2.2.6. Microbial Insecticides

Microbial insecticides are based on microorganisms that cause di fferent pathological reactions (sometimes death) of target insects. They may be based on viruses, protozoa, bacteria, and fungi. There are many microbial biopesticides on the market. Here, we focus on bacteria and fungi as they are used to produce two of the probably most widespread and popular biopesticides, also for the usage against the CPB. One evolved from the bacterium *Bacillus thuringiensis* var. *tenebrionis (Btt)*, which has become increasingly accessible within the last years. *Btt* is basically only e ffective if it is ingested. Sprayed, it is most successful against newly hatched CPB larvae, so it should be used in the fields around this time [2,93]. Another bio-compound is derived from the entomopathogenic fungus *Beauveria bassiana* (Bals.-Criv.) Vuill. (1912). Unlike *Btt*, *B. bassiana* represents an e fficient control against adults and all larval stages of the CPB and it is able to continue propagating after the application. This presents a very high degree of control during the entire potato growing season. Most notable limitations of *B. bassiana* seem to be its vulnerability to high temperatures and drought. Therefore, *B. bassiana* might not be of high importance for growers in warm, dry regions [94]. Other options could be specific bacteria such as *Bacillus popillae* Dutky 1941 and *Bacillus lentimorbus* Dutky 1940 which have been positively tested against several pests in laboratory experiments, as well as certain species of protozoa [95]. However, to our knowledge, most of these species have not ye<sup>t</sup> been efficiently tested against the CPB in field trials.

2.2.7. Environmentally Friendly Insecticides, Synergists and Their Combinations with Classical Insecticides

Synthetic pesticides are not allowed in organic farming. The use of insecticides in IPM is only permitted if the pest population reaches an economic threshold. Among the registered insecticides, there are some that are less dangerous for the environment and humans than others. We consider these insecticides as environmentally friendly ones. The group of insecticides that are more suitable for CPB control (i.e., environmentally friendly) in the IPM program is represented by four active ingredients of di fferent origins: *Btt*, neem extract, natural pyrethrin, and spinosad. All of them are also approved for organic farming approaches [23,32]. Compared to classical insecticides, their use reduces environmental pollution and the impact on beneficial entomofauna. The addition of sub-lethal doses of chemical insecticides to biological insecticides to improve their e fficacy was investigated by Kovacevic (1960) and later discussed by Benz (1971) [96]. Barˇci´c et al. [96] found that combinations of environmentally friendly insecticides with classical insecticides can lead to di fferent benefits at lower doses: (i) ecological, because the use of lower doses decrease pollution, and (ii) biological, because the use of combinations might slow down the development of resistance. In addition, the combined action of the insecticides used could lead to a synergistic e ffect (iii) economically, because the cost per treatment is lower.

Commercial formulations of spinosad applied at three di fferent concentrations (0.2%, 0.1%, and 0.05%) and temperatures (15, 20, and 25 ◦C) were tested against the CPB in the laboratory. Spinosad intoxicated beetles both by contact and ingestion. Experiments revealed that a temperature of 15 ◦C with a concentration of 0.2% in combination caused significantly higher mortality of adult insects than other temperatures and concentrations [17]. Similar positive e ffects were experienced in other laboratory and field experiments for spinosad and combinations with other ecological insecticides [96–98]. In addition to spinosad, the mixture of avermectin B1 (80%) and avermectin B1b (29%), and avermectin C also reached strong e fficacy against third instar larvae and adults [96]. Concerning combinations with spinosad, mixtures with *Btt*, azadirachtinand pyrethrin proved to be very active in both laboratory and field studies (Barˇci´c et al., 2006). Furthermore, the e fficacy of low doses of spinosad, *Btt,* and azadirachtin has been detected and the e ffect of combinations for these three alternative insecticides has been proposed [97].

In addition, the e fficacy of neem and karanja oil in binary mixtures against CPB larvae was investigated [99]. The experiment demonstrated a synergistic e ffect in laboratory trials. The most effective blend with ratio 1:1 was similar or more e ffective than neem oil alone and increased with exposure time. It was also demonstrated that doses can be lowered but still achieve an improved mortality e ffect against larval stages of the CPB [99]. Moreover, neem is a potential insect growth regulator, especially in combination with *Btt* [100].

Further research evaluated the relevance of synergistic effects between capsaicin and organophosphate insecticides against the CPB [20]. The addition of capsaicin to the compound at various temperatures led to an increase in insect mortality by almost one quarter at higher, and three quarters at lower temperatures, compared to organophosphate alone. [101]. There are also several other combinations of environmentally friendly and conventional insecticides that are being tested constantly; some are more and some are less effective against the CPB.

