Egyptian Journal of
Biological Pest Control
Bayramoglu et al. Egyptian Journal of Biological Pest Control (2018) 28:17
DOI 10.1186/s41938-017-0021-0
RESEARCH
Open Access
Efficacy of native entomopathogenic
nematodes from Turkey against the alder
leaf beetle, Agelastica alni L. (Coleoptera:
Chrysomelidae), under laboratory
conditions
Zeynep Bayramoglu1, Ismail Demir1* , Cihan Inan2 and Zihni Demirbag1
Abstract
The alder leaf beetle, Agelastica alni L. (Coleoptera: Chrysomelidae), is one of the most defoliator pests of oak and alder
trees. In the present study, the efficacies of three native strains of entomopathogenic nematodes, Heterorhabditis
bacteriophora (ZET35), Steinernema feltiae (ZET31), and Steinernema websteri (AS-1), were tested against pre-pupae and
adults of A. alni. Experiments were conducted by four concentrations under laboratory conditions in 2015. Four different
temperature regimes were tested at concentration of 1000 infective juveniles (IJs)/ml under laboratory conditions. It was
observed that pre-pupae were more sensitive than adults in all tests. Based on screening tests, S. websteri was the most
effective isolate on both pre-pupae and adults of A. alni at concentration of 1000 IJs/ml with 79.17 and 71.11% mortality,
respectively. It caused the highest mortality values at all temperatures, except for 30 °C against pre-pupae and adults.
Results of the present study suggested that S. websteri and H. bacteriophora had significant potentials against A. alni.
Keywords: Entomopatogenic nematodes, Biological control, Agelastica alni, Forest pests
Background
The alder leaf beetle, Agelastica alni L. (Coleoptera:
Chrysomelidae), is widely distributed in the Europe,
Caucasus, Siberia, North-Eastern Kazakhstan, and the
USA (Sezen et al. 2004). A. alni feeds on variety of broadleaf species including hazelnut (Corylus spp.) and alder
(Alnus spp.) during spring and summer seasons and occasionally damages other plant species and genera such as
Betula pendula (Fagales: Betulaceae), Salix caprea
(Malpighiales: Salicaceae), Populus spp. (Malpighiales:
Salicaceae), and Tilia spp. (Malvales: Malvaceae) (Medvedev
1983). Since the pest has high reproductive rate, it causes
severe defoliation to host plants in native habitats. Adults
and larvae of A. alni attack host plant or its products with
significant commercial value and cause mortality or predispose host to infestation by other pests. They cause loss of
* Correspondence: idemir@ktu.edu.tr
1
Karadeniz Technical University, Faculty of Science, Department of Biology,
Trabzon, Turkey
Full list of author information is available at the end of the article
markets due to quarantine status. The final damage of the
pest is unsightly and repeated heavy defoliation and can
cause growth loss in large trees and mortality of young
plants. In addition, foliar injury can be unsightly in residential areas, parks, and forest recreation sites.
Pests are generally controlled by chemical pesticides
that may lead to developing resistance by the target pest
in addition to causing harms to human and environment
(Ffrench-Constant et al. 2004). Therefore, researchers
have been studying to develop alternatives for pesticides.
An alternative method to chemical pesticides is biocontrol and the microbial biocontrol agents with no harmful
effects on human health and environment. The common
microbial biocontrol agents are viruses, bacteria, fungi,
and nematodes (Vega and Kaya 2012).
Entomopathogenic nematodes (EPNs) from the families
Steinernematidae and Heterorhabditidae are among such
alternatives as biological control agents against insect pests,
especially the ones inhabiting soil or in the cryptic habitats
(Kaya and Gaugler 1993). EPNs have been tested
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
Bayramoglu et al. Egyptian Journal of Biological Pest Control (2018) 28:17
successfully as potential biological control agents of insect
pests in Turkey (Kepenekci and Susurluk 2006; Yilmaz
et al. 2009; Gokce et al. 2013; Erbas et al. 2014; Kepenekci
et al. 2015).
