Galleria mellonella, the greater wax moth has always been an important pest against honeybees and has remained a nightmare for beekeeping farmers. Management of G. mellonella in live honeybee colonies is very difficult because most current management practices can destroy whole honeybee colonies. In the present study, experiments were conducted to isolate and characterize Bacillus thuringiensis from infected greater wax moth cadavers and to evaluate their biocontrol ability against G. mellonella. The bioefficacy of these isolates has been evaluated against greater wax moth along with the standard strain HD-1. Among all the strains tested, NBAIR BtGa demonstrated higher efficacy compared to other strains, with an LC50 value of 125.17 µg/ml, whereas HD-1 exhibited a significantly higher LC50 value of 946.61 µg/ml. Considering the economic importance of NBAIR BtGa we performed whole genome sequencing of this strain resulting in the identification of a genome size of 5.96 Mb consisting of 6888 protein-coding genes. Gene ontology analysis categorized these genes into three groups based on their roles, i.e., biological functions (2169 genes), cellular components (1900 genes), and molecular functions (2774 genes). Through insecticidal toxicity-related genes (ITRG) profiling of our strain across the genome by Bt toxin scanner and cry processor resulted in the identification of several Cry proteins namely Cry1Ab11, Cry1Ia44, Cry1Aa2, Cry2Af1, Cry1Da2, Cry1Eb1, Cry1Ab5, Cry1Cb2, Cry1Ac2. Besides Cry proteins, other ITRG genes, viz. Vip3Bb2, Zwittermicin A resistance proteins, Chitinase C, Mpp46Ab1, immune inhibitor A, Bmp1, Vpb4Ca1, and Spp1Aa1 were also reported, which show toxicity against lepidopteran pests. The studies were also conducted to test the biosafety of Bt toxins against honeybee larvae and adults, which showed strain NBAIR BtGa was more than 99

Abstract Background Melon fruit fly, Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae), is a devastating polyphagous pest attacking large number of fruits and vegetables causing huge economic yield losses across the world. Management of this notorious pest is very challenging as the larvae feed inside the fruit. Hence, the present research study aimed to screen the indigenous Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strains causing toxicity to larvae and to identify the insecticidal toxicity-related genes present in respective strains. In the present study, 50 indigenous B. thuringiensis (Bt) strains along with one reference strain were screened against second-instar larvae Z. cucurbitae. All the strains were analyzed for presence of 21 dipteran active cry genes. Results Mortality in Z. cucurbitae larvae due to Bt strains ranged from 16 to 92%. PCR results revealed that each strain tested positive for a minimum of three cry genes to maximum of nine cry genes. Among the cry genes, cry1A, cry2A, cry1C, cry19, cry11 and cry70 were detected in high frequency of 100, 88, 84, 74, 58 and 54%, respectively. Bioassay studies revealed that ten out of fifty strains displayed more than 50% mortality. Hence, these ten strains, along with the reference strain, were further tested for mortality for the calculation of the median lethal concentration (LC50). The LC50 values ranged between 38.48 and 105.18 μg/ml. The lowest LC50 found for the strain NBAIR Bt107 was 38.48 μg/ml and was on par with the reference strain (Bti 4Q1) (31.3 μg/ml). Conclusion Indigenous Bt strains displayed a toxicity against the larvae of Z. cucurbitae. The probable dipteran active cry genes responsible for toxicity were interpreted. Thus, the Cry toxins from Bt can play a very important role in the management of Z. cucurbitae.

