Interaction between Endemic and Introduced Entomopathogenic Nematodes in Conventional-Till and No-Till Corn

Biological Control 22, 235–245 (2001) doi:10.1006/bcon.2001.0978, available online at http://www.idealibrary.com on

Interaction between Endemic and Introduced Entomopathogenic Nematodes in Conventional-Till and No-Till Corn Leah C. Millar 1 and Mary E. Barbercheck Entomology Department, North Carolina State University, Raleigh, North Carolina 27607 Received October 27, 2000; accepted June 29, 2001

INTRODUCTION We used entomopathogenic nematodes as a model to address the issue of environmental impact of introduced biological control agents in the soil. The study was conducted during three field seasons (1997, 1998, and 1999) in no-till and conventional-till corn near Goldsboro, North Carolina. The main objective was to evaluate the interaction of two endemic nematodes, Steinernema carpocapsae and Heterorhabditis bacteriophora, and an introduced exotic nematode, Steinernema riobrave (Texas). Two baiting methods with Galleria mellonella were used to evaluate the nematodes with regard to infected insects and nematode persistence when alone or in cohabitation in the field. We also examined the effects of soil depth on the nematodes’ interactions, infectivity, and persistence. The results of the two baiting methods generally agreed with each other. The detection of H. bacteriophora was significantly suppressed in the presence of S. riobrave and slightly more so in conventional-till than in no-till. However, this endemic nematode was not completely displaced 1 and 2 years after the introduction of S. riobrave. Detection of S. carpocapsae and S. riobrave was not affected by the presence of each other, and detection of S. riobrave was not affected by the presence of H. bacteriophora. H. bacteriophora had the strongest tendency to be detected deeper in the soil profile, followed by S. riobrave and then S. carpocapsae. The nematodes’ differences in environmental tolerances, differences in tendencies to disperse deeper in the soil profile, and patchy distributions may help explain their coexistence. © 2001 Academic Press Key Words: Steinernema riobrave; Steinernema carpocapsae; Heterorhabditis bacteriophora; entomopathogenic nematode; biological control; exotic organism; nontarget risk.

1 To whom correspondence should be addressed. Fax: (919) 5131995. E-mail: [email protected].

Biological control is one of the most important tools in integrated pest management and has been labeled the foremost alternative to chemical pesticides (Greathead, 1986; Wratten, 1987). Compared to chemical pesticides, biological control agents have generally been considered environmentally safe and risk-free (Hokkanen and Pimental, 1989; Hoy, 1992). However, there has been a growing debate about the potential nontarget effects of introduced biological control agents (Howarth, 1983, 1991; Simberloff and Stiling, 1996a,b; Lockwood, 1993; Malakoff, 1999). This debate is hampered by the fact that data to support the assumption of lack of environmental impacts are not regularly collected (Howarth, 1991). The use of entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae as biological control agents inundatively applied against soil insect pests is rapidly expanding. They have a relatively broad host range (Gaugler, 1988), are safe for vertebrates and plants (Bathon, 1996), are easily applied with standard spray equipment (Georgis, 1990), and are compatible with many chemical pesticides (Rovesti and Deseo¨, 1990). In contrast to other insect pathogens, entomopathogenic nematodes are exempt from registration in the United States by the Environmental Protection Agency (Gorsuch, 1982). However, Ehlers and Hokkanen (1996) recommend that the release of exotic entomopathogenic nematodes should be regulated to reduce potential adverse effects on endemic nematode species and to reduce other environmental risks. Several countries, such as the United Kingdom, restrict importations of exotic nematodes (Kaya and Gaugler, 1993). These nematodes have a natural worldwide distribution (Hominick et al., 1996). Therefore, there is a possibility for displacement of native nematodes by introduced nematodes applied inundatively as biological control agents. One possible outcome of this could be disrupted natural control of soil insect pests. Exotic organisms may negatively affect native natural ene-

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mies (Bennett, 1993), and it should not be assumed that entomopathogenic nematodes are exempt from this possibility. For example, introduced steinernematid nematodes applied in a field trial to control southern corn rootworm in peanuts reduced detection of a naturally occurring population of Heterorhabditis bacteriophora Poinar (Lewiston strain) (M. E. Barbercheck, unpublished data). Here we report the results of studies designed to evaluate the interaction of two endemic nematode species and an inundatively applied introduced nematode species in a corn agroecosystem in North Carolina. MATERIALS AND METHODS

The study was conducted during three field seasons (1997, 1998, 1999) in no-till (NT) and conventional-till (CT) corn plots (1997 and 1998: Zea mays L. cv. Dekalb 714; 1999: Z. mays cv. Dekalb 580) at the Center for Environmental Farming Systems (CEFS) near Goldsboro, North Carolina. The 36.6 ⫻ 73.2-m plots are in a corn–soybean rotation. The no-till plots receive a wheat cover crop during the winter and have been under a no-till regime since 1995. The conventional-till plots receive no winter cover crop and are chisel plowed and disked in the fall and disked in the spring. Portions of the research site used for this study received fertilizer and herbicides but did not receive insecticides. The soil type at this center has been classified as predominantly Wickham fine sandy loam. The following abiotic soil factors were monitored during the field seasons: texture, temperature, organic matter, pH, moisture, and water potential. Steinernema carpocapsae (Weiser) and H. bacteriophora are native to the site. Dr. Patricia Stock, University of California at Davis, confirmed the species identification of these nematodes via morphological and molecular analysis. Steinernema riobrave Cabanillas, Poinar, and Raulston served as the introduced nematode. This nematode originated from the Lower Rio Grande Valley of Texas (Cabanillas et al., 1994) and, therefore, is nonnative to North Carolina. We examined the outcome of the interaction of the endemic and introduced nematodes based on insect mortality and nematode persistence. The study was carried out in both no-till and conventional-till corn, because tillage practices can affect the activity of soil organisms (Lupwayi et al., 1998), including entomopathogenic nematodes (Brust, 1991). In addition, because entomopathogenic nematodes may differ in their tendencies to disperse within the soil profile (Alatorre-Rosas and Kaya, 1990), the detection of the nematodes at two different soil depths was investigated. All three of the nematodes, H. bacteriophora (CEFS strain), S. carpocapsae (CEFS strain), and S. riobrave (Texas strain, originally from biosys, Columbia, MD), were cultured in the laboratory with last instar Galle-

