2.2.3. Augmentative Control

The natural enemy complex for each pest, as well as for the CPB, is geographically specific. The CPB is attacked by different generalist predators, but their presence depends on the geographic area and crop field type. They are subject to conservation biological control. Contrary to that, specific predators are not widely distributed and are used as augmentative control and parasitoids. The augmentative biological control uses insect predators [48] as well as parasitoids. The CPB has several natural enemies, but they can be hardly found in most potato fields, especially with heavy usage of conventional insecticides [20]. Within alternative and organic farming systems, the abundance and richness of natural enemies are higher, but are unlikely to fully control the CPB, even though some generalist predators provide good control [20,49].

The objective of several studies to diminish CPB populations focuses on insects of the order Hemiptera, including the Nearctic stink bug *Perillus bioculatus* (F.) [4,50]. The use of this predator to control the CPB has been successful in laboratory and microplot consumption tests. This bug is obviously not naturally abundant in all areas with CPB occurrences, especially not outside of its natural territories in North America [49]. *P. bioculatus* is a natural enemy of CPB in and could potentially also help to diminish CPB populations outside of North America [51]. To solve this problem, predators would have to be released in very high numbers using, for instance, mechanical distributors [49]. However, the production of huge amounts of predators could be difficult as for the suitable control of eggs and early larval stages of CPB, certain temperatures and storage times of the predator nymphs are necessary to be an efficient pest control [50]. The main problem of newly introduced species is their unknown effect on the ecosystem and, therefore, they often not represent a safe and suitable option.

Finally, it was suggested and shown that the combination of sub-lethal effects of *P. bioculatus* with products based on *Bacillus thuringiensis* Berliner var. *tenebrionis (Btt)* could significantly increase CPB larval mortality in field experiments [52].

The spined soldier bug *Podisus maculiventris* Say is another Hemiptera species that can diminish populations of CPB. In 1997, O'Neill demonstrated in laboratory and field experiments predation behaviors of this predator towards the CPB. Due to higher prey–predator ratios in laboratories than in the wild, the experiments revealed a strong decrease in beetle populations [53]. Moreover, just as *P. bioculatus*, under natural conditions none of the investigated predators obtain large enough populations, or are even completely absent when the CPB starts into the new feeding season. This way, CPB outbreaks cannot be avoided [3]. Due to this and the fact that the distribution of *P. maculiventris* is relatively scarce in North America, one plausible strategy could be to collect and transfer the bugs from pheromone traps to nursery traps in the potato fields from where they start the suppression of CPB populations [4]. Hough-Goldstein and McPherson (1996) tested both *P. bioculatus* and *P. maculiventris* in experiments [49]. Although the latter showed less strong significant prey rates comparatively, older life stages were concentrated on larger CPB larval stages. So, the overall success rate between the two CPB predators could even be related to the predator's life stage and used accordingly.

In addition to Hemiptera, several other natural enemies of the Coleoptera order had been investigated to be successful CPB pest controls. Already in field experiments of the late 80s, Hazzard et al. illustrated significant effects of control in both early and late generations of the CPB using the 12-spotted ladybeetle *Coleomegilla maculate* Lengi in western Massachusetts [54]. For the same species during a bigger field experiment, Mallampalli et al. detected the impact of *C. maculata* on a composition of possible prey species [55]. They figured out through computer models that when (beside CPB) eggs of the European corn borer (*Ostrinia nubilalis* Hübner, Lepidoptera) were found in the area, predation on beetle eggs highly became positively density dependent. When larvae and adults of the green peach aphid *Myzus persicae* Sulzer (Homoptera) were present, similar results were obtained accompanying a significant decrease of CPB individuals. These results witnessed that present control, as well as resistance managemen<sup>t</sup> strategies, should also consider the composition of prey species when developing managemen<sup>t</sup> strategies, but more research is needed especially through field experiments [55].

