Biological Control 25 (2002) 1–11

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Effects of tillage practices on entomopathogenic nematodes

in a corn agroecosystem
Leah C. Millar* and Mary E. Barbercheck
Department of Entomology, North Carolina State University, Raleigh, NC 27695-7634, USA
Received 28 February 2001; accepted 20 March 2002

Abstract

Cultural practices such as tillage affect soil abiotic and biotic factors, which in turn may affect the survival and activity of en-
tomopathogenic nematodes. We investigated the relative sensitivity of an inundatively applied nematode species, Steinernema
riobrave (Texas), and two endemic nematode species, Steinernema carpocapsae and Heterorhabditis bacteriophora, to tillage practices
in no-till and conventional-till corn near Goldsboro, North Carolina. Two baiting methods using Galleria mellonella, one conducted
in the laboratory and the other in the field, were used to evaluate the nematodes in terms of infected insects and nematode per-
sistence. H. bacteriophora, which was only rarely detected, was not significantly affected by tillage. Tillage had a significant negative
effect on the detection of S. carpocapsae and a significant positive effect on the detection of S. riobrave. The nematodes’ dissimilar
sensitivities to tillage may be partly explained by differences in environmental tolerances and differences in tendencies to disperse
deeper in the soil profile. Ó 2002 Elsevier Science (USA). All rights reserved.

Keywords: Steinernema riobrave; Steinernema carpocapsae; Heterorhabditis bacteriophora; Entomopathogenic nematode; Biological control; Tillage
effects

1. Introduction ature, moisture, and nematode natural enemies (Kaya

The use of entomopathogenic nematodes in the
families Steinernematidae and Heterorhabditidae as in-
undatively applied biological control agents against soil
insect pests is rapidly expanding. However, field trials
using these nematodes as biological control agents have
often yielded highly variable results (Georgis and Gau-
gler, 1991) and this has been attributed in part to the
lack of understanding of their biology in natural and
agricultural ecosystems. Knowledge of the effects of soil
environmental factors on entomopathogenic nematodes
is needed to select the most appropriate species for op-
timal insect pest management. The efficacy of entomo-
pathogenic nematodes, as with other biological control
agents, can be affected by various abiotic and biotic
factors. These factors can include soil texture, temper-
*
Corresponding author. Present address: USDA-APHIS, Raleigh
Plant Protection Center, 1017 Main Campus Dr., Suite 2500, Raleigh,
NC 27606-5202, USA. Fax: +919-513-1995.
E-mail address: leah.c.millar@aphis.usda.gov (L.C. Millar).
and Gaugler, 1993).
Cultural practices such as tillage affect soil abiotic
and biotic factors. For example, reduced tillage en-
hances soil microbial diversity (Hassink et al., 1991;
Lupwayi et al., 1998), nematode diversity (Freckman
and Ettema, 1993), and soil arthropod diversity (House
and Alzugaray, 1989). Soil under reduced tillage has
lower temperatures and higher soil moisture levels
compared to conventional tillage regimes (Sprague and
Triplett, 1986), which may favor the development of
disease in soil insect populations (Sloderbeck and
Yeargan, 1983). Soil moisture is considered an impor-
tant factor in influencing the infectivity of entomo-
pathogenic nematodes (Kaya, 1990). Hence, reduced
tillage has been suggested as a method of conserving
entomopathogenic nematodes (Brust, 1991). The detec-
tion of endemic Heterorhabditis bacteriophora Poinar
was higher in no-till (NT) compared to conventional-till
(CT) in corn (Brust, 1991). Hummel (2000) found a
greater percentage of bait insects infected with endemic
Steinernema carpocapsae (Weiser) (Fletcher strain) in

1049-9644/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S 1 0 4 9 - 9 6 4 4 ( 0 2 ) 0 0 0 4 2 - 7
2 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11