Up to now, control strategies applied for A. alni are
still insufficient to prevent its damage. However, increasing interest in developing environmentally safe pest control methods has inspired us to study the potential of
different biological agents against the pest.
The present study aimed to evaluate the efficacy of
three EPNs isolated from Turkey against pre-pupae and
adults of A. alni under laboratory conditions.
Materials and methods
Collection of insects
A. alni adults and larvae were collected from infested
Alnus glutinosa trees in the vicinity of Trabzon, Turkey,
between March and June, 2015. The larvae were carefully
handpicked from undersides of leaves by a soft fine-tipped
paintbrush, and the adults were caught by a sweep net. Insect samples were placed into plastic boxes (20 cm deep
and 20 cm diameter) with ventilated lids and freshly
collected plane leaves as food. Afterwards, the collection
was transported to the laboratory. Healthy adults and prepupae were acclimated for 2 days to the laboratory conditions then healthy ones were used for bioassays.
Nematode isolates
Heterorhabditis bacteriophora (ZET35), Steinernema feltiae (ZET31), and Steinernema websteri (AS-1) isolates,
used in the experiments, were maintained in the collection
of the entomopathogens, Department of Biology, Faculty
of Science at Karadeniz Technical University (Erbas et al.
2014; Gokce et al. 2015). Nematode cultures were maintained in last instar greater wax moth larvae, Galleria mellonella L. (Lepidoptera: Pyralidae) (Woodring and Kaya
1988), and infective juveniles were stored in distilled water
Page 2 of 5
at 10 °C. Before starting the experiments, the nematodes
were kept at 25 °C.
Laboratory bioassay
Experiments were carried out for all isolates to determine
their pathogenicity against A. alni. Plastic boxes (4 cm
deep and 3.4 cm diameter) were used for the experiments.
Each box was filled with 40 g sterilized sandy soil and adjusted to 7% (w/v) moisture by adding distilled water.
The efficacy of EPNs was tested at three concentrations: 200, 500, and 1000 infective juveniles (IJs) in 1 ml
of water per plastic box (10, 25, and 50 IJs per individual
in 50 μl of distilled water). For the control groups, only
50 μl of water was added to each box. The treated boxes
were kept at room temperature for 1 h, and then a single
pre-pupa or an adult were placed on the sand surface in
the boxes capped with a lid. Screening tests were conducted at 25 °C, and mortality rates were assessed on
7 days after treatment. Dead insects were dissected
under the stereomicroscope to ascertain that mortality
resulted from nematodes’ infection.
To determine the effect of increasing temperature,
1000 IJs ml−1 were applied to the boxes, which were
then placed in incubators at 15, 20, 25, or 30 °C. Seven
days after nematode treatments, the sandy soil in each
box was poured out and mortality rate of the tested insects was recorded. Experiments were performed with
30 pre-pupae or adults for each nematode concentration
and temperature regimes. The experiments were repeated three times on different dates.
Data analysis
Mortality data were corrected by Abbott’s formula
(Abbott 1925). The data were subjected to ANOVA and
subsequently to Duncan multiple range tests (p < 0.001)
to compare isolates with each other and the control
group. Lethal concentrations (LC50) for EPNs against
Fig. 1 Efficacy of H. bacteriophora (ZET35), S. feltiae (ZET31), and S. websteri (AS-1) on pre-pupae of A. alni at 25 °C
Bayramoglu et al. Egyptian Journal of Biological Pest Control (2018) 28:17
Page 3 of 5
Fig. 2 Efficacy of H. bacteriophora (ZET35), S. feltiae (ZET31), and S. websteri (AS-1) on adults of the alder leaf, A. alni beetle at 25 °C
pre-pupae or adults of A. alni were calculated by probit
analysis. All analyses were performed using SPSS 23.0
statistical software (IBM Corporation, Armonk).
