Influence of humidity and a surfactant-polymer-formulationon the control potential of the entomopathogenic nematodeSteinernema feltiae against diapausing codling moth larvae(Cydia pomonella L.) (Lepidoptera: Tortricidae)
Thurkathipana Navaneethan • Olaf Strauch •
Samantha Besse • Antoine Bonhomme •
Ralf-Udo Ehlers
Received: 22 December 2009 / Accepted: 21 June 2010 / Published online: 30 June 2010
� International Organization for Biological Control (IOBC) 2010
Abstract The codling moth (Cydia pomonella L.) is
a serious pest of pome fruit. Diapausing cocooned
larvae overwinter in cryptic habitats in the soil or in
the bark of infested trees. The entomopathogenic
nematode Steinernema feltiae (Filipjev) (Rhabditida:
Steinernematidae) is used to control diapausing cod-
ling moth larvae. The objective of this study was to
define environmental conditions favouring the perfor-
mance of the nematodes. Cocooned larvae were more
susceptible than non-cocooned larvae. Susceptibility
of pupae was low. To determine the influence of
decreasing water activity (aw-value) on the activity of
the nematodes, mortality of codling moth larvae and
Galleria mellonella L. were tested in sand-sodium-
polyacrylate mixtures of variable water activity.
S. feltiae was able to infect both insects at aw-values
[0.9. Cocooned larvae of both insects died at lower
aw-values than non-cocooned larvae. Mortality of
cocooned larvae did not further increase after half an
hour of exposure to nematodes, whereas the mortality
of non-cocooned larvae increased with increasing
exposure time. LC50 and LC90 considerably decrease
with increasing RH. The negative influence of the
relative humidity (macro environment) was less
important than the effect of the water activity in the
bark substrate (micro environment). The micro envi-
ronment can be manipulated by applying S. feltiae
with higher volumes of water. A surfactant-polymer-
formulation significantly increased nematode efficacy
and can buffer detrimental environmental effects.
Keywords Apple � Biocontrol � Cocooned larvae �Relative humidity � Water activity � Bark
Introduction
The codling moth, Cydia pomonella L. (Lepidoptera:
Tortricidae), is the most serious pest of apples (Barnes
1991). In late spring, adults emerge from pupae and
begin laying eggs individually on leaves near the
fruits. After hatching, neonates enter the fruit feeding
on the fruit and seeds (Blomefield et al. 1997). The last
(fifth) instar exits from the fruit in search for a cryptic
habitat (soil or bark), where they cocoon and pupate
or spend the winter as cocooned, diapausing larvae
(Beers et al. 1993). Depending upon the temperature,
Handling Editor: Eric Wajnberg.
T. Navaneethan � O. Strauch � R.-U. Ehlers (&)
Department of Biotechnology and Biological Control,
Institute for Phytopathology, Christian-Albrechts-
University, Hermann-Rodewald Str. 9, 24118 Kiel,
Germany
e-mail: [email protected]
T. Navaneethan � R.-U. Ehlers
Department of Biology, Ghent University,
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
S. Besse � A. Bonhomme
Natural Plant Protection, Arysta Life Science EAM,
Parc d’Activites Pau Pyrenees, 64000 Pau, France
123
BioControl (2010) 55:777–788
DOI 10.1007/s10526-010-9299-5
codling moth may go through one to four generations
per growing season (Barnes 1991).
Control methods have been based predominantly
on the use of broad-spectrum insecticides (Beers et al.
1993). During the last decade, codling moth developed
simple, cross- and multiple-resistances to various
insecticides in Europe and North America (Stara and
Kocourek 2007; Mota-Sanchez et al. 2008). One of the
more environmentally friendly measures employed for
codling moth control is mating disruption (Vickers
and Rothschild 1991; Howell et al. 1992; Gut and
Brunner 1998; Calkins and Faust 2003) and the
codling moth granulosis virus (CpGV) (Arthurs and
Lacey 2004; Lacey and Shapiro-Ilan 2008; Jehle
2009). However, both methods have constrains: For
mating disruption to be effective, codling moth
population density must be sufficiently low (Carde
and Minks 1995). Although the CpGV has been used
successfully in the past, recently resistance has been
reported from orchards in Europe (Eberle and Jehle
2006; Asser-Kaiser et al. 2007).
These concerns have lead to an increasing interest
for the use of entomopathogenic nematodes (EPNs),
like Steinernema feltiae or S. carpocapsae (Nema-
toda: Rhabditida), to control codling moth. The
Steinernematids are obligatory associated with sym-
biotic bacteria Xenorhabdus spp., which contribute to
the rapid killing of their host insects (Han and Ehlers
2000). They can be easily mass cultured in vitro
(Ehlers 2001), have good safety records on non-target
organisms especially regarding their effects on pre-
dators and pollinators. In addition, no evidence exists
for mammalian pathogenicity (Ehlers 2003).
Codling moth larvae are susceptible to EPNs
(Weiser 1955; Kaya et al. 1984; Sledzevskaya 1987;
Nachtigall and Dickler 1992; Unruh and Lacey 2001).
Lacey et al. (2006a) obtained control of sentinel larvae
with EPN in field trials of up to 95%. The stage of the
codling moth best suited for control with EPNs is the
overwintering larva in cryptic habitats. Their signif-
icant reduction at this stage would provide substantial
protection to fruit in the following growing season.
Cryptic habitats are also favourable environments for
EPNs. Their potential for control of codling moth
larvae and environmental factors that limit or enhance
their activity in orchards have been elucidated by Kaya
et al. (1984), Sledzevskaya (1987), Nachtigall and
Dickler (1992), Unruh and Lacey (2001) and Lacey
et al. (2006a, b). Most of the research thus far
conducted on EPNs for codling moth control has been
done with S. carpocapsae (Weiser) and S. feltiae
(Filipjev) although other Steinernema sp. and Het-
erorhabditis bacteriophora (Poinar) have shown
promise in laboratory studies (Lacey and Unruh 1998).
