Sains Malaysiana 55(5)(2026): 936-948

http://doi.org/10.17576/jsm-2026-5505-15

 

Bio-Insecticidal Efficacy and Molecular Characterization of Cry Genes in Bacillus thuringiensis Isolated from South Punjab Districts, Pakistan, against Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

(Keberkesanan Bio-Insektisid dan Pencirian Molekul Gen Cry dalam Bacillus thuringiensis yang Dipencilkan dari Daerah Punjab Selatan, Pakistan terhadapSpodoptera litura(Fabricius) (Lepidoptera: Noctuidae)

 

HUMA KHURSHID, FAKHAR-UN-NISA YUNUS*, HAFSA ZAHEER, GHAZALA JABEEN & ASMA ABDUL LATIF

 

Department of Zoology, Lahore College for Women University, Lahore, Pakistan

 

Diserahkan: 22 Ogos 2025/Diterima: 13 Mei 2026

 

Abstract

Lepidopteran insects can severely reduce crop productivity through extensive defoliation during outbreaks. Increasing resistance to chemical insecticides has emphasized the need for environmentally safer alternatives such as Bacillus thuringiensis (Bt)-based biopesticides. Since the insecticidal activity and Cry gene diversity of Bt strains vary geographically, this study aimed to isolate and characterise indigenous Bt strains from South Punjab, Pakistan, active against Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Bt isolates were recovered from soil samples using the acetate enrichment method and characterised through morphological, proteomic, and molecular analyses. Scanning electron microscopy showed five crystal morphologies, predominantly bipyramidal crystals. SDS-PAGE analysis showed major protein bands at approximately 130, 60, and 40 kDa, indicating diversity in crystal toxin composition. Among 93 isolates, 20 (21.5%) exhibited toxicity against second instar larvae of S. litura in preliminary diet bioassays. At 400 µg/g crystal protein concentration, 14 isolates caused >50% larval mortality. Isolate JTO-9 from Muzaffargarh showed the highest pathogenicity, causing 100% mortality with an LC₅₀ value of 91.62 µg/g diet after 96 h exceeding the toxicity of reference strains HD-1 and HD-73. PCR analysis showed that 65% of active isolates carried Cry1 genes, while 70% possessed Cry2 genes. Detection of Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Cry2Ac genes identified eight distinct Cry gene profiles among nine isolates. This study provides the first comprehensive characterization of indigenous Bt isolates from South Punjab, Pakistan, and identifies JTO-9 as a promising candidate for the development of effective biopesticides against S. litura. These isolates may support pest management in agricultural ecosystems.

Keywords: Bacillus thuringiensis; biopesticides; Cry genes; indigenous isolates; Spodoptera litura

 

Abstrak

Serangga lepidoptera boleh mengurangkan produktiviti tanaman dengan ketara melalui pengguguran daun yang meluas semasa wabak. Peningkatan rintangan terhadap racun serangga kimia telah menekankan keperluan untuk alternatif yang lebih selamat dari segi alam sekitar seperti biopestisid berasaskan Bacillus thuringiensis (Bt). Memandangkan aktiviti insektisida dan kepelbagaian gen Cry bagi strain Bt berbeza-beza secara geografi, kajian ini bertujuan untuk memencilkan dan mencirikan strain Bt asli dari Punjab Selatan, Pakistan, yang aktif terhadap Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Pencilan Bt diperoleh daripada sampel tanah menggunakan kaedah pengayaan asetat dan dicirikan melalui analisis morfologi, proteomik dan molekul. Mikroskopi elektron imbasan mendedahkan lima morfologi kristal, terutamanya kristal bipiramida. Analisis SDS-PAGE menunjukkan jalur protein utama pada kira-kira 130, 60 dan 40 kDa, menunjukkan kepelbagaian dalam komposisi toksin kristal. Antara 93 pencilan, 20 (21.5%) menunjukkan ketoksikan terhadap larva instar kedua S. litura dalam bioasai diet awal. Pada kepekatan protein kristal 400 µg/g, 14 pencilan menyebabkan kematian larva >50%. Pengasingan JTO-9 dari Muzaffargarh menunjukkan kepatogenan tertinggi, menyebabkan kematian 100% dengan nilai LC₅₀ 91.62 µg/g diet selepas 96 jam melebihi ketoksikan strain rujukan HD-1 dan HD-73. Analisis PCR mendedahkan bahawa 65% pencilan aktif membawa gen Cry1, manakala 70% mempunyai gen Cry2. Pengesanan gen Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab dan Cry2Ac mengenal pasti lapan profil gen Cry yang berbeza antara sembilan pencilan. Kajian ini menyediakan pencirian komprehensif pertama pencilan Bt asli dari Punjab Selatan, Pakistan dan mengenal pasti JTO-9 sebagai calon yang berpotensi untuk pembangunan biopestisid yang berkesan terhadap S. litura. Pencilan ini mungkin boleh menyokong pengurusan perosak dalam ekosistem pertanian.

Kata kunci: Bacillus thuringiensis; biopestisid; gen Cry; pencilan indigenus; Spodoptera litura

 

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*Pengarang untuk surat-menyurat; email: fakhar.yunus@lcwu.edu.pk