Results and discussion
The virulence of the three native EPN isolates (H. bacteriophora (ZET35), S. feltiae (ZET31), and S. websteri
(AS-1)) against pre-pupae and adults of A. alni at four
different concentrations (0, 200, 500, or 1000 IJs ml−1)
and four different temperature regimes (15, 20, 25, and
30 °C) were studied under laboratory conditions. Also,
the detected effects of increasing concentration of infective juveniles and temperature on virulence were determined (Figs. 1 and 2).
Results showed that pre-pupae were more sensitive
than adults in all tests. Mortality rate of the pre-pupae
and adults increased by increasing gradually the concentration of nematodes. The highest and fastest mortality
(79.31%) in pre-pupae was obtained by S. websteri (AS1) at 50 IJs/individual (F = 3571, 86; df = 6, 35; p < 0.001)
(Fig. 1). Additionally, application of 25 and 50 IJs/individual of S. websteri during the same period resulted in
63.33 and 68.18% mortality, respectively. It was also observed that H. bacteriophora caused 52.22, 64.77, and
77.01% mortality at concentrations of 10, 25, and 50 IJs,
respectively (F = 1317, 40; df = 3, 35; p < 0.001). Adults of
A. alni were also found to be susceptible to S. websteri
(AS-1), with mean percentage mortality ranging between
44.44 and 71.11% (F = 2121, 4; df = 3, 35; p < 0.001)
(Fig. 2). Also, lethal concentration (LC50) of each entomopathogenic nematode for A. alni was determined
(Table 1). The isolates, H. bacteriophora (ZET35), S. feltiae (ZET31), and S. websteri (AS-1) killed pre-pupae at
LC50 values of 201, 8064, and 64 IJs per pre-pupa,
respectively. These results indicated that pre-pupae of
the pest were more susceptible than adults. Also, LC50
calculations showed that H. bacteriophora and S. websteri had better values against both pre-pupae and adult
of A. alni.
The virulence of H. bacteriophora, S. feltiae, and S.
websteri to pre-pupae and adults of A. alni with 1000
IJs ml−1 was determined, under laboratory conditions at
four different temperatures (15, 20, 25, and 30 °C). Different temperatures caused significant sensitivity rates
on pre-pupae. Mortality rates with H. bacteriophora increased with the increasing temperature. It reached
100% at 30 °C (F = 302, 07; df = 3, 35; p < 0.001). This
value was the highest mortality among all tests. The
highest mortality with S. feltiae was 70.11% at 15 °C (F
= 1185, 7; df = 3, 35; p < 0.001), and mortality rates decreased with the increasing of temperature. S. websteri
also caused approximately the same mortality rates
(from 70 to 85.56%) on pre-pupae of A. alni (Fig. 3).
Table 1 Calculated LC50 values for pre-pupae and adults of A.
alni treated with three EPN isolates, Heterorhabditis bacteriophora
(ZET35), Steinernema feltiae (ZET31), and Steinernema websteri
(AS-1)
EPNs
Stages
95% limit
Slope Intercept X2
LC50
(IJ ml−1)
Lower Upper
ZET35 Pre-pupae 201.6
df
101.8
399.2
1,6
1,1
0,3 1
6220.4
849.3
ND
0,5
2,8
0,5 1
ZET31 Pre-pupae 8604.6
429.1
Adult
ND
0,3
3,5
0,9 1
6926.7 ND
0,6
1,7
0,7 1
Pre-pupae 64.3
13.9
297.0
0,7
3,6
0,6 1
Adult
110.1
970.3
0,9
2,5
0,3 1
Adult
AS-1
ND not determined
ND
326.8
Bayramoglu et al. Egyptian Journal of Biological Pest Control (2018) 28:17
Page 4 of 5
Fig. 3 Mean mortality rate of three native strains on pre-pupae of A. alni treated at a concentration of 1000 IJs/ml at four tested temperatures
Adults exhibited less sensitivity at all tested temperatures. Mortality ratio by H. bacteriophopra increased with
increasing of temperature and reached 40% at 30 °C (F =
1588, 08; df = 3, 35; p < 0.001). As in the pre-pupae, mortality rates of adults by S. feltiae decreased as the
temperature increased. The highest mortality by S. websteri was provided at 25 °C on adults (68.54%) (F = 11, 35,
10; df = 3, 35; p < 0.001).