Lacey et al. (2006a, b) and Reggiani et al. (2008)
reported that S. feltiae has the ability to control
diapausing codling moth larvae in the period of early
spring (March) or mid October. During this time of
the year, the control potential of S. carpocapsae is
often reduced due to low temperature (Vega et al.
2000; Reggiani et al. 2008). S. feltiae has been
reported to be more effective than S. carpocapsae
when temperature is below 15�C (Grewal et al. 1996;
Lacey and Unruh 1998).
The main obstacles for successful codling moth
control with EPNs are low temperature but also low
relative humidity (RH) resulting in quiescence of the
nematodes and then lethal desiccation of the infective
dauer juveniles (DJs) before they have penetrated the
host. Webster (1973) proposed manipulation of
habitats where EPNs will be applied, to favour DJ
survival and infectivity. Manipulation of macro
environment (weather) is impossible. Maintaining
the micro climate (substrate humidity) may give
success in the control of codling moth using EPNs.
Thus application technology and possibly improved
formulation can help to overcome the problems with
low RH.
The overall objective of this investigation was to
test an innovative formulation technology (Schroer
and Ehlers 2005; Schroer et al. 2005) for S. feltiae to
improve their performance at low humidity. Our first
objective was to study the susceptibility of cocoon
and non-cocooned larvae and pupae of the codling
moth to S. feltiae. The determination of the ideal
susceptible stage is important to obtain optimum
control. Second, we studied the pathogenicity of
S. feltiae at different humidities of the bark measured
as water activity (aw-value) and determined the
humidity supporting nematode performance to cause
50% and 90% mortality in cocooned and non-
cocooned larvae of codling moth and Galleria
mellonella L. (Lepidoptera: Pyralidae). The water
activity is defined as the relative proportion of
unbound water in a sample. The third objective was
to study the time required by S. feltiae to infect
cocooned and non-cocooned larvae. In a next exper-
iment we studied the influence of relative humidity
778 T. Navaneethan et al.
123
(RH) on the lethal concentration for codling moth.
Finally the effect of a formulation was investigated in
a bark chip assay and under semi-natural conditions
on apple tree trunks.
Materials and methods
Insects
Several batches of codling moth last instars were
provided by Natural Plant Protection S. A. (France) in
boxes (30 cm length 9 20 cm width 9 4 cm height)
filled with artificial diet. Larvae were stored at 4�C and
kept at 15�C for 24 h before use. Within the artificial
medium, larvae would not cocoon. For formation of
cocoons, last instars were transferred to bark pieces for
72 h at room temperature (25�C). For formation of
pupae they were stored for additional 72 h. For assays
started with cocooned larvae, larvae from the diet were
first released between two layers of cotton wool
separated by tissue paper in plastic boxes (30 9
20 9 4 cm) at 25�C for 72 h for cocooning. Each
cocooned last instar was then separated from the cotton
wool using surgical scissors and kept at 15�C until used.
G. mellonella larvae were produced on non-sterile
artificial diet as described by Han and Ehlers (2000).
Entomopathogenic nematodes
A commercial strains of S. feltiae that had been
produced in liquid culture by e-nema GmbH (Sch-
wentinental, Germany) was used. DJs were stored in
water on a shaker (180 rpm) at 4�C until use. The
EPNs were counted using the method described by
Kaya and Stock (1997).
Bark piece assay
Blocks of apple bark were broken into pieces of
approximately 4 cm length and 2 cm width. The bark
originated from apple trees from a commercial
orchard (Obsthof Schuster, Schwentinental, Germany),
which had been felled two years before. The pieces
were kept at 70�C for two days for drying and
elimination of other organisms.
Twenty last instars of the codling moth were
transferred to each glass bottle of 8 cm diameter and
7 cm height filled with 10 g dried bark pieces and
incubated at 25�C for 72 h to allow for cocooning.
For nematode application an airbrush sprayer was
used at 2 atm pressure with different volumes of
water. Controls were treated in the same way but
without nematode. The outer surface of the bark
remained on the top to mimic natural condition of the
tree. The lid of the bottles was perforated for air
exchange. The treated bottles were stored for 72 h at
15�C at different RH in climate chambers (KBWF
720, Binder, Tuttlingen, Germany). Afterwards the
bottles were removed from the climate chambers and
stored at room temperature (25�C) under ambient RH
until the adults emerged.
Susceptibility of different codling moth stages
to S. feltiae
Last instar codling moth exit from the fruit and search
for a cryptic habitat to cocoon and overwinter. This
experiment was conducted, to investigate, which is
the most susceptible developmental stage of the
insect after reaching the last instar. The bark piece
bioassay was used and treated bottles were stored at
100% RH at 15�C. The experiment was carried out
three times, each with three replicates of 60 cocooned
larvae or pupae or non cocooned larvae. Controls
were treated in the same way but without nematode.
Effect of lower water activity on nematode
performance
In order to assess the activity of the nematodes at
reduced humidity, they were tested at different water
activity. As adjusting and maintaining different
aw-values in sand or bark was almost impossible, the
experiments were conducted in a sand-sodium poly-
acrylate mixture at a ratio of 20:1 (SSPA mixture).
The sodium polyacrylate (Evonik-Degussa, Essen,
Germany) has the ability to absorb water hundred
times of its mass (Zhang et al. 2009). Different aw-
values were established by adding different amounts
of water to the SSPA mixture. Table 1 illustrates the
relation between percentage of water in SSPA and
the aw-value. The aw-values were measured using the
Aqualab water-activity-meter (Decagon Devices Inc.,
Pullman, WA, USA) at constant temperature.
The experiments were carried out using cocooned
and non-cocooned last instars of G. mellonella and
C. pomonella. To form cocoons, G. mellonella larvae
Influence of humidity and a surfactant-polymer-formulation 779
123
were kept in the SSPA mixture for three days prior to
DJ application (unlike C. pomonella, last instars of
G. mellonella form cocoons in sand). Ten insects per
dish were tested in the SSPA mixture in 5 cm diameter
Petri dishes at aw of 0.6 to 1.0 for G. mellonella and
from 0.8 to 1.0 for C. pomonella. Fifty DJs/larva were
applied in 300 ll water into the centre of the Petri dish.