To address the fungal wilt of pigeon pea caused by Fusarium oxysporium f. sp. udum, farmers currently rely on chemical fungicides, despite their harmful effects. However, there is a growing need for safer alternatives like green pesticides. Bacterial biocontrol agents and their derivatives serve as potential green pesticides in the management of plant pathogens. In the present study, we aimed to identify indigenous Bacillus subtilis strains effective against F. oxysporium f. sp. udum. We used PCR and MALDI-TOF analysis to identify the active components responsible for the efficiency of efficient strain. Biochemical studies of cell-free extracts extracted from B. subtilis strains demonstrated the highest biosurfactant activity in NBAIR BSWG1, with an oil displacement of 2 cm and an emulsification index of 60%. Molecular characterization confirmed the presence of surfactin, fengycin, and iturin coding genes in the B. subtilis strains, among them, NBAIR BSWG1 showed the highest number of lipopeptide-producing genes. Meanwhile, NBAIR BSWG1 showed inhibition of 79.84 % against F. oxysporium f. sp. udum using cell-free extract. Further metabolite profiling of NBAIR BSWG1 using MALDI-TOF analysis further confirmed surfactin, fengycin, and iturin in the purified cell-free extract of NBAIR BSWG1. Two peaks with m/z of 923.77 and 1149.92 were identified as novel lipopeptide compounds which need further characterization. The present study identified NBAIR BSWG1 as an efficient bacterial strain for the inhibition of F. oxysporium f. sp. udum and its antifungal properties are mainly due to the production of cyclic lipopeptides.

Bacillus subtilis is a Gram-positive and endospore producing bacterium. Limited studies have shown that lipopeptides produced by B. subtilis can be inhibitory to phytopathogens. Sclerotinia sclerotiorum is a plant pathogenic fungus which causes various diseases like cotton rot, watery soft rot, stem rot, crown rot and blossom blight in vegetable crops. The objective of the study was to isolate lipopeptides from B. subtilis and study their inhibitory potential against S. sclerotiorum. So, the B. subtilis isolates were extracted from the collected soils of Western Ghats of India. They were initially characterized through morphological parameters followed by PCR amplification of the 16S rDNA gene and confirmation through BLAST algorithm in NCBI database. The lipopeptides produced by these isolates were tested against S. sclerotiorum. B. subtilis strains were effective against S. sclerotiorum and exhibited 18.33 to 29.5 % inhibition under dual culture bio-assay. The antagonistic activity of lipopeptides extracted from B. subtilis strains showed 21.56 to 88.89 % inhibition of S. sclerotiorum in the lowest to highest concentration of lipopeptide tested and was found to be significantly higher than the control. The present study has shown that B. subtilis strains vary in the production of lipopeptides and some of them could produce lipopeptides that are highly inhibitory to S. sclerotiorum. B. subtilis strain NBAIR BSWG1 showed the highest inhibition for S. sclerotiorum. Lipopeptide based poison food technique and the dual culture bioassay results showed that B. subtilis strain NBAIR BSWG1 has immense potential for use in the biological control of S. sclerotiorum. Further studies are being carried out in formulating the lipopeptides for field application.

Alternaria alternata wreaks havoc on fruit and vegetable production globally, threatening food security by causing black leaf spot disease. Bacillus subtilis, a natural inhabitant of soil, is a promising biological control agent for the management of A. alternata. In the present study, the antagonistic potential of B. subtilis NBAIR-BSWG1 was initially confirmed against A. alternata through a dual culture technique with 43.03% inhibition of mycelial growth. Subsequently, we extracted the cell-free extract from the NBAIR-BSWG1 pure culture and assessed its impact on A. alternata through the poison food technique and found mycelial growth inhibition of 85.82%. Identification of secondary metabolites biosynthetic genes using specific PCR markers showed the presence of surfactin genes (sfp, srf AA) with an amplicon size of 675 bp and 201 bp, respectively. Amplification of fengycin (fenB) and iturin (ituD) at 670 bp and 423 bp respectively, by using a specific PCR primer confirms the contribution of fengycin and iturin for the antagonistic potential of NBAIR-BSWG1. This study identifies NBAIR-BSWG1 as an effective bacterial biocontrol agent for control of A. alternata, unlocks the genetic basis of antifungal activity NBAIR-BSWG1, depicts molecular mechanisms involved in biological suppression of A. alternata by NBAIR-BSWG1 paving the way for the development of bioformulations for management of A. alternata.