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Beauveria bassiana (Balsamo) Vuillemin is one of the most important entomopathogenic fungi (EPF) and has a key role in eco-friendly pest management. Among different strains of B. bassiana, NBAIR-Bb-5a is a promising strain currently being used against a wide range of insect pests in diverse agroecosystems of Kerala. The pathogenic process in EPF is mainly mediated by an array of bioactive metabolites that provide valuable insights into the host-pathogen interaction. Hence, the study entitled "Bioactive metabolites of Beauveria bassiana (Balsamo) Vuillemin and its efficacy on tobacco caterpillar, Spodoptera litura (Fab.)" was carried out at the Department of Agricultural Entomology, College of Agriculture, Vellanikkara, during 2020–2022 to identify and assess the effectiveness of metabolites from B. bassiana against S. litura

The secondary metabolites of B. bassiana (NBAIR-Bb-5a) were extracted from two different growth media viz., potato dextrose broth (PDB) and Czapek -Dox broth (CDB) and from B. bassiana infected S. litura. The crude toxin was separated by solvent extraction of culture filtrate and concentrated through a rotary vacuum evaporator, yielding crude toxin of 0.28 and 0.23 g mL-1 of PDB and CDB culture filtrates, respectively. The third-instar larvae of S. litura infected with B. bassiana @ 1x108 spores mL-1 yielded a crude toxin of 0.18 g mL-1 of larval extract. The spectral analysis of crude toxins extracted through standard procedures could detect an array of metabolites. HR-LCMS and HR-GCMS analysis identified 85 compounds in PDB, compared to 79 compounds each in CDB and infected S. litura

The bioactivity of metabolites produced by B. bassiana was identified through the PubChem and KEGG databases. Among the metabolites detected from three sources (PDB, CDB, and infected host), 18 were insecticidal, 16 of them were antimicrobial, and 6 were nematicidal compounds. HR-LCMS analysis detected the presence of predominant insecticidal compounds, viz., beauvericin, 1,2-dipalmitin, á- D-glucopyranose, 1,6- anhydro, 2-palmitoylglycerol, ethylenediamine-N, N'- dipropionic acid, harmine, cyclohexylmethyl undecyl ester, cordycepin, piperidinine and hexadecanoic acid. The volatile insecticidal compounds detected through HR- GCMS were harmine, hexadecenoic acid, and 2,6-pyridine dicarboxylic acid in the

crude toxin extracted from both PDB and CDB. Coumarin-6-carboxaldehyde, dioctyl phthalate, 6-hydroxy nicotinic acid from PDB, and N-nitroso-2,4,4- trimethyloxazolidine and vanillic acid from CDB is found to have antimicrobial activity. Antibacterial metabolites detected from PDB and CDB were cyclohexanone, 2-(2-butynyl)-, hydroxydocosahexaenoic acid and pyrrolo (1,2) pyrazine 1,4- dinonehexahydro. HR-GCMS analysis revealed the presence of dihydrothiophenone and nicotinic acid in PDB and octadecanoic acid and harmine in CDB, which are known to be nematicidal in action.

The insecticidal metabolites identified in the infected S. litura through HR- LCMS were beauvericin, hydroxyquebrachamine, monobutyl phthalate, and diisooctyl phthalate was detected through HR-GCMS. Other metabolites like 2,5- dihydroxybenzaldehyde, mesaconic acid, and (+)-aphidicolin possess antibacterial, antifungal, and antiviral activities, respectively. The metabolites specifically detected in crude toxin extracted from infected S. litura were hydroxyquebrachamine, 12-oxo phytodienoic acid, 9-oxo-10(E),12(E)-octadecadienoic acid, (+)-aphidicolin, diisooctyl phthalate, 1-octadecene, (E)-, 7-hexadecene, (Z)- and heptadecanoic acid.

The bioefficacy of the crude toxin was evaluated against third-instar larvae of
S. litura through topical bioassay using Potters Tower. Bioefficacy studies revealed dose-dependent mortality. The crude toxin at 100 ppm caused 100 per cent mortality at 60 h after treatment. The 90, 70 and 50 ppm concentrations caused 100 per cent mortality at 72, 84 and 96 h after treatment, respectively. The LC50 and LT50 were calculated using PoloPC software. Probit analysis revealed LC50 value of 109.76 ppm at 24 h whereas it was 87.51 ppm, 37.90 ppm and 20.41 ppm for an exposure time of 36, 48 and 60 h, respectively. LT50 values of crude toxin at 100 ppm was 22.77 h. At the same time, lower concentrations of 90 ppm, 70 ppm, 50 ppm, 30 ppm, and 10 ppm were 32.40, 38.66, 43.20, 45.20 and 50.60 h, respectively.

In silico molecular docking studies carried out with the 20 metabolites as ligands and AChE of as the target protein using CB-Dock software revealed the insecticidal potential of metabolites of Beauveria bassiana (NBAIR-Bb-5a). Among the 20 metabolites, 18 interacted with AChE at specific sites, viz., serine, histidine, and glutamate. Interactions with binding energies ranging from -5 to -10 kcal mol-1 were considered to have the best interaction with the active sites of AChE. The

highest interaction was recorded by N-[2-(2-benzoxazolylthio)- ethyl]benzenesulfonamide with a binding energy of -8.5 kcal mol-1, followed by hydroxydocosahexaenoic acid and coumarin-6-carboxaldehyde (-7.5 kcal mol-1), 9,12-octadecadienoic acid (Z, Z) and cyclohexylmethyl undecyl ester (-7.4 kcal mol-1), phenobarbital and octadecanoic acid (-7.1 kcal mol-1). The lowest interaction was exhibited by hydrothiophenone with a binding energy of -3.9 kcal mol-1.

Wet lab studies to validate the results of molecular docking of secondary metabolites of Beauveria bassiana revealed acetylcholinesterase activity of secondary metabolites. The Beauveria-infected larvae recorded a significantly high protein content of 0.0795 mg mL-1 with a lower specific activity of acetylcholinesterase of 0.0445 µMol min-1 mg-1 protein compared to the protein content of 0.0559 mg mL-1 and the specific activity of acetylcholinesterase of 0.0912 µMol min-1 mg-1 protein in healthy larvae. Acetylcholinesterase enzyme inhibition was 69.57 per cent in Beauveria-infected larvae compared to the inhibition of AChE in healthy larvae (11.30%) after 96 h of treatment.

The present study reveals that Beauveria bassiana (Bb5a) is a treasure trove of secondary metabolites that can be used as templates for the synthesis of novel insecticidal molecules for pest management. The wide array of metabolites identified in the crude toxin must be further characterised and studied for various bioactivities.

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