Untreated controls received water only. Treated dishes
were sealed with Parafilm and placed at 100% RH at
15�C. After exposure for 72 h, larvae were assessed
for mortality. Cocooned larvae were removed from
the silk before assessment. The experiments were
repeated three times with three replicates at the
different water activity values. The controls were kept
in the same way but without nematode.
Influence of exposure time on mortality
Depending on the ambient humidity of the air (RH) or
substrate (aw-value), DJs can rapidly die after appli-
cation, and nematode performance much depends on
the time the DJs need to penetrate an insect. The
influence of exposure time on mortality at a given
number of DJs was assessed in bark chip bioassays.
Mortality of ten cocooned or ten non-cocooned
codling moth larvae was assessed after different
exposure time to 1,000 DJs (100 DJs/larva) in 4 ml
water at 15�C and 100% RH. Assays were performed
in bark chips not larger than 5 9 5 9 2 mm in size.
Ten g bark was filled into each bottle and then ten
larvae were placed on the top of the bark, on which
another 10 g of bark was lined above the larvae in
order to best resemble the natural condition of the
cryptic habitat. After application of S. feltiae DJs,
larvae were removed at 30, 60, 180, 240, 480 min and
placed between two layers of blotting-paper in 15 cm
Petri dishes for rapid desiccation at 25�C. In order to
prevent the larvae from escaping, the dishes were
covered with perforated lids and sealed with Parafilm.
After 72 h the mortality was assessed. This bioassay
was carried out three times for each exposure time
with three bottles containing ten cocooned or non-
cocooned larvae.
Efficacy at different nematode concentrations
and relative humidities
Efficacy was measured as LC50 (concentration of DJs
killing 50% of the insects at a given time) at different
RH in the bark piece assay. Maintenance of different
levels of RH was done according to Winston and Bates
(1960). Two bottles filled with either glycerol (60%
RH), KNO3 (80% RH), Ca(HPO4)2 (90% RH) or
distilled water (100% RH) were transferred to humid-
ity chambers (25 9 15 9 20 cm) and kept at 15�C for
one week in the climate chamber before the assay was
started to allow the build-up of a stable RH. Then 20
cocooned codling moth were exposed to 5, 10, 20, 40
and 80 S. feltiae infective juvenile/larva sprayed with
an airbrush sprayer at 2 atm pressure in 2 ml of water
(20% water content of the bark). Controls were treated
with the same amount of water. After application the
bottles were stored at 60, 80 and 100% RH. This
bioassay was carried out three times with three bottles
each, containing 20 cocooned codling moths.
Effect of formulation at different relative
humidities and water activity
The effect of a surfactant-polymer-formulation (SPF)
on the efficacy against codling moth larvae was
assessed at different relative humidity and application
volume in the bark piece bioassay. SPF was prepared
using 0.3% Rimulgan� (Temmen GmbH, Hatters-
heim, Germany) and 0.3% Xanthan (UD Chemie
GmbH, Worrstadt, Germany) according to Schroer
and Ehlers (2005). The bark was treated with 0.5, 1
and 2 ml of water or SPF, with and without nema-
todes, establishing a moisture content of 5, 10 and 20%
of moisture (v/w) in the bark pieces and corresponding
to aw-values of 0.6, 0.8 and 0.99. The water activity
was measured throughout the experiment to ensure
that the bark maintained the particular level of
moisture. Measurements were done as described
above. The treated bottles were stored at 15�C at 60,
Table 1 The percentage of water and corresponding aw-value
in sand-sodium polyacrylate mixtures
aw Value % Water aw Value % Water
0.6 1.5 0.94 7.0
0.7 2.0 0.95 8.0
0.8 3.0 0.96 9.0
0.9 5.0 0.97 10.0
0.91 5.5 0.98 11.0
0.92 5.75 0.99 12.0
0.93 6.0 1.00 [12.0
780 T. Navaneethan et al.
123
80 and 100% RH. This bioassay was carried out three
times with three bottles containing 20 cocooned
codling moth last instars.
Effect of the surfactant-polymer-formulation
in tree trunk assay
To test the formulation under semi-natural conditions,
tests were conducted with apple tree trunks of 20 cm
length at 15�C and 60% and 80% RH. Trunks were
obtained from apple trees from the commercial
orchard, which had been felled two years before. They
were heat treated in the same way as described for the
bark. Then both edges of the tree trunk were covered
with wax to avoid entry of the larvae into the wood
instead of the bark. Single tree trunks were then
transferred into plexiglass cylinders (25 cm high and
16 cm diameter) in pots and covered with gauze on the
top of the cylinders to prevent the larvae from escaping.
Then 25 mature codling moth larvae were allowed to
cocoon in the bark of the trunk at 25�C for 72 h.
Afterwards the tree trunks were treated with 2,500 DJs
of S. feltiae in 10 ml SPF or water. Controls were
treated either with water or SPF only. The tests were
repeated three times with three replicates in each
repetition.
Data analysis
Abbott’s formula was used to correct the data for
control mortality (Abbott 1925). Data obtained as
percentages were arcsine transformed prior to the
statistical analysis using XLStat version 7 and R-
Project version 2.9.2 (University of Auckland, New
Zealand). ANOVA was used to identify general effects
and interactions. Tukey’s HSD (Honestly Significant
Difference) test was performed for multiple compar-
isons (P B 0.05). Probit analysis (Finney 1971) was
used to calculate the aw-value or lethal concentration
needed to cause 50% and 90% mortality of insects.
Results
Susceptibility of different codling moth stages
to S. feltiae
The susceptibility of codling moth pupae and coco-
oned and non-cocooned larvae differed significantly
(F2, 26 = 131.7, P \ 0.0001). Cocooned last instars
were most susceptible (mortality of 78.0% ± 1.0
SD), followed by non-cocooned larvae (54.1% ± 0.2
SD). Pupae were the least susceptible against the
S. feltiae (4.5% ± 2.5 SD).
Effect of lower water activity on nematode
performance
In untreated controls, the water activity (aw-values) in
the sand-polyacrylate mixture had no significant effect
on insect mortality (data not shown). S. feltiae was
unable to infect C. pomonella and G. mellonella larvae
at aw values B0.9. The water activity which allowed
90 % nematode efficacy was C0.98 against both
insects and cocooned and non-cocooned stages.
