereafter, we examined the entire surface of each dish at 50–1403 magnification with a dissecting microscope. Dishes with conidia or traps of the fungi were scored as positive for the respective fungus; we used conidial and trap morphology to identify the species. The most probable number of propagules per gram soil was determined using published tables (Alexander, 1982). Soil Experiments In two experiments, we tested the effect of the addition of fungus inoculum to soil on H. hepialus penetration into wax moth larvae. In the first experiment, each fungus species was studied in a separate trial consisting of a fungus treatment and a control in pasteurized or raw soil. In these trials, the wax moth larvae were added to the soil 2 days after H. hepialus. This experiment was performed once. The second experiment had 12 treatments, consisting of the five fungi and a control for pasteurized and raw soil. The wax moth larvae were added to the soil 4 days after H. hepialus. This experiment was performed twice. In both experiments, each treatment had 12 replicates of which 8 were used for the bioassay with wax moth larvae and 4 for the determination of fungal population density. Statistical Analysis In the agar experiment, the proportion of nematodes trapped or colonized at 36, 72, and 108 hr after addition of nematodes to the plates was transformed (arcsin square root). In the soil experiments, variations in IJ penetration efficiency between trials of the same experiment were compensated for by dividing the data by the mean of the control in pasteurized soil of the respective trial; data are presented in this form. Data from all experiments were analyzed using analysis of variance, and means were separated with Tukey test (SAS Insti-

249

TRAPPING FUNGI/ENTOMOPATHOGENIC NEMATODE

ments with or without fungus inoculum, nematode penetration was significantly less in raw soil than in pasteurized soil for each fungus (F $ 6.6; df 5 1,29; P , 0.02); reduction ranged between 15 and 27%. When data from raw and pasteurized soil for each fungus were combined, the addition of fungal inoculum reduced nematode penetration between 4% for A. oligospora and 15% for G. paucispora. The reduction was significant for G. paucispora (F 5 6.2; df 5 1,29; P , 0.02) and M. eudermatum (F 5 17.8; df 5 1,29; P , 0.01) but was not significant for the other fungi (P $ 0.14). In the first trial of the second soil experiment (4 days between addition of IJs and wax moth larva), nematode penetration averaged across the treatments with or without fungal inoculum was significantly less in raw soil than in pasteurized soil (60 6 2% vs 69 6 3%) (F 5 4.45; df 5 1,95; P , 0.05). Nematode recovery was significantly greater in the control than in the fungus treatments when data were averaged across pasteurized and raw soil (F 5 5.23; df 5 5,91; P , 0.001); we observed no differences among the treatments with fungi (Fig. 1A). The observations in trial 2 were very similar. The relative effect of N. concurrens in raw and pasteurized soil, however, was reversed in trial 2 compared to trial 1 (Figs. 1A vs 1B), and the effect was significantly different between trials both in pasteurized (t 5 3.9; df 5 15; P , 0.001) and raw soil (t 5 5.1; df 5 15; P , 0.001). The most probable number data show that the fungi established in both raw and pasteurized soil in the first experiment (Table 3) and second experiment (Table 4) and allow us to determine whether fungal population densities were realistic (see Discussion). G. paucispora, however, was not detected in the second trial of the second experiment (Table 4). In raw soil, a constant background of nematode-trapping fungi was detected; A. oligospora was detected in all soil samples, but the other fungi were detected only occasionally. No background was detected in pasteurized soil.

tute, 1988). If data were similar between trials, they were combined for analysis and presentation. Differences were considered significant at P , 0.05. RESULTS

