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E L S E V I E R Applied Soil Ecology 4 (1996) 221-230

Applied Soil Ecology

Coexistence of two steinernematid nematode species (Rhabditida: Steinernematidae) in the presence of two host species

Albrecht M. Koppenhi3fer *, Harry K. Kaya Department ofNematology, University of California, Davis, CA 95616, USA

Accepted 15 March 1996

Abstract

Interactions between entomopathogenic nematode species with different foraging strategies were examined in the presence of two host species in soil. Based on differences in insect behavior and nematode pathogenicity, we hypothesized that black cutworm, Agrotis ipsilon (Hufnagel), larvae would be more likely to serve as hosts for Steinernema carpocapsae or Steinernema riobravis, whereas masked chafer grabs, Cyclocephala hirta LeConte, would be more likely to serve as hosts for Steinernema glaseri. In the laboratory, the highest mortality of and nematode penetration in A. ipsilon were observed for S. carpocapsae followed by S. riobravis and S. glaseri. In C. hirta, substantial mortality and nematode penetration were only observed for S. glaseri. After combined applications of two nematode species, S. carpocapsae dominated over S. glaseri in A. ipsilon, whereas S. glaseri and S. riobravis shared the host resources. In C. hirta, S. glaseri outcompeted each of the other species. In the greenhouse, containers with turfgrass were inoculated with S. glaseri, S. carpocapsae, or S. riobravis, or combinations of S. glaseri with either S. carpocapsae or S. riobravis. Four days later, each container received seven A. ipsilon and nine C. hirta larvae, and new insects were added at 30-day intervals. The densities of infective juvenile nematodes were monitored over 150 days by taking soil samples from each container. In the single species treatments, numbers of S. carpocapsae and S. riobravis increased after 30 days, decreased thereafter and remained low. Steinernema glaseri numbers fluctuated between low and high densities. In the combination of S. glaseri and S. carpocapsae, both species were depressed compared with the single species treatment. In the combination of S. glaseri and S. riobravis, both species coexisted and showed parallel fluctuations but S. glaseri dominated numerically. Our observations indicate that two entomopathogenic nematode species may successfully coexist in an area by having different foraging strategies that separate nematode species spatially, exhibiting host specificity, and occupying a different niche. In the field, patchy distribution and alternate hosts may also support nematode coexistence.

Keywords: Steinernema; Agrotis ipsilon; Cyclocephala hirta; Soil ecology; Foraging strategy; Biological control

1. Introduction

Entomopathogenic nematodes (Rhabditida: Stein- ernematidae and Heterorhabdit idae) are obligate par- asites of insects (Poinar, 1990) and occur in soils

* Corresponding author.

throughout the world (Kaya, 1990). The infective juveni les (IJs) of these nematodes persist in the soil until they find and enter a suitable host. The IJs kill their host with the help of species-specific symbiotic bacteria (Boemare et al., 1993). The nematode para- sitic stages feed on the propagating bacteria and host tissue and develop and reproduce within the host

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222 A.M. Koppenh6fer, H.K. Kaya / Applied Soil Ecology 4 (1996) 221-230

cadaver. As resources in the host cadaver are de- pleted, new Us are produced and emerge into the soil environment (Poinar, 1990).

These nematodes are used as biological insecti- cides against many soil-dwelling insect pests (Kaya and Gaugler, 1993) and are commercially available. Because the focus of research involving ento- mopathogenic nematodes has been primarily on ap- plied aspects, knowledge about many aspects of their ecology in soil is still limited. For example, the observation that two species of entomopathogenic nematodes may occur sympatrically in the field (Amarasinghe et al., 1994; Stuart and Gaugler, 1994; Campbell et al., 1995) suggests that interspecific competition may occur. Laboratory studies demon- strated that antagonism occurred between nematode species when they co-infected a host individual (Alatorre-Rosas and Kaya, 1990, 1991). Although two steinernematid species can coexist in the same host (Kondo, 1989; KoppenhiSfer et al., 1995), even- tually one species will dominate. Outside the host, differences in foraging strategy between nematode species may allow different species to coexist.