Cocooned last instars were more susceptible than
the non-cocooned last instars in both insects tested
(Table 2). The SH50 (supporting humidity to cause
50% nematode-caused mortality) and SH90 were
always significantly lower for cocooned than for
non-cocooned instars (F1, 23 = 142.21, P \ 0.0001).
Significant differences were also obtained between the
insect species (F1, 23 = 17.95, P = 0.001) indicating
that nematodes need a higher humidity to infest
C. pomonella than G. mellonella larvae. Significant
differences were obtained for mortality between the
aw-values (F14, 89 = 38.8, P \ 0.001).
Influence of exposure time on mortality
Exposure time to S. feltiae in bark pieces generally
had a significant effect on insect mortality (F4, 89 =
9.33, P \ 0.0001). A strong difference was recorded
between cocooned and non-cocooned larvae (Fig. 1).
Exposure of cocooned larvae for half an hour was
sufficient for an infection of nearly 80% of the larvae,
whereas less than 20% were infected of the non-
cocooned after the same exposure time. The differ-
ences between cocooned and non-cocooned larvae
were highly significant (F1, 89 = 242.79, P \0.0001).
Efficacy at different nematode concentrations
and relative humidities
The mortality in untreated controls was not signif-
icantly affected by the varying relative humidities
(data not shown). The nematode concentrations
(F4, 119 = 65.6, P \ 0.0001) (Fig. 2) and the relative
Influence of humidity and a surfactant-polymer-formulation 781
123
humidity (F3, 119 = 7.64, P \ 0.0001) (Fig. 3) had a
significant effect on nematode efficacy against
C. pomonella in bark pieces. There was no significant
interaction between concentration and relative humid-
ity (F12, 119 = 1.03, P = 0.42). The LC50 and LC90
significantly increased with lower relative humidity
(Fig. 3). The major increase of the LC50 and LC90 was
observed by lowering the RH from 80% to 60%.
Effect of formulation at different relative
humidities and water activity
In general, the formulation (nematodes in water
compared to nematodes in surfactant-polymer), the
RH (60, 80 and 100%) and the water activity (5, 10 and
20% moisture of the bark) significantly influenced the
nematode efficacy in this trial (Fig. 4C, D; Table 3). In
untreated controls no significant effect of the formu-
lation, RH or water activity of the bark on the mortality
was observed (Fig. 4A, B) (F8, 80 = 0.55, P = 0.81).
No significant interaction occurred between the for-
mulation, RH and the water activity of the bark
(Table 3). The strongest and consistent effects were
observed for the SPF formulation and the water activity
of the bark. Nematodes applied in SPF always were
more effective than nematodes in water (Fig. 4C, D).
Increasing moisture of the bark significantly improved
nematode performance. Increasing RH also increased
nematode efficacy, but this effect was less pronounced
(Table 3).
Effect of the surfactant-polymer formulation
in tree trunk assay
When trunks had been sprayed with water or SPF
only, no significant difference in mortality was
observed (Fig. 5). Only the SPF-formulated nema-
todes caused a significant effect on cocooned codling
(F3, 71 = 3.45, P \ 0.0001). Nematodes applied in
water had no significant effect compared to the
untreated controls. No significant effect of the
relative humidity was recorded.
Discussion
Steinernema feltiae is the most promising nematode
for the control of the codling moth (Lewis et al. 1995,
Table 2 Water activity (aw) at which 50% (SH50) and 90% (SH90) of non-cocooned and cocooned last instars of Galleria mellonellaand Cydia pomonella died after exposure to Steinernema feltiae (50DJs/larva) in 300 ll of water in sand-sodium polyacrylate mixture
72 h at 100% RH and 15�C
SH50 (mean ± SE) SH90 (mean ± SE) R2 Pearson v2 df Probit model
Intercept Slope
Galleria mellonella
Non-cocooned larvae 0.95 ± 0.003 0.99 ± 0.01 0.96 24.0 9 -29.4 30.9
Cocooned larvae 0.93 ± 0.003 0.98 ± 0.01 0.97 20.7 9 -25.0 26.8
Cydia pomonella
Non-cocooned larvae 0.96 ± 0.02 1.00 ± 0.002 0.94 31.8 9 -30.6 31.9
Cocooned larvae 0.95 ± 0.001 0.99 ± 0.002 0.97 21.2 9 -30.2 31.8
For Probit analysis data from three replicates with different nematode and insect batches were combined. Insect mortality was
assessed at 11 different aw-values (in total 90 insects per aw-value, 30 per replicate). The regression coefficient was always
significantly different from 0 (Pearson v2 test, P \ 0.01)
0
20
40
60
80
100
1/2 1 2 4 8
Mor
talit
y (%
)
Exposure time (h)
Fig. 1 Abbott corrected mortality (mean ± SE) of cocooned
and non-cocooned Cydia pomonella larvae after exposed to
Steinernema feltiae (100 DJs/larva) for different time intervals
at 80% RH and 15�C. Different letters above bars indicate
significant differences according to Tukey’s HSD test
(P B 0.05). Mortality assessed 72 h after exposure at 25�C
782 T. Navaneethan et al.
123
Campbell and Gaugler 1997, Lacey et al. 2006a, b;
Reggiani et al. 2008). Therefore the study was
performed with this species only. Another reason to
prioritize S. feltiae over for instance S. carpocapsae
is its activity down to 8�C (Grewal et al. 1994,
Reggiani et al. 2008), as temperature can drop below
15�C in autumn during the time of control of
overwintering codling moth and S. carpocapsae is
active only above 15�C (Vega et al. 2000).
