3.3.5. Fungi

In the field, fungal pathogens can occasionally influence wireworm or click beetle survival greatly. In Switzerland, *Zoophthora elateridiphaga* was described by Turian [136] as attacking *A. sputator*. According to Keller [137], infection rates of *A. sputator* click beetles in Switzerland with *Zoophthora elateridiphaga* (Entomophthoraceae) varied between 72.6% and 100%. The same fungal pathogen occurred at one location near Braunschweig, Northern Germany, in *A. obscurus* and *A. sputator* click beetles, but with only about 10% becoming infected (Lehmhus personal observation 2013, determination of pathogen R. Kleespiess). Entomophthoraceae are comparatively sensitive, difficult to preserve and propagate, and unsuitable for most spray applications, and thus achieving long-term viability is often quite difficult [138]. However, as Keller [139] observed *Z. elateridiphaga* also attacking adults of *Notostira elongata* (Miridae) and achieving growth of colonies on Sabouraud Dextrose Agar (SDA), the host range of this fungus may be less narrow and cultivation less difficult than generally thought. A remaining problem is that attacks by this fungus are directed at adults.

More promising are the entomopathogenic fungi *Beauveria bassiana* (Cordicipitaceae) and *Metarrhizium anisopliae* sensu lato (Clavicipitaceae), including related forms like *M. brunneum* (Figure 4D). These naturally soil-inhabiting fungi are widely recognized as interesting biological control agents against several insect pests [140] and have been known to kill wireworms for more than 100 years [105]. The mechanisms involved in the infection process in wireworms have already been described in detail (e.g., [141,142]). Trials have been conducted with several different strains of both fungi (*Beauveria bassiana* and *Metarrhizium anisopliae* sensu lato, including *M. brunneum*) at different application rates and with different wireworm species both in the field and in the laboratory. The results were quite variable. A commercial product containing a *Beauveria bassiana* strain reached efficacy values between 54% and 94% against *Agriotes* spp. in the field in Northern Italy [143], but in other regions, no differences between potato plots treated with this product and untreated plots were observed [144,145]. Eckard et al. [146] showed differences in mortality for three different strains of *Metarrhizium brunneum* in the three most common European species: *Agriotes lineatus, Agriotes obscurus*, and *Agriotes sputator*. Species and stages of five North American elaterid species differed markedly in resistance to attack by a strain of each of the two entomopathogenic fungi *Metarrhizium anisopliae* and *Beauveria bassiana* [147]. Kabaluk et al. [122] tested 14 isolates of *Metarhizium anisopliae* against three species of wireworms. The North American *Ctenicera pruinosa* was susceptible to most isolates, while *Agriotes obscurus* was highly susceptible to four isolates, and *Agriotes lineatus* was the least susceptible species. Under these circumstances, it is clear that a suitable combination of wireworm species found in the field and EPF strain used is needed to achieve high control effects. A further constraint may be that some bacterial symbionts of wireworms could actively suppress the infection by entomopathogenic fungi [148], which may explain control failures when environmental conditions and the combination of species and strain seem to fit.

#### 3.3.6. EPN and EPF Use Generally

Both environmental and behavioral factors will further affect the infection of wireworms with entomopathogens (EPN and EPF likewise). The retaining of sufficient moisture is indispensable for the growth of entomopathogenic fungi [149] and has to be solved somehow for reliable control. According to Kabaluk et al. [122], Rogge et al. [150], and Kabaluk and Ericcson (2007), additional factors such as temperature, exposure time, conidia soil concentration, and food availability also affect mortality rates of wireworms when exposed to *Metarrhizium anisopliae*. However, while lower temperatures slow down the spread of wireworm infection [151], the desiccation commonly experienced under summer conditions might affect the viability of EPF in soil. Additionally, wireworms can perform seasonal movements to forage in favorable conditions and to avoid unfavorable ones [9,32,50,114,152–155], meaning that wireworms may escape a biocontrol agen<sup>t</sup> used when the lethal potential of an entomopathogen is not reached shortly after application. For example, infection late in the potato-growing season would probably not prevent damage to daughter tubers. Therefore, the temperature conditions under which the infection cycle of an isolate has its optimum must be considered. For early season applications, a more northern fungal isolate adapted to lower temperatures [156] might even enable crop protection early in the year for such crops that need to be protected as young plants. A temperature effect on the pathogenicity of EPN is also known [157,158], albeit not documented for pathogenicity against wireworms. A further constraint is that the soil type can also influence the effectiveness of biological wireworm control [159]. This has also been described for other insects, partly with contradicting results of higher EPN efficacy in sandy soil than in clay soil, or vice versa [160–162].

In contrast, the origin of inoculum had no significant effect on the virulence of a *Metarrhizium brunneum* strain. The mortality of wireworms treated with spores from host cadavers was similar to the mortality of wireworms treated with spores from a modified Sabouraud Dextrose Agar (SDA) after ten sub-cultivations [146]. Therefore, in general, virulence should not be affected by the conidia production method.

According to Ericsson et al. [163], the biological insecticide Spinosad interacted synergistically with *Metarhizium anisopliae* against *Agriotes lineatus* and *A. obscurus,* indicating that combinations with a second stressor (insecticide, EPN, or EPF) might enhance biological control. <the synergistic or additive effects of combined use of EPN, EPF, and EPB have been shown for several other pests [164–167].

Several studies [168–172] show that the application pattern is another important point to consider, with banded or spot application being particularly useful.

