When Competitors Join Forces: Consortia of Entomopathogenic Microorganisms Increase Killing Speed and Mortality in Leaf- and Root-Feeding Insect Hosts.

Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to be compatible and ideally work together. We studied the interaction of a previously selected BCA consortium of entomopathogenic pseudomonads (Pseudomonas chlororaphis), nematodes (Steinernema feltiae associated with Xenorhabdus bovienii), and fungi (Metarhizium brunneum). We monitored the infection course in a leaf- (Pieris brassicae) and a root-feeding (Diabrotica balteata) pest insect after simultaneous application of the three BCA as well as their interactions inside the larvae in a laboratory setting. The triple combination caused the highest mortality and increased killing speed compared to single applications against both pests. Improved efficacy against P. brassicae was mainly caused by the pseudomonad-nematode combination, whereas the nematode-fungus combination accelerated killing of D. balteata. Co-monitoring of the three BCA and the nematode-associated Xenorhabdus symbionts revealed that the four organisms are able to co-infect the same larva. However, with advancing decay of the cadaver there is increasing competition and cadaver colonization is clearly dominated by the pseudomonads, which are known for their high competitivity in the plant rhizosphere. Altogether, the combination of the three BCA increased killing efficacy against a Coleopteran and a Lepidopteran pest which indicates that this consortium could be applied successfully against a variety of insect pests.

Influence of Wireworm Diet on its Susceptibility to and Control With the Entomopathogenic Fungus Metarhizium brunneum (Hypocreales: Clavicipitaceae) in Laboratory and Field Settings.

Entomopathogenic fungi (EPF) represent promising control agents against wireworms but success in field experiments is inconsistent. The physiological condition of the targeted insect is crucial for its ability to withstand fungal infection. In particular, nutritional status is among the most important determinants of the insects' immune defense. In this study, we investigated the effects of diet on the development of the wireworm Agriotes obscurus (L.) (Coleoptera: Elateridae) and its subsequent susceptibility to the fungal pathogen Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) in a pot experiment. After being reared on one of five plant diets for eight weeks, wireworms were exposed to an environment inoculated with the EPF and monitored for their susceptibility to fungal infection. We then performed a field experiment in which three plant diets (clover, radish, and a cover crop mix), selected according to the insects' performance in the laboratory experiment, were grown as a cover crop with EPF application. Plant diet influenced growth and development of larvae, but there were no strong differences in susceptibility toward fungal infection in the laboratory experiment. Damage levels in EPF-treated plots in the field varied depending on the cover crop. Damage was highest in plots planted with a mix of cover crop species, whereas damage was lowest in plots with clover or radish alone. This agrees with the laboratory results where insect performance was inferior when fed on clover or radish. Cover crop effects on wireworm damage in the subsequent cash crop may thus vary depending on the cover crop species selected.

From lab to field: biological control of the Japanese beetle with entomopathogenic fungi.

The Japanese beetle, Popillia japonica, is an invasive scarab and listed as quarantine organism in many countries worldwide. Native to Japan, it has invaded North America, the Azores, and recently mainland Europe. Adults are gregarious and cause agricultural and horticultural losses by feeding on leaves, fruits, and flowers of a wide range of crops and ornamental plants. Larvae feed belowground and damage grassland. To date, no efficient and environmentally friendly control measure is available. Larval populations of other scarab species such as Phyllopertha horticola and Melolontha melolontha are controlled by applying spores of the entomopathogenic fungi Metarhizium brunneum and Beauveria brongniartii to larval habitats. Here, we tested this control strategy against Japanese beetle larvae in grasslands, as well as spore spray applications against adults in crops. Using both, large-scale field experiments and inoculation experiments in the laboratory, we assess the efficacy of registered fungal strains against Japanese beetle larvae and adults. Metarhizium brunneum BIPESCO 5 established and persisted in the soil of larval habitats and on the leaves of adult's host plants after application. However, neither larval nor adult population sizes were reduced at the study sites. Laboratory experiments showed that larvae are not susceptible to M. brunneum ART 212, M. brunneum BIPESCO 5, and B. brongniartii BIPESCO 2. In contrast, adults were highly susceptible to all three strains. When blastospores were directly injected into the hemolymph, both adults and larvae showed elevated mortality rates, which suggests that the cuticle plays an important role in determining the difference in susceptibility of the two life stages. In conclusion, we do not see potential in adapting the state-of-the-art control strategy against native scarabs to Japanese beetle larvae. However, adults are susceptible to the tested entomopathogenic fungi in laboratory settings and BIPESCO 5 conidiospores survived for more than three weeks in the field despite UV-radiation and elevated temperatures. Hence, control of adults using fungi of the genera Beauveria or Metarhizium is more promising than larval control. Further research on efficient application methods and more virulent and locally adapted fungal strains will help to increase efficacy of fungal treatments for the control of P. japonica.