The presented results provide useful information
for future improvement of EPN application in
orchards to control overwintering larvae of the
codling moth. To achieve optimum control of codling
moth, EPNs have to be applied against the most
0
20
40
60
80
100
5 10 20 40 80
a a a a b
0
20
40
60
80
100
5 10 20 40 80
a a b bc c
0
20
40
60
80
100
5 10 20 40 80
a a b b c
0
20
40
60
80
100
5 10 20 40 80
a a b bc c
M
orta
lity
(%)
Mor
talit
y (%
)
Concentration (IJs/larva)Concentration (IJs/larva)
B
C
A
D
Fig. 2 Abbott corrected mortality (mean ± SE) of cocooned
Cydia pomonella larvae after 72 h exposure to different
concentration (DJs/larva) of Steinernema feltiae at 60% (A),
80% (B), 90% (C) and 100% (D) relative humidity at 15�C
in 20% moist bark pieces. Different letters above bars indicate
significant differences according to Tukey’s HSD test
(P B 0.05)
0
5
10
15
Relative humidity (%)
0
20
40
60
80
50 60 70 80 90 100 50 60 70 80 90 100
Relative humidity (%)
Infe
ctiv
e ju
veni
les
/ lar
va
a b c d a b c cA B
Fig. 3 Influence of the relative humidity on the lethal
concentration at which 50% (LC50) (A) or 90% (LC90) (B)
of cocooned Cydia pomonella larvae died after 72 h and 15�C
in 20% moist bark pieces. Error bars indicate the 95%
confidence limits obtained by Probit analysis. Different letters
above the data points indicate significant differences according
to Tukey’s HSD test (P B 0.05)
Influence of humidity and a surfactant-polymer-formulation 783
123
susceptible stage. Lacey et al. (2005) reported that
pupae are less susceptible than cocooned last instars,
when the insects were exposed to S. carpocapsae at
25�C in perforated cardboard strips fixed to tree
trunks in the field. Our findings corroborate these
data, however, they reported 63.1 ± 1.7% mortality
of pupae with S. carpocapsae, whereas in this study
obtained only 4.5% with S. feltiae. This might have
two reasons. Cardboards have higher water holding
capacity than bark, probably providing prolonged
conditions of favourable humidity. Also, EPN per-
form better at 25�C than at 15�C (Mahar et al. 2007),
the temperature used in this study. Henneberry et al.
(1995) reported lepidopteran pupal stages to be less
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
60 80 100
D
Mor
talit
y (%
)M
orta
lity
(%)
Relative humidity (%) Relative humidity (%)
BA
C
60 80 100
60 80 100 60 80 100
Fig. 4 Mortality of
cocooned Cydia pomonellalarvae in bark pieces at
15�C in controls (A, B) 72 h
after application of water
(A) or surfactant polymer
formulation (SPF) (B) and
in treatments (C, D) with
100 DJs/larva Steinernemafeltiae in water (C) or SPF
formulated (D). The tests
were carried out at a RH of
60, 80 and 100% and 5, 10
and 20% moisture of the
bark pieces
Table 3 Statistical analysis (ANOVA) of the effect of relative humidity (RH), moisture in the bark pieces and formulation on the
efficacy of Steinernema feltiae against cocooned Cydia pomonella larvae assessed after Abbott correction of mortality
Effect df F value P value
Main effects
Relative humidity (RH) 2, 143 3.31 0.0392*
Moisture in the bark 2, 143 34.53 5.822e-13*
Formulation 1, 143 18.69 2.855e-05*
Interactions
RH 9 moisture in the bark 4, 143 1.16 0.3310
RH 9 formulation 2, 143 0.17 0.8407
Moisture in the bark 9 formulation 4, 143 2.11 0.1248
Combined effect
RH 9 moisture in the bark 9 formulation 4, 143 1.2 0.3139
EPNs in water corrected with control water, EPNs in SPF corrected with control SPF
P values followed by * indicate significant effects at P B 0.05
784 T. Navaneethan et al.
123
susceptible to EPNs. During this stage the insect is
well protected by it exoskeleton, which functions as a
barrier for EPNs penetration. Consequently, over-
wintering codling moth should be controlled with
EPN before they pupate in spring.
The active mature last instars come out from the
fruit during the month of August searching for cryptic
habitats to spin cocoons for hibernation. With the
beginning of October all larvae have left the apple
fruits (van Frankenhuyzen and Stigter 2002). In all
tests, cocooned larvae were more susceptible than
non-cocooned. Thus the control of codling moth
larvae should not start before the majority of the
larvae have cocooned, which, in Europe, is not before
late September.
EPNs are adapted to the soil environment, where
they are not exposed to extreme temperature changes,
fluctuation of moisture and ultraviolet light like on
the foliage. These factors can considerably reduce the
efficacy of EPNs (Kung et al. 1991; Lello et al. 1996).
However, EPNs can also be used to control foliar
insect pests (Kaya 1985; Baur et al. 1995; Schroer
and Ehlers 2005). But foliar application of EPNs is
only effective under specific conditions. Several
studies have noted the importance of maintaining
moisture for DJs survival and infectivity (Kaya et al.
1984; Nachtigall and Dickler 1992; Lacey and Unruh
1998; Unruh and Lacey 2001; Lacey et al. 2006a).
Lacey et al. (2006b) have reported effective control
of codling moth when combining irrigation and
mulch to extend the survival of DJs. Thus irrigation
and mulching the orchard is a possibility to increase
EPNs efficacy. EPN application in the evening will
also enhance their performance, as temperatures drop
during the night and humidity raises.
Another factor influencing EPN performance is the
available water in the substrate (bark), which can be
assessed by measuring the water activity (aw-value).
This value best indicates the microclimate in the bark
of the tree trunk. It is common understanding that
nematodes need a water film to migrate. For the first
time the influence of the water activity on nematode
efficacy was tested. In order to fine-tune the aw-value
it was necessary to use sand supplemented with
sodium polyacrylate, because the absorptive capacity
of either sand or bark was too low to produce
differential water activity values. The results indicate
that S. feltiae is not depending on a water film
(aw-value \1) to infest a potential host but can still
infest insects at aw-values down to 0.9. However, a
higher aw-value of 0.99 was necessary to obtain
control of 90%. Humidity requirements were slightly
lower for cocooned larvae and also for G. mellonella.
