**4. The role of root exudates on beneficial soil organisms – Indirect defense against soil herbivores**

#### **4.1. Entomopathogenic Nematodes (EPNs)**

Tritrophic interactions, which include a host plant, a harmful organism and its natural enemy, have been documented only recently for the underground parts of a plant. Some studies have shown that damaged roots of different plant species release into environment VOCs which can influence the movement of EPNs both as attractans [8, 9, 10, 44] and as repellents [53].

Soil is the natural habitat of EPNs (Steinernematidae and Heterorhabditidae) (Figure 1), and their application in pest management has been primarily used against soil-inhabiting insect pests [54]. EPNs are lethal pathogens of insects. These pathogens contribute to the regulation of natural populations of insects, but the main interest in them is an inundatively applied biocontrol agent [55]. Their success in this role can be attributed to the unique partnership between a host-seeking nematode and a lethal insect-pathogenic bacterium. Because of their biocontrol potential, considerable attention has been directed over the past few decades to genus, *Heterorhabditis* and *Steinernema* and their respective bacterial partners, *Photorhabdus* and *Xenorhabdus*.

Although heterorhabditids and steinernematids are not closely related [56], they share many features in common. These similarities, including their association with insect-pathogenic bacteria, are presumed to have arisen through convergent evolution [57]. In both *Steinernema* and *Heterorhabditis*, there is a single free-living stage, the infective juvenile (IJ) that carries in its gut, bacteria of the genus *Xenorhabdus* and *Photorhabdus* [58]. On encountering a suitable insect, the IJ enters through the mouth, anus, or spiracles and makes its way to the haemocoel [59]. Some species may also penetrate through the intersegmental membranes of the insect The Role of Volatile Substances Emitted by Cultivated Plant's Roots in Indirect Defense Against Soil Herbivores http://dx.doi.org/10.5772/61369 399

**Figure 1.** Infective juveniles of entomopathogenic nematode *Steinernema feltiae* (photo: J. Rupnik)

important signals in the soils are the emissions of CO2 by roots [48]. [48] reported that detection of CO2 seems to be dose-dependent, and soil insect are able to detect very small differences in the concentration of CO2. Besides CO2, plants emit various volatile compounds upon herbivore attack. The study of [49] investigated on-line VOC emissions by roots of *Brassica nigra* plants under attack by cabbage root fly larvae, *Delia radicum*. The investigation showed that several sulfur-containing compounds, such as methanethiol, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide and glucosinolate breakdown products such as thiocyanates and isothio‐ cyanates, were emitted by the roots in response to infestation [49]. [50] reported that fatty acids in oaks (*Quercus* sp.) and monoterpenes in carrot (*Daucus carotta* ssp. *sativus*), and potato (*Solanum tuberosum*) plants triggered the attraction of forest cockchafer larvae (*Melolontha hippocastani*) and wireworms (*Agriotes* spp.). Volatiles of fresh perennial ryegrass roots attracted larvae of *Costelytra zealandica* [51], and roots of *Medicago sativa* and *Trifolium pra‐ tense* attracted larvae of *Sitona hispidulus* [52]. Furthermore, [8] reported that maize (*Zea mays*) roots release ß-caryophyllene in response to feeding by larvae of the beetle *Diabrotica virgifera virgifera*. In a related research, [10] reported that mechanically damaged maize roots release

**4. The role of root exudates on beneficial soil organisms – Indirect defense**

Tritrophic interactions, which include a host plant, a harmful organism and its natural enemy, have been documented only recently for the underground parts of a plant. Some studies have shown that damaged roots of different plant species release into environment VOCs which can influence the movement of EPNs both as attractans [8, 9, 10, 44] and as repellents [53]. Soil is the natural habitat of EPNs (Steinernematidae and Heterorhabditidae) (Figure 1), and their application in pest management has been primarily used against soil-inhabiting insect pests [54]. EPNs are lethal pathogens of insects. These pathogens contribute to the regulation of natural populations of insects, but the main interest in them is an inundatively applied biocontrol agent [55]. Their success in this role can be attributed to the unique partnership between a host-seeking nematode and a lethal insect-pathogenic bacterium. Because of their biocontrol potential, considerable attention has been directed over the past few decades to genus, *Heterorhabditis* and *Steinernema* and their respective bacterial partners, *Photorhabdus* and

Although heterorhabditids and steinernematids are not closely related [56], they share many features in common. These similarities, including their association with insect-pathogenic bacteria, are presumed to have arisen through convergent evolution [57]. In both *Steinernema* and *Heterorhabditis*, there is a single free-living stage, the infective juvenile (IJ) that carries in its gut, bacteria of the genus *Xenorhabdus* and *Photorhabdus* [58]. On encountering a suitable insect, the IJ enters through the mouth, anus, or spiracles and makes its way to the haemocoel [59]. Some species may also penetrate through the intersegmental membranes of the insect

linalool, ß-caryophyllene, and α-caryophyllene.