Efficiency of natural substances to protect Beauveria bassiana conidia from UV radiation.

BACKGROUND: Solar radiation is assumed to be a major factor limiting the efficacy of entomopathogenic fungi used as biocontrol agents in open field applications. We evaluated 12 natural UV-protective co-formulants for their effect on the survival of UV-exposed Beauveria bassiana spores on agar plates, colza leaf discs and in the field. RESULTS: Colony-forming unit (CFU) counts of unformulated conidia on agar plates and leaf discs dropped to ≤ 50% after exposure to UV radiation. The highest UV protection was achieved with humic acid, which provided > 90% protection of UV-B-exposed conidia in laboratory experiments. In the field, 10% humic acid increased spore persistence up to 87% at 7 days after application. Sesame and colza oil also provided high UV protection in both assays (> 73% and > 70%, respectively). CONCLUSIONS: This study shows that it is possible to increase the persistence of B. bassiana spores under exposure to UV radiation by formulation with natural UV-protective additives. UV protectants might, therefore, increase the efficacy of entomopathogenic fungi as biocontrol agents in open field applications. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Assessing effects of the entomopathogenic fungus Metarhizium brunneum on soil microbial communities in Agriotes spp. biological pest control.

The release of large quantities of microorganisms to soil for purposes such as pest control or plant growth promotion may affect the indigenous soil microbial communities. In our study, we investigated potential effects of Metarhizium brunneum ART2825 on soil fungi and prokaryota in bulk soil using high-throughput sequencing of ribosomal markers. Different formulations of this strain, and combinations of the fungus with garlic as efficacy-enhancing agent, were tested over 4 months in a pot and a field experiment carried out for biological control of Agriotes spp. in potatoes. A biocontrol effect was observed only in the pot experiment, i.e. the application of FCBK resulted in 77% efficacy. Colony counts combined with genotyping and marker sequence abundance confirmed the successful establishment of the applied strain. Only the formulated applied strain caused small shifts in fungal communities in the pot experiment. Treatment effects were in the same range as the effects caused by barley kernels, the carrier of the FCBK formulation and temporal effects. Garlic treatments and time affected prokaryotic communities. In the field experiment, only spatial differences affected fungal and prokaryotic communities. Our findings suggest that M. brunneum may not adversely affect soil microbial communities.

Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont.

The new C7N aminocyclitol kirkamide (1) was isolated from leaf nodules of the plant Psychotria kirkii by using a genome-driven (1)H NMR-guided fractionation approach. The structure and absolute configuration were elucidated by HRMS, NMR, and single-crystal X-ray crystallography. An enantioselective total synthesis was developed, which delivered kirkamide (1) on a gram scale in 11 steps and features a Ferrier carbocyclization and a Pd-mediated hydroxymethylation. We propose that kirkamide is synthesized by Candidatus Burkholderia kirkii, the obligate leaf symbiont of Psychotria kirkii. Kirkamide (1) was shown to be toxic to aquatic arthropods and insects, thus suggesting that bacterial secondary metabolites play a protective role in the Psychotria/Burkholderia leaf nodule symbiosis.

Host tracking or cryptic adaptation? Phylogeography of Pediobius saulius (Hymenoptera, Eulophidae), a parasitoid of the highly invasive horse-chestnut leafminer.

Classical biological control is often advocated as a tool for managing invasive species. However, accurate evaluations of parasitoid species complexes and assessment of host specificity are impeded by the lack of morphological variation. Here, we study the possibility of host races/species within the eulophid wasp Pediobius saulius, a pupal generalist parasitoid that parasitize the highly invasive horse-chestnut leaf-mining moth Cameraria ohridella. We analysed the population genetic structure, host associations and phylogeographic patterns of P. saulius in Europe using the COI mitochondrial gene. This marker strongly supports a division into at least five highly differentiated parasitoid complexes, within two of which clades with differing degrees of host specialization were found: a Balkan clade that mainly (but not only) attacks C. ohridella and a more generalist European group that attacks many hosts, including C. ohridella. The divergence in COI (up to 7.6%) suggests the existence of cryptic species, although this is neither confirmed by nuclear divergence nor morphology. We do not find evidence of host tracking. The higher parasitism rates observed in the Balkans and the scarcity of the Balkan-Cameraria haplotypes out of the Balkans open the possibility of using these Balkan haplotypes as biological control agents of C. ohridella elsewhere in Europe.