The reason probably is that the microclimate
inside the cocoon provides higher humidity. Larval
G. mellonella are much bigger than C. pomonella and
could therefore also have produced a more humid
environment than the smaller codling moth. In
general, the results indicate the importance of the
microclimate for the performance of EPNs.
Bark used for the assays was obtained from
relatively old and dead trees, which had been felled
two years before the bark was taken. After collection
of the bark from the field in June, but before heat
treatment, a water activity of 0.935 was assessed,
which is too low to support EPNs activity. Bark from
a living tree had an aw-value of 0.965, which is higher
and would be enough to obtain 50% mortality of
cocooned larvae, but still not sufficient to support
EPNs activity to obtain 90%. However, with the
appropriate amount of water applied, the water
activity should be sufficiently high to obtain higher
control than 50% in bark of living trees. Thus results
from the laboratory assays provided with this study
can be considered to be ‘‘worst-case-scenarios’’.
When EPNs are applied in open fields, a loss of
infectivity due to desiccation can rapidly occur
(Solomon et al. 2000). However, as water is used to
apply EPNs, supportive conditions can probably
prevail long enough for successful penetration of
the insect. The success of an application will then
depend on the time DJs need to reach the insect.
0
10
20
30
40
50
60
70
W SPF W+N SPF+N
Mor
talit
y (%
)
Fig. 5 Mortality of cocooned Cydia pomonella larvae
(mean ± SE) in controls (W and SPF) and treatments
(W ? N and SPF ? N) 72 h after application of water (W),
surfactant-polymer formulation (SPF), Steinernema feltiae(100 DJs/larva) in water (W ? N) or SPF (SPF ? N) at
relative humidity of 60% and 80% at 15�C in tree trunks
Influence of humidity and a surfactant-polymer-formulation 785
123
Schroer and Ehlers (2005) working with larvae of
Plutella xylostella observed that S. carpocapsae was
able to cause maximum mortality already within 1 h
after application. Prolongation of the exposure to
even 20 h after application did not result in any
increase in insect mortality. In this study similar
results were obtained, but only for cocooned larvae.
Exposure of cocooned larvae for half an hour to the
nematodes was sufficient for the infection of nearly
80% of the last instars of the codling moth and no
significant increase was recorded even after 8 h of
exposure. In contrast, mortality of non-cocooned
larvae reached less than 20% after half an hour and
increased significantly over time. However, 30 min
might not necessarily be sufficient time for the DJs to
infect the cocooned larvae. Inside the cocoon humid-
ity is probably maintained for a longer period of time
than near the non-cocooned individuals. After remov-
ing the insects from the sprayed bark, DJs can have
stayed inside the cocoon and infected later. There-
fore, these results again indicate that the cocoon
provides favourable microclimatic conditions for
S. feltiae to cause mortality.
The sustainable use of S. feltiae against overwin-
tering codling moth in apple orchards will depend on
an exact definition of environmental conditions that
support control at levels [80%. From the previously
discussed results it is obvious that the water activity
should be [0.98 and that these conditions should at
least last for 1 h after spraying. The objective of the
tests in bark was to further investigate the influence
of the ambient RH on the LC50 of the nematodes.
Tests applied with 2 ml of water resulted in a water
content of 20% and a water activity of 0.99. The
efficacy was positively correlated with an increasing
number of DJs and an increasing RH. Significant
differences were obtained between the concentrations
at equal conditions of RH. The number of S. feltiae
DJs necessary to cause 50% (LC50) or 90% (LC90)
mortality of cocooned codling moth larvae increased
with decreasing RH. A comparison of the results
obtained at different RH at equal DJ concentration
indicates a significant difference in mortality at a RH
of 60% with all other conditions of RH, but not
between 80, 90 and 100% (analysis not shown).
When analysing the concentration-response, no sig-
nificant differences were observed in the mortality of
codling moth larvae between 5 and 40 DJs at
60%RH. These results corroborate with results
reported by Baur et al. (1995). At a RH \76% the
mortality of P. xylostella was not correlated to the
number of DJs (S. carpocapsae) when applied to
the leaf surface of cabbage. From a practical point of
view, the results imply that EPNs should only be
sprayed at a RH [80%. When sufficient water is
used, then relatively high mortality can be expected.
Increasing amounts of DJs cannot provide better
control when environmental conditions are
unfavourable.
Additives like humectants can help to reduce the
negative impact of low RH or low water activity on
nematode performance (Baur et al. 1995; Lacey et al.
2005). We tested the SPF formulation developed by
Schroer et al. (2005), which not only prolonged
survival of the DJs, but also produced favourable
environmental conditions for invasion of P. xylostel-
la. The effect was correlated to the viscosity of the
adjuvant. An application of EPNs with this adjuvant
can extend the life period on a glabrous surface and
significantly increase the mortality compared to the
application of water only. This was also observed in
the bark piece assays. A significantly higher mortality
was recorded when S. feltiae were applied with SPF
in bark pieces. Increasing moisture of the bark also
increased the efficacy and the effect was even more
pronounced. At 20% moisture content of the bark,
mortality significantly increases in treatment with or
without SPF. The least pronounced effect was
recorded for the RH. At low RH the water applied
with the EPNs evaporates quickly, the bark dries
inhibiting movement of the DJs. This effect can be
reduced when applying EPNs with SPF. As a
consequence for practical application, the amount of
water applied with the nematodes should be high and
the use of the formulation is recommended.
In order to test the efficacy on tree trunks, S. feltiae
was tested at 60% and 80% RH. A significant effect
was recorded only for EPN in the SPF formulation,
supporting the results in the bark piece assay.
Mortality reached only 32% at 80% RH. Tree trunks
were obtained from older trees, which had dried
already in the field and their humidity was even more
reduced by drying them at 70�C before use in the
experiments. The amount of only 10 ml water for
EPN application could not provide favourable con-
ditions for higher larval control. The trunk pieces had
several cracks, which provided cryptic habitats for
the larvae to cocoon in the core of the tree trunk.