**4.1. Entomopathogenic Nematodes (EPNs)**

**against soil herbivores**

398 Insecticides Resistance

*Xenorhabdus*.

cuticle [60]. In the haemocoel, the IJ releases cells of its bacterial symbiont from its intestine. Bacteria multiply rapidly in haemolymph and produce toxins and other secondary metabo‐ lites, which contribute to the weakening of the host's defense mechanism. The host attacked by EPNs usually dies because of poisoning or failure of certain organs in 24–72 hours after the infection [61]. Two developmental cycles thus occur in the host – one of nematodes and the other of bacteria. The first-generation nematodes pass into the second generation. After the larvae cast off the fourth sheath and enter into the adult period, nematodes pass into the third generation, which thrives in the host as long as there is availability of food. The host is by then already dead – being killed by the toxins secreted by bacteria. The third-generation nematodes are thus already saprophagic [62]. Bacteria also produce toxins, such as 3,5 *dihydroxy-4 isopropyl-stilben*, which deter other microorganisms from settling in the carcass [63]. When the developmental cycle is finished, nematodes leave the parts of carcasses that have not decom‐ posed, and return to the ground. Nematodes cannot develop without a host (an insect) [64], without which they survive in the ground for only a very brief period of time [65].

The importance of EPNs and biological plant protection against harmful organisms was first established in the USA in the 1930s. In 1923, Glaser and Fox discovered a nematode which attacked and caused death of the beetle, *Popillia japonica* Newman [66]. Glaser introduced a method of growing EPNs *in vitro*. With such nematodes, he, in 1939, carried out the first field experiment in New Jersey to suppress the species, *P. japonica* [67].

When EPNs were first discovered, a hypothesis was proposed that nematodes alone cause death of the insects being attacked. In 1937, Bovien first hypothesized the possibility of the existence of symbiotic bacteria that live with EPNs in a mutualistic relationship. His hypothesis was, in 1955, confirmed by Dutky and Weiser [68]. However in 1982, Boemare proved that nematodes from the genus *Steinernema* produce toxic substances which negatively influence the immune system of infected insects and can themselves alone – without the presence of symbiotic bacteria – cause death of the host. For EPNs from the genus *Heterorhabditis*, it has not yet been established that they can alone produce toxic substances that would diminish the vitality of infected insects [69].

The use of EPNs in biological plant protection was until some years ago still traditionally connected with suppressing soil-inhabiting insect pests [70]. The research results in the last two decades indicate they have also potential to suppress aboveground insect pests, but only in certain circumstances [71, 72]. Lesser efficiency of EPNs in suppression of aboveground insect pests is primarily due to inappropriate (insufficient) moisture [73], exposure to thermal extremes [74], and ultraviolet radiation [75]. These factors are of crucial importance for the survival of nematodes [65]. For this reason nematodes are less efficient against aboveground insect pests outdoors, though the previous laboratory tests showed much higher efficiency [76].

To lay nematodes on plants, equipments intended for spraying plant protection products, manuring, or irrigation can be used. *Backpack manual or tractor sprayers*, sprinklers, and also planes are suitable for this purpose. IJs can be passed through spray tubes with diameter of at least 500 μm, capable to withstand pressure up to 2000 kPa [77].

IJs can tolerate short-term exposure (2–24 hours) to many chemical and biological insecticides, fungicides, herbicides, fertilizers, and growth regulators and can thus be tank-mixed and applied together [78, 79, 80, 81]. Nematode–chemical combinations in tank-mixes could offer a cost-effective alternative to foliar integrated pest management (IPM) systems.

Due to the sensitivity of nematodes to ultraviolet radiation, nematodes have to be applied to plants in the evening, early in the morning, or during a cloudy weather, when the radiation is not so intense [73]. Nematode survival and efficacy on foliage has also been shown to be enhanced to varying degrees by addition of various adjuvants to the spray mixture, which have antidesiccant (e.g., glycerol, various polymers) or UV-protective (brighteners) properties [82], although additional measures are required to enhance post-application survival. The greatest potential for using EPNs against foliar pests is almost certain in IPM programs, in conjunction with other biocontrol agents [83] or selective chemicals [78, 84].

EPNs are considered exceptionally safe biological agents [85]. Because their activity is specific, their environmental risk is considerably lower than that of chemical agents for plant protection [86]. Since the first use of EPNs for suppressing beetles of the species *P. japonica* in the USA [66], until now, no case of environmental damage due to these biological agents has been docu‐ mented. The use of nematodes is safe for users. EPNs and their bacteria are not harmful for mammals and plants [87].

#### **4.2. Movement of EPNs**

The ability of EPN IJs to disperse actively through soil and locate a host is a key element for the successful application of some EPN species in pest management [88]. When an EPN locates its host, it can enter it through natural openings. By excreting its symbiotic bacteria, which release toxins into the host's body, it causes the death of the insect in 24–72 hours after the infection [55].

EPNs have through the evolution developed different ways of searching for hosts, which is a species-specific characteristic [89, 90, 91]. The species *Heterorhabditis bacteriophora* and *Steiner‐ nema kraussei* actively search for hosts (cruisers). Some species of EPNs wait for a host in an ambush (ambushers). The passive way of searching for a host is characteristic for the species *S. carpocapsae*. Some species (*S. feltiae*) combine both ways of searching for a host and are categorized as intermediates [89, 90, 91].