ria mellonella (L.) (Lepidoptera: Pyralidae, greater wax moth) (Northern Bait, Chetek, WI and Nature’s Way, Hamilton, OH) (Kaya and Stock, 1997). Infective juveniles (IJ) were stored in suspension at 10°C no longer than 2 weeks before field application. Two experiments were conducted, the first involving soil baiting with G. mellonella in the lab (Experiment 1) and the second involving soil baiting with G. mellonella in the field (Experiment 2). Lab Soil Baiting (Experiment 1) This experiment was conducted for all three field seasons (1997, 1998, 1999). For each year, the experiment was replicated at four locations (blocks), each containing both no-till and conventional-till plots. For each block and tillage regime, there were different nematode treatments in 3 ⫻ 3-m assay areas located a minimum of 3 m apart. In 1997, the nematode treatments were (1) untreated control (only naturally occurring nematodes) and (2) S. riobrave (exotic nematode introduced) (R). In 1998, the nematode treatments were (1) untreated control, (2) S. riobrave (R), (3) S. riobrave and S. carpocapsae (native nematode S. carpocapsae augmented) (RC), and (4) S. riobrave and H. bacteriophora (native nematode H. bacteriophora augmented) (RH). In 1999, the nematode treatments were (1) untreated control, (2) H. bacteriophora (H), (3) S. carpocapsae (C), (4) S. riobrave (R), (5) S. riobrave and S. carpocapsae (RC), and (6) S. riobrave and H. bacteriophora (RH). For all 3 years, the control treatment received only water. To simulate a commercial application, S. riobrave IJ were inundatively applied in a liquid suspension with a watering can at a rate of 250,000 nematodes/m 2/4 liters water. An aliquot of S. riobrave IJ used in the field application was used to infect G. mellonella to confirm their viability. Approximately 1 week prior to S. riobrave application, S. carpocapsae and H. bacteriophora were applied by burial of plastic biopsy cassettes (4 ⫻ 3 cm, with 1-mm holes) (Histosette II, Simport, Beloeil, Canada), each containing two cadavers of G. mellonella that had been exposed to H. bacteriophora or S. carpocapsae 2 weeks earlier. Nine cassettes were buried at approximately 5 cm below the soil surface at a randomly chosen location within each square meter (1 cassette/m 2) in the plot to simulate natural foci of infection. Even though H. bacteriophora and S. carpocapsae are endemic to the site, they have a very patchy distribution (Stuart and Gaugler, 1994; Spiridonov and Voronov, 1995); therefore, we augmented the native nematodes to assure their presence in the treatment plots. Nine (1997) or six (1998, 1999) soil cores were collected from a randomly chosen location within six of the nine square meters (1 core/m 2) from each 3 ⫻ 3-m assay area the day before S. riobrave application (pre-

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treatment dates 5/14/97, 5/20/98, and 5/19/99), the day after nematode application, then weekly for 1 month, and then once monthly. In total, there were nine sampling dates each for 1997 and 1998, and eight sampling dates for 1999. For all assay areas, except for the 1998 and 1999 no-till control and R treatment, 1.5 cm diameter ⫻ 10 cm soil cores were collected. For the 1998 and 1999 no-till control and R treatment, the six soil cores collected were 1.5 cm diameter ⫻ 20 cm, which were then divided into two depths: 0 –10 cm and 10 –20 cm. The soil samples (cores) (216/date in 1997, 240/date in 1998, and 336/date in 1999) were placed individually into plastic zip-lock bags. These bags were taken in a cooler to the lab for baiting with three last instar G. mellonella. The samples were stored at room temperature (22–25°C) for 3 to 4 days, after which the number of living and dead insects was recorded. In 1997 and 1998, this baiting technique was repeated for each bag until there was no longer 100% larval mortality (that is, at least one larva was still alive following the 3- to 4-day baiting period). In 1999, this baiting technique was repeated for each bag until there was no larval mortality. Determination of nematode genus infecting the dead insects was by the color of the insect cadaver (reddish color for H. bacteriophora; ochre or tan color for S. carpocapsae and S. riobrave). Preliminary sampling during the winter of 1997 did not reveal the presence of the endemic nematode S. carpocapsae. Consequently, the presence of the endemic steinernematid was realized too late to allow distinction between S. carpocapsae and S. riobrave in 1997. In 1998 and 1999, the cadavers with steinernematids were dissected in Ringer’s solution 4 to 5 days after initial exposure to soil samples, and the adult nematodes were identified to species. Nematode male genitalia and the presence/ absence of a mucron (for males and females) were used to distinguish between S. carpocapsae and S. riobrave. Keys and descriptions (Cabanillas et al., 1994; Kaya and Stock, 1997), in addition to advice from Dr. Khuong B. Nguyen of the University of Florida at Gainesville and Dr. Enrique Cabanillas of the USDA/ ARS in Weslaco, Texas, were used to distinguish the two steinernematids. In 1998 and 1999, the treatment areas were marked with survey equipment. This enabled the location of the 3 ⫻ 3-m treatment areas 1 and 2 years later in 1999 and 2000. These areas were resampled on two dates in 1999 (7/1/99, 7/12/99) and one date in 2000 (7/6/00), with the same soil baiting method as above, to determine whether the introduced nematode, S. riobrave, could persist in the soil and to detect any long term effects on the interaction between the introduced and the endemic nematodes. In 1999, the number of male S. carpocapsae and S. riobrave in each cadaver was also recorded. Preliminary laboratory assays in soil with these two nematode

