| Summary: | Biological control agents are a promising substitute for chemical pesticides in agricultural pest management. In this study, <i>Penicillium</i> sp. with high pathogenicity to the agricultural pests oriental leafworm moth (<i>Spodoptera litura</i>) and diamondback moth (<i>Plutella xylostella</i>) were isolated from naturally infected insects and grown on different agricultural residues as an inexpensive substrate for their sporulation. Ten strains of <i>Penicillium</i> (P.01~P.10) were identified as <i>P. citrinum</i> based on morphological features and molecular studies, with sequence analysis using an internal transcribed spacer region. Different fungal isolates exhibited a varying degree of pathogenicity against <i>S. litura</i> and <i>Pl. xylostella</i>, and strains P.04 and P.09 showed the highest pathogenicity to <i>S. litura</i>, with a mortality rate of 92.13% after 7 days of treatments, while strain P.06 resulted in the highest mortality of <i>Pl. xylostella</i> (100%) after 6 days of treatment. Moreover, among ten isolates infected with both <i>S. litura</i> and <i>P. xylostella</i>, P.06 showed potential virulence against <i>S. litura</i> and <i>Pl. xylostella</i>, with lethal time for 50% mortality (LT<sub>50</sub>) values of 4.5 days and 3.0 days, respectively. The ten isolates showed higher virulence to <i>Pl. xylostella</i> than to <i>S. litura</i>. The agro-industrial-based medium showed efficiency for the cultivation of isolates for sporulation on an industrial scale, suggesting that the newly isolated <i>P. citrinum</i> is a potential biological control agent for controlling insect pests and could be further developed for microbial pesticide production.
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