CC BY
107Russian Journal of Nematology, 2020, 28 (1), 29 - 39
Are synthetic VOC, typically emitted by barley (Hordeum vulgare L.) roots, navigation signals for entomopathogenic nematodes (Steinernema and
Heterorhabditis)?
Anamarija Jagodic1, Ivana Majic2, Stanislav Trdan1 and Ziga Laznik1
'Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia 2Section of Entomology and Nematology, Faculty of Agriculture, University of Osijek, Vladimira Preloga 1, 31000, Osijek, Croatia
e-mail: ziga.laznik@bf.uni-lj.si
Accepted for publication 13 March 2020
Summary. We tested the chemotactic response of infective juveniles (IJ) of the entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae to the synthetic volatile compounds (VOC) (dimethyl sulfide, hexanal, 2-pentylfuran, and (E)-non-2-enal) typically emitted by barley roots. For the purpose of our investigation, we used single VOC and their blends. We hypothesised that attraction behaviour exhibited by the EPN toward the tested VOC could be related to the species/strains and would vary with foraging strategy and VOC. Heterorhabditis bacteriophora was the most mobile species in our assay. We confirmed differences among commercial and soil-isolated strains of EPN. The movement of EPN toward different VOC was influenced by the species/strain of EPN. Our investigation showed synergistic effect of dimethyl sulfide as an attractant for EPN. The data showed that chemosensation is more a species/strain-specific trait than a host searching strategy. All compounds tested in our assay influenced the movement of IJ, suggesting that synthetic VOC, typically emitted by barley roots, could play an important role in EPN navigation.
Key words: barley, chemosensation, dimethyl sulfide, (E)-non-2-enal, hexanal, infective juveniles, 2-pentylfuran.
A complex mixture of volatile organic compounds (VOC) is emitted by various plant organs (seeds, flowers, leaves, stems and roots), ranging from terpenoids, fatty acid derivatives and sulfur compounds to phenylpropanoids (Qualley & Dudareva, 2009; Gfeller et al, 2013). The plant emission of VOC can be induced by physiological or environmental stresses. The composition and quantity of released VOC vary depending on the stress type (herbivory attack, dehydration, heat, etc.) (Ferry et al., 2004; Jansen et al., 2011).
In recent decades, many studies have shown that VOC emitted by plants upon a herbivory attack are signalling compounds at different trophic levels. These signals are utilised by plants for the development of host plant resistance (Dicke & Baldwin, 2010; Danner et al., 2015). However, due to technical limitations, the belowground VOC potentially responsible for such interactions have been partially neglected (Rasmann et al., 2005; Crespo et al., 2012; Jagodic et al., 2017).
The release of root VOC can mediate different plant interactions: direct or indirect defence of roots against soil herbivores (Rasmann et al., 2005), resistance of roots against pathogens (Vilela et al., 2009), plant-plant competition (Ens et al., 2009) and symbiotic relationships (Asensio et al., 2012). Furthermore, VOC emitted by roots can also attract herbivores (Weissteiner et al., 2012; Gfeller et al., 2013) and their natural enemies (Rasmann et al., 2005; Ali et al., 2011; Laznik & Trdan, 2016a, b).
Ali et al. (2010) found that citrus roots emit several terpenes in the surrounding soil in response to feeding by the root weevil Diaprepes abbreviatus. Gfeller et al. (2013) reported the identification of 29 different VOC from isolated 21-d-old barley (Hordeum vulgare L.) roots. Interestingly, in the olfactometer assay, these VOC proved to be attractants for an economically important insect pest, wireworms (larvae of Agriotes sordidus, Coleoptera: Elateridae). On the other hand, several studies proved (Ali et al., 2011;
© Russian Society of Nematologists, 2020; doi: 10.24411/0869-6918-2020-10002
Laznik & Trdan, 2016a, b; Jagodic et al., 2017) that these VOC affect the behaviour of entomopathogenic nematodes (EPN), insects' natural enemies (Dillman et al., 2012). Crespo et al. (2012) reported that Brassica nigra roots damaged by cabbage root fly larvae emit different VOC (several sulfur-containing compounds and glucosinolate breakdown products). Interestingly, Jagodic et al. (2017) proved that these VOC are repellents to the EPN S. kraussei.
