The effect of Xenorhabdus bacteria metabolites on Colorado potato beetle (Leptinotarsa decemlineata) adult feeding and larval survival.

Colorado Potato Beetle (CPB) is one of the most destructive potato pests that can quickly develop resistance to insecticides. Therefore, new safe and effective control strategies that are less susceptible to the development of resistance by CPB are urgently needed. Due to their complex mode of action, the likelihood of resistance development by target pests is generally low with antifeedants. In the present study, we assessed the effect of secondary metabolites of various Xenorhabdus bacteria species and strains on CPB adult feeding and on larval development. The metabolites were applied in the form of cell free supernatants (CFSs) from Xenorhabdus cultures. In bioassay 1, leaves treated with ten Xenorhabdus cultures were fed to CPB adults, and their feeding was assessed daily for one week. In bioassay 2, CPB egg masses were placed on the leaves treated with five bacterial cultures, and larval development to pupae was monitored. Out of the ten Xenorhabdus cultures tested, two strains exhibited a significant reduction in the feeding behavior of Colorado Potato Beetle adults, with reductions of up to 70% compared to the control. The effect of CFSs on larval development was variable, and when treated with X. khoisanae SGI 197, over 90% of larvae died in the first few days before reaching the 2nd instar, and complete mortality was achieved on the 8th day of the experiment. Our study is the first study to demonstrate the antifeedant effect of Xenorhabdus cultures towards herbivorous beetles, and the metabolites of these bacteria may have potential for CPB control. Clearly, the metabolites produced by X. khoisanae SGI-197 may be a promising tool for CPB larvae control with the potential to significantly decrease damage to potato plants.

The use of Phasmarhabditis nematodes and metabolites of Xenorhabdus bacteria in slug control.

Many species of slugs are considered serious pests in agriculture and horticulture around the world. In Europe, slugs of the genera Arion and Deroceras are the most harmful pests in agriculture. Therefore, the main goal of this study was to evaluate the effect of the whole-cell metabolites of 10 strains of five Xenorhabdus and three slug-parasitic nematodes (Phasmarhabditis hermaphrodita, Phasmarhabditis bohemica, and Phasmarhabditis apuliae) on the feeding behaviour and repellent effect on target slugs and evaluate a new possible means of biocontrol of these pests. The repellent and anti-feedant effects of nematode-killed insects, metabolites, slug-parasitic nematodes and a combination of metabolites and nematodes were studied through experimental designs: sand-filled plastic boxes divided into two parts in several modifications: with dead Galleria mellonella killed by nematodes, lettuce treated with bacterial metabolites and lettuce placed on the treated sand. We found that slugs avoid eating G. mellonella killed by nematodes, while they eat freeze-killed G. mellonella. Similarly, they avoid the consumption of lettuce in areas treated with bacterial metabolites (the most effective strains being Xenorhabus bovienii NFUST, Xenorhabdus kozodoii SLOV and JEGOR) with zero feeding in the treated side. All three Phasmarhabditis species also provided a significant anti-feedant/repellent effect. Our study is the first to show the repellent and anti-feedant effects of metabolites of Xenorhabdus bacteria against Arion vulgaris, and the results suggest that these substances have great potential for biocontrol. Our study is also the first to demonstrate the repellent effect of P. apuliae and P. bohemica. KEY POINTS: • Slugs avoid eating G. mellonella killed by entomopathogenic nematodes. • Bacterial metabolites have a strong repellent and antifeedant effect on slugs. • Presence of slug parasitic nematodes increases the repellent effect of metabolites.

Virulence of Beauveria bassiana Strains Isolated from Cadavers of Colorado Potato Beetle, Leptinotarsa decemlineata.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a serious, widely distributed pest of potato and other crops. This pest is able to defoliate the host plant and cause severe yield loss. Moreover, the pest quickly becomes resistant to many chemical pesticides. Therefore, the development of novel biopesticides targeting this pest is urgently needed. The purpose of this study was to obtain new strains of the entomopathogenic fungus Beauveria bassiana and assess their efficacy against L. decemlineata adults under laboratory conditions. Twelve strains were isolated from cadavers of Colorado potato beetles collected in potato fields in the Czech Republic. Test beetles were treated by suspensions of conidia at the concentration of 1 × 107 spores per milliliter and their survival was recorded daily for three weeks. The results of the bioassays revealed that all new native strains were pathogenic to L. decemlineata adults and caused mortality up to 100% at the end of the trial period with an LT50 of about 7 days. These strains were more virulent than a reference strain GHA and some of them can be recommended for the development of a new mycoinsecticide against L. decemlineata. Our findings also highlight the importance of searching for perspective strains of entomopathogenic fungi among naturally infected hosts.

Efficacy of the Applied Natural Enemies on the Survival of Colorado Potato Beetle Adults.