NT compared to clean-till corn, tomatoes, and cabbage. 2.1. Laboratory soil baiting (Experiment 1)
The presence of crop residue, which is greater under
reduced tillage, favored the persistence of inundatively This experiment was conducted for all three field sea-
applied S. carpocapsae (Mexican strain) (Shapiro et al., sons (1997, 1998, and 1999) on corn. For each year, the
1999). Nevertheless, because of the considerable inter- experiment was replicated at four locations (blocks), each
and intraspecific variability in environmental tolerances containing both NT and CT plots. For each block and
among entomopathogenic nematodes (Poinar, 1990), it tillage regime, there were different nematode treatments in
cannot be assumed that all nematodes species and 3 m
3 m assay areas within the corn fields (Table 1). For
strains will respond similarly to different tillage prac- all three years, the control treatment received only water.
tices. For example, other types of soil nematodes, such To simulate a commercial application, S. riobrave IJs
as plant-parasitic and free-living nematodes, vary in were inundatively applied in a water suspension with a
their response to tillage regimes (Stinner and Crossley, watering can at the rate of 250,000 nematodes/m2 =4 li-
1982; Parmelee and Alston, 1986). ters of water. An aliquot of S. riobrave IJ used in the field
The objective of our research was to investigate the application was used to infect G. mellonella to confirm
relative sensitivity of an inundatively applied nematode their viability. Because entomopathogenic nematodes
and natural populations of two endemic nematodes to have a patchy distribution (Akhurst et al., 1992; Stuart
tillage practices in a corn agroecosystem. and Gaugler, 1994), we augmented the native nematodes
to assure their presence in the treatment plots. Ap-
proximately one week prior to S. riobrave application,
2. Materials and methods S. carpocapsae and H. bacteriophora were applied by
burial of plastic biopsy cassettes (4 cm
3 cm cages with
The study was conducted during three field seasons 1 mm holes, Histosette II, Simport, Beloeil, Canada),
(1997, 1998, and 1999) in NT and CT corn plots (1997 each containing two cadavers of G. mellonella that had
and 1998: Zea mays L. cv. Dekalb 714; 1999: Z. mays been exposed to H. bacteriophora 11–14 days earlier or
cv. Dekalb 580) at the Center for Environmental S. carpocapsae 7–8 days earlier. At this time, IJs were
Farming Systems (CEFS) near Goldsboro, NC. The just beginning to emerge from the cadavers. Nine cas-
36:6 m
73:2 m plots were in a corn–soybean rotation settes were buried at approximately 5 cm below the soil
(Millar, 2000). The NT plots were planted with a wheat
Table 1
cover crop during the fall and had been under a NT
Summary of nematode treatments for Experiments 1 and 2. Nematode
regime since 1995. The CT plots received no winter treatments (H. bacteriophora, S. carpocapsae, and S. riobrave applied
cover crop and were chisel plowed and disked in the fall singly or in combination with each other) were applied to 3 m
3 m
and spring. The research site used for this study received assay areas within both NT and CT corn fields
fertilizer and herbicides typical for corn production in Experiment Year Nematode treatment
this region but did not receive insecticides. The soil type Laboratory soil 1997 Untreated control (water only)
in the plots is classified as predominantly Wickham fine baiting H. bacteriophora
sandy loam. (Experiment1) S. riobrave
Three nematodes were studied in our experiments. S. 1998 Untreated control (water only)
carpocapsae and H. bacteriophora are endemic to the S. riobrave
S. riobrave + S. carpocapsae
site. Dr. Patricia Stock, University of Arizona, Tuscan, S. riobrave + H. bacteriophora
confirmed the species identification via morphological 1999 Untreated control (water only)
and molecular analysis. The commercially available H. bacteriophora
nematode S. riobrave Cabanillas, Poinar, and Raulston S. carpocapsae
(Texas strain, originally from Biosys, Columbia, MD) S. riobrave
S. riobrave + S. carpocapsae
was chosen as a nematode that could potentially be S. riobrave + H. bacteriophora
used as an inundatively applied biocontrol agent in
corn. All of the nematodes were cultured in the labo- Field soil baiting 1997 Untreated control (water only)
(Experiment 2) H. bacteriophora
ratory using last instar Galleria mellonella (L.) (Lepi- S. riobrave
doptera: Pyralidae, greater wax moth) (Northern Bait, S. riobrave + H. bacteriophora
Chetek, WI and Nature’s Way, Hamilton, OH) (Kaya 1998 Untreated control (water only)
and Stock, 1997). Infective juveniles (IJs) were stored H. bacteriophora
in tap water at 10 °C for no longer than two weeks S. carpocapsae
S. riobrave
before field application. S. riobrave + S. carpocapsae
Two experiments were conducted, the first one in- S. riobrave + H. bacteriophora
volving soil baiting with G. mellonella in the laboratory Preliminary sampling during the winter of 1997 did not reveal the
(Experiment 1) and the other involving soil baiting with presence of the native S. carpocapsae; therefore, this nematode was not
G. mellonella in the field (Experiment 2). augmented during the 1997 field season.