EPN species respond distinctly to cues associated with hosts (insects) or plants, depending primarily on their foraging strategy [89]. The cruisers spend most of their time searching for resource-associated cues as they move through their environment [89, 91]. In contrast, ambushers do not respond as strongly as cruisers and spend little time actively moving and searching for volatile cues. Ambushers are thought to wait for resources to come to them [10, 89]. Several EPN species adopt both (cruise and ambush) foraging strategies and are classified as intermediates [89].

EPNs uses chemosensation to find host, avoid noxious conditions, develop appropriately, and mate. Several authors report that IJs respond to CO2 [89], temperature, changes in pH, bacterial symbionts [92], electrical field [93], and different plant VOCs [8, 9, 10, 44].

#### **4.3. Indirect defense against soil herbivores**

#### *4.3.1. Potato*

existence of symbiotic bacteria that live with EPNs in a mutualistic relationship. His hypothesis was, in 1955, confirmed by Dutky and Weiser [68]. However in 1982, Boemare proved that nematodes from the genus *Steinernema* produce toxic substances which negatively influence the immune system of infected insects and can themselves alone – without the presence of symbiotic bacteria – cause death of the host. For EPNs from the genus *Heterorhabditis*, it has not yet been established that they can alone produce toxic substances that would diminish the

The use of EPNs in biological plant protection was until some years ago still traditionally connected with suppressing soil-inhabiting insect pests [70]. The research results in the last two decades indicate they have also potential to suppress aboveground insect pests, but only in certain circumstances [71, 72]. Lesser efficiency of EPNs in suppression of aboveground insect pests is primarily due to inappropriate (insufficient) moisture [73], exposure to thermal extremes [74], and ultraviolet radiation [75]. These factors are of crucial importance for the survival of nematodes [65]. For this reason nematodes are less efficient against aboveground insect pests outdoors, though the previous laboratory tests showed much higher efficiency [76]. To lay nematodes on plants, equipments intended for spraying plant protection products, manuring, or irrigation can be used. *Backpack manual or tractor sprayers*, sprinklers, and also planes are suitable for this purpose. IJs can be passed through spray tubes with diameter of at

IJs can tolerate short-term exposure (2–24 hours) to many chemical and biological insecticides, fungicides, herbicides, fertilizers, and growth regulators and can thus be tank-mixed and applied together [78, 79, 80, 81]. Nematode–chemical combinations in tank-mixes could offer

Due to the sensitivity of nematodes to ultraviolet radiation, nematodes have to be applied to plants in the evening, early in the morning, or during a cloudy weather, when the radiation is not so intense [73]. Nematode survival and efficacy on foliage has also been shown to be enhanced to varying degrees by addition of various adjuvants to the spray mixture, which have antidesiccant (e.g., glycerol, various polymers) or UV-protective (brighteners) properties [82], although additional measures are required to enhance post-application survival. The greatest potential for using EPNs against foliar pests is almost certain in IPM programs, in

EPNs are considered exceptionally safe biological agents [85]. Because their activity is specific, their environmental risk is considerably lower than that of chemical agents for plant protection [86]. Since the first use of EPNs for suppressing beetles of the species *P. japonica* in the USA [66], until now, no case of environmental damage due to these biological agents has been docu‐ mented. The use of nematodes is safe for users. EPNs and their bacteria are not harmful for

The ability of EPN IJs to disperse actively through soil and locate a host is a key element for the successful application of some EPN species in pest management [88]. When an EPN locates its host, it can enter it through natural openings. By excreting its symbiotic bacteria, which

a cost-effective alternative to foliar integrated pest management (IPM) systems.

conjunction with other biocontrol agents [83] or selective chemicals [78, 84].

least 500 μm, capable to withstand pressure up to 2000 kPa [77].

vitality of infected insects [69].

400 Insecticides Resistance

mammals and plants [87].

**4.2. Movement of EPNs**

Here, we describe our study of the chemotactic behavior of *Steinernema feltiae* (Filipjev), *Steinernema carpocapsae* Weiser, *Steinernema kraussei* (Steiner), and *Heterorhabditis bacteriophora* Poinar toward Decanal; Nonanal; Octanal; Undecane; 1,2,4-trimethylbenzene; 2-ethyl-1 hexanol; and 6-methyl-5-hepten-2-one; compounds released from insect (*M. hippocastani* grubs) damaged and undamaged potato tubers (*S. tuberosum*) [50]. The aims of our research were (1) to study the effect of different EPN foraging strategies (ambush, intermediate, or cruise) toward the tested VOCs, (2) to determine whether chemotaxis is species-specific, (3) to assess whether the VOCs from damaged potato tubers have any behavioral effects on the EPNs studied, and (4) if VOCs are a part of an indirect plant defense.