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species showed a highly significant correlation between the number of males and the number of females infecting G. mellonella larvae (R ⫽ 0.8834; P ⫽ 0.0001). Therefore, the number of male nematodes in larvae is an indicator of the total number of nematodes infecting G. mellonella. Field Soil Baiting (Experiment 2) This experiment was conducted during the 1997 and 1998 field seasons in the same no-till and conventionaltill corn plots used in Experiment 1. For these 2 years, the results of the two different baiting experiments, field baiting and laboratory baiting, generally agreed with each other. This plus the fact that the field baiting method is much more labor intensive than the lab baiting method are the reasons that we did not carry out the field baiting experiment during the 1999 field season. For each block and tillage regime, nematode treatments were applied to 1 ⫻ 1-m assay areas. In 1997, the nematode treatments were (1) untreated control, (2) H. bacteriophora (H), (3) S. riobrave (R), and (4) S. riobrave and H. bacteriophora (RH). In 1998, the nematode treatments were (1) untreated control, (2) S. riobrave (R), (3) S. carpocapsae (C), (4) H. bacteriophora (H), (5) S. riobrave and S. carpocapsae (RC), and (6) S. riobrave and H. bacteriophora (RH). The control plots received only water. S. riobrave was applied with the same method used in Experiment 1. Approximately 1 week prior to S. riobrave application, H. bacteriophora and S. carpocapsae were applied by burial, at approximately 5 cm below the soil surface in the center of the assay area, with one biopsy cassette containing two cadavers of G. mellonella that had been exposed to nematodes 2 weeks earlier. On six dates (1997) and five dates (1998) throughout the growing seasons, live G. mellonella larvae in biopsy cassettes were buried, at approximately 5 cm below the soil surface, in the treatment plots to detect nematodes. Eight (1997) or six (1998) individually caged insects were buried in each assay area, four on two edges of the assay area and four (1997) or two (1998) in the center. After 4 days, the caged insects were retrieved and taken back to the laboratory in a cooler. The number of living and dead insects was recorded, and the mortality by each nematode species was determined with the same method used in Experiment 1. Data Analysis All analyses to evaluate the effects of nematode treatment on the three nematode species were carried out with only the posttreatment sampling dates. Also, analyses for effects on S. riobrave were carried out with only the R, RC, and RH treatments. Following square root transformations, the nematode data for both field experiments were subjected to split-split plot analyses

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of variance with the General Linear Models Procedure in SAS (SAS Institute, 1988). For each year, for all data except for the depth data, tillage was the whole-plot factor, nematode treatment was the subplot factor, and sample date was the sub-subplot factor. To examine the effect of depth, data from the no-till control and R plots were analyzed as a split-split plot with nematode treatment as the whole-plot factor, depth as the subplot factor, and sampling date as the sub-subplot factor. For the treatments that were replicated across the different years, additional split-split plot analyses of variance were performed, which included year as an additional factor. Where appropriate, means for significant factors were separated with pairwise comparisons on least squares means. Analysis of correlation on nontransformed data was used to assess the spatial association of the different nematode species and the relationship between the number of nematode-killed insects and the number of male nematodes. A 0.05 level of significance was used in all analyses. Also, the index of aggregation (k) was calculated as k ⫽ x 2/(s 2 ⫺ x), where x is the arithmetic mean and s 2 is the sample variance. Data in text and figures are presented as nontransformed means with the standard errors of the means. RESULTS

Correlation between Infected Insects and Male Nematodes The number of insects infected by each nematode species was an appropriate indicator of the relative number of nematodes present in the field. In 1999, in both the current and the plots treated in the previous year, there was a significant positive correlation between the number of infected insects and the number of male S. carpocapsae (R ⫽ 0.77090; P ⫽ 0.0001) and S. riobrave (R ⫽ 0.50508; P ⫽ 0.0001) counted in insects from each soil sample. Interaction of Nematodes: Experiment 1 S. riobrave was successfully introduced to the field for all 3 years in both tillage regimes. In 1997, S. riobrave and S. carpocapsae infections were not distinguished from each other, but the significant increase in total infection for the R treatment compared to the control (F ⫽ 92.03; df ⫽ 1, 6; P ⫽ 0.0001) for both no-till and conventional-till indicated the successful introduction of S. riobrave. In 1998 and 1999 (Fig. 1), S. riobrave was detected throughout the growing season in both tillage regimes in all treatments where it was applied. To evaluate the effect of the introduced nematode, S. riobrave, on the two endemic nematodes, H. bacteriophora and S. carpocapsae, preplanned treatment com-

FIG. 1. Infections by treatment in 1999 (Experiment 1): mean numbers ⫾ SE of Galleria mellonella larvae infected per soil sample by Steinernema carpocapsae, Heterorhabditis bacteriophora, and S. riobrave in the six nematode treatments [untreated control, S. riobrave (R), H. bacteriophora (H), S. riobrave and H. bacteriophora (RH), S. carpocapsae (C), and S. riobrave and S. carpocapsae (RC)] in (A) no-till and (B) conventional-till.