786 T. Navaneethan et al.
123
S. feltiae could not cause infection as the cores of the
tree trunk were completely dry (aw-value in the core
was 0.4 to 0.5). Many of the adult insects were even
unable to emerge from the core after hatching,
probably the reason why the mortality in the controls
was high as well. But more important, the positive
effect of the SPF was confirmed. Host seeking of
S. feltiae is an active process and SPF supported
infestation of cocooned codling moth larvae com-
pared to EPN application with water only. SPF can
reduce detrimental environment conditions on the
bark. Introduction of a biological control method,
which is highly dependant on favourable weather
conditions (80% RH) is less accepted by growers.
The study indicates that the macro environment (RH)
is of less importance for EPN performance than the
micro environment of the bark (water activity). The
microclimate can be manipulated by either increasing
the amounts of application water and by using the
SPF formulation.
Acknowledgements These results are part of the Master
thesis of the first author, who is grateful to the Flemish
Interuniversity Council—University Development Cooperation
(VLIR-UOS) for granting a scholarship to carry out studies
within the Postgraduate International Nematology Course
(http://www.pinc.ugent.be). Thanks are also due to Ms. Mar-
tina Wittke, Mr. Michel Wingen for their technical support and
to Dr. Mario Hasler (University Kiel) for support with the
statistical analysis.
References
Abbott WS (1925) A method for computing the effectiveness
of an insecticide. J Econ Entomol 18:265–267
Arthurs SP, Lacey LA (2004) Field evaluation of commercial
formulations of the codling moth granulovirus: persis-
tence of activity and success of seasonal applications
against natural infestations of codling moth in Pacific
Northwest apple orchards. Biol Control 3:388–397
Asser-Kaiser S, Fritsch E, Undorf-Spahn K, Kienzle J, Eberle
KE, Gund NA, Reineke A, Zebitz CPW, Heckel DG,
Huber J, Jehle JA (2007) Rapid emergence of baculovirus
resistance in codling moth due to dominant sex-linked
inheritance. Science 317:1916–1918
Barnes MM (1991) Tortricids in pome and stone fruits codling
moth occurrence host race formation and damage. In: van
der Geest LPS, Evenhuis HH (eds) Tortricid pests their
biology natural enemies and control. Elsevier, Amster-
dam, NL, pp 313–327
Baur ME, Kaya HK, Thurston GS (1995) Factors affecting
entomopathogenic nematode infection of Plutella xylo-stella on a leaf surface. Entomol Exp Appl 77:239–250
Beers EH, Brunner JF, Willett MJ, Warner GM (1993) Orchard
pest management: a resource book for the Pacific North-
west Yakima Washington. Good Fruit Grower, Wash-
ington, pp 276
Blomefield TL, Pringle KL, Sadie A (1997) Field observations
on oviposition of codling moth Cydia pomonella (Lin-
naeus) (Lepidoptera: Olethreutidae) in an unsprayed apple
orchard in South Africa. Afr Entomol 5:319–336
Calkins CO, Faust RJ (2003) Overview of area wide programs
and the program for suppression of codling moth in the
western USA directed by the United States Department of
Agriculture—Agricultural Research Service. Pest Manag
Sci 59:601–604
Campbell JF, Gaugler RR (1997) Inter-specific variation in
entomopathogenic nematode foraging strategy: dichotomy
or variation along a continuum? Fundam Appl Nematol
20:393–398
Carde RT, Minks AK (1995) Control of moth pests by mating
disrution: successes and constrains. Ann Rev Entomol
40:559–585
Eberle KE, Jehle JA (2006) Field resistance of codling moth
against Cydia pomonella granulovirus (CpGV) is autoso-
mal and incompletely dominant inherited. J Invertebr
Pathol 93:201–206
Ehlers R-U (2001) Mass production of entomopathogenic
nematodes for plant protection. Appl Microbiol Biotech-
nol 56:623–633
Ehlers R-U (2003) Biocontrol nematodes In: Hokkanen HMT,
Hajek AH (eds) Environmental impacts of microbial
insecticides need and methods for risk assessment. Kluwer
Acadamic Press, NL, pp 177–220
Finney DJ (1971) Probit analysis. Cambridge Univ Press,
London, UK
Grewal PS, Lewis EE, Gaugler R, Campbell JF (1994) Host
finding behaviour as a predictor of foraging strategy in
entomopathogenic nematodes. Parasitology 108:207–215
Grewal PS, Gaugler R, Wang Y (1996) Enhanced cold toler-
ance of the entomopathogenic nematodes Steinernemafeltiae through genetic selection. Ann Appl Biol 129:
335–341
Gut LJ, Brunner JF (1998) Pheromone-based management of
codling moth (Lepidoptera: Tortricidae) in Washington
apple orchards. J Agric Entomol 15:387–405
Han R, Ehlers R-U (2000) Pathogenicity development and
reproduction of Heterorhabditis bacteriophora and
Steinernema carpocapsae under axenic in vivo condition.
J Invertebr Pathol 75:55–58
Henneberry TJ, Lindegren JE, Jech LF, Burke RA (1995) Pink-
bollworm (Lepidoptera: Gelechiide) cabbage-looper and
beet army worm (Lepidoptera: Noctuidae) pupal suscep-
tibility to steinernematid nematodes (Rhabditida: Stein-
ernematidae). J Econ Entomol 88:835–839
Howell JF, Knight AL, Unruh TR, Brown DF, Krysan JL, Sell
CR, Kirsch PA (1992) Control of codling moth in apple
and pear with sex pheromone-mediated mating disruption.
J Econ Entomol 85:918–925
Jehle JA (2009) The promise, practice and prospects of bac-
uloviruses in biocontrol. IOBC/WPRS Bull 45:16
Kaya HK (1985) Soil ecology. In: Gaugler R, Kaya HK (eds)
Entomopathogenic nematodes in biological control. CRC
Press, Boca Raton, FL, pp 93–116
Influence of humidity and a surfactant-polymer-formulation 787
123
Kaya HK, Stock SP (1997) Techniques in insect nematology.