 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11
surface at a randomly chosen location within each
square meter (1 cassette/m2 ) in the plot to simulate nat-
ural foci of infection. Additional biopsy cassettes were
prepared to monitor emergence of IJs in the laboratory.
The day before application of S. riobrave (pretreat-
ment dates 5/14/1997, 5/20/1998, and 5/19/1999), the day
after application of this nematode, then weekly for one
month, and then once monthly, nine (1997) or six (1998,
1999) soil cores were collected in a regular pattern
(1 m2 ) from each 3 m
3 m assay area. In total, there
were nine sampling dates each for 1997 and 1998 and
eight sampling dates for 1999. For all assay areas,
1:5 cm dia:
10 cm soil cores were collected. The soil
samples (cores) (216/date in 1997, 192/date in 1998, and
288/date in 1999) were placed individually into plastic
zip-lock bags. These bags were taken in an insulated
cooler to the laboratory for baiting with three last instar
G. mellonella. The samples were stored at room tem-
perature (22–25 °C) for 3–4 days, after which the num-
ber 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 re-
peated 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
for H. bacteriophora; ochre or tan for S. carpocapsae
and S. riobrave). In 1997, the presence of native stein-
ernematids was realized too late to allow for distinction
between S. carpocapsae and S. riobrave. In 1998 and
1999, the insects killed by steinernematids were dissected
in Ringer’s solution 4–5 days after initial exposure to
soil samples and the adult nematodes were identified to
species. Nematode male genitalia and the presence/ab-
sence 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; Poinar, 1990), as well as advice from
Dr. Khuong B. Nguyen of the University of Florida
at Gainesville and Dr. Enrique Cabanillas of the USDA/
ARS in Weslaco, TX, were used to distinguish the two
steinernematids. The number of insects infected by each
nematode species was an appropriate indicator of the
relative number of nematodes present in the field, be-
cause a significant positive correlation between the
number of infected insects and the number of nematodes
was determined in the field for the two steinernematid
species (Millar and Barbercheck, 2001). Other authors
have confirmed a linear relationship between insect
mortality and number of nematodes penetrating into
bait insects as well (Koppenh€ ofer et al., 1998).
In 1998, the treatment areas were marked using sur-
vey equipment. This enabled us to locate the 3 m
3 m
treatment areas one year later in 1999. These areas were
resampled on two dates (7/1/1999, 7/12/1999), using the
3
same soil baiting method as above, to determine any
multiple season effects of tillage on the nematodes.
2.2. Field soil baiting (Experiment 2)
This experiment was conducted during the 1997 and
1998 field seasons in different areas of the same NT and
CT corn fields as for Experiment 1. For each block and
tillage regime, nematode treatments (Table 1) were
applied to 1 m
1 m assay areas. The control plots re-
ceived only water. S. riobrave was applied using the same
method as for Experiment 1. Approximately one 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,
of one plastic biopsy cassette containing two cadavers of
G. mellonella that had been exposed to H. bacteriophora
11–14 days earlier or S. carpocapsae 7–8 days earlier.
On six dates (1997) and five dates (1998) throughout
the growing seasons, live G. mellonella larvae in biopsy
cassettes were buried 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 four 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 of each nematode species was determined us-
ing the same method as for Experiment 1.
2.3. Soil characteristics
Soil samples of 5 cm diameter and 5 cm deep were
taken for evaluation of soil characteristics for Experi-
ment 1. In 1997 and 1998, gravimetric percentage of
soil moisture (mass of water/dry weight of soil) and
percentage of organic matter were monitored in each
block/tillage combination on each sampling date. In
1998, soil pH and texture (percentages of sand, silt,
and clay) were also determined for each block/tillage
combination on one sampling date. In 1999, the above
soil characteristics plus soil water potential ()kPa)
were monitored for each 3 m
3 m treatment area
within each block/tillage combination: soil moisture
and water potential on every sampling date, organic
matter on three sampling dates (early, middle, and late
seasons), and pH and texture on one sampling date.
Soil pH was measured from a 1:2 soil:water suspen-
sion, soil organic matter was measured using a dry
ashing procedure (described by D.A. Storer, Agro-
nomic Service Laboratory, Agrico Chemical Company,
unpublished), soil water potential was measured using
the filter paper method (Kaya and Stock, 1997), and
the percentages of sand, silt, and clay were determined
using the hydrometer method (Gee and Bauder, 1986).
Soil temperature was monitored all three years
4 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11
throughout the field season in both tillage regimes in
one of the four blocks (block 4 in 1997, block 3 in