The results of our research showed that the movement of EPNs was conditioned by the type of VOCs excreted by damaged/undamaged potato tubers (see Figures 2 and 3). VOCs Nonanal, Octanal, and Decanal proved to have a greater influence on the movement of EPNs as other tested volatiles in our investigation. Nonanal and Decanal are among other indicator substan‐ ces for degradation processes [50]. Decanal is also described to be induced by mechanical and herbivore damage [46, 94, 95]. [50] reports that damaged potato tubers excrete the substances Nonanal, Octanal, and Decanal. The results of our research showed that the said substances acted as attractants in regard to the movement of EPNs. Decanal in our experiment proved as an attractant for the species *H. bacteriophora* and *S. kraussei* at both studied concentrations (pure concentration and 0.03 ppm concentration) (see Figures 2 and 3). Octanal proved an attractant for the species *H. bacteriophora* and as a weak attractant for the species *S. carpocapsae*. Nonanal proved to be a weak attractant for the species *S. carpocapsae*. Thus we confirm the thesis that damaged plant roots release into the environment substances that influence the movement of beneficial organisms – indirect plant defense.

In our investigation two distinct VOC concentrations were used. A pure concentration, which does not reflect a concentration found near plant roots [96], had a bigger influence on IJ movement than a concentration of 0.03 ppm, which is the average concentration of volatile compounds found in soil, 10 cm away from the root system [12]. In our experiment, the difference in responsiveness of EPNs in regard to the concentration of VOC was most sub‐ stantially expressed in the case of the chemical substance Undecane. At pure concentration, the said substance proved to be an attractant for the species *S. kraussei* and as a repellent for the species *S. feltiae*. At the concentration 0.03 ppm, the said substance did not have any influence on the movement of EPNs in our experiment (see Figures 2 and 3). We also found out that the duration of exposure of an EPN to VOCs is of key importance for perceiving chemical stimuli. After 24 hours we detected the movement of EPN in 32%, while the move‐ ment after 2 hours was detected only in 3% (see Figures 2 and 3). Similar findings were produced by our earlier research [10].

The results of our research showed that the movement of EPNs toward the selected VOC is substantially determined also by their foraging strategy. In regard to the way of searching the host EPNs fall into three types. Cruisers (*H. bacteriophora* and *S. kraussei*) actively move toward their prey by perceiving stimuli from the environment [97], while the so-called ambushers (*S. carpocapsae*) wait for their prey in an ambush [90]. Some species (*S. feltiae*) combine both ways of searching for the host and are classified as the so-called intermediates [89]. The VOC Decanal in our experiment proved to be an attractant for the species *H. bacteriophora* and *S. kraussei* (see Figures 2 and 3), which are classified as the so-called cruisers. We also found out that the movement of the nematodes classified as cruisers and intermediates was more pronounced than in the species *S. carpocapsae*, which proved to be the least mobile species of EPNs in our research. [98] says that the movement of cruisers at longer distances is conditioned by perceiving chemical stimuli, which, however, is not characteristic for the nematodes classified as ambushers. In some related studies the species *H. bacteriophora* proved to be very susceptible to perceive chemical stimuli from the environment [97, 99]. This was also confirmed in our research for the substances Decanal and Octanal, which affected the said species as attractants. The ambusher *S. carpocapsae* in comparison with other studied species in our experiment displayed a high degree of susceptibility to the VOC 6-Methyl-5-hepten-2-one. On the basis of some of our earlier research [10] and the current one, we conclude that the movement of EPNs toward the selected VOC is influenced primarily by the species and not so much by the way of searching the host. Our hypothesis is confirmed with the fact that Octanal acted as an attractant for the nematode *H. bacteriophora*, while the nematode *S. kraussei*, which is also classified as a cruiser, was not affected by it. Similar conclusions were reached also in the study by [91] who studied the reaction of EPNs on damaged citrus roots. Susceptibility to perceiving chemical stimuli from the environment is a species-specific characteristic prevailing over the foraging strategy [10].

for the species *H. bacteriophora* and as a weak attractant for the species *S. carpocapsae*. Nonanal proved to be a weak attractant for the species *S. carpocapsae*. Thus we confirm the thesis that damaged plant roots release into the environment substances that influence the movement of

In our investigation two distinct VOC concentrations were used. A pure concentration, which does not reflect a concentration found near plant roots [96], had a bigger influence on IJ movement than a concentration of 0.03 ppm, which is the average concentration of volatile compounds found in soil, 10 cm away from the root system [12]. In our experiment, the difference in responsiveness of EPNs in regard to the concentration of VOC was most sub‐ stantially expressed in the case of the chemical substance Undecane. At pure concentration, the said substance proved to be an attractant for the species *S. kraussei* and as a repellent for the species *S. feltiae*. At the concentration 0.03 ppm, the said substance did not have any influence on the movement of EPNs in our experiment (see Figures 2 and 3). We also found out that the duration of exposure of an EPN to VOCs is of key importance for perceiving chemical stimuli. After 24 hours we detected the movement of EPN in 32%, while the move‐ ment after 2 hours was detected only in 3% (see Figures 2 and 3). Similar findings were