The foraging strategies of the IJs of these nema- todes range from sit-and-wait (ambusher) to widely foraging (cruiser) (Campbell and Gaugler, 1993). Steinernema carpocapsae (Weiser) is a typical am- busher that does not disperse far and accumulates near the soil surface (Moyle and Kaya, 1981). Its nictating behavior (Kondo and Ishibashi, 1986) and unresponsiveness to host cues (Lewis et al., 1992) indicate an adaptation to infect mobile hosts on the soil surface (Campbell and Gaugler, 1993). Stein- ernema glaseri (Steiner), on the other hand, is a cruiser that disperses actively and is distributed more uniformly in the soil (Georgis and Poinar, 1983; Schroeder and Beavers, 1987), responds to host cues (Lewis et al., 1992), and is adapted to infect seden- tary hosts (Campbell and Gaugler, 1993). Stein- ernema riobravis Cabanillas, Poinar and Raulston appears to share characteristics of both ambush (nic- tation) and cruise foragers (high motility) (Cabanillas et al., 1994; Grewal et al., 1994a).

Another factor that may allow different nematode species to coexist is their differential pathogenicity to various insect species. Thus, S. carpocapsae is more pathogenic than S. glaseri to many lepi- dopteran species (Morris et al., 1990), whereas S.

glaseri is more pathogenic than S. carpocapsae to scarabaeid larvae (Klein, 1990; Wang et al., 1994).

Our hypothesis was that two species of ento- mopathogenic nematodes with different foraging strategies will coexist if provided with two insect host species that differ in behavior and susceptibility to the two nematode species. We used the following system to test this hypothesis. Both S. carpocapsae and S. glaseri have been isolated from turfgrass and use different foraging strategies. Two turfgrass in- sects that are commonly found in the same locality are the larvae of the black cutworm (BCW), Agrotis ipsilon (Hufnagel; Lepidoptera: Noctuidae) and white grubs (WG), the larvae of the masked chafer, Cyclo- cephala hirta LeConte (Coleoptera: Scarabaeidae) (Tashiro, 1987). These two species of insects differ in their location in the soil and their behavior. BCW are mobile and feed on foliage of grasses and other agricultural crops at the soil surface (Rings and Musick, 1976) and are therefore more likely to come into contact with ambush foragers like S. carpocap- sae. WG feed on grass roots in the soil and a cruise forager such as S. glaseri is more likely to contact this species. The interactions between these nema- todes and their hosts in laboratory and greenhouse trials were determined. After comparing nematode pathogenicity to and the penetration efficiency into these insects, we observed nematode population dy- namics in the absence and presence of another nema- tode species when they were provided periodically with BCW and WG as hosts.

2. Materials and methods

2.1. Nematodes and insects

Steinernema carpocapsae all strain, S. glaseri NC strain, and S. riobravis Texas strain were cul- tured in last instar larvae of the greater wax moth, Galleria mellonella (L.), and the emerging IJs were harvested from White traps and stored in sterilized distilled water at 10°C (Woodring and Kaya, 1988) for 5-21 days before use. BCW were obtained from Dow Elanco (Indianapolis, IN) as eggs and reared to fourth and fifth instars on a casein/wheat germ diet for the experiments. Third instar WG were collected at Mace Meadows golf course (Pioneer, CA). They

A.M. Koppenh6fer, H.K. Kaya /Applied Soil Ecology 4 (1996) 221-230 223

were kept in a mixture of organic compost and a sandy loam soil with perennial rye grass seeds at 10°C. One week before use in experiments, WG were placed individually in 30-ml plastic containers filled with soil and grass seeds at 23 ___ 2 °C and only actively feeding larvae were selected for use. Throughout the study, fourth to fifth instar BCW and third instar WG were used.

2.2. Soil

Throughout the study, a mixture of sandy loam soil amended with 2% ( w / w ) peat moss (final com- position: 84% sand, 12% silt, 4% clay; 0.9% organic matter; pH 7.6). The mixture was autoclaved (121°C, 2 h) at least 2 weeks before use. The soil was prepared at 13% ( w / w ) moisture ( - 6 kPa water potential).

2.3. Nematode pathogenicity

The pathogenicity of the nematodes was tested at 23 + 2 °C in 30-ml plastic containers filled with 20 cm 3 of soil to which seeds of perennial rye grass were added. Then, 0.1 ml of sterilized distilled water was added to the cups containing no IJs, or 100 IJs of S. glaseri, 100 IJs of S. carpocapsae, 100 IJs of S. riobravis, 50 IJs each of S. glaseri and S. car- pocapsae, or 50 IJs each of S. glaseri and S. riobravis. After 24 h, half of the cups received one BCW, the other half one WG. Each combination of nematode treatment and insect species had 12 repli- cates. For 10 days, the cups were checked daily to assess insect mortality. Cadavers from the single nematode species treatments were dissected to verify nematode penetration, while cadavers from the mixed treatments were set individually on small White traps to observe IJ production. The IJs of different species were easily separated by their lengths with S. glaseri being about twice as long as S. carpocapsae and S. riobravis (Poinar, 1990; Cabanillas et al., 1994).