1998, and block 2 in 1999) using a Campbell CR-21
data logger (probe buried at 10 cm soil depth). In ad-
dition, data on rainfall during the field seasons were
obtained from a weather station at the farm office.
2.4. Data analysis
Analysis for effects of tillage on H. bacteriophora was
carried out for all three years, whereas analyses for
effects on S. carpocapsae and S. riobrave were carried out
only for 1998 and 1999. Analyses for effects on S. rio-
brave were carried out using only data from post-treat-
ment dates and treatments where this nematode was
applied. Following square-root transformations, the
nematode data for both field experiments were subjected
to split–split plot analyses of variance using the General
Linear Models Procedure of SAS (SAS Institute, 1988).
For each year, tillage regime was the whole plot factor,
nematode treatment the subplot factor, and sample date
a sub–sub plot factor. When combining years, additional
split–split plot analyses of variance were performed,
which included year as an additional factor. In 1997 and
1998, the soil moisture and organic matter variables were
subjected to a split plot analysis of variance, with tillage
as the whole plot factor and sample date as the subplot
factor. In 1999, the variables, soil moisture and water
potential, were subjected to a split–split plot analysis of
variance, with tillage as the whole plot factor, nematode
treatment as the subplot factor, and date as the sub
subplot factor. In 1999, organic matter, pH, and texture
variables were subjected to a split plot analysis of vari-
ance, with tillage as the whole plot factor and nematode
treatment as the subplot factor. When combining years
for soil characteristics, additional split plot analyses of
variance were performed which included year as an ad-
ditional factor. Soil water potential was log transformed
for analyses and all other soil characteristics were ana-
lyzed using non-transformed data. A significance level of
a ¼ 0:05 was used in all analyses of variance. A poly-
nomial regression was performed on the 1999 data to
determine the relationship between soil water potential
(log transformed) and the number of bait insects infected
by each nematode species (Abacus Concepts, 1989).
Data in text and figures are presented as non-trans-
formed means with the standard errors of the means.
3. Results
3.1. Laboratory soil baiting (Experiment 1)
3.1.1. Effect of tillage regimes on nematode infections
Although the number of infections by H. bacterio-
phora was more often numerically higher in CT
compared to NT across sampling dates, we did not
detect a significant effect of tillage on the number of
infections by H. bacteriophora (P > 0:05) (Figs. 1A
and 2).
In Experiment 1 in 1998 and 1999, the mean number
of infections by S. carpocapsae was numerically higher
in NT compared to CT. This difference was not sig-
nificant in 1999 (P > 0:05). However, when combining
the 1998 and 1999 Experiment 1 data, plus the data for
the 1998 plots resampled in 1999, there were signifi-
cantly more S. carpocapsae infections in NT com-
pared to CT (F ¼ 6:87; df ¼ 1; 6; P ¼ 0:0395) (Figs. 1B
and 2).
S. riobrave infections were detected more frequently
in CT compared to NT for Experiment 1 in 1999
(F ¼ 62:25; df ¼ 1; 3; P ¼ 0:0042) (Fig. 1C).
3.1.2. Nematode infections between years
For Experiment 1, the overall (NT and CT combined)
mean numbers (averaged over all sample dates) of bait
insects infected by S. carpocapsae and H. bacteriophora
per sample were both slightly higher in 1999 (S. carpo-
capsae: 0:198  0:027; H. bacteriophora: 0:064  0:011)
compared to 1998 (S. carpocapsae: 0:171  0:020; H.
bacteriophora: 0:040  0:011), but these differences were
not significant (P > 0:05). In contrast, the mean number
of S. riobrave infections per sample was significantly
higher in 1998 (1:024  0:050) compared to 1999
(0:220  0:023) (F ¼ 61:56; df ¼ 1; 6; P ¼ 0:0002). The
treatment by year interaction was not significant
(P > 0:05).
3.1.3. Effect of soil characteristics on nematode infections
For the 1999 data, analyses were carried out to
detect a relationship between the measured soil char-
acteristics and the number of nematode infections, re-
gardless of tillage regime. Because soil characteristics
were not monitored for each 3 m
3 m treatment area
in 1997 and 1998, the data for these years were not
included in the analyses. Polynomial regression on the
non-zero (i.e., using only soil samples with at least
one insect infected by the particular nematode species)
1999 data showed a significant quadratic relationship
between soil water potential (log transformed) and
the number of insects infected by S. carpocapsae
(P ¼ 0:0106) (F ¼ 3:408; df ¼ 2; P ¼ 0:0374) and S.
riobrave (P ¼ 0:0124) (F ¼ 3:591; df ¼ 2; P ¼ 0:0332).
The polynomial regression model was non-significant
for H. bacteriophora infections (P > 0:05). The optimal
water potential (maximum point on the quadratic
curve) was calculated to be 398:11 kPa for S. carpo-
capsae and 794:33 kPa for S. riobrave. No significant
relationship was detected between soil organic matter,
pH, or texture and the number of nematode infections
by each species.
 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11 5