Colorado potato beetle Leptinotarsa decemlineata is among the most destructive pests of potatoes quickly developing resistance to traditional insecticides. In the present study, we tested the effect of various species and strains of entomopathogenic nematodes on CPB adults, and subsequently, the most effective nematodes were applied alone and in combination with entomopathogenic fungus B. bassiana in pots with potato plants and in the field and their effect on the number of emerging adults was evaluated. In the experimental infections, both the nematode invasion and pathogenicity were variable, and, in several strains, the mortality reached 100%. In pot experiments, soil application of nematodes S. carpocapsae 1343 and S. feltiae Jakub and fungus significantly decreased numbers of emerging CPB adults, while, after the application on leaves, only fungal treatment was effective. The field application of fungus B. bassiana significantly decreased the number of emerging CPB adults in comparison to control sites by ca. 30% while the effect of nematodes and the nematodes-fungus combination was not significant. In conclusion, we demonstrate the necessity of thorough bioassays to select the most effective nematode strains. Entomopathogenic nematodes have the potential to effectively decrease the emergence of CPB adults, but further research is needed to improve the effectiveness in the field.

The Effect of Artificial Media and Temperature on the Growth and Development of the Honey Bee Brood Pathogen Ascosphaera apis.

Ascosphaera apis is a causative agent of chalkbrood, which is one of the most widespread honey bee diseases. In our experiments, the influence of several artificial media and cultivation under different temperatures was evaluated. Concretely, the radial growth of separated mating types was measured, reproductive structures in a Neubauer hemocytometer chamber were counted simultaneously, and the morphometry of spore cysts and spore balls was assessed. The complex set of experiments determined suitable cultivation conditions. A specific pattern between reproductive structure size and temperature was found. The optimal temperature for both mating types was 30 °C. SDA and YGPSA media are suitable for fast mycelial growth. Moreover, the effect of bee brood on fungus growth and development in vitro was investigated by modification of culture medium. The newly modified medium PDA-BB4 was most effective for the production of the reproductive structures. The result suggests that honey bee brood provides necessary nutrients for proper fungus development during in vitro cultivation. As there is no registered therapeutic agent against chalkbrood in most countries, including the European Union, the assessment of A. apis growth and development in different conditions could help to understand fungus pathogenesis and thus control chalkbrood disease.

Dissemination of Isaria fumosorosea Spores by Steinernema feltiae and Heterorhabditis bacteriophora.

Entomopathogenic nematodes and fungi are globally distributed soil organisms that are frequently used as bioagents in biological control and integrated pest management. Many studies have demonstrated that the combination of biocontrol agents can increase their efficacy against target hosts. In our study, we focused on another potential benefit of the synergy of two species of nematodes, Steinernema feltiae and Heterorhabditis bacteriophora, and the fungus Isaria fumosorosea. According to our hypothesis, these nematodes may be able to disseminate this fungus into the environment. To test this hypothesis, we studied fungal dispersal by the nematodes in different arenas, including potato dextrose agar (PDA) plates, sand heaps, sand barriers, and glass tubes filled with soil. The results of our study showed, for the first time, that the spreading of both conidia and blastospores of I. fumosorosea is significantly enhanced by the presence of entomopathogenic nematodes, but the efficacy of dissemination is negatively influenced by the heterogeneity of the testing arena. We also found that H. bacteriophora spread fungi more effectively than S. feltiae. This phenomenon could be explained by the differences in the presence and persistence of second-stage cuticles or by different foraging behavior. Finally, we observed that blastospores are disseminated more effectively than conidia, which might be due to the different adherence of these spores (conidia are hydrophobic, while blastospores are hydrophilic). The obtained results showed that entomopathogenic nematodes (EPNs) can enhance the efficiency of fungal dispersal.

Low Efficacy of Isaria fumosorosea against Box Tree Moth Cydalima perspectalis: Are Host Plant Phytochemicals Involved in Herbivore Defence against Fungal Pathogens?

Buxus sp. is an important native and ornamental tree in Europe threatened by a serious invasive pest Cydalima perspectalis. The larvae of this moth are able to defoliate box trees and cause their death. The development of novel biopesticides targeting this pest might help protect Buxus trees grown wildly or in city parks. Laboratory experiments were conducted to assess the efficacy of entomopathogenic fungus Isaria fumosorosea strain CCM 8367 against C. perspectalis. The last-instar larvae of the box tree moth were treated by the suspension of fungus conidia at concentrations ranging from 1 × 104 to 1 × 108 spores per 1 mL. Fungus infection was observed mostly in pupae, but the maximum mortality did not exceed 60%, indicating a very low susceptibility of C. perspectalis to I. fumosorosea. Furthermore, a number of ungerminated fungal conidia were found on larval cuticles using a low-temperature scanning electron microscopy. Our data also reveal that the hydroalcoholic extract from B. sempervirens leaves significantly inhibits both the germination of I. fumosorosea conidia and fungus growth. It can be speculated that the strain CCM 8367 of I. fumosorosea is not a potent biocontrol agent against C. perspectalis and low virulence of the fungus might be due to the accumulation of host plant phytochemicals having antimicrobial activity in larval cuticle of the pest.