2.4. Penetration efficiency

To study the penetration efficiency of these nema- todes into the two insect pests, 500-ml styrofoam cups were filled to a height of 10 cm with the soil mixture (400 cm 3) and sown with perennial rye grass

seeds. The cups were kept at 23 + 2 °C and watered with distilled water as needed. Three weeks after planting, the grass was cut to 3 cm in height. On the following day, 5 ml of distilled water was added to the cups containing either no IJs, 1500 IJs of S. glaseri (corresponding to an application rate of 2.5 × 109 IJs ha- l ) , 1500 IJs of S. riobravis, 3000 IJs of S. carpocapsae, 750 IJs of S. glaseri and 1500 IJs of S. carpocapsae, or 750 IJs each of S. glaseri and S. riobravis. Each treatment had 12 replicates. Steinernema carpocapsae was applied at double the rate of the other species because preliminary studies had shown a higher post-application loss in this species (see also Curran, 1993). The cups were then watered with 10 ml of distilled water to wash the IJs into the soil and covered with petri dish lids with five ventilation holes.

After 3 days, each cup received one BCW and one WG. The BCW was placed on the grass surface whereas the WG was introduced through a hole in the side of the cup bored with a cork borer at 7 cm depth. Four days after introduction, the insects were recovered and rinsed in tap water. Dead insects were dissected in a 0.5% pepsin solution and incubated for 2 h at 37°C to digest the insect tissues (Mauleon et al., 1993) before counting the number of nematodes that had penetrated into the insects. Insects that were still alive after the first exposure period were incu- bated individually in 30-ml plastic cups filled with soil (WG) or in petri dishes (100 mm × 15 mm) on moist filter paper with some casein/wheat germ diet (BCW). After 3 days, dead insects were processed as described above.

2.5. Coexistence on BCW and WG in greenhouse

We wanted to determine whether different forag- ing behavior would allow two nematode species to coexist in the presence of two insect host species. Plastic containers (15 liters) were filled with 11 liters of soil mixture (soil height 15 cm) and sown with perennial rye grass seeds. The grass was cut to approximately 3 cm in height at 4-week intervals. Five weeks after sowing, 50 ml of distilled water were applied to each container containing no nema- todes (control), or S. glaseri, S. riobravis, S. car- pocapsae, S. glaseri and S. carpocapsae, or S. glaseri and S. riobravis (Table 1). Before being

224 A.M. KoppenhOfer, H.K. Kaya / Applied Soil Ecology 4 (1996) 221-230

Table 1 Summary of experimental design for coexistence of two ento- mopathogenic nematode species in soil under greenhouse condi- tions

IJ inoculum Insects Treatment ( × 1000) added a code b

- + -

4 4 Sc - C - 44 Sc + C + 22 Sg - G - 22 Sg + G + 22 Sr - R - 22 Sr + R + 22 Sc + 11 Sg + CG + l l S r + l l Sg + RG+

Sc, S. carpocapsae; Sg, S. glaseri; Sr, S. riobravis. a Seven fourth to fifth instar black cutworms and nine third instar Cyclocephala hirta were added to each container. b Treatments with two nematode species had four replicates, all others three replicates.

efficiency experiment. To maximize the nematode extraction efficiency, the samples were baited a sec- ond time for 3 days and processed as above. In the combination of S. glaseri and S. carpocapsae, species were determined by the shape of the tail and absence/presence of a mucron (Poinar, 1978, 1986). Because adult S. glaseri and S. riobravis are very difficult to distinguish, the soil samples from the treatment combining these two species were baited for three 1.5-day periods. All wax moth larvae were dissected immediately after the conclusion of each baiting period. In this way, the two nematode species could be identified by the size of the IJs. Both baiting methods have approximately the same recov- ery efficiency (Koppenhtifer, unpublished observa- tions).

