Bioprospecting native Bacillus thuringiensis strains with insecticidal activity against Holotrichia serrata and in vitro plant growth–promoting traits

Abstract

ABSTRACT White grubs (Holotrichia serrata) are destructive soil-dwelling pests causing significant root damage and yield losses in diverse cropping systems. The limitations associated with chemical insecticides, including environmental hazards, non-target toxicity, and resistance development, necessitate sustainable alternatives. In this study, 25 native Bacillus thuringiensis strains were isolated from the Western Ghats of Karnataka and characterised through morphological, microscopic, and molecular approaches, including 16S rRNA gene sequencing. Phylogenetic analysis clustered isolates into five distinct clades, indicating considerable genetic diversity. Microscopic observations confirmed the presence of bipyramidal, pyramidal, and cuboidal crystal inclusions, suggesting variability in Cry toxin profiles. Screening for extracellular enzymes showed protease, lipase, lecithinase, and chitinase activities in 76%, 64%, 52%, and 28% of strains, respectively. All strains exhibited plant growth-promoting traits, with indole-3-acetic acid (IAA) production ranging from 10.30 to 42.14 µg/mL and ammonia production from 11.84 to 58.72 µg/mL, while 32% produced siderophores. Selected strains further enhanced maize seed germination and seedling vigour under in vitro conditions, indicating expression of PGP traits. Bioassays against second instar larvae of H. serrata identified nine strains exhibiting >50% mortality, with strain NBAIR Bt274 showing the highest efficacy (73.33% mortality) and lowest LC₅₀ value (152.3 µg/mL). PCR-based screening further confirmed the presence of insecticidal genes, including cry1, cry3, cry9, and cry18. The integration of insecticidal Cry proteins, lytic enzyme activity, and plant growth-promoting traits highlights the multifunctional potential of strain NBAIR Bt274. These findings emphasise the potential of native B. thuringiensis isolates as eco-friendly bioinoculants for integrated pest management and sustainable crop production. GRAPHICAL ABSTRACTA infographic workflow diagram showing sequential laboratory and bioassay steps used to isolate, characterize, and test Bacillus thuringiensis strains.The figure shows a circular workflow infographic illustrating procedural steps for working with Bacillus thuringiensis. At the top left, a drawing of a sample container with soil represents collection of soil samples. To the right, a cartoon culture plate with bacterial colonies indicates isolation of Bacillus thuringiensis from soil. Farther right, a micrograph style square with rod shaped cells shows confirmation of isolates through crystal staining. Next, a stylized deoxyribonucleic acid sequence trace labeled with letters represents 16 S ribosomal ribonucleic acid sequencing for National Center for Biotechnology Information accession numbers. To the upper right, a circular radial chart shows a phylogenetic tree used for molecular characterization. Moving downward along curved arrows, a photograph of test tubes in a rack and a culture plate signifies evaluation of plant growth promoting traits of Bacillus thuringiensis. Below, a set of four culture plates with zones around colonies depicts evaluation of enzyme activity. At the lower left, a tube containing spores illustrates preparation of spore crystal suspension. Above that, a tray holding sliced carrot pieces with larvae indicates bioassay of Bacillus thuringiensis strains against second instar white grubs. In the center, a drawing of a plant with roots and an enlarged larva beside it emphasizes multifaceted capabilities of Bacillus thuringiensis NBAIR Bt274 for pest management and crop production.