EPN are soil-inhabiting, lethal insect parasites from the families Steinernematidae and Heterorhabditidae, and they have been proven to be very effective in biological control of soil and above-ground insect pests (Koppenhoffer et al., 2004; Laznik & Trdan, 2011). EPN are mutually associated with bacteria of the family Enterobacteriaceae; the bacterium carried by Steinernematidae is usually a species of the genus Xenorhabdus, and Photorhabdus is carried by Heterorhabditidae (Koppenhofer, 2007). The third juvenile stage of EPN is referred to as the infective juveniles (IJ). IJ of both genera release their bacterial symbionts in the insect host body and develop into fourth-stage juveniles and adults. The insects die mainly due to a septicaemia (Dillman et al., 2012). When the food source is depleting, next-generation IJ leave the insect cadavers into the surrounding environment and search for a new host.
Nematodes use chemotaxis as the main sensory mode for an orientation toward their hosts. CO2 has proved to be one of the most important cues and has been shown to attract several species of EPN (Hallem et al., 2011). Furthermore, several investigations showed that VOC released from insect-damaged roots influence the movement of EPN as attractants (e.g., [E]-B-caryophyllene, 4,5-dimethylthiazole) or repellents (e.g., hexanal, terpinolene) (Rasmann et al., 2005; Hallem et al., 2011; Jagodic et al., 2017). Movement of EPN can be influenced also by electrical fields with comparable electrical potentials to those observed on the surface of some insects or the membrane potentials of the roots (Shapiro-Ilan et al., 2012).
More than two decades ago, several papers were published on the host-finding behaviour of IJ of EPN in terms of ambush and cruise foraging strategies (Grewal et al., 1994; Campbell & Gaugler, 1997). According to their results, cruise foragers tend to move actively through the substrate and use distant volatile cues to assist in host-finding. Ambush foragers, by contrast, tend to remain near the substrate surface where they lift their body into the air, which facilitates attachment to passing insects. Most species, including all Heterorhabditis
species, are regarded as cruisers, a few such as S. carpocapsae are classed as ambushers, while others such as S. feltiae employ an intermediate foraging strategy (Campbell & Gaugler, 1997). Recently, however, several studies indicate that this distinction may be over-simplified (Kruitbos et al., 2009; Wilson et al., 2012). Wilson et al. (2012) proposed that many species will show different behaviours depending on the substrate in which they forage, and these differences may be related to different volatile signals (e.g., VOC, CO2) used by the EPN as foraging cues. Although the ambush-cruise foraging strategy paradigm may be imperfect, it is premature to replace it with a habitat specialisation paradigm to explain interspecific differences in EPN behaviour.
The choice of the VOC used in our investigation was based on the research of Gfeller et al. (2013), who analysed VOC emissions by barley roots. The major VOC were fatty acid-derived compounds. To increase our knowledge of belowground-induced VOC, we describe our study of the chemotactic behaviour of S. feltiae, S. carpocapsae and H. bacteriophora toward four VOC released from barley roots. EPN are natural enemies of wireworms (Ansari et al., 2009) and our goal was to provide useful information about the impact of VOC released by insect-damaged barley roots also on EPN. For the purpose of our investigation, we used single VOC and their blends. The aims of our investigation were: i) to study the effect of different EPN foraging strategies (ambush, intermediate, or cruise) toward the tested VOC; ii) to determine the behaviour difference between natural EPN population and laboratory-produced strains; iii) to determine whether chemotaxis is species-specific; iv) to assess whether the VOC from barley roots have any effect on the behaviour of the tested EPN, and v) to determine if there is any synergistic effect of different VOC mixed together on EPN behaviour.