In: Lacey LA (ed) Manual of techniques in insect
pathology. Academic Press, London, UK, pp 281–324
Kaya HK, Joos JL, Falcon LA, Berlowitz A (1984) Suppres-
sion of the codling moth (Lepidoptera: Olethreutidae)
with the entomogenous nematode Steinernema feltiae(Rhabditida: Steinernematidae). J Econ Entomol 77:
1240–1244
Kung SP, Gauger R, Kaya HK (1991) Effect of soil tempera-
ture moisture and relative humidity on entomopathogenic
nematode persistence. J Invertebr Pathol 57:242–249
Lacey LA, Shapiro-Ilan DI (2008) Microbial control of insect
pests in temperate orchard systems: Potential for incor-
poration into IPM. Annu Rev Entomol 53:121–144
Lacey LA, Unruh TR (1998) Entomopathogenic nematodes for
control of codling moth Cydia pomonella (Lepidoptera:
Tortricidae): effect of nematode species, concentration,
temperature and humidity. Biol Control 13:190–197
Lacey LA, Neven LG, Headrick HL, Fritts R (2005) Factors
affecting entomopathogenic nematodes (Steinerneniati-
dae) for control of overwintering codling moth (Lepi-
doptera: Tortricidae) in fruit bins. J Econ Entomol
98:1863–1869
Lacey LA, Arthurs SP, Unruh TR, Headrick H, Fritts R (2006a)
Entomopathogenic nematodes for control of codling moth
(Lepidoptera: Tortricidae) in apple and pear orchards:
Effect of nematode species and seasonal temperatures,
adjuvants, application equipment, and post-application
irrigation. Biol Control 37:214–223
Lacey LA, Granatstein D, Arthurs SP, Headrick H, Fritts R
(2006b) Use of entomopathogenic nematodes (Steinerne-
matidae) in conjunction with mulches for control of
overwintering codling moth (Lepidoptera: Tortricidae).
J Entomol Sci 41:107–119
Lello ER, Patel MN, Mathews GA, Wright DJ (1996) Appli-
cation technology for entomopathogenic nematodes
against foliar pests. Crop Prot 15:567–574
Lewis EE, Grewal PS, Gaugler R (1995) Hierarchical order of
host cues in parasite foraging strategies. Parasitol
110:207–213
Mahar AN, Jan ND, Mahar GM, Hullio MH, Lanjar GL, Buriro
AH (2007) Effectiveness of entomopathogenic nematodes
against the larvae of mustard beetle Phaedon cochleriae at
different temperatures. Int J Agric Biol 9:851–856
Mota-Sanchez D, Wise JC, Poppen RV, Gut LJ, Hollingworth
RM (2008) Resistance of codling moth Cydia pomonella(L) (Lepidoptera: Tortricidae) larvae in Michigan to
insecticides with different modes of action and the impact
on field residual activity. Pest Manag Sci 64:881–890
Nachtigall G, Dickler E (1992) Experiences with field appli-
cations of entomoparasitic nematodes for biological con-
trol of cryptic living insects in orchards. Acta Phytopathol
Entomol Hung 27:485–490
Reggiani A, Curto G, Vergani S, Caruso S, Boselli M (2008)
Effectiveness of entomopathogenic nematodes in the
control of Cydia pomonella overwintering larvae in
Northern Italy. IOBC/WPRS Bull 31:287–293
Schroer S, Ehlers R-U (2005) Foliar application of the
entomopathogenic nematode Steinernema carpocapsaefor the biological control of diamond back moth larvae
(Plutella xylostella). Biol Control 33:81–86
Schroer S, Ziermann D, Ehlers R-U (2005) Mode of action of a
surfactant-polymer formulation to support performance of
the entomopathogenic nematodes Steinernema carpocap-sae for control of diamondback moth larvae (Plutellaxylostella). Biocontrol Sci Technol 15:601–613
Sledzevskaya ER (1987) Study of the factors determining the
activity of the nematode Neoaplectana carpocapsae and
its efficacy against orchard insect pests. In: Sonin MD (ed)
Helminths of insects. Amerind Publishing Co, New Delhi,
India, pp 152–155
Solomon A, Salomon R, Paperna I, Glazer I (2000) Desiccation
stress of entomopathogenic nematodes induces the accu-
mulation of a novel heat-stable protein. Parasitol
121:409–416
Stara J, Kocourek F (2007) Insecticidal resistance and cross-
resistance in populations of Cydia pomonella (Lepidop-
tera: Tortricidae) in Central Europe. J Econ Entomol
100:1587–1595
Unruh TR, Lacey LA (2001) Control of codling moth Cydiapomonella (Lepidoptera : Tortricidae) with Steinernemacarpocapsae: Effects of supplemental wetting and pupa-
tion site on infection rate. Biol Control 20:48–56
Van Frankenhuyzen A, Stigter H (2002) Schadliche und nut-
zliche Insekten und Milben an Kern-und Steinobst. Ulmer
Verlag, Stuttgart, Germany, p 288
Vega FE, Lacey LA, Reid AP, Herard F, Pilarska D, Danova E,
Tomov R, Kaya HK (2000) Infectivity of a Bulgarian and
an American strain of Steinernema carpocapsae againstcodling moth. BioControl 45:337–343
Vickers RA, Rothschild GLH (1991) Use of sex pheromones
for control of codling moth. In: van der Geest LPS,
Evenhuis HH (eds) Tortricid pests their biology natural
enemies and control. Elsevier, Amsterdam, NL, pp 339–
354
Webster JM (1973) Manipulation of environment to facilitate
use of nematodes in biocontrol of insects. Exp Parasitol
33:197–206
Weiser J (1955) Neoaplectana carpocapsae n. sp. (Anguillu-
lata: Steinernematidae) novy cizopasnik housenek obalece
jablecneho Carpocapsae pomonella L. Vestnik Ces-
koslovaenske Zoolo Spolecynosti 19:44–52
Winston PW, Bates DH (1960) Saturated solutions for the
control of humidity in biological research. Ecology
41:232–237
Zhang CX, Zhang WX, Pan ZY, Zhang XY, Liu J, Yue CW
(2009) Research on various factors influencing the mois-
ture absorption property of sodium polyacrylate. Sci
China Chem 52:1000–1008
788 T. Navaneethan et al.
123
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