Trapping on Agar After 108 hr at either 15 or 20°C, all five fungi had trapped .98% of the IJs of both nematode species. At 36 hr, we observed significant differences between the fungi in rate of trapping of both nematode species at both temperatures (F $ 21.1; df 5 4,25; P , 0.001). G. paucispora trapped both nematode species significantly slower than all other fungi, while N. concurrens trapped both nematode species faster than the other fungi, although not always significantly faster (Table 2). In contrast to the other fungi, N. concurrens did not colonize the trapped H. hepialus IJs; the IJs remained trapped and died after 7–14 days. Among the other fungi, we observed significant differences in colonization rate at 72 hr at both temperatures for both S. glaseri (F $ 29.9; df 5 4,25; P , 0.001) and H. hepialus (F $ 58.4; df 5 3,20; P , 0.001). Colonization rate by G. paucispora was significantly slower than that of the other fungi at both temperatures for both nematode species (Table 2). Because the trapping rates were generally similar at 15 and 20°C for all fungi, the soil experiments were conducted at 20°C. Suppression of H. hepialus in Soil In the first soil experiment (2 days between addition of IJs and wax moth larva), IJ recovery from wax moth larvae was greatest in pasteurized soil without fungal inoculum followed by pasteurized soil with inoculum, the raw soil without inoculum, and the raw soil with inoculum (Table 3). With all fungi, IJ penetration was significantly less in the raw soil with fungal inoculum than in the pasteurized soil without inoculum (F . 3.0; df 5 3,27; P , 0.05). When averaged across the treat-

TABLE 2 Experiment 1: Percentage of Nematodes Trapped after 36 hr and Colonized after 72 hr by Five Species of Nematode-Trapping Fungi at Two Temperatures a 15°C H. hepialus Fungus Arthrobotrys oligospora Geniculifera paucispora Monacrosporium eudermatum Monacrosporium cionopagum Nematoctonus concurrens a

Trapped ab

87 6 4 38 6 10 b 93 6 3 a 96 6 2 a 98 6 1 a

20°C S. glaseri

H. hepialus

Colonized

Trapped

Colonized

Trapped

Colonized

Trapped

Colonized

56 6 5 a 24 6 8 b 71 6 5 a 73 6 4 a 0c

90 6 2 b 16 6 3 d 74 6 4 c 79 6 5 bc 98 6 1 a

58 6 5 b 21 6 3 c 69 6 7 b 67 6 5 b 91 6 1 a

83 6 5 b 25 6 3 c 97 6 1 a 99 6 1 a 99 6 1 a

44 6 10 b 16 6 4 c 68 6 4 a 75 6 3 a 0b

79 6 5 b 19 6 3 c 91 6 2 b 88 6 4 b 99 6 1 a

71 6 5 b 17 6 4 c 71 6 6 b 81 6 2 ab 90 6 1 a

Each petri dish received infective juveniles of Steinernema glaseri (500) or Heterorhabditis hepialus (1000) (n 5 6). Means 6 SE followed by same letter within columns were not significantly different (P . 0.05; Tukey). c Data were not normally distributed and were excluded from analysis. b

S. glaseri

¨ FER ET AL. KOPPENHO

250 DISCUSSION

The five fungi selected for this study suppressed H. hepialus penetration into wax moth larvae in raw and in pasteurized soil. Because the fungi established in both soils and efficiently trapped H. hepialus on agar, we assume that the suppression was due to parasitism TABLE 3 Experiment 2: Recovery of Heterorhabditis hepialus Infective Juveniles (% of Control in Pasteurized Soil 6 SE) from Bait Insect after 2 Days Exposure in Raw and Pasteurized Soil Inoculated or Noninoculated with Five Species of Nematode-Trapping Fungi and Most Probable Number (MPN) of Fungus Propagules per Gram Soil a Trial

Fungus

Soil

1

None Arthrobotrys oligospora None Arthrobotrys oligospora None Monacrosporium eudermatum None Monacrosporium eudermatum None Geniculifera paucispora None Geniculifera paucispora None Monacrosporium cionopagum None Monacrosporium cionopagum None Nematoctonus concurrens None Nematoctonus concurrens