MATERIAL AND METHODS
Source and maintenance of entomopathogenic
nematodes. We tested three commercial EPN species (S. feltiae, S. carpocapsae and H. bacteriophora), obtained from Koppert B.V. (Berkel en Rodenrijs, the Netherlands 2017). One H. bacteriophora strain (HbV) isolated from the soil was included in our investigation as well. HbV strain was isolated in the forest soil near the ancient city, today's archeological site Vucedol (NE Croatia, 45°20'13.4" N; 19°03'29.8" E). All EPN strains were reared using the final-instar larvae of lesser wax moth (Achroia grisella [Fabricius, 1794])
(Lepidoptera: Pyralidae) (Khatri-Chhetri et al., 2011). Lesser wax moth larvae were placed in plastic boxes containing a standard synthetic diet (Sehnal, 1966) and reared in growth chamber (BTES-e - frigomat, Termo Medicinski Aparati Bodalec) under a 12:12 h L:D photoperiod at 25°C and 70% RH. The IJ were stored at 4°C at a density of 2000 IJ ml-1. We used only IJ that were less than 2 weeks old (Laznik & Trdan, 2013). The concentration of the EPN suspension was calculated according to Laznik et al. (2010). The nematode viability was determined prior to the initiation of the chemotaxis experiment, and only nematode stocks with > 95% survival were used.
Tested volatile compounds. The choice of the VOC used in our investigation was based on the research of Gfeller et al. (2013), who analysed VOC emissions by roots of H. vulgare plants. Root-emitted VOC were detected using head-space solid-phase-microextraction (HS-SPME) and gas chromatography mass spectrometry (GC-MS). The results of their investigation showed that 21-d-old barley roots release 29 different VOC. According to their results on the concentration estimation of different VOC, we decided to test four VOC; [1] dimethyl sulfide, [2] hexanal, [3] 2-pentylfuran, and [4] (E)-non-2-enal. To perform our investigation, we used synthetically produced compounds (Merck Life Science UK Limited), which were tested at a concentration of 0.03 ppm (the average concentration of the VOC in soil located 10 cm from the root system) (Weissteiner et al., 2012). The 0.03-ppm concentration was reached by dissolving pure VOC in dimethyl sulfoxide. The suspension was then mixed in the vortexer and immediately used in the laboratory bioassay. For the purpose of our investigation, we used single VOC and their blends in a ratio 1:1.
Chemotaxis assay. The chemotaxis assay was based on an assay developed by O'Halloran & Burnell (2003) and modified by Jagodic et al. (2017). The assay plates were Petri dishes (diam. = 9 cm) containing 25 ml of 1.6% technical agar (Biolife, Milano, Italy), 5 mM potassium phosphate (pH 6.0), 1 mM CaCl2 and 1 mM MgSO4. The experimental arena is presented in Figure 1. Each treatment included five replicates, and the experiment was repeated three times. The Petri dishes were kept in dark, in a rearing chamber (RK-900 CH, Kambic Laboratory equipment, Semic, Slovenia) at 20°C and 75% RH. The nematodes were allowed to move freely for 24 h, after which they were immobilised by placing the Petri dishes in a freezer at -20°C for 3 min. The number of nematodes in the treatment and control areas were
Fig. 1. Three circular marks (diam. = 1 cm) were made on the bottom of the plate: first in the centre, then on the right and on the left side of the Petri dish at 1.5 cm from its edge. A 10-^l drop of a tested substance (or their blends) at a concentration of 0.03 ppm was placed on the right side of the agar surface (treated area), and 10 ^l of distilled water (control area) was placed on the left side of the agar surface (both sides are considered outer circles) (Jagodic et al., 2017). The volatile organic compounds (VOC) were immediately applied to the agar plates before the application of the nematodes (Laznik & Trdan, 2016b); 50-^l drop of 100 infective juveniles (IJ) was placed in the centre of the agar surface (inner circle). In the control treatment, 10 ^l of distilled water was applied to the control and treated area and a 50-^l drop of 100 IJ was placed in the centre of the agar surface.
counted using a binocular microscope (Nikon C-PS) at x25 magnification. The specific chemotaxis index (CI) was based on an assay developed by Bargmann & Horvitz (1991) and modified by Laznik & Trdan (2016b). CI was calculated as follows:
(% of IJ in the treatment area - % of IJ in the control area) (100%)-1
The CI varied from 1.0 (perfect attraction) to -1.0 (perfect repulsion), and in the experiments described here, the compounds are classified as follows: > 0.2 as an attractant, from 0.2 to 0.1 as a weak attractant, from 0.1 to -0.1 as having no effect, from -0.1 to -0.2 as a weak repellent and <-0.2 as a repellent to EPN (Laznik & Trdan, 2016b; Jagodic et al., 2017).