The results of our research showed that the movement of EPNs toward the selected VOC is substantially determined also by their foraging strategy. In regard to the way of searching the host EPNs fall into three types. Cruisers (*H. bacteriophora* and *S. kraussei*) actively move toward their prey by perceiving stimuli from the environment [97], while the so-called ambushers (*S. carpocapsae*) wait for their prey in an ambush [90]. Some species (*S. feltiae*) combine both ways of searching for the host and are classified as the so-called intermediates [89]. The VOC Decanal in our experiment proved to be an attractant for the species *H. bacteriophora* and *S. kraussei* (see Figures 2 and 3), which are classified as the so-called cruisers. We also found out that the movement of the nematodes classified as cruisers and intermediates was more pronounced than in the species *S. carpocapsae*, which proved to be the least mobile species of EPNs in our research. [98] says that the movement of cruisers at longer distances is conditioned by perceiving chemical stimuli, which, however, is not characteristic for the nematodes classified as ambushers. In some related studies the species *H. bacteriophora* proved to be very susceptible to perceive chemical stimuli from the environment [97, 99]. This was also confirmed in our research for the substances Decanal and Octanal, which affected the said species as attractants. The ambusher *S. carpocapsae* in comparison with other studied species in our experiment displayed a high degree of susceptibility to the VOC 6-Methyl-5-hepten-2-one. On the basis of some of our earlier research [10] and the current one, we conclude that the movement of EPNs toward the selected VOC is influenced primarily by the species and not so much by the way of searching the host. Our hypothesis is confirmed with the fact that Octanal acted as an attractant for the nematode *H. bacteriophora*, while the nematode *S. kraussei*, which is also classified as a cruiser, was not affected by it. Similar conclusions were reached also in the study by [91] who studied the reaction of EPNs on damaged citrus roots. Susceptibility to perceiving chemical stimuli from the environment is a species-specific characteristic prevailing over the

beneficial organisms – indirect plant defense.

402 Insecticides Resistance

produced by our earlier research [10].

foraging strategy [10].

**Figure 2.** Effects of time of exposure to VOCs on the chemotactic response of EPN species (A-G), at a concentration of 0.03 ppm. Each data point represents the mean chemotaxis index ± S.E. Bars with the same letter are not significantly different (P>0.05). The small letters indicate statistically significant differences among different EPN species with the same time of exposure. The capital letters indicate statistically significant differences among different times of expo‐ sure within the same EPN species. Hb – *H. bacteriophora*; Sk – *S. kraussei*; Sf – *S. feltiae*; Sc – *S. carpocapsae*. The substan‐ ces in our research were with the chemotaxis indexes divided into the following intervals: >0.2 (attractant); from 0.2 to 0.1 (weak attractant); from 0.1 to -0.1 (no effect); from -0.1 to -0.2 (weak repellent); < -0.2 (repellent) [10]

**Figure 3.** Effects of time of exposure to VOCs on the chemotactic response of the EPN species (A-G), at pure concentra‐ tion. Each data point represents the mean chemotaxis index ± S.E. Bars with the same letter are not significantly differ‐ ent (P>0.05). The small letters indicate statistically significant differences among different EPN species at the same time of exposure. The capital letters indicate statistically significant differences among different times of exposure within the same EPN species. Hb – *H. bacteriophora*; Sk – *S. kraussei*; Sf – *S. feltiae*; Sc – *S. carpocapsae*. The substances in our research were with the chemotaxis indexes divided into the following intervals: >0.2 (attractant); from 0.2 to 0.1 (weak attractant); from 0.1 to -0.1 (no effect); from -0.1 to -0.2 (weak repellent); < -0.2 (repellent) [10]

### *4.3.2. Carrot*

**Figure 3.** Effects of time of exposure to VOCs on the chemotactic response of the EPN species (A-G), at pure concentra‐ tion. Each data point represents the mean chemotaxis index ± S.E. Bars with the same letter are not significantly differ‐ ent (P>0.05). The small letters indicate statistically significant differences among different EPN species at the same time of exposure. The capital letters indicate statistically significant differences among different times of exposure within the same EPN species. Hb – *H. bacteriophora*; Sk – *S. kraussei*; Sf – *S. feltiae*; Sc – *S. carpocapsae*. The substances in our research were with the chemotaxis indexes divided into the following intervals: >0.2 (attractant); from 0.2 to 0.1 (weak

attractant); from 0.1 to -0.1 (no effect); from -0.1 to -0.2 (weak repellent); < -0.2 (repellent) [10]

404 Insecticides Resistance

Here, we describe our study of the chemotactic behavior of *S. feltiae*, *S. carpocapsae*, *S. kraussei*, and *H. bacteriophora* toward α-Pinene, Bornyl acetate, Borneol, 2,4-Di-tetra-butylphenol, 2- Ethyl-hexanol, and Terpinolene; compounds released from insect (wireworms and grubs) damaged carrot (*Daucus carota* ssp. *sativus*) roots [50, 100]. The aims of our research were (1) to study the effect of different EPN foraging strategies (ambush, intermediate, or cruise) toward the tested VOCs, (2) to determine whether chemotaxis is species-specific (3) to assess whether the VOCs from damaged and undamaged carrot roots have any behavioral effects on the EPNs studied, and (4) if VOCs are a part of an indirect plant defense.