The carabid beetle *Lebia grandis* Say is one of many natural enemies native to North America. The larvae of these beetles are obligate parasitoids, while also the adults feed on CPB eggs and larvae [16]. This predator represents one of the most important natural control species of CPBs in North America. As the activity of this predator peaks at night and it rarely appearance in pitfall trap studies, an insufficient amount of research about this species was conducted so far [16,50]. Other field studies from Idaho indicated that the introduced generalist ground beetle *P. melanarius* might be a useful biocontrol within CPB pest management. Large numbers of eggs and larvae were consumed by adult predators. Hence, the pest population decreased more in untreated than in insecticide-treated fields, because a higher abundance of predator individuals fed on pest individuals [56].

Larval stages of the lacewing (*Chrysoperla carnea* (Stephens)), a Neuroptera species, might represent another good alternative to control CPB populations. Even if field assays are still needed, laboratory studies highlighted a valuable ability to control beetle larvae, with greatest efficacy on the younges<sup>t</sup> stages [57].

Within the Hymenoptera order, the parasitic wasp *Edovum puttleri* Grissell seems to be an effective weapon against CPB damage in potato fields. First, a computer model was built to calculate the possible parasitism of CPB eggs. This algorithm incorporated the specific attack behavior of *E. puttleri*, and the development time for parasitized egg masses [58].

The use of natural enemies may be another option to control CPB populations, but many of those are not abundant in nature and manual release in large areas is not very practical [59]. One promising solution could be the mechanical (physical) distribution of predators. In a test study, huge amounts of predators were mixed in containers with a carrier material. In the field, the containers were opened mechanically at various spots and all predators released at once [60]. Obtained results indicated that the mechanical release of predators ended in a better control of beetle populations and egg masses than manual release [2,60].

Augmentative control could be a valuable approach due to its low negative impact on ecosystem health and biodiversity. We focused on some of the most investigated arthropod parasites and parasitoids in research studies. Species of mites, phalangids, spiders, and parasitic flies (Tachinidae) were discovered to be able to control CPB populations [49]. Still, it has to be mentioned that many (described) experiments were conducted in the laboratory and only have a theoretical value if not also tested in the fields and large geographical scales.

#### 2.2.4. Use of the Plant Extracts and Botanical Insecticides

Since 1990, biopesticides are slowly but steadily replacing synthetic, conventional pesticides and are even used commercially [61–64]. They are often (besides hydrolytic compounds or primary metabolites) generated from compounds that are produced by plants in secondary metabolites, often after pest infestation or in harsh environmental situations [65]. They appear as repellents or antifeedants and help to resist against a broad range of pest species by increasing their mortality or decreasing the reproduction ability [66]. Although biopesticides seem to be a promising way to replace conventional, chemical insecticides, they are still underexplored and the practical application is still limited [67].

Plant extracts and botanical insecticides represent two di fferent types of products: homemade products as well as commercially available botanical insecticides. Even if they originate from nature, their properties are not always acceptable for plant protection [10,23]. However, commercially available botanical insecticides have been subject to the same registration procedure as chemical pesticides. If they are approved, they are, therefore, considered safe for use with all restrictions as stated on the label. A comprehensive review of 48 di fferent plant extracts and botanical insecticides tested against the CPB (including only a few widely used commercial products) shows that some of the plant extracts have a high potential for CPB control and should be investigated further [10].

A small number of commercial and widely used botanical products are on the market for use against CPB. Rotenone is a biopesticide that is one root extract from several species within the Fabaceae family [68]. Since it kills pests rather slowly, it can be associated with pyrethrum for a more rapid effect, lasting for up to two days [69]. It should be used carefully as it is also poisonous to non-target insects as well as to domestic and farm animal species. The European Union (EU) began a phase-out of rotenone in 2008 [70]. The final authorization was withdrawn on 31 October 2011. Therefore, rotenone is not approved for use in the EU or any EU member state [71].

The e ffects of *Origanum vulgare* L. extracts have been discussed in several papers [14,72]. Experiments demonstrated that extracts gained from the dry and fresh matter at the highest concentrations contributed to the greatest reduction of females and males feeding on potato plants. Similar results were observed after the application of lower concentrations, but only in females [14]. Moreover, the morbidity of the essential oil of Iranian lemongrass, *Cymbopogon citrates* Stapf, was positively assessed against adults and third instar larvae of CPBs. The higher the concentration, the stronger the e ffect against the pest [73].