Statistical analysis. For all of the treatments and controls, preferential movement of nematodes from
the inner to the outer circle of the Petri dish (i.e., a directional response) was determined using a paired t-test comparing the number of IJ in the inner versus the outer circles (Statgraphics Plus for Windows 4.0; Shapiro-Ilan et al., 2012; a = 0.05). Additionally, to compare the response levels among the foraging strategies, the average percentage of IJ that moved to the outer circle or stayed in the inner circle was calculated for each dish. The data were compared through an analysis of variance (ANOVA, a = 0.05) (Laznik & Trdan, 2016b). Additionally, ANOVA was performed on the CI values to compare the response among the different EPN species to the tested VOC and their blends; the means were separated by Duncan's multiple range test with a significance level of P < 0.05 (Laznik & Trdan, 2013). The data are presented as the mean ± S.E. All of the statistical analyses were performed using Statgraphics Plus for Windows 4.0 (Statistical Graphics Corp., Manugistics, Inc., Rockville, MD, USA). A multivariate cluster analysis (hierarchical clustering, Ward's minimum variance method) was performed to investigate linkage of EPN species and VOC based on CI. A constellation plot was carried out to visualise these clusters using JMP ver. 13 (SAS Institute, Cary, NC, USA).
RESULTS
Diversity of movement among EPN species and their foraging strategies. The analyses of the pooled results showed that directional movement in response to VOC and their blends from inner (central part of the Petri dish) to the outer test circles were influenced by different factors (foraging strategy, EPN species and VOC) and their interactions (Table 1). Based on the t-test results (t = 76.21; P < 0.0001; a = 0.05), statistically significant differences were observed among the average percentage of IJ in the inner (77.7 ± 0.9%) and outer (22.3 ± 0.8%) circles after 24 h. The different foraging strategies of the tested EPN species influenced the movement of IJ to the outer circles; the greatest movement was observed among cruisers (28.2 ± 1.3%), while, on the other hand, only 15.4 ± 1.0% of intermediates moved from inner to outer circles. Interestingly, cruisers (23.3 ± 1.0%) were more mobile than intermediates. Among the tested EPN species, the most mobile species was H. bacteriophora. The results showed that there were no statistically significant differences in the movement from inner to outer circles among both
H. bacteriophora strains tested in our investigation (commercial strain: 29.4 ± 1.2%; HbV strain: 27.0 ±
I.4%). The least mobile species was S. feltiae (Fig. 2).
Table 1. ANOVA results for the directional movement in
response to volatile organic compounds (VOC) by infective juveniles of the entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae from the inner to the outer circle of the Petri dish (df for the error term: 959).
Source F df P
Foraging strategy 244.35 2 <0.0001
Species 290.23 3 <0.0001
VOC 34.03 15 <0.0001
Temporal replication Spatial replication 0.38 0.73 4 2 0.8230 0.4821
VOC x species 22.4 45 <0.0001
Foraging strategy x VOC 7.64 30 <0.0001
Fig. 2. Percentage of different infective juveniles (IJ) of the entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae in outer circles after 24 h. Error bars are corresponding to SE. The capital letters indicate statistically significant differences (P < 0.05) among different EPN species. Sc - S. carpocapsae, Sf -S. feltiae, Hb - H. bacteriophora (commercial strain), HbV - H. bacteriophora (Vucedol strain).
An important factor in the movement of EPN in our investigation proved to be the type of the VOC. The highest movement of EPN was observed in the mixture of 2-pentylfuran and dimethyl sulfide where we recorded 31.3 ± 2.7% of IJ in the outer circles. Only 15.5 ± 1.3% of IJ movement was recorded in hexanal (Fig. 3).
