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Abstract
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This study aimed to achieve two main objectives: first, to determine whether the virulence factors of symbiotic
bacteria of entomopathogenic nematodes (EPNs) against insect hosts are cell-associated or secreted, and to shed
light on the underlying mechanisms of pathogenicity; and second, to identify and evaluate the standalone
pathogenicity of symbiotic bacteria associated with entomopathogenic nematodes against Tenebrio molitor. Three
bacterial species, Xenorhabdus nematophila (A41, SC, A18 and SF), Photorhabdus kayaii, and P. thracensis, were
isolated and characterized via phylogenetic analysis of 16S-rRNA and gyrB genes. Bioassays involved injecting
T. molitor larvae with bacterial suspensions, cell-free supernatants, immersing them in bacterial suspensions, and
incorporating various bacterial treatments into their diet. Lethal concentrations (LC50) and lethal times (LT50)
were determined based on observed mortality trends within specified periods. No mortality was recorded in
T. molitor larvae when they were immersed in bacterial suspensions or fed diets incorporating various bacterial
treatments. Among injection-based trials, X. nematophila strains A41, SC, and SF demonstrated remarkable
virulence through low LC50 values in bacterial suspension tests, whereas X. nematophila A18 stood out in
intracellular suspension trials. Interestingly, P. thracensis triggered mortality exclusively during extracellular
suspension testing. However, no discernible effects emerged when employing non-invasive techniques such as
soaking or spiking the insect’s diet. Overall, our investigation highlights significant intra-species/inter-species
variation in the pathogenicity profiles of nematode-associated bacteria towards T. molitor across diverse appli-
cation modes, emphasizing the importance of tailored implementation approaches in utilizing microbials as
effective biocontrol agents. The variation in susceptibility of host to cell suspension, cellular extract, and cell-free
suspe
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