Various insect bacterial associates are involved in pathogeneses caused by entomopathogenic fungi. The outcome of infection (fungal growth or decomposition) may depend on environmental factors such as temperature. The aim of this study was to analyze the bacterial communities and immune response of Galleria mellonella larvae injected with Cordyceps militaris and incubated at 15 °C and 25 °C. We examined changes in the bacterial CFUs, bacterial communities (Illumina MiSeq 16S rRNA gene sequencing) and expression of immune, apoptosis, ROS and stress-related genes (qPCR) in larval tissues in response to fungal infection at the mentioned temperatures. Increased survival of larvae after C. militaris injection was observed at 25 °C, although more frequent episodes of spontaneous bacteriosis were observed at this temperature compared to 15 °C. We revealed an increase in the abundance of enterococci and enterobacteria in the midgut and hemolymph in response to infection at 25 °C, which was not observed at 15 °C. Antifungal peptide genes showed the highest expression at 25 °C, while antibacterial peptides and inhibitor of apoptosis genes were strongly expressed at 15 °C. Cultivable bacteria significantly suppressed the growth of C. militaris. We suggest that fungi such as C. militaris may need low temperatures to avoid competition with host bacterial associates.

Beauveria and Metarhizium fungi are facultative plant endophytes that provide plant growth-stimulating, immunomodulatory, and other beneficial effects. However, little is known about the level of plant colonization by these fungi under natural conditions. We assessed the endophytic colonization of potatoes (Solanum tuberosum) with entomopathogenic fungi at their natural load in soils (10²–10⁴ colony-forming units per g). Microbiological analyses of soils and plant organs, as well as a metagenomic analysis of potato roots and leaves, were conducted in three locations in Western Siberia, consisting of conventional agrosystems and kitchen gardens. The fungi were isolated at a relatively high frequency from unsterilized roots (up to 53% of Metarhizium-positive plants). However, the fungi were sparsely isolated from the internal tissues of roots, stems, and leaves (3%). Among the genus Metarhizium, two species, M. robertsii and M. brunneum, were detected in plants as well as in soils, and the first species was predominant. A metagenomic analysis of internal potato tissues showed a low relative abundance of Beauveria and Metarhizium (<0.3%), and the communities were represented primarily by phytopathogens. We suggest that colonization of the internal tissues of potatoes occurs sporadically under a natural load of entomopathogenic fungi in soils. The lack of stable colonization of potato plants with Beauveria and Metarhizium may be due to competition with phytopathogens.

Ricin-B-lectins (RBLs) have been identified in many groups of organisms, including coleopterans insects, particularly the Colorado potato beetle Leptinotarsa decemlineata (LdRBLs). We hypothesized that one of these LdRBLs (LdRBLk) may be involved in the immune response to fungal infections. We performed a theoretical analysis of the structure of this protein. Additionally, the expression levels of the LdRBlk gene were measured in L. decemlineata in response to infections with the fungi Metarhizium robertsii and Beauveria bassiana. The expression levels of LdRBlk in the L. decemlineata cuticle and fat body were increased in response to both infections. The induction of LdRBlk expression was dependent on the susceptibility of larvae to the fungi. Upregulation of the LdRBlk gene was also observed in response to other stresses, particularly thermal burns. Elevation of LdRBlk expression was frequently observed to be correlated with the expression of the antimicrobial peptide attacin but was not correlated with hsp90 regulation. Commercially available β-lectin of ricin from Ricinuscommunis was observed to inhibit the germination of conidia of the fungi. We suggest that LdRBLk is involved in antifungal immune responses in the Colorado potato beetle, either exerting fungicidal properties directly or acting as a modulator of the immune response.