Chemotaxis index. The results show that CI values were influenced by different factors and their
100
LLj 30
& 30
j 70
E so £
o 50
C D DN H HD HDN HN HP HPD HPDN HPN N P PD PDN PN
VOCs and their blends
Fig. 3. Percentage of infective juveniles (IJ) of the entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae in outer circles at different volatile organic compounds (VOC) and their blends. Error bars are corresponding to SE. The capital letters indicate statistically significant differences (P < 0.05) among different VOC and their blends. D = dimethyl sulfide, H = hexanal, P = 2-pentylfuran, N = (E)-non-2-enal, C = control.
Table 2. ANOVA results for the CI values (df for the error term: 959).
Source F df P
66.86 2 <0.0001
177.37 3 <0.0001
74.29 15 <0.0001
0.62 4 0.6483
1.12 2 0.3267
39.06 45 <0.0001
9.80 30 <0.0001
VOC = volatile organic compounds.
interactions (Table 2). The effect of different VOC and their blends on the chemotactic response of the EPN is presented in Table 3. The mixture of hexanal, dimethyl sulfide and (E)-non-2-enal proved to be an attractant for the commercial strain of H. bacteriophora (CI = 0.24 ± 0.03) and S. carpocapsae (CI = 0.22 ± 0.0), and the weak attractant for S. feltiae (CI = 0.15 ± 0.0). Interestingly, the soil-isolated H. bacteriophora strain HbV was not influenced by this mixture. The movement of commercial EPN species was different compared to soil-isolated species when EPN were
exposed to the mixture of 2-pentylfuran and dimethyl sulfide. As presented in Table 3, we can see that the soil-isolated H. bacteriophora strain HbV was attracted by this mixture (CI = 0.38 ± 0.02), while commercial strains of H. bacteriophora (CI = -0.15 ± 0.01) and S. carpocapsae (CI = -0.16 ± 0.02) were both repelled. We found a difference among commercial and soil-isolated strains of EPN also in the case of dimethyl sulfide and the combination of dimethyl sulfide and (E)-non-2-enal. In both cases, VOC served as attractants to H. bacteriophora strain HbV, while there was no effect on other tested strains. Results presented in Table 3 prove that the movement of IJ toward VOC can be strain-specific as well as a species-specific trait.
Cluster analysis has been based on CI classified groups of EPN species and VOC into six clusters, which is presented as a constellation plot in Figure 4. Clusters with members marked green (Cluster 1) and purple (Cluster 2) were the most dissimilar to other ones. Data with the highest average CI were grouped in Clusters 1 and 2 (Table 3). We can see that members of these clusters consist mostly of two H. bacteriophora strains (78%). With one exception, they are grouped with blends of VOC that contain dimethyl sulfide. When associated with dimethyl sulfide and 2-pentylfuran, the soil-isolated strain H. bacteriophora (HbV) was grouped in the subcluster showed an attraction behaviour toward this blend of VOC. Steinernema carpocapse was
Foraging strategy
Species
VOC
Temporal replication Spatial replication
VOC x species
Foraging strategy x VOC
also grouped in Cluster 1 with a blend of VOC that contain dimethyl sulfide, and S. feltiae with pure 2-pentylfuran.
VOC were classified into three clusters based on CI, and it was presented as a constellation plot in Figure 5. VOC grouped in a cluster with blue colour have the highest average CI (Table 3). Dimethyl
sulfide and 2-pentylfuran and four combinations of their blends grouped together as attractants. Blend of hexanal and dimethyl sulfide forms one cluster (marked green), and can be interpreted as VOC with weak influence on EPN. The third cluster with VOC and their blends marked red can be considered to have no effect on the chemotactic response of EPN.
Table 3. Effect of different volatile organic compounds (VOC) on the chemotactic response of infective juveniles of the entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae at 20°C after 24 h.