Nevertheless, in many cases, results achieved by biological control are not ye<sup>t</sup> satisfactory compared to an effectiveness between 50–90% achieved by plant protection products based on the insecticides carbosulfan, fonofos, findane, fipronil, imidacloprid, thiamethoxam, or bifenthrin used in the past [143,173,174]. However, even an array of insecticides tested on five different wireworm species in three elaterid genera demonstrates that there are clear differences in mechanisms, symptoms, and mortality, with even chemical insecticides failing to remove all wireworms [175].

#### 3.3.7. Attract and Kill—A Possible Solution?

The key issue is how the effect of an entomopathogen could be further enhanced for reliable control. One idea is the development of an attract-and-kill strategy that exploits the foraging behavior of herbivorous insects. This means the combination of a compound attracting the wireworms directly to the product and a killing compound that disposes of them effectively. Such attract-and-kill formulations could be used to enhance the effect of both EPN and EPF, as the wireworms are lured directly to the entomopathogen.

CO2 is a known attractant for wireworms [74,176] and other soil insects [177,178]. Barsics et al. [179] summarized earlier research that demonstrated the existence of CO2 perception and research on a shorter range working chemosensory sensillae in wireworms. Brandl et al. [172] developed an attract-and-kill system with an alginate capsule with yeas<sup>t</sup> and starch producing CO2 as an attractant, with a *Metarrhizium brunneum* strain as kill component; as a result, they were able to reduce tuber damage significantly when compared to the control in three out of seven field trials in potato. In four out of seven trials, the potato tuber damage appeared lower in the attract-and-kill when compared to kill

treatment, but differences were not significant. However, different application scenarios were tested, so the trials are not directly comparable. A resulting formulation is currently the only product in potato against wireworm damage in Germany (emergency registration, restricted acreage). According to Küppers et al. [171], a reduction in damage was achieved with this product at low-to-medium wireworm infestation.

Wireworms are also attracted to plant- or root-produced volatile aldehydes when they are actively foraging [180], similar to several other soil-inhabiting insect herbivores [181]. This or other organic plant compounds could be exploited for an attract-and-kill strategy. La Forgia et al. [182] encapsulated entomopathogenic nematodes with potato extracts as an attractant and feeding stimulant in alginate beads against the wireworm *Agriotes sordidus*. When compared to conventional EPN application and to beads containing only potato extract, the beads with both potato extract and *S. carpocapsae* or *H. bacteriophora* increased mortality rates, significantly only for the latter, indicating the importance of a suitable attractant for effective wireworm control. However, these are the first steps towards an attract-and-kill formulation, and it is possible that a combination of CO2 and root volatiles may enhance the efficacy of the method even further.

Additionally, attract-and-kill strategies using entomopathogens (again *Metharizium brunneum* spores) as alternatives to chemical pesticides may also be used effectively to interfere with click-beetle populations and subsequent wireworm ones, as suggested by Kabaluk et al. [183]. This might be pursued by using modified pheromone traps that allow beetles to enter back and forth traps containing spore powder. This strategy does not require a 100% catch, or the vast majority of male beetles to be caught in a short space of time, since the killing agen<sup>t</sup> would spread through the population, coming into increasing contact with both male and female adult beetles. At least in some click beetles, sex pheromones also perform as aggregation pheromones, and they can also attract significant numbers of females, as demonstrated for *A. sordidus, A. brevis*, and *A. ustulatus* [184–187]. This may be an additional pathway to increasing entomopathogen infections in click-beetle populations and further reducing wireworm pressure on crops.

#### 3.3.8. Problem: Different Species of Wireworms

A general problem in biological control of wireworms is the involvement of several different wireworm species with mixed populations often at the same site [13,32,40,42,46,152,188–190]. When observing the differences in efficacy of a specific EPN or EPF strain against common wireworm species (e.g., [122,132,146]), it becomes clear that for biological control in a certain location, we need to know the wireworm species involved. This is not an easy task. Considerable time and expertise are needed for a reliable identification of wireworm species. Both the molecular method (PCR) and the morphological methods have their difficulties, but both produce reliable results for most individuals [13,39,40,72,191]. Additionally, recent molecular research suggested a possible occurrence of cryptic species in some North American wireworms [72,192], which may also affect the efficacy of a biological product. Furthermore, the activity pattern and damage potential of different wireworms in a crop may differ, which could affect the risk a certain species poses for a certain crop [35,153].

Early on, a specific key only for the Elateridae harming agriculture and horticulture needed to be established, as a major part of wireworms are not relevant in agriculture [193] and could be omitted. Keys for wireworms of economic importance only have been provided early in some countries [37,38,45]. Recently, a simple morphological key has been proposed for more common middle European genera in agricultural fields [41], which may be useful for farmers and plant protection service field workers without access to molecular methods. A final solution could be a combined product involving different strains of entomopathogens with sufficient growth at low temperatures, high efficacy against the commonest wireworm species in a region, and additionally an attractant source, applied in furrow at planting.

#### *3.4. Naturally Derived Insecticides*

Biocidal meals are practical options for controlling wireworm populations, both as prevention structural measures (wireworm population reduction at a suitable rotation period) and as rescue treatments just before the sowing of susceptible crop; after that, the occurrence of a wireworm density exceeding the threshold has been assessed [85]. They contain the same glusosinolate–myrosinase system described for biocidal cover crops (Section 3.1.1). Their potential can be considered comparable to that of chemical insecticides [85]. In laboratory [10] and pot trials [85], *Brassica carinata* seed meals caused a larval mortality higher than 80% and complete maize seedling protection. At large-field scale, both potato and maize crops have been effectively protected. In order to obtain successful practical results in the field, the same conditions described for biofumigant cover crops (Section 3.1.1) need to be fulfilled concurrently. Biocidal meals have become commercial products available for farmers, and practical implementation has already taken place.