parisons were made within each tillage regime for treatments with and without the addition of S. riobrave: control versus R (1997, 1998, and 1999), H versus RH (1999), and C versus RC (1999). For H. bacteriophora in 1997, there was no significant effect (P ⬎ 0.5) of nematode treatment on its detection in either tillage regime. In 1998, there was an overall (NT and CT combined) effect of nematode treatment on the number of H. bacteriophora infections (F ⫽ 7.45; df ⫽ 3, 18; P ⫽ 0.0019). This effect was due to the increase in H. bacteriophora infections in the treatment where it was augmented (RH) compared to the control treatment. Despite this effect, mean separation tests showed no significant difference (P ⬎ 0.1) in the number of H. bacteriophora infections for the control versus R in either tillage regime. In 1999, there was an overall (NT and CT combined) effect of nematode treatment on the number of H. bacteriophora infections (F ⫽ 10.74; df ⫽ 5, 30; P ⫽ 0.0001) (Fig. 1). This effect was due, in part, to the increase in H. bacteriophora infections in the treatments where it was augmented (H, RH) compared to the treatments where it occurred only naturally. Furthermore, mean separation tests showed overall (NT and CT combined) more infections by H. bacteriophora in H compared to RH (P ⫽ 0.0273). However, there was no significant difference (P ⬎ 0.1)

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in the number of H. bacteriophora infections for H versus RH in each tillage regime separately or for the control versus R and C versus RC overall (NT and CT combined). In a combined analysis of the 1997, 1998, and 1999 data for the control and R, there was no significant difference (P ⬎ 0.5) in H. bacteriophora infections for the control versus R comparison. For S. carpocapsae in 1998, there was no significant effect (P ⬎ 0.1) of nematode treatment on its infection rate in either tillage regime. In 1999, there was an effect of nematode treatment on the number of S. carpocapsae infections (F ⫽ 14.20; df ⫽ 5, 30; P ⫽ 0.0001) (Fig. 1). This effect was due to the increase in S. carpocapsae infections in the treatments where it was augmented (C, RC) compared to the treatments where it occurred only naturally. However, mean separation tests showed no significant treatment effect (P ⬎ 0.5) for the control versus R, H versus RH, or C versus RC for either tillage regime in 1999. When the 1998 and 1999 data for the control, R, RC, and RH treatments were combined, treatment affected the number of S. carpocapsae infections (F ⫽ 8.84; df ⫽ 3, 36; P ⫽ 0.0002). Again, this effect was due to the increase in S. carpocapsae infections in the treatment where it was augmented (RC) compared to the other treatments. However, mean separation tests showed no significant treatment effect (P ⬎ 0.1) for the control versus R comparison. For both years and both tillage regimes, the pretreatment mean number of S. carpocapsae infections was higher in the control treatment than in the R treatment, although, these numbers were not significantly different. In 1999, nematode treatment affected the number of S. carpocapsae males (F ⫽ 11.5; df ⫽ 5, 30; P ⫽ 0.0001) in a host. This effect was due to the increase in S. carpocapsae males in the treatments where they were augmented compared to treatments where they occurred only naturally. However, mean separation tests showed no significant treatment effect (P ⬎ 0.5) for the control versus R, H versus RH, or C versus RC in either tillage regime. S. riobrave was detected on all dates for both years. To evaluate the effect of the native nematodes on S. riobrave, preplanned treatment comparisons (R versus RC, R versus RH) were made within each tillage regime in 1998 and 1999. There was no significant effect (P ⬎ 0.1) of nematode treatment in either tillage regime on the number of infections by S. riobrave for 1998, 1999 (Fig. 1), or for 1998 and 1999 combined. In 1999, there was no significant effect (P ⬎ 0.1) of nematode treatment on the number of S. riobrave males for either tillage regime. Interaction of Nematodes: Experiment 2 S. riobrave was successfully introduced into the field in both 1997 and 1998. As with Experiment 1, in 1997,

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FIG. 2. Infections by treatment in 1998 (Experiment 2): mean numbers ⫾ SE of Galleria mellonella larvae infected per 1 ⫻ 1-m treatment area by Heterorhabditis bacteriophora, Steinernema carpocapsae, and S. riobrave in the six nematode treatments [untreated control, S. riobrave (R), H. bacteriophora (H), S. riobrave and H. bacteriophora (RH), S. carpocapsae (C), and S. riobrave and S. carpocapsae (RC)] in (A) no-till and (B) conventional-till.

S. riobrave and S. carpocapsae infections were not distinguished from each other, but a significant increase in total infections for the R and RH treatments compared to the treatments not receiving S. riobrave (control and H) indicated successful introduction of S. riobrave. In 1997, nematode treatment affected the total number of infections (F ⫽ 13.93; df ⫽ 3, 18; P ⫽ 0.0001). The total number of infections was higher in R than in the control (P ⫽ 0.0003) and in RH than in H (P ⫽ 0.0015). In 1998, S. riobrave was detected in both tillage regimes in all the treatments where it was applied (Fig. 2). To evaluate the effect of the native and introduced nematodes on each other, the same preplanned treatment comparisons used in Experiment 1 were carried out. In 1997, nematode treatment affected the number of H. bacteriophora infections (F ⫽ 5.70; df ⫽ 3, 18; P ⫽ 0.0063), and there was a tendency for a treatment by tillage interaction (F ⫽ 2.90; df ⫽ 3, 18; P ⫽ 0.0635). This treatment main effect was, in part, due to the increase in H. bacteriophora infections in one of the treatments where it was augmented (H) compared