Our results show that the chemosensation of IJs toward and away from insect-induced carrot root volatile compounds [50, 100] varied depending on the EPN species, VOC, concentration of VOC, time of exposure and interaction between EPN species and time of exposure (Figures 4 and 5). Our results indicate that all tested EPN species exhibited attraction (or repulsion) to volatiles irrespective of their foraging strategy (in our investigation, terpinolene was a repellent for EPN species classified in all three foraging groups) (Figures 4 and 5). Similar conclusions were also reported in recent research from [91] in which a cruiser *H. indica* [89], ambusher *S. carpocapsae* [89], and two other species thought to exhibit an intermediate foraging strategy [89] were all attracted to root weevil *Diaprepes abbreviatus*-damaged roots of the Swingle rootstock. Furthermore, [10] reported that responses to different volatile cues are a strain-specific characteristic rather than a different host-searching strategy. Similar conclu‐ sions were also made by [9, 91]. Our current results suggest that responsiveness to different volatile cues is a species-specific characteristic.

In our investigation two distinct VOC concentrations were used. A pure concentration, which does not reflect a concentration found near plant roots [96], had a bigger influence on IJ movement than a concentration of 0.03 ppm, which is the average concentration of VOCs found in soil, 10 cm away from the root system) [12]. However, we are aware that such laboratory studies do not reflect a nematode's true behavior in nature because of exposure to different conflicting chemical signals [44, 101].

Plant roots emit an incredible variety of compounds, which are known to affect interactions between plants and other organisms [11]. The active role plants play in recruiting natural enemies, like belowground herbivores, has been recently demonstrated in a few plant species [8, 10, 88, 96, 102, 103]. EPN host finding is mediated by both long-range cues that facilitate root zone finding, as well as shorter-range cues that facilitate host localization within the root zone [8, 63, 91, 102]. Recently, [53] reported positive chemotaxis of the two EPN species *H. bacteriophora* and *S. carpocapsae* to several VOCs such as methyl salicylate, hexanol, heptanol, undecyl acetate, and 4,5-dimethylthiazole. Interestingly, they showed that several volatiles repelled the nematodes. Similar effects of VOCs on the behavior of EPNs were also observed in our investigation (see Figures 4 and 5). Terpinolene repelled both *Steinernema* and *Hetero‐ rhabditis* species in our investigation. [100] reported that terpinolene is a VOC released from the undamaged roots of cultivated carrots. Our results suggest that healthy plant roots release specific VOCs into the soil, which signal to natural insect enemies (EPNs) to keep away. Our findings could support the theory of [91]. [91] suggest that selection of a herbivore-induced signaling response should be directionally stronger toward channeling resources for produc‐ tion of a distress signal only when necessary because a constant release would likely carry a high physiological cost [104, 105]. Our conclusion is also supported by the VOC α-pinene (released from undamaged carrot roots) [100], which was a weak repellent of *S. carpocapsae* and *S. kraussei*. The other tested VOCs in our investigation (Bornyl acetate, Borneol, 2,4-Ditetra-butylphenol, and 2-Ethyl-hexanol) acted inconsistently (as a weak repellents or weak attractants) (see Figures 4 and 5).

**Figure 4.** Effects of time of exposure to VOCs on the chemotactic response of EPN species (A-F), at a concentration of 0.03 ppm. Each data point represents the mean chemotaxis index ± S.E. Bars with the same letter are not significantly different (P>0.05). The small letters indicate statistically significant differences among different EPN species with the same time of exposure. The capital letters indicate statistically significant differences among different times of expo‐ sure within the same EPN species. Hb – *H. bacteriophora*; Sk – *S. kraussei*; Sf – *S. feltiae*; Sc – *S. carpocapsae*. The substan‐ ces in our research were with the chemotaxis indexes divided into the following intervals: >0.2 (attractant); from 0.2 to 0.1 (weak attractant); from 0.1 to -0.1 (no effect); from -0.1 to -0.2 (weak repellent); < -0.2 (repellent) [10]

The Role of Volatile Substances Emitted by Cultivated Plant's Roots in Indirect Defense Against Soil Herbivores http://dx.doi.org/10.5772/61369 407

**Figure 5.** Effects of time of exposure to VOCs on the chemotactic response of the EPN species (A-F), at pure concentra‐ tion. Each data point represents the mean chemotaxis index ± S.E. Bars with the same letter are not significantly differ‐ ent (P>0.05). The small letters indicate statistically significant differences among different EPN species at the same time of exposure. The capital letters indicate statistically significant differences among different times of exposure within the same EPN species. Hb – *H. bacteriophora*; Sk – *S. kraussei*; Sf – *S. feltiae*; Sc – *S. carpocapsae*. The substances in our research were with the chemotaxis indexes divided into the following intervals: >0.2 (attractant); from 0.2 to 0.1 (weak attractant); from 0.1 to -0.1 (no effect); from -0.1 to -0.2 (weak repellent); < -0.2 (repellent) [10]

#### *4.3.3. Maize*

tion of a distress signal only when necessary because a constant release would likely carry a high physiological cost [104, 105]. Our conclusion is also supported by the VOC α-pinene (released from undamaged carrot roots) [100], which was a weak repellent of *S. carpocapsae* and *S. kraussei*. The other tested VOCs in our investigation (Bornyl acetate, Borneol, 2,4-Ditetra-butylphenol, and 2-Ethyl-hexanol) acted inconsistently (as a weak repellents or weak