In additional studies conducted in Turkey, potato leaves were prepared with three different extract solutions of five different plant species (*Arctium lappa* L., *Bifora radians* (M.Bieb), *Humulus lupulus* L., *Xanthium strumarium* L., *Verbascum songaricum* Schrenk) and then exposed to the larvae of CPB. Observations of larval behavior during one day of exposure revealed that the plant blends significantly influenced the interaction between beetles and leaf tissue. This was not the case for very low concentrations—only the medium (except *V. songaricum*) and high extract (all species) concentrations [74]. In another similar experiment, extracts of *Acanthus dioscoridis* L., *Achillea millefolium* L., *Bifora radians*, *Heracleum platytaenium* Boiss, *H. lupulus*, and *Phlomoides tuberosa* L. were tested against different larval stages for two days. For second to fourth instar larval stages, *H. lupulus* and *H. platytenium* reached the highest CPB mortality rate while the first larval stage was more susceptible [75].

Di fferent essential oils of *Eugenia caryophyllus* (Sprengel), *Mentha spicata* L., *Myrtus communis* L., *Ocimum basilicum* L., *Satureja khuzistanica* Jamzad, and *Thymus daenensis* Celak were tested for their nutritional indices and mortal e fficacy against adults and fourth instar larvae of CPB. All essential oils showed a deterrent e ffect, with the most e fficient oil coming from *S. khuzistanica* [18]. Several authors examined the e ffects of ethanolic extracts obtained from various parts of *Liquidambar orientalis* L., *Buxus sempervirens* L., *Alnus glutinosa* (L.) Gaertn., *Artemisia absinthium* L., *Aesculus hippocastanum* L., and *Rhus coriaria* L. on the egg-laying behavior of CPBs in the laboratory. Afterward, the antifeedant and toxic e ffects of the two most e ffective extracts leading to the smallest number of egg-laying, *L. orientalis* and *B. sempervirens*, were tested in a field study. Both extracts indicated significant decreases in egg numbers of CPBs and seemed to be potentially successful in the field as an alternative to chemical pesticides [76].

In the laboratory, first-generation CPB adults were treated with *Artemisia vulgaris* L. and *Satureja hortensis* L. The extracts of both plants had no lethal e ffect on adult mortality. Nevertheless, in both cases, the aqueous extract solutions induced a higher percentage of sterility of eggs compared to the alcoholic extract, while the e ffect on eggs treated with *Artemisia* variants was higher than that with *Satureja* [77]. Furthermore, the e ffects of compounds from two Piperaceae species, *Piper nigrum* L. and *Piper tuberculatum* Jacq., against adults and larval CPBs were assessed with several di fferent plant extract concentrations [78]. It was found that early larval stages of few days were most vulnerable

in both plant species. Additionally, the activity of *P. nigrum* indicated that contact toxicity was most effective when early instar larvae were targeted. Late instar larvae could be knocked down with higher concentrations and 50% of the adults could be killed with a high application. *P. nigrum* lost much of its repellent function under pure sunlight. Nevertheless, pepper species could be suitable biopesticides since they are among the most traded species, they are relatively safe to use and store, and seed and leaf material are universally available [78,79].

In an experiment with many different plant species, the contact and residual toxicity of 30 plant extracts was investigated on third instar CPB larvae. The insects were sprayed and the effectiveness was measured every 24 h for one week. After a 24-h incubation, blends of *Artemisia vulgaris* L., *Hedera helix* L., *H. lupulus*, *Lolium temulentum* L., *Rubia tinctoria* L., *Salvia o*ffi*cinalis* L., *Sambucus nigra* L., *Urtica dioica* L., *V. songaricum*, and *X. strumarium* killed significantly more beetles than the control. In general, a longer incubation time than 24 h did not show higher values. The *H. lupulus* extract was the most toxic of all products, causing 99% beetle mortality [4]. Fresh and dry matter of wild thyme (*Thymus serpyllum* L.) in different concentrations was tested on the feeding behavior of CPB adults and larvae. For efficient control of adults, a dry matter extract with the highest tested concentration (10%) should be used, while larvae at the fourth instar appeared to be significantly more vulnerable [80].

