1. Introduction
The ermine moths (Lepidoptera: Yponomeutidae) include about 500 species (28 in Poland), whose larvae feed on a range of species and varieties of fruit and ornamental trees. Ermine moths may be monophagous, oligophagous, or polyphagous insects [
1]. Species
Yponomeuta are significant pests throughout Europe [
2]. The orchard ermine moth,
Yponomeuta padella (Linnaeus, 1758), defoliates fruit and ornamental trees, whereas the small ermine moth,
Yponomeuta cagnagella, (Hübner, 1813) feeds on the spindle tree. The larvae of ermine moths build characteristic webs at the tips of twigs and join together neighboring leaves, which constitute their feed. In spring, they feed mainly on buds, while in the early summer, they consume massive amounts of leaves, thus markedly limiting the trees’ fruit development [
3].
Yponomeuta padella and
Y. cagnagella are closely related and have very similar morphology and growth cycle. In summer, ermine moth females secrete pheromones to attract males [
4]. Fertilized eggs are then laid on leaves near the tips of twigs or buds. After several days, caterpillars hatch from the eggs but do not start feeding and, instead, overwinter. In spring, the caterpillars begin feeding on leaves and twigs and produce dense, protective webs. After achieving an appropriate body size, they build cocoons, in which they pupate, with the imagines flying out in summer [
5,
6,
7,
8,
9]. In 1972,
Y. padella spread widely across Northern Ireland, destroying about 150,000 km of hawthorn hedges [
10,
11]. Since then, the species has been considered an environmental threat because of its remarkable ability for defoliation, but also due to the insecticides that are commonly used to control the pest. There is a variety of commercially available pesticides used to control ermine moths in large orchard plantations, which contain various active ingredients: cypermethrin from the pyrethroid group; emamectin benzoate, a compound from the macrocyclic lactones group; chlorantraniliprole, a compound from the anthranilic diamide group; abamectin, a compound from the of macrocyclic lactone group; and azadirachtin A, a compound belonging to the limonoid group [
12]. Beyond the chemical means of pest control, there are also biological methods available that contain
Bacillus thuringiensis var.
aizawai. Ermine moths are resistant to chemical insecticides used on crops, but only for a short time; if the motile larvae do not have webs and feed on young leaves, they can be controlled with preparations containing
B. thuringiensis [
13]. Hence, environmentally safe methods need to be found to control these pests [
14,
15]. So far, research on the use of biological methods for combating
Yponomeuta has focused mainly on searching for their natural enemies. As a result, dozens of species have been identified, most of which are parasitoids (e.g.,
Ageniaspis fuscicollis (Dalman)) and predators (e.g.,
Agria mamillata (Pandellé)). Various viruses and microorganisms (e.g., nuclear polyhedrosis virus,
Microsporidum sp.) have also been isolated [
11,
16,
17,
18,
19,
20,
21]. At present, however, none of the above-mentioned organisms have found practical application in the control of species of the genus
Yponomeuta.
Recently, more attention has been paid to the potential use of entomopathogenic nematodes (EPNs) to control populations of many insect species pests. Entomopathogenic nematodes of the genera
Heterorhabditis and
Steinernema are insect obligate parasites. These nematodes have a symbiotic relationship with bacteria of the genera
Photorhabdus and
Xenorhabdus, respectively. Infective juveniles (IJs) enter the host through natural openings such as the mouth, anus, or spiracles, but the IJs of some species can also enter through the cuticle. After penetrating the host’s hemocoel, nematodes release their symbiotic bacteria, which usually kill the host [
22,
23]. Entomopathogenic nematodes of the genera
Steinernema and
Heterorhabditis have been extensively used as biological agents against pest insects [
24,
25,
26]. It was found that about 250 insect species representing 10 orders were sensitive to the nematode
Steinernema feltiae (Filipjev, 1934) [
25]. The native isolate of
S. feltiae ZAG15 was tested in our previous studies against various insect species (Lepidoptera, Coleoptera) and always showed high efficacy. Since we assumed that this isolate would also be effective against the species
Yponomeuta, we made two hypotheses:
The native isolate of S. feltiae shows high activity against Yponomeuta larvae and pupae, whether they are in webs or not;
The native isolate of S. feltiae causes mortality of over 50% of the Yponomeuta population during its outbreak under field conditions.