#### 2.2.8. Conservation Biological Control

Conservation biological control is the implementation of practices that maintain and enhance the e fficacy of natural enemies. As it was mentioned earlier, there are just a few specific natural enemies that attack the CPB, but often are only spread at a limited geographic range. Therefore, the complex of generalist predators that attack di fferent developmental stages of the CPB is location-specific and not well investigated [10,102]. Not many papers are dealing with the complex of natural enemies for potato plants, but generally, authors agree that CPB populations are commonly preyed upon by a variety of generalist arthropod predators, including predatory bugs of the genera *Orius* and *Geocoris*, as well as in general Carabidae, Cantharidae, and Opiliones [102–104].

Plant diversity in the vicinity of or on potato fields, e.g., through margins, improves the habitats for natural enemies of the CPB near, outside, or directly inside the fields [7]. Refuge strips often contain both grasses and herbs that provide shelter and resources for predatory arthropods, and flowering plants which are inviting to generalist predators and parasitoids feeding on organic material. This can have a positive e ffect on crop growth [105,106]. Stripes can also help to minimize the use of synthetic chemicals in potato farming as they reduce the likelihood that action thresholds are reached. Encouraging the increases in enemy abundances and diversity can strengthen pest managemen<sup>t</sup> and help to conserve and improve agroecosystems [107]. Increasing the habitat of natural enemies by providing food sources such as leaves, pollen, and nectar within the field or along field boundaries can, therefore, improve the overall e fficacy of conservation biological controls [105].

Focusing directly on the CPB, research showed that increased biodiversity can provide better ecosystem services for e ffective pest control, as alternative methods can lead to increased species richness, evenness, and even larger potato plants [108]. This knowledge is supported by findings that also predators are more abundant in communities with high evenness as they have to compete less with others than with individuals of their species. That is direct proof that a higher diversity of natural enemies leads to improved ecosystem services and functional diversity [108,109]. It was also proposed that companion planting, also labeled "agronomic pendant of plant biodiversity", diminishes the successive colonization of the CPB into potato fields, particularly on organic farms due to increased botanical background noise. That makes host-finding for the pest more di fficult [20]. In a study by Johnson and colleagues, they found no significant di fferences in the number of CPB adults, but more larval individuals in control than in straw plots—possibly again due to di fferent predation rates of natural enemies [110].

Finally, mulch generates microenvironments that benefit CPB predators. In the first half of the season, soil predators—mainly ground beetles—climb on potato plants to feed on second and third instar larvae of CPBs. In the second half of the season, ladybirds and lacewings are the main predators, feeding on eggs and younger larval stages of CPBs. On mulch, there were more predators than on non-mulched plots, following in significantly less damage of potato plants by the beetles [20]. Interestingly, it can often be an advantageous solution to use a healthy ecosystem with improved living conditions for a species-rich environment (including natural enemies of the pest) instead of conventional insecticides to protect crop yields from high pest populations.

In general, there are still many open questions and knowledge is very vague. In most cases, it is not known how single alternative approaches influence specific ecology factors, such as biodiversity, ecosystem services, functional diversity, or pollination success. This shows that a lot more research is needed here to disentangle specific methods and their exact impacts, negative and positive. All conservation biological methods (beside all other alternative ones) are listed in Table 1.








**Table 1.** *Cont.*

#### **3. E** ff**ects of Alternative Control Methods on the Environment**

The relationship between alternative pest control methods and biodiversity is not a one-sided relationship. Some studies have indicated that the utilization of commercial, synthetic compounds for CPB managemen<sup>t</sup> seems to have direct toxic impacts on vulnerable target individuals, but also indirect e ffects on non-target species from various phyla, such as soil-dwelling arthropods [110]. This can disrupt entire food webs leading to communities that are dominated by very few resistant species, while most others disappear from the system [109]. The dynamics of foliar biodiversity are to be negatively a ffected by insecticide treatments on various crops [104,114,115]. Observations in experiments found that the abundance of foliar arthropods was at its lowest level in years when plantations were sprayed with di fferent insecticides five times a year. On the other hand, insect abundance was much higher in years with only two foliar sprays [106]. In another study, the biomass, the number of earthworms, and the abundance and richness of soil collembolas and mites had decreased significantly after one season of conventional potato cultivation. After that, it usually takes about 3–4 years until the biodiversity reaches the same level as it used to before the use of conventional insecticides [48,112].