D DN H HD
Hb 0.02 ± 0.03 A 0.07 ± 0.01 B 0.04 ± 0.02 C -0.12 ± 0.01 A
HbV 0.25 ± 0.01 C 0.32 ± 0.0 C -0.07 ± 0.01 A 0.05 ± 0.02 B
Sc 0.0 ± 0.0 A -0.01 ± 0.02 A -0.03 ± 0.01 B -0.14 ± 0.01 A
Sf 0.09 ± 0.01 B 0.02 ± 0.01 A -0.01 ± 0.01 B -0.01 ± 0.01 A
HDN HN HP HPD
Hb 0.24 ± 0.03 C 0.03 ± 0.02 C -0.02 ± 0.02 A 0.19 ± 0.01 C
HbV 0.0 ± 0.02 A -0.04 ± 0.01 B 0.04 ± 0.02 A 0.20 ± 0.01 C
Sc 0.22 ± 0.0 C -0.14 ± 0.02 A 0.09 ± 0.03 B 0.0 ± 0.01 A
Sf 0.15 ± 0.0 B 0.06 ± 0.02 D 0.05 ± 0.01 B 0.03 ± 0.0 B
HPDN HPN C N
Hb 0.01 ± 0.01 B -0.06 ± 0.02 A -0.01 ± 0.01 AB 0.11 ± 0.01 C
HbV -0.05 ± 0.01 A 0.05 ± 0.02 C 0.01 ± 0.02 B 0.05 ± 0.02 B
Sc -0.05 ± 0.02 A -0.07 ± 0.02 A 0.0 ± 0.01 B -0.16 ± 0.01 A
Sf 0.02 ± 0.0 B 0.0 ± 0.0 B -0.03 ± 0.01 A 0.06 ± 0.02 B
P PD PDN PN
Hb 0.0 ± 0.02 A -0.15 ± 0.01 A -0.01 ± 0.02 B 0.01 ± 0.01 A
HbV 0.25 ± 0.01 B 0.38 ± 0.02 C 0.04 ± 0.02 D 0.16 ± 0.01 D
Sc 0.03 ± 0.01 A -0.16 ± 0.02 A -0.07 ± 0.01 A 0.12 ± 0.01 C
Sf 0.22 ± 0.03 B 0.04 ± 0.01 A 0.01 ± 0.0 C 0.09 ± 0.01 B
Each data point represents the mean CI value ± SE. Data point with the same letter are not significantly different (P > 0.05) from each other. The capital letters indicate statistically significant differences among different EPN species with the same VOC. Sc - S. carpocapsae, Sf - S. feltiae, Hb - H. bacteriophora (commercial strain), HbV - H. bacteriophora (Vucedol strain). D = dimethyl sulfide, H = hexanal, P = 2-pentylfuran, N = (E)-non-2-enal, C = control.
CI values are classified as follows: > 0.02 as an attractant, from 0.2 to 0.1 as a weak attractant, from 0.1 to -0.1 as no effect, from -0.1 to -0.2 as a weak repellent and < -0.02 as a repellent to EPN (Jagodic et al., 2017).
DISCUSSION
The chemosensation of IJ to different VOC and their blends from barley roots varied depending on different factors. EPN species, VOC, foraging strategy, and several interactions proved to influence the movement and orientation of IJ in our investigation. Results of this study support our previous related studies, where we investigated IJ chemosensation toward mechanically-damaged corn
roots (Laznik & Trdan, 2013), and insect-damaged potato tubers, carrot roots and black mustard roots (Laznik & Trdan, 2016a, b; Jagodic et al., 2017). However, the CI values in our investigation were relatively low compared to other related studies (Hallem et al., 2011; Dillman et al., 2012). Both authors reported CI values above ± 0.5 for Steinernema and Heterorhabditis species. The highest CI value (0.38 ± 0.02) was observed, when H. bacteriophora strain HbV was exposed to
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Fig. 4. Constellation plot of the entomopathogenic nematodes (EPN) Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae grouped with volatile organic compounds (VOC) based on CI. Sc - S. carpocapsae, Sf -S. feltiae, Hb - H. bacteriophora (commercial strain), HbV - H. bacteriophora (Vucedol strain). D = dimethyl sulfide, H = hexanal, P = 2-pentylfuran, N = (E)-non-2-enal, C = control.
Fig. 5. Constellation plot of volatile organic compounds (VOC) based on CI. Sc - S. carpocapsae, Sf - S. feltiae, Hb - H. bacteriophora (commercial strain), HbV - H. bacteriophora (Vucedol strain). D = dimethyl sulfide, H = hexanal, P = 2-pentylfuran, N = (E)-non-2-enal, C = control.