Fig. 1. Infections by tillage regime (Experiment 1): mean numbers  SEM of G. mellonella larvae infected per soil sample by (A) H. bacteriophora, (B)
S. carpocapsae, and (C) S. riobrave in NT and CT during each year’s growing season.

Fig. 2. 1998 plots resampled in 1999 (Experiment 1): mean numbers  SEM of G. mellonella larvae infected per soil sample by H. bacteriophora,
S. carpocapsae, and S. riobrave in the 1998 NT and CT plots resampled in 1999, one year after nematode field applications.
6 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11

3.2. Field soil baiting (Experiment 2): effect of tillage
For Experiment 2 in 1997 and 1998, combining all
nematode treatments and sampling dates, the mean
numbers of infections by H. bacteriophora and S. rio-
brave were numerically higher in CT compared to NT,
whereas the mean number of infections by S. carpo-
capsae was numerically higher in NT compared to CT
(Fig. 3). However, these differences were not significant
(P > 0:05).
3.3. Soil characteristics
Percentage of soil moisture was higher in NT com-
pared to CT for all three years (Table 2). In 1999, soil
water potential was higher in NT compared to CT
(Table 2). Percentage of organic matter was higher in
NT compared to CT in 1998 and 1999, but was not
significantly higher in NT in 1997 (Table 2). Soil pH was
higher in NT compared to CT for 1998 and 1999 com-
bined (F ¼ 23:15; df ¼ 1; 6; P ¼ 0:0030) (mean  SE for
each year presented in Table 2). In 1999, the soil mois-
ture, water potential, organic matter, and pH were not
significantly different between the different 3 m
3 m
assay areas (P > 0:05). There was no significant differ-
ence in percentage sand, silt, and clay between the two
tillage regimes and the four blocks in 1998 and 1999 nor
between the different 3 m
3 m assay areas in 1999
(P > 0:05). Comparing the 1998 and 1999 field seasons,
there was no significant effect of year on soil moisture or
organic matter (P > 0:05).
For all three years, the average maximum soil tem-
perature (°C) across dates was higher in CT compared to
NT (Table 2). For 1997 and 1998, the average minimum
soil temperature across dates was higher in NT
compared to CT. In 1999, the average minimum soil

Fig. 3. Infections by tillage regime (Experiment 2): mean numbers  SEM of G. mellonella larvae infected per 1 m
1 m treatment plot by (A) H.
bacteriophora in 1997 and 1998, (B) S. carpocapsae in 1998, and (C) S. riobrave in 1998 in NT and CT.
 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11 7