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2). S. riobrave was detected on all dates in all treatments where it was applied. Long-Term Persistence

FIG. 3. Infections by Heterorhabditis bacteriophora in 1997 (Experiment 2): mean numbers ⫾ SE of Galleria mellonella larvae infected per 1 ⫻ 1-m treatment area by H. bacteriophora in the nematode treatments, H. bacteriophora (H) and S. riobrave and H. bacteriophora (RH), in (A) no-till and (B) conventional-till. Mean separation tests showed more H. bacteriophora infections in H than in RH in conventional-till (P ⫽ 0.0020) and overall (no-till and conventional-till combined) more H. bacteriophora infections in H than in RH (P ⫽ 0.0133).

to the treatments where it occurred only naturally. Furthermore, mean separation tests showed more H. bacteriophora infections in H than in RH in CT (P ⫽ 0.0020) and overall (NT and CT combined) more H. bacteriophora infections in H than in RH (P ⫽ 0.0133) (Fig. 3). However, there was no significant difference (P ⬎ 0.5) in the number of infections by H. bacteriophora for the control versus R in either tillage regime and H versus RH in NT. In 1998 (Fig. 2), there was no significant effect (P ⬎ 0.1) of nematode treatment on the number of H. bacteriophora infections in either tillage regime, although, as in 1997, the mean number was higher in H than in RH in both tillage regimes. With the 1997 and 1998 control, R, H, and RH data combined, there was an effect of nematode treatment on the number of H. bacteriophora infections (F ⫽ 5.44; df ⫽ 3, 36; P ⫽ 0.0035). Mean separation tests showed more H. bacteriophora infections in H than in RH for CT (P ⫽ 0.0024), but no significant difference (P ⬎ 0.05) for H versus RH in NT or for the control versus R for either tillage regime. In 1998, there was no significant effect (P ⬎ 0.1) of nematode treatment on the number of S. carpocapsae and S. riobrave infections in either tillage regime (Fig.

In 1999 and 2000, 1 and 2 years after the introduction of S. riobrave in 1998 and 1 year after the introduction of S. riobrave in 1999, treatment plots were resampled to determine whether S. riobrave was still present in the field and to determine any long-term effects on the endemic nematodes, S. carpocapsae and H. bacteriophora. In particular, we wanted to know whether the endemic nematodes could have been completely displaced by S. riobrave. In 1999, S. riobrave was detected in 10% of the samples in the treatments where it was applied in 1998 (R, RC, RH). It was detected in all three of these treatments and in both tillage regimes. Native H. bacteriophora was detected only in the control and RH treatments from 1998. Native S. carpocapsae was detected in all four nematode treatments (control, R, RC, and RH) from 1998. Among the R, RC, and RH treatments, there was no significant effect (P ⬎ 0.05) of nematode treatment on the number of infections by S. riobrave in either tillage regime. Across all treatments (control, R, RC, and RH), there was no significant effect (P ⬎ 0.05) of treatment on the number of infections by S. carpocapsae and H. bacteriophora in either tillage regime. Among the R, RC, and RH treatments, there was no significant effect (P ⬎ 0.05) of nematode treatment on the number of S. riobrave males in either tillage regime. Across all treatments (control, R, RC, and RH), there was no significant effect (P ⬎ 0.05) of nematode treatment on the number of S. carpocapsae males in either tillage regime. For the one resampling date in 2000, S. riobrave was detected in 13% of the samples in the treatments where it was applied in 1998 and in 10% of the samples in the treatments where it was applied in 1999 (R, RC, RH). It was detected in all three of these treatments and in both tillage regimes. In addition, S. riobrave was detected in 8% of the samples in two of the treatments where this nematode had not been applied in 1999 (control, C). H. bacteriophora was detected only in the RH treatment from 1998 and in the C treatment from 1999. S. carpocapsae was detected in all four treatments from 1998 (control, R, RC, RH), but was not detected in any of the treatment plots from 1999. Among the R, RC, and RH treatments, there was no significant effect (P ⬎ 0.05) of nematode treatment on the number of infections by S. riobrave in either tillage regime. Across all treatments (control, C, H, R, RC, and RH), there was no significant effect (P ⬎ 0.05) of treatment on the number of infections by S. carpocapsae and H. bacteriophora in either tillage regime.

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FIG. 4. Negative spatial association (Experiment 1): frequency of single and multiple nematode species detection in individual soil samples in treatments with Steinernema riobrave [S. riobrave (R), S. riobrave and Heterorhabditis bacteriophora (RH), and S. riobrave and S. carpocapsae (RC)]. Includes data from 1998, 1999, 1998 plots resampled in 1999, both tillage regimes, and both soil depths (0 –10 cm, 10 –20 cm); N ⫽ 2783.

