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Abstract: BACKGROUND: Conidiobolus obscurus is a widespread fungal entomopathogen with aphid biocontrol potential. This study focused on a de novo transcriptomic analysis of C. obscurus. RESULTS: A number of pathogenicity‐associated factors were annotated for the first time from the assembled 17 231 fungal unigenes, including those encoding subtilisin‐like proteolytic enzymes (Pr1s), trypsin‐like proteases, metalloproteases, carboxypeptidases and endochitinases. Many of these genes were transcriptionally up‐regulated by at least twofold in mycotized cadavers compared with the in vitro fungal cultures. The resultant transcriptomic database was validated by the transcript levels of three selected pathogenicity‐related genes quantified from different in vivo and in vitro material in real‐time quantitative polymerase chain reaction (PCR). The involvement of multiple Pr1 proteases in the first stage of fungal infection was also suggested. Interestingly, a unique cytolytic (Cyt)‐like δ‐endotoxin gene was highly expressed in both mycotized cadavers and fungal cultures, and was more or less distinct from its homologues in bacteria and other fungi. CONCLUSION: Our findings provide the first global insight into various pathogenicity‐related genes in this obligate aphid pathogen and may help to develop novel biocontrol strategy against aphid pests. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]