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<p>Pigeonpea (<i>Cajanus cajan</i> L. Millsp.) is an important legume crop worldwide. However, its productivity is severely constrained by insect pests, particularly <i>Helicoverpa armigera</i>, which damages flowers and pods, leading to significant yield losses. To develop eco-friendly pest management strategies, six bacterial strains (A–F) were isolated from naturally dead larvae, and their protein extracts were evaluated for effects on larval growth and mortality. Protein from strain B caused the highest mortality (68.75%), followed by strain C, while strains A, D, E, and F showed no significant effect. Strain B was identified as <i>Bacillus subtilis</i>, a gram-positive, rod-shaped, motile, spore-forming bacterium with optimal growth at pH 7–8 and 25–35&#xa0;°C, and positive for citrate utilization, casein, starch, and gelatin hydrolysis, nitrate reduction, catalase, and esculin hydrolysis. Proteomic analysis revealed differential protein expression among strains, with strain B exhibiting a novel ~ 46&#xa0;kDa band associated with insecticidal activity. Crude and ammonium sulphate-fractionated proteins (60% and 70%) were purified by gel filtration chromatography, with peak protein content in fractions 18–20. SDS-PAGE confirmed a ~ 46&#xa0;kDa protein in the 70% fraction, which was selected for N-terminal sequencing. 2D gel electrophoresis and MALDI-MS identified seven protein spots (26–60&#xa0;kDa), which were functionally annotated, and bioassays demonstrated maximum larval mortality (93.75%) with the 70% fraction. Gene-specific PCR amplified a 488&#xa0;bp cDNA fragment, which, upon cloning and sequencing, showed 100% similarity to a GHKL domain-containing protein of <i>Bacillus pumilus</i>, and homology analysis linked protein spot 2 to this cloned protein, confirming its role in larval mortality. These findings indicate that <i>B. subtilis</i>-derived proteins, particularly the ~ 46&#xa0;kDa GHKL domain protein, represent effective, eco-friendly biocontrol agents for sustainable management of <i>H. armigera</i> in Pigeonpea.</p>

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Molecular and biochemical characterization of a novel entomotoxic protein from Bacillus subtilis Isolated from naturally killed pod borer (Helicoverpa armigera) larvae in pigeonpea

  • 期刊论文

  • 发表日期：

    2026年3月5日

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  • Pradeep Kumar Bharti,
  • Nawaz Ahmad Khan,
  • Anurag Mishra,
  • Saurabh Pandey,
  • Raza Husain,
  • Md. Shamim,
  • Ashutosh Singh

摘要

Pigeonpea (Cajanus cajan L. Millsp.) is an important legume crop worldwide. However, its productivity is severely constrained by insect pests, particularly Helicoverpa armigera, which damages flowers and pods, leading to significant yield losses. To develop eco-friendly pest management strategies, six bacterial strains (A–F) were isolated from naturally dead larvae, and their protein extracts were evaluated for effects on larval growth and mortality. Protein from strain B caused the highest mortality (68.75%), followed by strain C, while strains A, D, E, and F showed no significant effect. Strain B was identified as Bacillus subtilis, a gram-positive, rod-shaped, motile, spore-forming bacterium with optimal growth at pH 7–8 and 25–35 °C, and positive for citrate utilization, casein, starch, and gelatin hydrolysis, nitrate reduction, catalase, and esculin hydrolysis. Proteomic analysis revealed differential protein expression among strains, with strain B exhibiting a novel ~ 46 kDa band associated with insecticidal activity. Crude and ammonium sulphate-fractionated proteins (60% and 70%) were purified by gel filtration chromatography, with peak protein content in fractions 18–20. SDS-PAGE confirmed a ~ 46 kDa protein in the 70% fraction, which was selected for N-terminal sequencing. 2D gel electrophoresis and MALDI-MS identified seven protein spots (26–60 kDa), which were functionally annotated, and bioassays demonstrated maximum larval mortality (93.75%) with the 70% fraction. Gene-specific PCR amplified a 488 bp cDNA fragment, which, upon cloning and sequencing, showed 100% similarity to a GHKL domain-containing protein of Bacillus pumilus, and homology analysis linked protein spot 2 to this cloned protein, confirming its role in larval mortality. These findings indicate that B. subtilis-derived proteins, particularly the ~ 46 kDa GHKL domain protein, represent effective, eco-friendly biocontrol agents for sustainable management of H. armigera in Pigeonpea.