Table 2
Comparison of soil characteristics (mean  SE) in the NT and CT regimes during the 1997, 1998, and 1999 growing seasons
Soil characteristic Year NT CT P value
Percentage of soil moisture 1997 15:4  0:92 9:77  0:82 0.0090
1998 16:63  1:17 11:73  0:98 0.0070
1999 17:8  0:49 11:73  0:47 0.0023
Soil water potential (kPa) 1999 1610:93  335:45 14359:28  3588:39 0.0023
Percentage of organic matter 1997 2:54  0:13 1:94  0:1 0.0614
1998 2:64  0:13 1:44  0:01 0.0233
1999 2:72  0:12 2:03  0:10 0.0370
Soil pH 1998 5:43  0:19 4:86  0:21 > 0:05
1999 4:81  0:06 4:47  0:05 > 0:05
Mean max temperature (°C) 1997 28:26  0:86 30:63  1:26 N/Aa
1998 29:47  0:62 30:90  0:56 N/Aa
1999 25:00  0:72 27:29  0:62 N/Aa
Mean min temperature (°C) 1997 22:78  0:87 22:41  1:03 N/Aa
1998 24:96  0:49 24:46  0:53 N/Aa
1999 20:95  0:37 23:60  0:55 N/Aa
a
Statistical comparison of temperatures between the two tillage regimes was not possible because the soil temperature was only monitored in one of
the four blocks (replicates) during the study.