The highest pathogenic effect was recorded by S. feltiae on pre-pupae at 15 °C, while the mortality caused
by S. feltiae on both pre-pupae and adults decreased by
increasing the temperature. This exhibits that S. feltiae
is more active and effective at lower temperatures. An
optimal biological activity of S. feltiae was detected in
the temperature at 25 °C (Belair et al. 2003). Besides
these two situations, the pathogenic effect of S. websteri
increased to 16% on pre-pupae and changed from 70.4
to 86.66% mortalities by increasing the temperature. S.
websteri caused high mortality of pre-pupae of A. alni at
different temperatures. Some groups also investigated
the efficacy of some EPN species/strains on A. alni.
Doucet et al. (1996) reported that H. bacteriophora was
found to be effective at temperature between 18 and 30 °C
with an optimum range of 22–26 °C. Tomalak (2004)
tested the infectivity and biocontrol potential of H. megidis and S. feltiae on A. alni under the laboratory and
Fig. 4 Mean mortality rate of three native strains on adults of A. alni treated at a concentration of 1000 IJs/ml at four tested temperatures
Bayramoglu et al. Egyptian Journal of Biological Pest Control (2018) 28:17
semi-field experiment conditions and reported that H.
megidis caused a significant mortality against last instar
larvae of A. alni. He also demonstrated that 50 IJs of S. feltiae (ScP) strain against last instar larvae of A. alni caused
56–66% mortality. Our results reported that S. feltiae
caused the highest mortality (69.6%) at 15 °C. Also, H. bacteriophora and S. websteri had 98.51 and 86.66% mortality
on pre-pupae of A. alni at 30 °C at the same period, respectively. Also, Choo et al. (2002) demonstrated that S.
carpocapsae and H. bacteriophora were found to be highly
virulent against different larval stages (first, second, and
third larval instars) of Agelastica coerulea, where the both
isolates had high mortality rates, while H. bacteriopohora
caused 100% mortality on all larval stages. In agreement
with other groups, our results showed that 15 °C recorded
lower mortality than 20 or 25 °C (Choo et al. 2002; Belair
et al. 2003; Trdan et al. 2009).
Trdan et al. (2009) performed laboratory studies to determine the effectiveness of S. feltiae, S. carpocapsae, H.
bacteriophora, and H. megidis on Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) which is another
member of Chrysomelidae family at three different temperatures (15, 20, and 25 °C). Although his studies showed
that the lowest efficacy against all stages of the insect was
at 15 °C, in the present tests, S. feltiae (ZET31) gave 70.11
and 64.44% mortality on pre-pupae of A. alni at 15 and
20 °C, respectively. Also, our study showed that the highest mortality on pre-pupae of A. alni provided as 98.51%
mortality for H. bacteriophora at 30 °C.
The effectiveness of EPNs in controlling chrysomelids
is affected by biotic and abiotic conditions. One of the
most important abiotic factors is temperature, which influenced the activity of the nematodes. Increasing the
temperature from 15 to 30 °C caused a significant increase in pre-pupae and adult mortality rates of A. alni
after the treatments of S. websteri and H. bacteriophora
isolates. In contrast, mortality caused by S. feltiae ZET31
was significantly lower at 30 °C than at 15 °C against
pre-pupae and adult of A. alni (Figs. 3 and 4).
Conclusions
Obtained results may suggest that H. bacteriophora
(ZET35) and S. websteri (AS-1) can be used as biological
control agents against pre-pupae and adults of A. alni.
Future field studies are recommended with the aim of
finding a better biological control agent against A. alni
and for using these nematode isolates as biopesticides.
Authors’ contributions
All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Page 5 of 5
Author details
1
Karadeniz Technical University, Faculty of Science, Department of Biology,
Trabzon, Turkey. 2Karadeniz Technical University, Faculty of Science,
Department of Molecular Biology and Genetics, Trabzon, Turkey.
Received: 27 June 2017 Accepted: 6 December 2017
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