2- pentylfuran and dimethyl sulfide. Different methodological approaches in other related studies could influence the achieved range of CI. Furthermore, several authors suggested that chemotaxis toward volatiles is a strain-specific trait of EPN (Ali et al., 2011; Laznik & Trdan, 2016a, b; Jagodic et al., 2017). Hallem et al. (2011) reported a difference in the attraction behaviour among five EPN strains of H. bacteriophora (HP88, GPS11, NCA, M31e and BU). In this study, we report the difference in movement between the commercial (obtained by Koppert) and soil-isolated strains (isolated from soil near Vucedol, Croatia) of the same species, as well as a difference in behavioural traits when comparing the results of the Hb strains tested by Hallem et al. (2011).
Different EPN species were attracted by different VOC in our investigation. Heterorhabditis bacteriophora (the cruiser nematode) was the most mobile species in our assay, and after 24 h, 28% of H. bacteriophora IJ moved to the outer circles. The least mobile species was S. feltiae (the intermediate nematode); only 15% of IJ moved toward the outer circles in our assay. This finding only partially supports the theory of EPN foraging strategy (Grewal et al., 1994; Campbell & Gaugler, 1997). According to their results, cruise foragers tend to move actively through the media and use distant volatile cues to assist in host-finding, while ambushers only lift their body into the air which facilitates attachment to passing insects. In our case, the movement of S. carpocapsae (the ambusher nematode) was more prominent than S. feltiae. Our results support recent investigations made by Kruitbos et al. (2009) and Wilson et al. (2012). Wilson et al. (2012) proposed that many species will show different behaviours depending on the substrate in which they forage. In our investigation the EPN response to VOC was species-specific rather than foraging strategy-specific.
Behavioural difference of both studied H. bacteriophora strains was found when exposed to several VOC. For instance, the commercial H. bacteriophora strain was repelled by a mixture of 2-pentylfuran and dimethyl sulfide, while the soil-isolated strain was attracted. Furthermore, the mixture of hexanal, dimethyl sulfide, and (E)-non-2-enal proved to be an attractant for the commercial strain of H. bacteriophora, S. carpocapsae and S. feltiae. Interestingly, the soil-isolated H. bacteriophora strain HbV was not influenced by this mixture. We believe that the difference is due to the different origins of EPN rather than to different maintenance of the strains, as follows from work by Gaugler et al. (2006). These authors reported a
possible loss of natural behavioural traits of EPN when cultivated in vitro. Genotypic variation among IJ of H. bacteriophora and S. carpocapsae in heat, desiccation, ultraviolet tolerance and the host-finding ability was assessed by comparing the performance of inbred lines of these EPN. The results for host-finding ability varied considerably within each line. The authors concluded that within 6-12 generations in vitro produced EPN can change their behaviour compared to natural populations. Further research of the influence of VOC on EPN natural population strains is required in order to confirm our hypothesis.
Results of our current study showed that dimethyl sulfide and 2-pentylfuran and four combinations of their blends attracted EPN. In a related study (Jagodic et al., 2017), the authors found that dimethyl sulfide acted as a repellent on the EPN. Again, we can explain the differences in results obtained in both investigations by the hypothesis of the strain-specific behaviour of EPN. In a related study, Jagodic et al. (2017) used commercial strains obtained from BASF, while in our current study the Koppert strains and the soil-isolated strain of EPN were used. Until now, VOC 2-pentylfuran had never been used in EPN behaviour studies. Results of our investigation showed the synergistic effect of dimethyl sulfide as an attractant for EPN. Based on the results of our two related studies, we can conclude that understanding the behaviour of EPN toward VOC is very complex. Most VOC that are involved in the belowground tritrophic interactions remain unknown but an increasing effort is being made in this field of science.
