Influence of humidity and a surfactant-polymer-formulation on the control potential of the...

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


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


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


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)


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


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


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.


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.

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