temperature across dates was higher in CT compared to
NT. Combining both tillage regimes over approximately
the same time period per year 5/25–8/22, 1998 had the
highest mean minimum and maximum soil temperatures
(min ¼ 24:7  0:4; max ¼ 30:2  0:44), followed by
1997 (min ¼ 22:6  0:7; max ¼ 29:5  0:8), and then
1999 (min ¼ 22:3  0:4; max ¼ 26:2  0:5).
3.4. Rainfall
Over the same time period (5/20–8/14) for each year,
the 1999 field season received the highest total amount
of rainfall (54.7 cm), followed by 1998 (22.9 cm), and
then 1997 (14.9 cm). In 1997 and 1998, there was a
moderate amount of rain through most of the season
and then a heavy rain near the end of the season in
August (1997: 7.6 cm 8/17–8/23; 1998: 11.9 cm 8/23–8/29,
8.4 cm 8/30–9/5) (Figs. 4A and B). In contrast, in 1999,
there was a very heavy rain (>16 cm) in the middle of
the season (6/13–6/19) and then two more heavy rains
during the second half of the season (9.4 cm 7/11–7/17;
8.4 cm 8/8–8/14) (Fig. 4C).
4. Discussion
The effects of tillage practices on soil abiotic factors
were consistent with those of other studies (National
Research Council, 1989). Compared to the CT regime,
the NT regime was characterized by higher organic
matter, soil moisture content, soil pH, and soil water
potential. The maximum soil temperatures were lower
and the soil temperatures were more stable in NT
compared to CT. There was no significant difference in
soil texture between the tillage regimes.
Our study suggests that the three nematodes have
different sensitivities to the conditions created by tillage.
The different application techniques used for the en-
demic and the introduced nematodes may have influ-
enced their sensitivities to tillage. However, the objective
of this study was to evaluate the effect of tillage on an
inundatively applied, commercially available nematode,
and on naturally occurring endemic nematodes. The
application of the introduced nematode simulated a
commercial application, whereas the application of the
endemic nematodes simulated natural foci of infection.
We only rarely detected the native nematode, H.
bacteriophora, and did not detect any effect of tillage on
this nematode, although the mean number of infected
bait insects was almost always numerically higher in CT
compared to NT. The percentage of infection by H.
bacteriophora in our study was less than 0.05%. One
hypothesis for this low infection rate is that soil fauna,
microarthropods, and/or predatory nematodes may
have consumed much of the nematode-killed Galleria
and/or the emerging IJs and contributed to the low es-
tablishment potential of this nematode (Kerry, 1995).
This hypothesis is currently being investigated by one of
us (M.E. Barbercheck).
Endemic S. carpocapsae appeared to be negatively
affected by tillage. In contrast, the data suggest that
tillage favored the inundatively applied nematode, S.
riobrave. Several abiotic and biotic soil factors, i.e.,
structure, temperature, moisture, organic matter,
chemistry, and abundance and distribution of antago-
nists and potential hosts, could have contributed to
these effects.
In another study on corn, Brust (1991) found that
NT, compared to CT, was associated with a significant
increase in detection of a population of endemic H.
bacteriophora using G. mellonella as an assay insect.
Because the population Brust studied was endemic to a
research site in Clayton, North Carolina, which is ap-
proximately 66 km from the present research site, we
8 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11
Fig. 4. Rainfall during the growing seasons: total amount of rainfall (cm) per approximately one week period during the (A) 1997, (B) 1998, and (C)
1999 growing seasons.
expected a similar outcome for the endemic H. bacte-
riophora population at our site. The fact that we found
no significant effect of tillage on this nematode may be
attributable to its relatively low detection rate in our
study. The percentage infection of G. mellonella by H.
bacteriophora ranged from 39.2% to 87.2% in Brust’s
study, which is much higher than the infection rate of
less than 0.05% in our study. The different results be-
tween our study and the study by Brust could be at-
tributed to a difference in the geographic isolates, which
may vary in a number of characteristics, including sen-
sitivity to tillage. Also, the sites of the two studies may
differ in crop history, soils, antagonists, hosts, flora, etc.
The results of our study agree with those of other
studies in terms of S. carpocapsae infections (Hummel,
2000; Shapiro et al., 1999). The greater complexity of the
soil environment associated with higher levels of organic
matter and the presence of more weeds in NT may have
been factors influencing the more frequent detection of
S. carpocapsae in NT. This type of environment may
provide more hosts for endemic nematodes (Brust, 1991;
Kaya and Gaugler, 1993). Also, under a CT regime, the
soil surface has higher temperatures and lower moisture
compared to the rest of the soil profile. Hence, the fact
that S. carpocapsae has been categorized as an ‘‘am-
busher’’ (Campbell et al., 1996; Grewal et al., 1995;
Kaya and Gaugler, 1993), which is a nematode that
typically remains nearly sedentary at or near the soil
surface, may explain its relatively higher sensitivity to
tillage. On the other hand, a ‘‘cruiser’’ nematode, which
is typically more mobile and moves deeper in the soil
profile, may not be as affected by tillage because it can
move to a more stable environment of the deeper soil
profile. H. bacteriophora is considered to be a ‘‘cruiser’’
(Campbell et al., 1996; Grewal et al., 1995). The
difference in foraging behavior of these two endemic
 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11
nematodes, S. carpocapsae and H. bacteriophora, was
supported by a separate study at the same field site, for
it suggested that H. bacteriophora had a higher tendency
than S. carpocapsae to move deeper in the soil profile
(Millar and Barbercheck, 2001).
The introduced nematode, S. riobrave, appears to
share characteristics of both ambushers and cruisers
(Cabanillas et al., 1994; Grewal et al., 1995; Grewal
et al., 1994). This nematode has vertical and horizontal
dispersal abilities intermediate to those of S. carpocap-
sae and H. bacteriophora and it parasitizes both mobile