The use of the above-described alternatives to synthetic insecticides can decrease the broad spectrum of negative environmental e ffects on the ecosystem's health that the use of synthetic insecticides causes. Often this corresponds to high species richness, composition, evenness, and ecosystem services [116]. Through many studies, it is highly recognized that organic farming promotes healthier and more diverse ecosystems than conventional farming [117]. This was confirmed by results of several studies that were conducted on small plots, entire fields, or even big farms [108]. In organic fields, a high proportion of flowering plants in the upper canopy might be able to improve the feeding conditions for many arthropods. The presence of a variety of visible flowers and visiting insects can make organic fields much more attractive to people who seek relaxation in agricultural areas [117]. Organic farming also increases the functional diversity, including important functional groups of plants, pollinators, and predators that can improve natural pest control [106,118,119]. For example, research on birds, small mammals, insects, invertebrates, and various soil organisms almost exclusively showed higher levels and diversity on organic farmland compared to conventional farms, including a higher proportion of rare or threatened species [116,119].

MacFadyen et al. found that organic farms contain higher species richness at three trophic levels: plant, herbivore, and parasitoid [120]. In a later study, they concluded that insect network modules on conventional farms have fewer connections among themselves than on organic ones, which could decrease the stability of these networks. Geiger found higher soil–seed density and weed biomass on organic farms, while seed density positively correlated with both bird species richness and abundance [121]. High community evenness, which is much more present on organic than conventional farms, was found to be positively correlated to stable interactions between soil microbes and plants [122]. Moreover, farming practices that deploy alternative methods of CPB control can result in higher soil organic matter, increased microbial activity, less erosion due to thicker topsoil, cleaner ground and surface water quality, less nitrogen pollution, fewer greenhouse gas emissions, and reduced energy consumption (e.g., fossil energy and pesticides) [123,124]. At the local level, field-margin habitats favored a more diverse carabid beetle fauna than pure potato fields. On a larger scale, beetle diversity on potato fields rose while community composition changed with the increase of natural area that was present within a 1.5 km radius around the field [113]. In another experiment, the number of coccinellids found was significantly higher in fields inter-planted with dill and coriander than in control fields without additional flowering plants. The survival rate of CPB larvae was much lower in dill fields than in the control. Strip-intercropping with properly flowering species is able to significantly improve the conservation of CPB predators and increase biodiversity and reproduction in vegetable production systems [125].

Perhaps the most important aspect of avoiding conventional, synthetic insecticide applications is the fact that it can even have long-term negative impacts on ecologically friendly pest control [106,118]. Conventional broad-spectrum synthetic insecticides can disrupt the communities of natural enemies of pests—resulting in less e fficient control results. For example, the former use of carbofuran to control the European corn borer and the CPB has suppressed or even eliminated predator communities, including ground beetles, in eastern North Carolina with negative e ffects on CPB control and potato plants [116]. Crowder et al. carried out a study of biological pest control in agricultural systems and investigated whether organic farming promotes pest control by increasing the biodiversity of the natural enemy community in potato fields in Washington [108]. They found a higher species evenness in organic fields where natural enemies were relatively evenly distributed, compared to conventional fields dominated by one enemy species that accounted for up to 80% of individuals. In line with this discovery is that a higher evenness of natural enemies particularly decreased CPB abundance through direct attacks either against adults by various insect predators or against the larvae by entomopathogenic nematodes in the soil. Alyokhin and Atlihan, and Alyokhin et al. partially supported the findings on potato plants by demonstrating that CPB populations on manure-amended plots were not only lower but also took longer to develop than on chemically fertilized ones [8,126]. Natural enemy populations increased in further research with rising overall insect diversity and suppressed the pest population [127]. Due to mulching, carabids were found to be more abundant and diverse compared to un-mulched fields, especially in potato fields. Mulching increased the total amount of captured beetles with 17% more on hay mulched and 14% on leaf litter mulched plots [111]. Higher abundances of natural enemies on mulched than on non-mulched plots were very likely responsible for an expansion in yield due to a reduction of CPB leaf feeding [128,129]. Therefore, an increase in overall biodiversity in a healthy ecosystem is often accompanied by an increase in the number of natural enemies of the pest. Ideally, this can lead to a self-reinforcing e ffect, i.e., a subsequent decrease in pest numbers increases the yield and profit of farmers, improves acceptance of natural control methods, and slowly eliminates synthetic insecticides [108].