2.6. Statistical analysis

covered with a nylon screen, the containers were irrigated with 100 ml of water to wash the IJs into the soil. Four days later (day 0 of the experiment), to most containers nine WG and seven BCW were added as nematode hosts. Containers that did not receive insects had been inoculated with either one of the nematode species (Table 1) and were used to observe nematode persistence in the absence of BCW and WG. The WG were placed in the sampling holes which were then refilled with moist soil mixed with rye grass seed, whereas the BCW were set on the grass surface. Additional insects were added on days 30, 60, 90, and 120. The treatment combinations are listed in Table 1.

The nematode populations were estimated in soil samples taken on days 0, 30, 60, 90, 120, and 150. On each sampling day, five soil cores were randomly taken from each container to a depth of 10 cm with cork borers (2.25 cm diameter), consolidated, and divided into two 100 cm 3 soil samples. Between sampling each container, the borers were decontami- nated with hot water. The nematode population in the samples was estimated using a baiting method described in detail by KoppenhiSfer et al. (1996). Thus, the samples were baited with five wax moth larvae for 3 days at 23 + 2°C, the larvae were recov- ered, and the number of nematodes that penetrated was evaluated as described above for the penetration

The number of days until insects were killed by the nematodes in the 30-ml containers, excluding surviving insects, and the square root transformed numbers of nematodes of each species that had penetrated into insects were subjected to analysis of variance (ANOVA) and means separation by Tukey's test (Statistical Analysis Systems Institute Inc., 1988). In the coexistence experiment, the average numbers of nematodes recovered per 100 cm 3 soil in the two samples taken per replicate were logl0(x + 1) trans- formed and analyzed by species. The data from the first sampling day were analyzed separately with ANOVA, whereas the data from the remaining sam- pling days were analyzed with repeated measures ANOVA. Means were separated by Tukey's test. All numbers are reported as untransformed data and, where appropriate, standard errors of the means (SE) are given.

3. Results

3.1. Nematode pathogenicity

No mortality was observed in the control insects of either species. The mortality and the time required for insects to die from nematode infection (in paren- theses) were as follows. In BCW, S. carpocapsae alone, S. glaseri + S. carpocapsae, and S. glaseri

A.M. Koppenhiifer, H.K. Kaya / Applied Soil Ecology 4 (1996) 221-230 225

+ S. riobravis caused 100% mortality (2.4 _ 0.4 days, 2.6 + 0.3 days, and 6.6 +_ 0.8 days, respec- tively). Steinernema glaseri alone caused 83% mor- tality (6.1 + 0.8 days), and S. riobravis alone caused 75% mortality (3.7 + 0.4 days). The mean number of days until BCW died was significantly different among treatments ( F = 10.2; d.f. = 4,45; P < 0.001). Steinernema glaseri alone and S. glaseri + S. rio- bravis took significantly longer to kill BCW than S. carpocapsae alone and S. carpocapsae + S. rio- bravis. In WG, S. glaseri alone caused 83% mortal- ity (5.9-t-0.6 days), S. glaseri with S. riobravis caused 75% mortality ( 6 . 7 _ 0.7 days), S. glaseri with S. carpocapsae caused 50% mortality (5.8 + 0.7 days), S. carpocapsae alone caused 17% mortality (4.5 + 1.2 days), and S. riobravis caused no mortal- ity, No significant differences were observed in the mean number of days until WG died ( F = 0.62; d.f. = 3,23; P = 0.6).

With regard to the mixed nematode treatments, the following progeny type was observed. When exposed to S. glaseri with S. carpocapsae, eight BCW produced only S. carpocapsae progeny, three BCW produced S. carpocapsae and a few S. glaseri (20 -50 IJs), and one BCW produced only S. glaseri. When exposed to S. riobravis with S. glaseri, six BCW produced only S. glaseri, and six BCW only S. riobravis. In all treatments, WG produced only S. glaseri.