Pasteurized Pasteurized

2

3

4

5

Raw Raw Pasteurized Pasteurized

Raw Raw

Pasteurized Pasteurized Raw Raw Pasteurized Pasteurized

Raw Raw

Pasteurized Pasteurized Raw Raw

% H. hepialus recovered b MPN c 95% CL 100 6 6 ad 94 6 4 ab

0 121

0 37–399

79 6 6 b 66 6 10 b

27 121

8–90 37–399

100 6 4 a 94 6 4 a

0 11

0 3–37

91 6 2 a 74 6 4 b

0 5

0 2–16

100 6 5 a 104 6 6 a

0 42

0 13–138

79 6 2 b 50 6 4 c

0 11

0 3–37

100 6 8 a 83 6 9 ab

0 32

0 10–106

71 6 4 b 67 6 5 b

0 32

0 10–106

100 6 7 a 84 6 5 a 78 6 6 ab 75 6 5 b

0 321

0 97–1059

0 321

0 97–1059

a Inoculum consisted of 40 infective juveniles of Steinernema glaseri colonized by the respective fungus per 15 cm3 soil. Background fungi in raw soil (and their average MPNs/g soil) were A. oligospora (24), A. musiformis (1), and G. paucispora (2). b Compared to control in pasteurized soil without fungal inoculum, which therefore equals 100%. Approximately 200 infective juveniles were added to each vial; actual number of infective juveniles recovered per wax moth larva in pasteurized control in trials 1–5: 178, 137, 179, 180, and 191, respectively. c Only the MPN of the treatment fungus is shown. d Means 6 SE followed by the same letter within a column for each fungus species are not significantly different (P . 0.05; Tukey).

FIG. 1. Recovery of Heterorhabditis hepialus infective juveniles (% of control in pasteurized soil 6 SE) from wax moth larva after 4 days exposure in raw or pasteurized soil in two trials. The soil was inoculated with five species of nematode-trapping fungi in the form of 40 infective juveniles of Steinernema glaseri colonized by the respective fungus per 15 cm3 of soil. Approximately 200 infective juveniles were added to each vial. Actual number of IJs recovered per wax moth larva in pasteurized control: 132 in trial 1 (A) and 124 in trial 2 (B). Note that recovery in the control is 100% by definition.

by the fungi. Although N. concurrens did not colonize H. hepialus on agar, the nematodes never escaped from its traps, and we assume that similar interactions occurred in soil. Perhaps the J2 cuticle, which is typically retained by heterorhabditid IJs, protected H. hepialus IJs from infection by N. concurrens (Timper and Kaya, 1989). The suppressiveness of most fungus treatments tended to be greater in raw soil than in pasteurized soil. This may be due to several factors. Most obviously, the presence of background nematophagous fungi, especially A. oligospora, in the raw soil may have added to the suppression. Other background antagonists of nematodes such as viruses, bacteria, protozoa, mites, and insects (Stirling, 1991; Kaya and Koppenho¨fer, 1996) also may have contributed to the stronger suppression in raw soil. None of these was observed during the

TRAPPING FUNGI/ENTOMOPATHOGENIC NEMATODE

TABLE 4 Experiment 3: Most Probable Number (MPN) of Fungus Propagules per Gram of Soil after Inoculation with Five Different Species of Nematode-Trapping Fungi and 14 Days Incubation at 20°C in Raw or Pasteurized Soil a Trial 1 Soil

Fungus

Pasteur- N. concurrens ized A. oligospora M. eudermatum M. cionopagum G. paucispora Raw N. concurrens A. oligospora M. eudermatum M. cionopagum G. paucispora

MPN b 3949 42 23 27 42 864 64 11 10 9

Trial 2

95% CL

MPN b

95% CL

1197–13,031 13–138 7–76 8–90 13–138 262–2851 19–212 3–37 3–33 3–29

2270 54 17 19 0 864 57 11 32 0

688–7493 16–179 5–55 6–64 0 262–2851 17–187 3–37 10–106 0

a Inoculum consisted of 40 infective juveniles of Steinernema glaseri colonized by the respective fungus per 15 cm3 of soil. Background fungi in raw soil (and their MPNs/g soil) were A. oligospora (22), A. musiformis (2), and M. eudermatum (2) in Trial 1 and A. oligospora (21), G. paucispora (1), and A. musiformis (1) in Trial 2. b Only the MPN of the treatment fungus is shown.