In conventional potato farming, the main interest is to evaluate under which circumstances a high benefit (yield) could be achieved most e fficiently including minimum negative e ffects on the farm business [48]. For several years already, synthetic chemical insecticides have continued to be marked as bad or even evil in public opinion; thus, they have a bad reputation. The ability of insect pests to develop resistances against many insecticides as one major aspect forces continual renewal of the existing insecticide inventory which noticeably reduces the overall absolute benefit of conventional production [48,106]. Turnbull and Hector illustrated that escalating resistance can lead to increased insecticide spraying including an increasing, ever-wider variety of chemicals, and spiraling costs for farmers [109]. It was further suggested that alternative and ecologically friendly methods which increased natural enemy diversity and evenness of CPBs lead to an increase in potato yield through fewer pests and larger plants as well as the option for farmers to use less cost-intensive insecticides [108,130]. In a review about several crop types planted under various conventional and alternative control conditions, it was detected slightly, often not significant, lower yields in sustainable production. The authors explain the low advantage of conventional methods by the fact that the best conditions for sustainable farming often lack suitable know-how for best application procedures, showing high potential for improvement. In addition, most data from conventional farming was found in developed areas with high-input farming and, thus, over-average yields [131]. Sustainable and ecologically based agriculture can improve the quality of the environmental and natural resource bases upon which the agriculture depends. This can also maintain the (economic) sustainability of farms, and considerably improve the well-being of farmers and the whole society [48]. The higher the acceptance by farmers to use more alternative and ecologically friendly methods, the healthier the ecosystem, as well as the biodiversity of the pest enemies, could be expected. This can potentially reduce pest abundance, further increase yields and profits, and increase farmers' acceptance of using fewer conventional and more alternative control methods (Figure 2).

**Figure 2.** Self-reinforcing effects between alternative control methods and biodiversity: + indicates the increase, − the decrease of its corresponding expression next to it. The increased usage of alternative methods leads to less usage of conventional insecticides, which improves the general biodiversity and ecosystem health. The following higher diversity of natural enemies decreases the pest abundance which leads to a higher yield and profit for the farmer. By that, the acceptance for alternative methods improves including the increase of its usage instead of conventional ones.

In addition, pure alternative control methods and the development of new, ecologically friendly compounds, integrated pest managemen<sup>t</sup> (IPM) could be an alternative to pure conventional techniques. Here, conventional pesticides are being substituted by biological or behavioral control methods. One of the main goals of IPM is to limit the number of pesticides used by decreasing the amount or concentrations. Even if it requires effort to fully understand the synergistic effects between various mechanisms, it has been demonstrated that it can benefit farmers and the environment [132]. For instance, laboratory results indicated that entomopathogenic nematodes tended to reproduce better in CPBs fed on potato plants with high levels of bio-fertilizer [9]. Other results indicated that it is possible to lower the doses of neem and karanja oils and even receive improved efficacy against CPB larval vitality when combining [99], and, under field conditions, the biopesticides spinosad and avermectin caused almost as high mortalities as the conventional insecticide tiamethoxan and higher ones than pirimiphos-methyl [97].

Nevertheless, alternative methods are not free from criticism, for various reasons. First, producers and consumers are still reluctant to accept the approach, because of its complexity and unpredictability [133]. Decision-makers may find it hard to overcome the obstacles of implementation that could result from a lack of tools and know-how to handle the unpredictability of ecologically friendly control methods [48]. This naturally reduces the general acceptance of environmentally friendly methods. Preventing insect outbreaks by manipulating biotic interactions can also be tricky, because it additionally elevates the complexity and difficulty of insect protection systems [133]. Moreover, the micro-climate, local soil properties, and managemen<sup>t</sup> history of a site heavily impact biota interactions and make it difficult to create proper pest control solutions appropriate for large geographical areas and available for every farmer [48,134]. As most invasive pests have intentionally or unintentionally been introduced through humans and often lack natural predators in their new environment, biological control can be challenging or almost impossible [48]. Additionally, a high predator density in organic fields does not necessarily result in low numbers of pests always and everywhere, as has also been shown in tests with the CPB [106]. If alternative methods act differently than expected, it is, therefore, possible that they may cause even more damage than the existing conventional pesticides they are supposed to eliminate [133]. Some techniques are at present time more promising than others, which might also have to do with the complexity of certain alternative methods as well as their under-investigation so far.

Overall, the focus on pure profit is slowly decreasing while the focus on health and the environment is growing for various reasons and due to public opinion [48]. High costs for new conventional insecticides, pest resistance, and loss of biodiversity in a world of constant species loss under the threat of global warming are just some of the reasons to gradually shifting the focus to alternative pest control methods, such as for CPBs. Further research is needed to improve farming and pest control conditions, so that farmers and nature benefit equally by switching from conventional to alternative pest control methods worldwide.