**Figure 4.** Effects of time of exposure to VOCs on the chemotactic response of EPN species (A-F), at a concentration of 0.03 ppm. Each data point represents the mean chemotaxis index ± S.E. Bars with the same letter are not significantly different (P>0.05). The small letters indicate statistically significant differences among different EPN species with the same time of exposure. The capital letters indicate statistically significant differences among different times of expo‐ sure within the same EPN species. Hb – *H. bacteriophora*; Sk – *S. kraussei*; Sf – *S. feltiae*; Sc – *S. carpocapsae*. The substan‐ ces in our research were with the chemotaxis indexes divided into the following intervals: >0.2 (attractant); from 0.2 to

0.1 (weak attractant); from 0.1 to -0.1 (no effect); from -0.1 to -0.2 (weak repellent); < -0.2 (repellent) [10]

attractants) (see Figures 4 and 5).

406 Insecticides Resistance

Here, we describe our study [10] of the chemotactic behavior of *S. feltiae* (strain B30, strain C76, and strain 3162), *S. carpocapsae* (strain B49, strain C67, and strain C101), *S. kraussei* (strain C46), and *H. bacteriophora* (strain D54) toward linalool, α-caryophyllene, and ß-caryophyllene, compounds released from the mechanically damaged root systems of different *Zea mays* hybrids [106]. In a related study, [9] reported that mechanically damaged citrus roots attracted less nematodes than insect-damaged roots. The aims of our research were (1) to study the effect of different foraging strategies (ambush, intermediate, or cruise) of EPNs to the tested VOCs, (2) to determine whether chemotaxis is species- and strain-specific, and (3) to assess whether the VOCs from mechanically damaged maize roots have any behavioral effect on the studied EPNs.

The results of our current laboratory investigation showed that the movement and chemo‐ sensation of IJs toward and away from damaged maize root VOCs [106] varied depending on the species, strain, foraging strategy, VOC, and interaction between the EPN strain and volatile compound (see Figure 6). The intermediate foragers (*S. feltiae*) proved to be less active in their movement toward the VOCs in comparison with the ambushers (*S. carpocapsae*) and cruisers (*S. kraussei* and *H. bacteriophora*); ß-caryophyllene proved to be the most attractive compound of the three substances tested in our experiment (see Figure 6). The results of our investigation showed that the cruisers were more attracted to β-caryophyllene than the ambushers and intermediates. The foraging strategy did not influence the IJ movement toward the other tested volatile compounds and the control (see Figure 6). Similar conclusions were also reported in the recent research of [91] in which the ambusher *S. carpocapsae* [89], the cruiser *H. indica* [89], and two species thought to exhibit an intermediate foraging strategy [89] were all attracted to *Diaprepes abbreviatus*-damaged roots of Swingle rootstock. Some related studies on the foraging strategies of EPNs have been conducted in nonsoil systems [107]; however, we are aware that such studies do not reflect the nematode's true behavior in nature, whereby they are exposed to a myriad of conflicting chemical signals [44, 63]. In our experiment, pure compounds were applied to agar [107], which does not reflect the concentration near the roots of plants [96]. [96] reported that the total sesquiterpene hydrocarbon content in the herbivore-damaged roots of *Zea mays* was 81 ng g-1, whereas the control plants contained only 25 ng g-1, and the relative amount of ß-caryophyllene among several other different terpenes in the maize roots was less than 5%. Moreover, [9] reported that roots damaged by insect larvae attracted more nematodes than mechanically damaged roots and sand controls. The speed of the nematode's response to the chemical stimuli in its natural environment largely depends on the diffusion rate of the chemical compound and on the soil structural heterogeneity [108]. When a foraging nematode is confronted with an array of signals originating from the same general area, the response may depend on the strength and exposure time and on the nature of the stimuli [63].

[89] reviewed the literature on foraging and host recognition in *Heterorhabditis* and *Steinerne‐ ma* IJs and proposed that ambusher nematodes respond to host (insect) cues in a hierarchical order, with the volatile cues only becoming important after the IJ had made contact with the insect cuticle, whereas remote volatile cues are more important for cruiser nematodes. Several related studies have also shown that IJs exhibit a preference for different volatile root com‐ pounds [8, 9, 63, 91]. ß-caryophyllene is a common compound and has been identified from various plant species [8, 44, 106]; however, its function, as for most plant volatiles, remains unclear. As [8] reported that ß-caryophyllene strongly attracted *H. megidis*, attraction has been confirmed for all of the tested species, with the exception of *S. feltiae* (see Figure 6).