Effect of Depth The effect of depth was evaluated in the NT control and R nematode treatments in 1998 and 1999. The number of S. carpocapsae infections was greater in the upper 10 cm than in the lower 10 cm for 12 of the 15 sample dates, and the mean number for all dates combined for both years was higher in the upper 10 cm than in the lower 10 cm (F ⫽ 11.00; df ⫽ 1, 12; P ⫽ 0.0061). Thirty-one percent of the S. carpocapsae infections occurred in the lower 10 cm. The number of S. riobrave infections was higher in the upper 10 cm than in the lower 10 cm for 9 of the 13 posttreatment dates, and the mean number for all dates combined for both years was higher in the upper 10 cm than in the lower 10 cm (F ⫽ 9.41; df ⫽ 1, 6; P ⫽ 0.0220). Thirty-nine percent of the S. riobrave infections occurred in the lower 10 cm. H. bacteriophora was detected on only 4 of 15 sample dates in 1998 and 1999 in the control and R treatments in NT. On 3 of 4 of those dates (5/20/98, 9/12/98, 6/3/99), H. bacteriophora was detected only in the lower 10 cm, and on the fourth date (7/16/99) it was detected only in the upper 10 cm. The overall mean number of H. bacteriophora infections was higher in the lower 10 cm than in the upper 10 cm; however, this difference was not significant (P ⬎ 0.1). Spatial Distribution All three nematode species have a clumped distribution. No entomopathogenic nematodes were detected in the majority of the samples. For Experiment 1, over all dates and treatments, H. bacteriophora was detected in 1.7% of soil samples in 1997 and 1998 and in 2.6% in 1999. S. carpocapsae was detected in 8.2% of the soil samples in 1998 and in 5.7% in 1999. For the treatments where S. riobrave was applied (R, RC, RH), this nematode was detected in 42.5% of the soil samples in 1998 and in 12.1% in 1999. With regard to overall nematode infection, nematodes were detected in 32.5%

of the soil samples in 1997, in 36.4% in 1998, and in 13.1% in 1999. For clumped distributions, the variance is larger than the mean (that is, the variance-to-mean ratio is ⬎1), and the index of aggregation, k, is small (less than 8) (Pedigo and Zeiss, 1996). The smaller the value of k, the greater the intensity of aggregation. With regard to the number of infections by each nematode species, k was less than 2 and the variances were much larger than the means on almost every date for all 3 years (Millar, 2000). With regard to the number of males detected for S. riobrave and S. carpocapsae, k was less than 1 and the variances were much larger than the means on almost every date in 1999 (Millar, 2000). The variance was not larger than the mean only when no nematodes of the particular species were detected or when the number detected was so low that it made the variance and the mean equal to each other. In 1999, across all treatments, the maximum number of males per soil sample was much larger at the beginning of the season than at the end. For S. carpocapsae, the maximum was 927 on 5/19/99 and then fell to 3 on 8/11/99. For S. riobrave, the maximum was 81 on 5/27/99 and then fell to 2 on 8/11/99. In the 1998 plots resampled in 1999, the maximum number of S. carpocapsae males per soil sample was 6 on 7/1/99 and 227 on 8/5/99. The maximum for S. riobrave males was 186 on 7/1/99 and 32 on 8/5/99. In all the treatments where S. riobrave was applied (R, RC, and RH), we determined the frequency of samples in which no, single, and multiple nematode species were detected (Fig. 4). Where nematodes were detected, the occurrence in a single soil sample of multiple species was rare relative to the number of samples in which only one nematode species was detected. For both the upper 10 cm and the lower 10 cm soil depths, there were no samples in which all three nematodes were detected together, and there were no samples in which a single host was coinfected by more than one species. At the lower depth, multiple species detection

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occurred only in one sample, in which S. riobrave and S. carpocapsae were detected together. These data suggest that the nematodes’ distributions rarely overlapped. An analysis of correlation (with only soil samples with at least one species detected) showed a highly significant negative association for all three nematodes for both years and for both soil depths. In 1998, at 0 –10 cm, there was a negative association between S. riobrave and S. carpocapsae (R ⫽ ⫺0.20979; P ⫽ 0.0001), S. riobrave and H. bacteriophora (R ⫽ ⫺0.15577; P ⫽ 0.0004), and S. carpocapsae and H. bacteriophora (R ⫽ ⫺0.23565; P ⫽ 0.0020). In 1999, at 0 –10 cm, there was a negative association between S. riobrave and S. carpocapsae (R ⫽ ⫺0.30799; P ⫽ 0.0001), S. riobrave and H. bacteriophora (R ⫽ ⫺0.30400; P ⫽ 0.0002), and S. carpocapsae and H. bacteriophora (R ⫽ ⫺0.27714; P ⫽ 0.0001). In 1998, at 10 –20 cm, there was a negative association between S. riobrave and S. carpocapsae (R ⫽ ⫺0.34356; P ⫽ 0.0067). H. bacteriophora was not detected in the R treatment at 10 –20 cm in 1998, so there was no analysis with this nematode. In 1999, at 10 –20 cm, there was a negative association between S. riobrave and S. carpocapsae (R ⫽ ⫺0.80178; P ⫽ 0.0053) and S. riobrave and H. bacteriophora (R ⫽ ⫺1.0000; P ⫽ 0.0001). The number of nonzero data points was too low to allow analysis for the association of S. carpocapsae and H. bacteriophora, but lack of detection of these two nematodes in the same sample indicated a negative spatial association. DISCUSSION