This study was designed to assess the sensitivity of Y. padella and Y. cagnagella larvae and pupae to a native strain of S. feltiae ZAG15 nematodes under laboratory conditions and to test the biological activity of these nematodes against the larvae and pupae of these species in field studies. This present study is the first attempt to evaluate the efficacy of native EPN isolate in controlling Yponomeuta species.
3. Results
The highest mortality was found in
Y. padella pupae (88.3%) in the Petri dish tests, while the lowest was for
Y. cagnagella pupae (15.4%) in the container tests. All the results are presented in
Table 2.
No significant (
p > 0.05) differences were found in the mortality of larvae and pupae of
Y. padella three days after application in both Petri dish and container tests (
Table 3). In the Petri dish tests, no significant differences (
p > 0.001) between the mortality of
Y. cagnagella larvae and pupae were noted. The container tests, however, showed statistically significant differences; the mortality of the larvae was higher (44.19%) than that of the pupae (15.38%) (
Table 4).
The comparison of Petri dish and container tests for
Y. padella showed significant differences in the mortality of both larvae and pupae (
p ≤ 0.001). A higher mortality was noted for the Petri dishes. No significant differences were found in
Y. cagnagella (
Table 5).
Significant differences were also found when comparing the mortality of
Y. padella and
Y. cagnagella larvae and pupae. Both larvae and pupae (81.7% and 88.3%, respectively) had greater mortality for
Y. padella (
Table 6).
In the container tests, however, significant differences were noted only for pupae: the mortality of pupae was greater (35.6%) for
Y. padella (
Table 7).
Regardless of the stage (larva, pupa) and species of Yponomeuta, all insects survived and showed no signs of infection with nematodes. The application of EPNs in field trials appeared ineffective. The survival of both larvae and pupae was 100%.
4. Discussion
Testing alternative pest control means such as EPNs is needed to curb the use of chemical pesticides on fruit, ornamental trees, and shrubs. The results of our study show that both the larvae (81.7%) and pupae (88.3%) of
Y. padella had a greater susceptibility to EPNs than that of
Y. cagnagella (50% and 33.3%, respectively). Such an effect may result from the fact that the physical structure and immunological resistance of various host species affect their susceptibility to infection by EPNs [
36]. There are a few examples in the literature showing that two closely related host species have very different susceptibility to EPNs. A similar observation was made in a study by Mazurkiewicz et al. [
37], where the difference in the mortality of three species of
Pieris (
P. brassicae L. and
P. rapae L.) after applying
S. feltiae ZAG15 isolate reached 20%. Studies on the use of EPNs to control moths of the genus
Yponomeuta were carried out by Kepenekci et al. [
38]. The application of
S. feltiae to infect two species (
Y. padella and
Y. malinellus Zell.) resulted in mortality ranging from 33.3 to 49.9%. In our study, the mortality of
Y. padella was much greater and amounted to 81.7%. Kepenekci et al. [
38] observed similar mortality (88.9%) in the larvae of
Y. padella after applying the isolate
Heterorhabditis bacteriophora Poinar. The diverse difference in results between Kepenekci et al. [
38] and our studies may indicate differences in pathogenicity, both for isolates within one species and also between nematode species. The dosage does not seem to matter; Kepenekci et al. [
38] used a double dose of IJs and obtained a much lower mortality rate when using