Belowground multitrophic interactions could be exploited to create a more efficient biological control strategy of insects. Plants often utilise these interactions as part of their indirect defence mechanism against herbivory (Bonkowski et al., 2009; Ali et al., 2011; Hiltpold et al, 2013). Hallem et al. (2011) reported attraction/repulsion behaviour in steinernematids and heterorhabditids toward different VOC. Our study was based on the results of Gfeller et al. (2013) who identified VOC emitted by barley roots and assessed their role in the chemical ecology of wireworms. Wireworms were attracted to the cues emanating from barley seedlings (Gfeller et al., 2013). Our results showed that EPN were attracted to the same VOC as well. Previously (Ali et al., 2011; Hiltpold et al., 2013) multitrophic interactions between plants, herbivores and their natural enemies have been reported as well.
Wireworms are polyphagous and important soil-dwelling pests (Ansari et al., 2009), with demanding
control strategy options. After decades of application of very efficient broad-spectrum insecticides, due to its cancellation of registration, integrated wireworms control measures are the focal point of state-of-the-art research (Reddy et al., 2014). Our goal was to provide useful information about the possible tri-trophic interactions between barley roots, wireworms and EPN. We confirm that insect natural enemies, EPN, recognise and navigate toward the same compounds emitted by barley roots as insect herbivores, wireworms. Furthermore, our results confirm the synergistic effect of dimethyl sulfide with different VOC mixed together on the behaviour of EPN.
Our results raised several questions to be further answered in the understanding of plant volatile induced signal orientation of different EPN species and strains. Further experiments involving different EPN strains and species could help us to get answers to these questions. Furthermore, exploring the hypothesis of Wilson et al. (2012) that many species will show different behaviours depending on the substrate in which they forage, better results can be obtained with the use of belowground olfactometers instead of agar Petri dishes. Hiltpold et al. (2010) reported that selected strains of EPN responded to a greater extent in laboratory experiments compared to field conditions. Nonetheless, our results reveal a potential of selecting beneficial organisms in combination with selected VOC for making biological control of insect pests more efficient.
ACKNOWLEDGEMENTS
This work was conducted within Horticulture no. P4-0013-0481, a programme funded by the Slovenian Research Agency. Part of this research was funded within Professional Tasks from the Field of Plant Protection, a programme funded by the Ministry of Agriculture, Forestry, and Food of Phytosanitary Administration of the Republic Slovenia. Special thanks are given to Vanja Resnik, Jaka Rupnik, Dr Tamas Lakatos, Dr Timea Toth, and Dr Branimir Njezic for their technical assistance. We would like to thank Koppert for providing the commercial strains of EPN.
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A. Jagodic, I. Majic, S. Trdan and Z. Laznik. Являются ли синтетические летучие органические соединения, в норме выделяемые корнями ячменя (Hordeum vulgare L.), навигационными сигналами для энтомопатогенных нематод (Steinernema и Heterorhabditis)?
Резюме. Была изучена хемотаксическая реакция инвазионных личинок (ИЛ) почвенных энтомопатогенных нематод (ЭПН) Heterorhabditis bacteriophora, Steinernema carpocapsae и S. feltiae на летучие органические соединения (ЛОС) (диметилсульфид, гексанал, 2-пентилфуран и (Е)-нон-энал) в норме выделяемые корнями ячменя. В рамках исследования использовали как отдельные ЛОС, так и их смеси. Предполагалось, что реакция привлечения ЭПН к исследованным ЛОС зависит от вида/изолята нематод и будет зависеть от пищевой стратегии нематоды и применяемого ЛОС. Heterorhabditis bacteriophora был наиболее подвижным видом в данном исследовании. Были выявлены достоверные различия между коммерческими и вновь выделенными из почвы изолятами ЭПН. Передвижение личинок ЭПН по направлению к источникам различных ЛОС достоверно зависело от вида и изолята нематод. Был выявлен синэргический эффект диметилсульфида как аттрактанта для ЭПН. Полученные данные также показали, что положительный хемотаксис более зависел от вида и изолята нематод, чем от характерной для вида пищевой стратегии. Все изученные летучие соединения оказывали влияние на двигательную активность ИЛ, что подверждает существенную роль синтетических ЛОС, в норме выделяемых корнями ячменя, в навигационном поведении личинок ЭПН.