In this study, the introduced nematode, S. riobrave, was able to establish and persist in both the no-till and the conventional-till regimes, and the number of infections by this nematode was not affected by the augmentation of the native nematodes. We detected S. riobrave in both tillage regimes and, overall, in 10% of the samples 1 year after its application in the field and in 13% of the samples 2 years after its application in the field. One year after its field application in 1998, in 288 soil samples, we detected 4 samples with ⬎10 S. riobrave male nematodes, with 186 male nematodes detected in 1 sample. With only 1 to 3 steinernematid males being found in the majority of soil samples during this study and the maximum number of S. riobrave males found in a single host soon after field application being 81, this high number of males (186) indicates that S. riobrave recycled in undetermined hosts in the soil. Despite its establishment, S. riobrave does not seem to have had any negative short-term or long-term effects on the detection of the native nematode, S. carpocapsae, at either soil depth and in either tillage regime. In contrast, although many factors affect detection, the data suggest that the population level of

the native nematode, H. bacteriophora, was negatively affected by the presence of S. riobrave, and this negative effect was slightly stronger in conventional-till than in no-till in 1997. The soil-mixing action of tillage probably makes substrates in the soil more homogeneous, which does not promote biological diversity (Lupwayi et al., 1998). Detection of H. bacteriophora was suppressed when comparing treatments in which H. bacteriophora was augmented (H versus RH), but not in treatments with only naturally occurring H. bacteriophora (control versus R, C versus RC). It may be that the population level of the naturally occurring H. bacteriophora was too low to allow detection of any effects or that S. riobrave did not have any effect if H. bacteriophora was below a certain population level. In a preliminary laboratory assay, the number of H. bacteriophora infections in G. mellonella was delayed and suppressed when in cohabitation with S. riobrave in a small, sterile soil assay arena (L. C. Millar, unpublished data). These laboratory results suggest that when the distributions of these two nematodes overlap, there may be a reduction in the detection of H. bacteriophora with the Galleria baiting method. Hence, by augmenting the population of H. bacteriophora in the field, we may have increased the probability of overlapping distributions of these two nematodes and, therefore, an effect of S. riobrave on the detection of H. bacteriophora. Despite the possible negative effect on this native nematode, monitoring of plots 1 and 2 years after nematode applications did not reveal complete displacement of H. bacteriophora in the treatment receiving both S. riobrave and augmented H. bacteriophora (RH). In summary, the data suggest that coexistence of the three nematode species in the field was possible and that risk of local extinction of the native nematodes was minimal. More than one species of entomopathogenic nematodes can naturally coexist in the field, for multiple species have been isolated from the same site (Akhurst and Brooks, 1984; Stuart and Gaugler, 1994). Such coexistence is possible if environmental factors and behavioral differences allow for a strong niche differentiation. In laboratory and greenhouse studies, for example, differences in foraging behaviors may reduce competition among nematodes, thereby permitting coexistence (Koppenho¨fer and Kaya, 1996a,b). Two main foraging behaviors have been described on a continuum. At one extreme is the “ambush” behavior, where the nematode typically remains nearly sedentary at or near the soil surface and waits for mobile surfaceadapted hosts. At the other extreme is the “cruise” behavior, where the nematode is typically highly mobile and seeks out relatively sedentary hosts deeper in the soil profile. The two native nematode species in our study have been categorized into these two behavioral extremes. S. carpocapsae has been categorized as an ambusher (Kaya and Gaugler, 1993) and H. bacterio-

INTERACTION OF ENTOMOPATHOGENIC NEMATODES

phora as a cruiser (Grewal et al., 1995). The facts that S. carpocapsae and H. bacteriophora appear to have two distinct foraging behaviors and that they naturally coexist in the present field site could be evidence of this niche differentiation. The introduced nematode, S. riobrave, appears to share characteristics of both ambushers and cruisers (Cabanillas et al., 1994; Grewal et al., 1995). Our study supports this categorization of these three species. The data suggest that the three nematodes have different tendencies to disperse through the soil profile, with H. bacteriophora having the strongest tendency to move into the soil, followed by S. riobrave and then S. carpocapsae. This behavioral difference among the three nematodes could help explain their apparent coexistence, by allowing them to seek out hosts at different locations in the soil profile. Their apparent coexistence could be further explained by their patchy distribution. Aggregated distribution of the nematodes probably did not result from an inability to detect all nematodes in the samples, because the loss of organisms from samples with a constant probability cannot transform a regular or Poisson distribution into an aggregated distribution (Spiridonov and Voronov, 1995). Entomopathogenic nematodes typically have a patchy or aggregated distribution (Stuart and Gaugler, 1994). Spiridonov and Voronov (1995) hypothesized that the field distribution pattern of S. feltiae infective juveniles is a combination of Poisson low-level background of “old” IJ and separate narrow peaks (at least five to seven nematodes) resulting from recent insect infestations. They concluded that the characteristic dimension of these IJ peaks is 15–20 cm. According to other studies, S. riobrave (Cabanillas and Raulston, 1994), S. carpocapsae (Campbell et al., 1998), and H. bacteriophora (Campbell et al., 1998) also have a patchy spatial pattern in the field. Koppenho¨fer and Kaya (1996a) suggested that this patchy distribution in the field may help support nematode coexistence. Indeed, the distributions of the nematodes in the current study rarely overlapped. It was rare to find two species in the same soil sample, and there were no samples in which all three species were detected. Sturhan and Lisˇkova´ (1999) suggested that mixtures of species in the same sample are often not discovered with the Galleria baiting technique. However, in laboratory assays, we were able to frequently detect multiple species in single soil samples with the Galleria baiting technique (L. C. Millar, unpublished data). We detected all three nematodes in single samples as long as there were multiple baitings per sample. The lack of overlap of the nematodes’ distributions in the field was also indicated by the absence of insects coinfected by multiple species. This lack of coinfection in the field occurred despite the fact that coinfection by S. carpocapsae and S. riobrave of single G. mellonella hosts was possible in preliminary laboratory experiments with mixed inoculations