S. feltiae isolates.
Despite the many studies carried out worldwide on the use of EPNs to control plant pests, only Kepenekci et al. [
38] analyzed Yponomeutidae from this aspect. Therefore, in the
Section 4, we refer to other moth species whose caterpillars build protective silk webs when feeding. Among moth species, most studies pertained to the fall webworm,
Hyphantria cunea Drury (Lepidoptera: Erebidae). In laboratory studies by Yüksel et al. [
39], an EPN dose of 50 IJs caused high mortality (75–85%) in the larvae of
H. cunea 96 h after application. In our study of
Y. padella, comparable mortality had already been reached after 48 h. Gözel [
40] used four native EPN isolates and obtained high mortality (66–100%) in
H. cunea after 48 h. Other studies on the sensitivity of the larvae of brown-tail moths,
Euproctis chrysorrhoea (L.) (Lepidoptera: Erebidae), to native EPN isolates, showed them to be highly effective, producing between 51.6 and 81.3% mortality [
41], being thus comparable to our findings. To our knowledge, this study is the first attempt to use nematodes to control
Yponomeuta in the field. The prospective results of laboratory experiments on the effectiveness of EPNs against insect pests do not often translate to comparable results in field trials [
42]. This was the case in our research, where laboratory tests showed that the isolates had a high pathogenicity towards
Yponomeuta; however, the field trials did not demonstrate this effect of the
S. feltiae isolate on
Y. padella and
Y. cagnagella. The ineffectiveness of the EPN application in the field may result from the influence of abiotic factors and also the IJs finding it more difficult to penetrate through the silk webs and pupal cocoons [
43]. This was confirmed by our results; the higher larvae and pupae mortality in the Petri dish tests (insects without webs) compared to the container tests (insects in webs) proves the protective role of these structures. On the other hand, the higher mortality of larvae compared to pupae in the container tests indicates that pupal cocoons provide an additional protective barrier against nematodes.
According to Lacey and Georgis [
44] and Yüksel et al. [
39], after the application of the EPNs, the survival rate and tolerance to drying differed markedly across species and EPN isolates. The most critical period for the survival of IJs is often counted in the minutes and hours directly after application. UV radiation and the associated fast drying are responsible for a rapid decline in the number of IJs (40–80%) during this period [
45]. Keeping in mind the high potential of EPNs and the results of the laboratory tests presented here, one should search for other solutions that might prove effective in future field trials. One of the ways to improve the effectiveness of nematode applications might be the use of adjuvants and new formulation technology of EPNs. Their use may slow down the rate at which nematodes dry, increase their effectiveness, and enable EPNs to persist longer on the surface of plants and silk webs [
46,
47]. One example of the application of adjuvants is found in the research of Beck et al. [
48], who showed that the use of the Addit adjuvant during nematode spraying increased the amount of EPNs retained on the leaves. In a study by Hoctor et al. [
49], the authors evaluated the effect of four surfactants (Revolution, Aqueduct, Cascade Plus, OARS) on the survival and virulence of
H. bacteriophora. The results of this study showed that adding these surfactants increased the survival rate of EPN larvae. Increasing the survival rate of IJs using adjuvants was also demonstrated in studies by Platt et al. [
50]. In field studies [
51] where Atpolan Bio 80 EC adjuvant was used with
S. feltiae ZAG15 isolates, a relatively high effectiveness was achieved with foliar application. Searching for new strains and species is also a feasible approach, which might lead to increased effectiveness based on innate differences in nematode virulence, environmental tolerance, and other properties. Using native species is also preferred in order to reduce environmental risk [
42]. Much EPN research has focused on the use of indigenous isolates, as they are expected to be better adapted to local conditions and thus more successful in practical applications [
52]. Our earlier studies clearly show that while some strains are always effective, the pathogenicity of others is more dependent on the host [
53]. Now, the most promising studies seem to be those by Yüksel et al. [
39], which have shown, under laboratory conditions, that the bacterial cell-free supernatants isolated from freshly emerged IJs, not the IJs themselves, have a high degree of effectiveness (20–87.5%) against
H. cunea.
Despite the failure in field application, new opportunities for the use of EPNs in pest control should continue to be sought because Yponomeuta is very sensitive to EPNs. The development of a biological method to control Yponomeuta based on EPNs could become an element of integrated pest management (IPM) against these pests. This is extremely important because they occur in places where the use of chemicals should be avoided.