Construction and Evaluation of Engineered Yersinia entomophaga for Stable Inheritance of trans-Cry3Aa-T-HasA Against Monochamus alternatus

<i>Monochamus alternatus</i> larvae, as concealed trunk-boring pests, evade conventional insecticide contact due to their cryptic feeding niche. To overcome this limitation, previous studies have engineered strains of the naturally entomopathogenic bacterium <i>Yersinia entomophaga</i>. The lethality of these strains against <i>M. alternatus</i> was enhanced by incorporating extracellular secretion systems and enriching insecticidal proteins within the larval midgut. However, plasmid loss occurs during serial subculturing. Here, we established an engineered strain that expresses the red fluorescent protein gene mCherry to explore the applicability of bacterial conjugation transfer to <i>Yersinia</i>. We then constructed a chromosomally integrated strain (CSLH88-pCHSW) that incorporates extracellular secretion systems. The results of stability assays demonstrated 100% retention of the mCherry and Cry3Aa-T-HasA genes over 78 generations. SDS-PAGE and Western blot analyses confirmed the extracellular secretion of the Cry3Aa-T protein in the CSLH88-pCHSW strain. Bioassays revealed that the CSLH88-pCHSW strain was significantly more virulent against <i>M. alternatus</i> larvae than both the wild-type strain (CSLH88) and the plasmid-transformed strain (CSLH88-pCHKW), and exhibited markedly faster insecticidal kinetics. Our study reveals the application of bacterial conjugation transfer technology for constructing biocontrol strains. This genomically stabilized <i>Yersinia</i> strain eliminates the risks of failure associated with plasmid loss in the field, enabling the sustainable control of <i>M. alternatus</i>.