3.2. Penetration efficiency

No mortality was observed in the control insects of either species. In BCW, S. carpocapsae alone and S. carpocapsae + S. glaseri caused 100% mortality, S. glaseri + S. riobravis caused 83% mortality, S. riobravis alone caused 67% mortality, and S. glaseri alone caused 58% mortality. The number o f nema- todes of each species penetrated into BCW was significantly different among treatments ( F = 48.8; d.f. = 6,64; P < 0.001) with S. carpocapsae having the highest penetration rates (Table 2). In the combi- nation of S. carpocapsae with S. glaseri, one BCW contained only S. carpocapsae, and 11 BCW con- tained both S. carpocapsae and S, glaseri. In the combination of S. riobravis with S. glaseri, five BCW contained S. glaseri only, four BCW con-

Table 2 Mean (5: SE) numbers of nematodes recovered from black cut- worms (BCW) and white grubs (WG) exposed for 96 h to different species of nematodes and their combinations

Treatment Nematode No. nematodes No. nematodes species per WG y per BCW

Control - 0 z 0 z

Sc (3000) x .Sc 66 z (!) 615.0+45.2 a (12) Sg (1500) Sg 200.7 +74.2 a (7) 14.8 :[:7.0 c (7) Sr (1500) Sr 0 z 57.6 :t: 27.4 c (8) Sg (750) Sg 136.8 + 17.6 a (6) 23.2 5:4.9 c (12) +Sc (1500)

Sc 3.3 ±0.6 b (6) 271.0+17,1 b(12) Sg (750) Sg 55.6+ 17.0 a (5) 24.3 + 15.6 c (10) + Sr (750)

Sr 9.8 5:3.0 b (5) 72.7 5:25.7 c (10)

Cups with grass were inoculated with infective juveniles (IJs) of S. carpocapsae (Sc), S. glaseri (Sg), S. riobravis (St), or combi- nations of Sc+Sg or Sr+Sg. After 3 days, one BCW and one WG were added to each cup. Means followed by the same letter within columns are not signifi- cantly different (P < 0.05). x The number of IJs per nematode species added to each cup is given in parentheses. Y Number of nematode-infected insects is given in parentheses. z Data were excluded from analysis.

tained S. riobravis only, and one BCW contained both nematode species.

In WG, S. glaseri alone caused 58% mortality, S. glaseri with S. carpocapsae caused 50% mortality, S. glaseri with S. riobravis caused 42% mortality, S. carpocapsae alone caused 8%, and S. riobravis alone caused 0% mortality. The number of nema- todes of each species that penetrated into WG was significantly different among the treatments ( F = 6.2; d.f. = 4,24; P < 0.01), and S. glaseri had the high- est penetration rates (Table 2). In the combinations S. glaseri + S. carpocapsae or S. glaseri + S. rio- bravis, all nematode-infected WG contained both nematode species.

3.3. Coexistence on BCW and WG in greenhouse

The average air temperature during the experi- ment was 24°C (range 12-31 °C); soil temperature averaged 24°C (range 13.5-29°C) at 5 cm depth. For each 30-day period, the average soil temperature did not deviate more than 0.5 °C from the overall average.

226 A.M. Koppenh6fer, H.K. Kaya / Applied Soil Ecology 4 (1996) 221-230

No nematodes were recovered from the control and these data were excluded from the analysis. As expected with the different inoculum levels, at day 0 only about half as many nematodes were recovered from the mixed treatments as from the single nema- tode species treatments. The differences were signifi- cant for S. glaseri ( F = 12.99; d . f .= 3,10; P < 0.001) but not for S. carpocapsae ( F = 2.59; d.f. = 2,7; P = 0.14) or S. riobravis ( F = 1.35; d.f. = 2,7; P = 0.32).

Steinernema carpocapsae became extinct, i.e. could no longer be recovered within 30 days in the containers to which no BCW and WG were added (C - ; see Table 1 for abbreviations) (Fig. I(A)). In C + , S. carpocapsae was recovered at similar num- bers at 0 and 30 days (Fig. I(D)). Thereafter, its numbers gradually declined. At 120 and 150 days, no nematodes were recovered from one and two containers, respectively. In CG + , S. carpocapsae

numbers declined gradually after 0 days (Fig. I(D)). Higher numbers at 120 days were due to high recov- ery from one container. At 150 days, no S. car- pocapsae were recovered from two containers, while only a few were recovered in the other two contain- ers. Because there was a great deal of variation, a significant difference between C + and CG + ( P < 0.05) was detected only at 30 days. The repeated measures ANOVA showed a significant interaction between time and treatment ( F = 9.37; d.f. = 8; P < 0.01).

