Bacterial extracellular vesicles from Chromobacterium subtsugae and Bacillus thuringiensis as cell-free bioinsecticidal nanocarriers against the soybean pest Euschistus heros.

Bacterial extracellular vesicles (bEVs) are membrane-enclosed nanoparticles that transport bioactive cargo and mediate bacterial interactions. Although increasingly recognized as natural delivery systems, their application in plant pest biocontrol remains poorly explored. Here, we evaluated bEV-enriched and soluble fractions from two entomopathogenic bacteria, Chromobacterium subtsugae and Bacillus thuringiensis var. kurstaki, against the soybean pest Euschistus heros. Preparations were characterized by tunable resistive pulse sensing, nano-flow cytometry, transmission electron microscopy, SDS-PAGE, MALDI-TOF mass spectrometry, and label-free quantitative proteomics. The C. subtsugae bEV-enriched fraction displayed a proteome remodeled relative to its soluble fraction, with enrichment of outer-membrane, secretion-associated, proteolytic, and membrane-active proteins. MALDI-TOF selectively detected a violacein-associated ion in this fraction. At the offered doses, this preparation reduced nymph survival relative to the negative control (HR = 5.56, padj = 0.0003) and its corresponding soluble fraction (HR = 5.56, padj < 0.0001). It also produced greater mortality than the particle-matched B. thuringiensis bEV-enriched fraction (HR = 2.73, padj = 0.0163). Neither B. thuringiensis fraction differed from the negative control after adjustment, and the fractions did not differ from each other (HR = 0.98, padj = 0.9611). A Cry1Ab-like protein was detected mainly in the B. thuringiensis soluble fraction. Together, the insecticidal activity, violacein-associated signal, and differential protein composition of the C. subtsugae preparation support a testable multi-component hypothesis but do not establish causality for specific components or intact vesicles. Mechanistic studies examining cargo depletion, vesicle disruption, reconstitution, and tissue uptake are required to identify the active component or combination.