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in both a petri dish and a soil assay arena (L. C. Millar, unpublished data). A combination of extrinsic and intrinsic factors is typically responsible for aggregated distributions (Campbell et al., 1998). Such factors may include spatial and temporal variability of the nematodes’ hosts and natural enemies, production of large numbers of IJ from single hosts, limited dispersal ability of IJ, and heterogeneity of soil microclimates (Stuart and Gaugler, 1994; Campbell et al., 1998). The lack of overlap of patches inhabited by different nematode species may be attributed to differences in susceptibility to natural enemies (Kerry, 1995; Huston, 1994), variation in infectivity for different insects (Bedding et al., 1983), and variation in adaptation to different abiotic conditions (Cabanillas and Raulston, 1996; Gouge et al., 1999). Because we did not find any naturally infected insects, we do not know the natural hosts of our endemic nematode species or the hosts utilized by our introduced nematode, but, because of their apparent differences in foraging behavior and differences in host preference found in laboratory studies (Koppenho¨fer and Kaya, 1996a; Gouge et al., 1999), these three species may have utilized different host species during this study. As mentioned above, multiple species have been isolated in field surveys from natural sampling sites, and Sturhan (1999) states that this supports evidence of differences in host preference. Entomopathogenic nematodes can be affected by soil abiotic factors such as soil texture, temperature, and moisture (Kaya and Gaugler, 1993). Because the introduced nematode, S. riobrave, originated from a semiarid subtropical region (Cabanillas et al., 1994), and the native nematodes are endemic to a humid temperate region, it is possible that S. riobrave has environmental tolerances and preferences different from those of the native nematodes. Indeed, a separate analysis of the data suggested that S. riobrave’s infectivity was greater in a drier, and possibly warmer, soil than infectivity of the native S. carpocapsae (L. C. Millar and M. E. Barbercheck, unpublished data). Other studies have shown different temperature tolerances for these two nematode species (Gouge et al., 1999; Cabanillas and Raulston, 1996). Competition between nematode species with similar resource needs may be minimal, thereby facilitating coexistence (Ettema, 1998). It appears that coexistence of nematode species is made possible by small-scale disturbance and predation, which reduce local population sizes and, therefore, interspecific competition. Even if competitive exclusion did occur, immigration may reestablish the extinct population (Huston, 1994). Active dispersal by entomopathogenic nematodes may be limited, but they can travel short or long distances on or in insects (Poinar, 1979), on earthworms (Shapiro et al., 1993), and on mites (Epsky et al., 1988). Because parasites of insects depend on a living host, their re-

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sources are very patchy and ephemeral, resulting in a low probability of colonization and a high probability of extinction in each patch (Price, 1984). The net result is that populations of parasites increase and decline in a patch in a very unpredictable fashion, and many resources remain unexploited. Hence, interspecific competition may frequently be an unimportant organizing force for parasites of insects (Price, 1984). This may apply not only to naturally occurring entomopathogenic nematodes, but also to inundatively applied nematodes because of their rapid decline in detectable numbers (Georgis, 1992) and rapid transformation to an aggregated distribution after application (Campbell et al., 1998). In our study, 1 week following its field application, S. riobrave was detected in less than half of the samples in treatments where it was applied (35.4% in 1998, 27.8% in 1999), and it had already attained a highly aggregated distribution. In summary, this study suggests that the risk of complete displacement of endemic entomopathogenic nematodes by an inundatively introduced nematode may be minimal. This was the case for both the lab soil baiting and the field baiting experiments. However, one should not assume that because there was little or no effect on the endemic nematodes in the short term, there can be no detrimental effects in the long term. Hence, long-term monitoring is needed to confirm our hypothesis of low risk of complete displacement of the endemic nematodes. This is especially so because S. riobrave was able to persist for more than one field season, and the number of infections by H. bacteriophora was suppressed in the presence of S. riobrave. The risk of displacement of endemic nematodes should be further evaluated by examination of the interactions of other species and strains of entomopathogenic nematodes, in addition to those evaluated in the current study. As the similarity of behavioral characteristics and/or microclimatic preferences increases, there may be a greater likelihood of overlap of the nematodes’ population distributions and of competition for the same hosts, thereby leading to increased risk of displacement. Partial or complete displacement of endemic entomopathogenic nematodes should be considered a possible consequence of the release of exotic species. Endemic nematodes are an important biological resource since they are probably the best adapted to the local habitat and perhaps to targeted insect soil pests. However, one must evaluate the relative risks and advantages of any pest management practice, including biological control, in light of other available control tactics. Any potential risks associated with the use of entomopathogenic nematodes as biological control agents are probably minor compared to the use of chemical pesticides, especially when vertebrate safety is considered (Boemare et al., 1996). Chemical insecticides used as an alternative to these nematodes to

control soil pests may have a negative impact on endemic entomopathogenic nematodes (Gordon et al., 1996), and this should be kept in mind when the risks and benefits of inundatively introduced nematodes are evaluated. Finally, compared to some other groups of biological control agents, these nematodes are thought to have relatively minor nontarget ecological risks (Ehlers and Peters, 1995). Reasons for this low risk include, among others, their rapid reduction to naturally occurring levels following inundative application, their susceptibility to many generalist antagonists, and their very low dispersal ability. ACKNOWLEDGMENTS Funding by the USDA’s National Research Initiative Competitive Grants Program (Grant No. 9702083) is gratefully acknowledged. We thank Fred Gould and David Orr for their comments on the manuscript; Cavell Brownie and Ronald Stinner for help with statistical analyses; George Naderman for providing use of research plots at the Center for Environmental Farming Systems; Ronald Sheffield, Jing Wang, Charles Warrick, Gregory Linville, Carla Hendrix, Joseph Stout, Jeremy Ashton, Grover Garcia, Nabor Mendizabel, and Tracy Morris for technical support; and Patricia Stock, Khuong Nguyen, and Enrique Cabanillas for guidance with nematode identifications.

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