The Role of Volatile Substances Emitted by Cultivated Plant's Roots in Indirect Defense Against Soil Herbivores http://dx.doi.org/10.5772/61369 409

hybrids [106]. In a related study, [9] reported that mechanically damaged citrus roots attracted less nematodes than insect-damaged roots. The aims of our research were (1) to study the effect of different foraging strategies (ambush, intermediate, or cruise) of EPNs to the tested VOCs, (2) to determine whether chemotaxis is species- and strain-specific, and (3) to assess whether the VOCs from mechanically damaged maize roots have any behavioral effect on the studied

The results of our current laboratory investigation showed that the movement and chemo‐ sensation of IJs toward and away from damaged maize root VOCs [106] varied depending on the species, strain, foraging strategy, VOC, and interaction between the EPN strain and volatile compound (see Figure 6). The intermediate foragers (*S. feltiae*) proved to be less active in their movement toward the VOCs in comparison with the ambushers (*S. carpocapsae*) and cruisers (*S. kraussei* and *H. bacteriophora*); ß-caryophyllene proved to be the most attractive compound of the three substances tested in our experiment (see Figure 6). The results of our investigation showed that the cruisers were more attracted to β-caryophyllene than the ambushers and intermediates. The foraging strategy did not influence the IJ movement toward the other tested volatile compounds and the control (see Figure 6). Similar conclusions were also reported in the recent research of [91] in which the ambusher *S. carpocapsae* [89], the cruiser *H. indica* [89], and two species thought to exhibit an intermediate foraging strategy [89] were all attracted to *Diaprepes abbreviatus*-damaged roots of Swingle rootstock. Some related studies on the foraging strategies of EPNs have been conducted in nonsoil systems [107]; however, we are aware that such studies do not reflect the nematode's true behavior in nature, whereby they are exposed to a myriad of conflicting chemical signals [44, 63]. In our experiment, pure compounds were applied to agar [107], which does not reflect the concentration near the roots of plants [96]. [96] reported that the total sesquiterpene hydrocarbon content in the herbivore-damaged roots of *Zea mays* was 81 ng g-1, whereas the control plants contained only 25 ng g-1, and the relative amount of ß-caryophyllene among several other different terpenes in the maize roots was less than 5%. Moreover, [9] reported that roots damaged by insect larvae attracted more nematodes than mechanically damaged roots and sand controls. The speed of the nematode's response to the chemical stimuli in its natural environment largely depends on the diffusion rate of the chemical compound and on the soil structural heterogeneity [108]. When a foraging nematode is confronted with an array of signals originating from the same general area, the response

may depend on the strength and exposure time and on the nature of the stimuli [63].

confirmed for all of the tested species, with the exception of *S. feltiae* (see Figure 6).

[89] reviewed the literature on foraging and host recognition in *Heterorhabditis* and *Steinerne‐ ma* IJs and proposed that ambusher nematodes respond to host (insect) cues in a hierarchical order, with the volatile cues only becoming important after the IJ had made contact with the insect cuticle, whereas remote volatile cues are more important for cruiser nematodes. Several related studies have also shown that IJs exhibit a preference for different volatile root com‐ pounds [8, 9, 63, 91]. ß-caryophyllene is a common compound and has been identified from various plant species [8, 44, 106]; however, its function, as for most plant volatiles, remains unclear. As [8] reported that ß-caryophyllene strongly attracted *H. megidis*, attraction has been

EPNs.

408 Insecticides Resistance

**Figure 6.** Effects of time of exposure to VOCs on the chemotactic response of the EPN species (A-C), at pure concentra‐ tion. Each data point represents the mean chemotaxis index ± S.E. The bars with the same letter are not significantly different (P>0.05). The small letters indicate statistical significant differences among the different EPN strains at the same time of the exposure. The capital letters indicate statistically significant differences among the different times of exposure for the same EPN strain. B30, C76, and 3162 = *S.feltiae*; B49, C67, and C101 = *S. carpocapsae*; C46 = *S. kraussei*; D54 = *H. bacteriophora*. The substances in our research were with the chemotaxis indexes divided into the following intervals: >0.2 (attractant); from 0.2 to 0.1 (weak attractant); from 0.1 to -0.1 (no effect); from -0.1 to -0.2 (weak repel‐ lent); <-0.2 (repellent) [10]

Our results suggest that the response to different volatile cues is more a strain-specific characteristic than a different host-searching strategy. Similar conclusions were also made in the research of [9, 91]. Indeed, *H. bacteriophora* and *S. carpocapsae* strain B49 showed strong chemotaxis to ß-caryophyllene, whereas the other two isolates of *S. carpocapsae* hardly reacted (see Figure 6). A similar conclusion can be made with regard to linalool, with only *S. carpo‐ capsae* strain B49 showing an attraction to this volatile compound from damaged maize roots (see Figure 6). One reason for the attraction of *S. carpocapsae* strain B49 to linalool and ßcaryophyllene may relate to its origin, as this strain was isolated in a grassland near a maize field [109], supporting the theory of [110] who concluded the possible genetic adaptation of EPNs to different biotic and abiotic factors. In related work, [111] reported that specialization rather than the foraging strategy may better explain the attraction of EPNs to different VOCs. The EPN strains in our experiment showed only a weak attraction to α-caryophyllene, suggesting that this compound could not have an important role in the orientation of IJs to the damaged roots of maize plants (see Figure 6). *S. kraussei* showed a retarded reaction to both ßcaryophyllene and α-caryophyllene in our experiment, suggesting a different host (insect) cue hierarchical order than the other cruisers (*H. bacteriophora*), with the volatile cues only becom‐ ing important after a long exposure.