Stilbenes are phenolic compounds that are produced in several vines in large quantities and function as plant defenses. Oligomeric forms were proven to be very efficient against a broad range of pests. The aim of a study conducted by Gabaston et al. was to explore the activity of a grapevine root extract containing a stilbene oligomer pool [81]. In the laboratory, the extracts showed toxic effects on larvae and slowed down their development and food intake, while in field experiments, high CPB mortality could be observed. In addition, the extract also killed non-targeted organisms, such as earthworms (*Eisenia fetida* Savigny). The authors emphasized that grapevine roots still represent promising sources of bioactive compounds to create alternative insecticides [81].

In the study by Trdan et al., refined rapeseed oil (*Brassica napus* L.) and slaked lime were tested under laboratory conditions for their efficacy against CPB larvae and adults at three different temperatures [82]. Heat or cold did not play a specific role as the tested substances caused significant damage to beetles at each temperature. Adults were the most sensitive developmental stage and revealed the highest mortality rate, while refined oil was discovered to be the stronger beetle repellent [82].

Various products of azadirachtin, which is produced from neem tree seeds, showed effects against the CPB. In one study, neem (along with several other bio-insecticides) was suggested as an effective control agen<sup>t</sup> against CPB larvae and adults [82]. On the plants treated with azadirachtin, between 8% and 32% of eggs were left unhatched. This effect was reported even 7–8 days after the end of hatching on the untreated control [83]. However, neem concentrations of more than 1% can potentially lead to phytotoxicity in potato plants. Although once considered benign to beneficial insects and effective against the CPB, neem products have also demonstrated some adverse effects as it was found to be poisonous to ladybirds, particularly in early larval stages [84]. Neem has also been found less effective than *Btt* [85].

A two seasons field experiment in Canada evaluated the use of spraying plant blends as an alternative control of the CPB. The herbs evaluated as companions to potato plants were bush beans, flax (*Linum usitatissimum* L.), French marigold (*Tagetes patula* L.), horseradish (*Armoracia rusticana* Gaertn., C.A.Mey. & Scherb.), and tansy (*Tanacetum vulgare* L.). A capsaicin extract, a garlic extract, a neem seed extract, a *Btt* product, and a pine extract were tested as controls [19]. This showed that plant individuals sprayed with neem extract experienced higher yields, lower beetle density, and less defoliation than each of the other treatments and the control. *Btt* controlled all, but mainly larval stages (less the adults) of CPBs, but in total less than neem. On the other hand, garlic and capsaicin extracts, as well as companion planting, did not diminish CPB densities in potatoes. This raised concerns about the use of companion plants without first testing their efficacy, but also demonstrated the potential success of this approach [19].

The best known and oldest botanical insecticide is a powder obtained by grinding the dried flowers of the Dalmatian pyrethrum plant, *Chrysanthemum cinerariaefolium* Trev. and related species *C. coccineum* Wild. It is a wide spectrum insecticide e ffective against many di fferent pests. Laboratory and field investigations demonstrated that the e fficacy of pyrethrin was between 83% and 86% in the laboratory and between 86% and 88.0% in field trials [86]. In the same trials, the e fficacy of neem extract was between 62% and 63% in laboratory trials and between 55% and 88% in field trials. The e fficacy of both insecticides was significantly lower than the e fficacy of the standard insecticide spinosad and at the same level as the e fficacy of *Btt* [86]. The main field advantage is that it dissolves quickly in direct sunlight without spreading widely in the crop plants [15].

Finally, pyola is a natural compound that contains canola oil and pyrethrins. It is applied not only against the CPB, but also against several other insect pests. Since a large part of rapeseed oil available commercially used comes from genetically modified plants, this product may not be in line with rules of organic farming despite its success in pest managemen<sup>t</sup> [23].