Steinernema glaseri numbers declined gradually in the absence of BCW and WG (G - ) and no more nematodes were recovered at 150 days (Fig. I(B)). In G + , S. glaseri numbers declined until 60 days, but fluctuated thereafter around intermediate densi- ties (Fig. I(E)). In CG + , S. glaseri numbers had increased drastically at 30 days, dropped below the 0 day level at 60 days, decreased thereafter at the same

300

100

0

400

300

200

100

0 60

S. carpocapsae S. glaseri

D

S. riobravis

, \\

0 60 120

o

oo

to ._=

.Q

E

l o

.= 8

6 Z

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120

F

0 60 120

Days after 1st host introduction

--l- O

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Fig. 1. Nematodes recovered from containers with 11 liters of sterilized soil and turfgrass. The containers were inoculated with S. carpocapsae alone, S. glaseri alone, S. riobravis alone, S. glaseri with S. carpocapsae, or S. glaseri with S. riobravis 4 days prior to the addition of insect hosts. At 30-day intervals, to each treatment container nine white grubs (WG) and seven black cutworm larvae (BCW) were added. Each column shows data from the various treatments for one nematode species as indicated by column heading. ((3) in absence of competitor; ( 0 ) combination of S. glaseri and S. carpocapsae (D, E); ( • ) combination of S. glaseri and S. riobravis (E).

A.M. KoppenhiJfer, H.K. Kaya / Applied Soil Ecology 4 (1996) 221-230 227

rate as in G - , and became almost extinct at 150 days (Fig. I(E)). In RG + , numbers of S. glaseri recovered fluctuated at lower levels after 60 days, with the exception of one container at 90 days which had extremely high numbers (Fig. I(E)). In this treatment, S. glaseri was recovered from each con- tainer at 150 days. Differences between treatments in numbers of S. glaseri recovered were significant only at 150 days ( F - - 12.7; d.f. = 3,10; P < 0.001) when numbers were significantly lower in G - and CG + than in G + and RG + ( P < 0.05). The re- peated measures ANOVA detected no significant times × treatments interaction ( F = 2.0; d.f. = 12; P = 0 . 0 8 6 ) .

Steinernema riobravis numbers declined gradu- ally in the absence of BCW and WG ( R - ) and no more nematodes were recovered at 150 days (Fig. I(C)). In R + , numbers of S. riobravis recovered had increased at 30 days, but fluctuated thereafter at low densities (Fig. I(F)). In RG + , S. riobravis fluctuated at low densities with the exception of one container which had high numbers at 90 days (Fig. I(F)). In this treatment, S. riobravis was recovered from each container at 150 days. Differences be- tween treatments were significant only at 150 days ( F = 12.7; d . f .=3,10; P < 0 . 0 0 1 ) when S. rio- bravis numbers were significantly lower ( F = 8.2; d.f. = 2,7; P < 0.05) in R - than in the other treat- ments. The repeated measures ANOVA detected no significant times X treatments interaction (F = 0.54; d.f. = 8; P = 0.8).

4. Discussion

Our observations on the susceptibility of BCW and WG to entomopathogenic nematodes concur with reports by other researchers. Late instar BCW are highly susceptible to S. carpocapsae on filter paper (Capineira et al., 1988; Morris et al., 1990) with more variable results in soil (Capineira et al., 1988; Kaya et al., 1994). On filter paper, Morris et al. (1990) observed a much lower susceptibility of BCW to S. glaseri than to S. carpocapsae, which corre- sponds with our observations in soil. Similarly, S. riobravis appears to be less pathogenic to BCW and this was also observed in other studies conducted under very similar conditions (Baur, unpublished

results). Although the temperature in these studies was not much lower than the optimal temperature for S. riobravis, this may have affected its efficiency (Grewal et al., 1994b). The relatively low suscepti- bility of WG to entomopathogenic nematodes may be explained by aggressive and evasive behaviors (Gaugler et al., 1994) and enhanced immune re- sponses (Wang et al., 1994) by scarabaeid larvae in response to entomopathogenic nematodes. The dif- ferent degrees of susceptibility of both BCW and WG to the three nematode species are also reflected by the nematode penetration efficiencies.

The laboratory observations confirmed our basic assumptions for the greenhouse study. As expected, WG were successfully used as a resource only by S. glaseri. In BCW, S. carpocapsae dominated but did not exclude S. glaseri completely, and S. riobravis and S. glaseri shared the resource. The relatively short nematode persistence in the absence of BCW and WG, especially of S. carpocapsae, allows us to assume that nematode recycling took place even if nematode numbers did not increase significantly be- tween 30-day periods. In sterilized soil, S. carpocap- sae and S. glaseri persist considerably longer (Kung et al., 1990; KoppenhiSfer and Kaya, 1996): In our study, the lower nematode persistence may be partly due to more variable conditions in the greenhouse, including temperature and soil moisture, especially in the upper soil layer. Although nematode antago- nists .(Kaya and Koppenh6fer, 1996) were not di- rectly observed, the experimental conditions did not exclude the establishment of such organisms.