and sedentary insects effectively (Grewal et al., 1995).
This categorization of its foraging behavior was sup-
ported by a separate study at the same field site of the
present study, for it suggested that this nematode had a
tendency to move deeper in the soil profile intermediate
to those of S. carpocapsae and H. bacteriophora (Millar
and Barbercheck, 2001). The foraging behavior of S.
riobrave may partially explain the fact that it was not
negatively affected by tillage, by enabling it to move to
the more stable environment deeper in the soil profile.
Active vertical migration may serve as a means of sur-
vival for this nematode in extended dry periods (Caba-
nillas et al., 1994).
Not only was S. riobrave not negatively affected by
tillage, but its detection was favored by tillage. Be-
cause S. carpocapsae was detected less frequently in
the CT compared to NT, one could hypothesize that
decreased interspecific competition for resources may
explain the increased success of S. riobrave in CT.
However, in another study, we found that these two
nematodes did not affect each other in terms of the
number of infected insects (Millar and Barbercheck,
2001).
The origin of this nematode may help explain the
greater success of S. riobrave in CT. The original cli-
matic locality tends to determine the temperatures at
which infective juveniles became inactive (Molyneux,
1985). S. riobrave is endemic to a semi-arid subtropical
region that receives about 60–70 cm of annual rainfall. It
was originally isolated from a soil with 1.1% organic
matter, soil pH 8.3, and daytime soil temperatures of
about 35  4 °C at 5 cm deep during the corn-growing
season (Cabanillas et al., 1994). Its ability to survive and
persist in the soil after extended dry periods indicates
that S. riobrave is well adapted to this semi-arid region
(Cabanillas et al., 1994). The site of our study is a humid
temperate region that receives about 122–127 cm of
annual rainfall (State Climate Office of NC, NC State
University). Over the three years of this study, the soil
temperatures and percentage of organic matter in CT
were closer than those in NT to the soil characteristics of
the original site of isolation of S. riobrave. In addition,
the lower soil moisture in CT was probably closer to the
soil moisture of the site of origin. In summary, the CT
regime probably matched more closely the native soil
9
environment of S. riobrave than that of NT thereby
favoring its infectivity and persistence.
In our study, the optimal water potential for detec-
tion of S. riobrave was lower than that of S. carpocapsae,
i.e., detection of S. riobrave was more frequent in a drier
soil than that for S. carpocapsae. The optimal temper-
ature for growth and development of S. riobrave appears
to be about 30 ° C and this nematode seems to be nat-
urally selected for biological control at high tempera-
tures (Cabanillas et al., 1994). Optimal survival and
pathogenicity were at 25 °C for S. carpocapsae (Kung
et al., 1991) and survival of this nematode in soil de-
creased with increasing temperatures above 25 °C
(Molyneux, 1985). The temperate origin of S. carpo-
capsae perhaps favors its persistence at low temperatures
(5–25 °C) (Kung et al., 1991). In corn, S. riobrave was
more effective in controlling corn earworm than
S. carpocapsae at high temperatures (Cabanillas and
Raulston, 1996). S. riobrave also performed better than
S. carpocapsae at higher temperatures in laboratory
studies (Gouge et al., 1999; Henneberry et al., 1996).
The higher number of infections by S. riobrave in
1998 compared to 1999 may have been a result of many
factors, which include the overall higher soil tempera-
tures in 1998 compared to 1999. Another reason,
though, for its lower infectivity in 1999 compared to
1998 could have been the more fluctuating weather
conditions for the 1999 growing season, during which
there was a drought followed by very heavy rains. Not
only may these differences in soil temperature and
weather conditions between years have affected the in-
fectivity of S. riobrave directly, they may have affected
the availability of insect hosts and/or the activity of
nematode antagonists. The availability of suitable hosts
is a crucial factor that determines the survival of nem-
atodes at a site (Mracek, 1979).
Knowledge of differences in sensitivity to production
practices, such as tillage regimes and soil environmental
conditions, may be used to better select cultural prac-
tices and nematode species or strains that enhance bio-
logical control. For instance, our study supports the
notion that persistence is greater in areas similar to the
nematode’s climatic origins (Kung et al., 1991). In a
humid temperate region that experiences seasonal
drought such as the coastal plain of North Carolina, a
nematode that is adapted to a relatively warm, dry cli-
mate, such as S. riobrave, may be more efficacious when
a CT regime is used. In contrast, if one wants to con-
serve naturally occurring nematodes in this region, es-
pecially predominantly surface dwelling ones like S.
caropocapsae, our study suggests that reduced tillage
may be more appropriate. Endemic nematodes are
adapted to abiotic and biotic characteristics of the na-
tive geographical area, which in our case may have been
approached more so by a NT regime (although still far
from being undisturbed) than a CT regime. Conserving
10 L.C. Millar, M.E. Barbercheck / Biological Control 25 (2002) 1–11
endemic populations of entomopathogenic nematodes
or matching production practices to enhance the sur-
vival of specific nematodes are areas of research that
have not received much attention. However, in light of
the results of this and other studies, plus these nema-
todes’ widespread occurrence, these areas of research
appear to offer possibilities for the management of soil
insect pests.
Acknowledgments
This research was funded in part by the USDA’s
Grant No. 9702083. We thank F. Gould and D. Orr for
comments on an earlier version of the manuscript; C.
Brownie and R. Stinner for assistance with statistical
analyses; G. Naderman for use of research plots; and R.
Sheffield, J. Wang, C. Warrick, G. Linville, C. Hendrix,
J. Stout, J. Ashton, G. Garcia, N. Mendizabel, and T.
Morris for technical support.
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