study but our procedures were not intended for detection of these other agents. Another possible reason for the stronger suppression in raw than in pasteurized soil may be that pasteurization adversely affected the introduced fungi. Autoclaving is known to change soil properties (Skipper and Westerman, 1973; Lotrario et al., 1995), and these changes may affect the ability of microorganisms to grow and compete in the soil matrix (Salonius et al., 1970). At the lower pasteurization temperature, however, changes in soil properties should be much less dramatic. The possible destruction of trap-inducing factors (Dijksterhuis et al., 1994 and references therein) by heat treatment (Pramer and Stoll, 1959) should have been compensated for in the present study by the addition of H. schachtii J2 to the soil. After determining the suppressive potential of the selected nematode-trapping fungi, our second objective was to relate the laboratory observations to the field using most probable number estimates. The fungus propagule numbers per gram soil observed in this study were similar to those observed in soil samples collected at BMR (Jaffee et al., 1996) for three of the fungi. The largest number in our laboratory experiments vs the largest number observed at BMR were 121 vs 686 for A. oligospora, 42 vs 32 for G. paucisporum, and 23 vs 15 for M. eudermatum. This suggests that the suppression observed in the laboratory is relevant to the field at BMR for these three fungi. The two other fungi, M. cionopagum and especially N. concurrens, were detected only sporadically in the field samples and at much lower densities than in the laboratory experiments.

251

It is difficult to judge the importance of individual fungi in the system at BMR. First, in our experiments the suppressiveness of the five fungi was similar. Second, the dilution method has an inherent low order of precision (Adams and Welham, 1995). Third and most important, we lack knowledge about the relationship between fungus densities and suppressiveness; thus, G. paucispora was not detected on the plates in the second trial of the second soil experiment, yet the suppression of nematode penetration was similar to that in the first trial. The suppression of H. hepialus increased as the time of IJ exposure to the fungus-inoculated soil increased (2 vs 4 days before addition of wax moth larvae to soil). Entomopathogenic nematodes with active foraging strategies (cruisers) (Campbell and Gaugler, 1993) continue to move through the soil in search of potential hosts, and IJs of H. hepialus seem to fall into this category (Koppenho¨fer, unpublished). This motility obviously increases the chances for contact between nematode and fungus over time (Timper et al., 1991). Jansson (1982) observed that suppression of Panagrellus redivivus in soil by A. oligospora increased until approximately 14 days exposure of the nematode. Similarly, H. hepialus suppression may have increased if the wax moth larvae had been added later. We do not know the optimal exposure time of the nematodes to the fungi nor do we know how realistic any exposure time would be. Our observations indicate that nematode-trapping fungi may influence the plant–insect–nematode system at BMR. Further investigations will have to clarify the relationship between fungal population densities in our bioassay and those in the field. We also need to understand the effects of fungal saprophytism and interactions between species on suppression of H. hepialus and detection of fungi. ACKNOWLEDGMENTS The authors appreciate the technical assistance of Lori Timm and Daralyn Pearson. A.M.K. was supported in part by the German Research Association (DFG). REFERENCES Adams, M. J., and Welham, S. J. 1995. Use of most probable number technique to quantify soil-borne plant pathogens. Ann. Appl. Biol. 126, 181–196. Alexander, M. 1982. Most probable number method for microbial populations. In ‘‘Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties—Agronomy Monograph No. 9,’’ 2nd ed., pp. 815–820. ASA-SSSA, Madison, WI. Amarasinghe, L. D., Hominick, W. M., Briscoe, B. R., and Reid, A. P. 1994. Occurrence and distribution of entomopathogenic nematodes in Sri Lanka. J. Helminthol. 68, 277–286. Barron, G. L. 1977. ‘‘The Nematode-Destroying Fungi.’’ Canadian Biological Publications, Guelph, Ontario, Canada. Barron, G. L., and Dierkes, Y. 1977. Nematophagous fungi: Hohenbue-

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