The number of S. carpocapsae recovered, both in the single species treatment and in the combination with S. glaseri, followed a similar trend as in Kop- penhBfer and Kaya (1996), where wax moth larvae were introduced at 0 and 10 cm soil depth. Slight differences are explained by the shorter nematode persistence under greenhouse conditions. Stein- ernema carpocapsae obviously cannot maintain high population levels over long periods if hosts are added at 30-day sampling intervals, which may be due to its low persistence. In natural systems, this surface adapted nematode may rely on a variety of surface- dwelling insect hosts which as a group are continu- ously available over long periods (Campbell et al., 1995).

When provided with wax moth larvae as hosts, S.

228 A.M. Koppenh6fer, H.K. Kaya /Applied Soil Ecology 4 (1996) 221-230

glaseri populations tended to decline over time, in both the presence and the absence of S. carpocapsae (KoppenhiSfer and Kaya, 1996); this decline was probably due to the high susceptibility of this nema- tode to intraspecific competition in the host (Kop- penhiSfer and Kaya, 1995). In the present study, the intraspecific competition may have been ameliorated by lower nematode persistence, the presence of two potential host species, and the lower susceptibility to nematode infection of both host species compared to wax moth larvae; this may have allowed S. glaseri populations to recover periodically and also explain the fluctuations in all treatments of numbers of S. glaseri recovered.

Steinernema riobravis showed an increase in two out of three containers during the first 30 days but fluctuated at low densities thereafter. Considering the relatively long persistence in the absence of BCW and WG, we must assume that S. riobravis did not recycle very well. As discussed above, this may, in part, be temperature related.

In the combination of S. carpocapsae and S. glaseri, both species were suppressed compared with the single species treatment. Steinernema glaseri showed a strong increase at 30 days while its num- bers dropped in the single species treatment. The lower starting inoculum in the mixed treatments may have reduced intraspecific competition. Because S. glaseri became almost extinct at 150 days, we as- sume that it did not recycle efficiently in WG. Although S. glaseri was outcompeted in BCW by S. carpocapsae in the laboratory experiments, in the greenhouse the longer persistence of S. glaseri may have enabled it to compete successfully with S. carpocapsae for BCW hosts.

In the combination of Steinernema riobravis and S. glaseri, both species followed parallel trends and coexisted in all treatment replicates. It is likely that they shared the BCW resource as in the laboratory experiments. Considering that S. riobravis can pro- duce approximately ten-fold higher progeny numbers from wax moth larvae than S. glaseri (Grewal et al., 1994b), S. glaseri clearly was the dominating species. At higher temperatures, the competitiveness of S. riobravis may be better. The fluctuation of S. glaseri numbers may be due to occasional recycling in WG.

In the present study, WG may have been very

resistant even to infection by S. glaseri which in- creased competition for BCW. In fields with natural populations of scarabaeids, some entomopathogenic nematode species have been observed to persist for many years (Campbell et al., 1995). However, even at very high host densities, the nematode distribution appeared to be patchy with sporadic hot spots (Campbell, unpublished observations). Epizootics have been observed (Akhurst et al., 1992; Kaya, unpublished observations), indicating that scarabaeid larvae are susceptible to nematode infection only under certain circumstances. Persistence of nematode populations over long periods may therefore occur only on a larger scale than available in the present study.

Our observations show that two ento- mopathogenic nematode species may successfully coexist in an area. Mechanisms allowing for such coexistence may be different foraging strategies that separate nematodes species spatially, different niches, or patchy distribution. Differential host susceptibili- ties may contribute to nematode coexistence. How- ever, availability of alternate hosts will also allow nematodes to overcome periods in which more com- mon hosts are not available (Burlando et al., 1993). Further research should consider competitive interac- tions between heterorhabditid species as well as ant- agonistic effects between nematode species outside the host (e.g. possible competition for space in the soil environment).

Acknowledgements

We thank M.E. Baur, J.F. Campbell, and J. Takeyasu for critically reading the manuscript, and T.M. Burlando, L.E. Nishimura, and J. Fisher for technical assistance. AMK was supported by the German Research Association (DFG).

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