Bt strains

A native Bt isolate, T121, and the reference strain B. thuringiensis subsp. tenebrionis (Btt) were acquired from the Bt laboratory at the Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu, India. The T121 isolate was chosen from a pool of 50 Bt isolates following a preliminary screening bioassay using a spore crystal mixture at a concentration of 25 µg/ml (unpubl. data). The acrystalliferous strain 4Q7 was initially procured from the Bacillus Genetic Stock Centre, Columbus, OH, USA. These cultures were grown either in T3 broth at 30°C with vigorous agitation for 12 h in an orbital shaker (Orbitek, Scigenics, Chennai, India) or on T3 media agar plates at 30°C.

Bacterial colony morphology and crystal pattern

The morphology of the Bt colonies was examined visually after growth on T3 media agar plates. To examine spore crystal inclusions, 2 µl of a liquid culture of the Bt was evenly spread on glass slides, heat fixed, stained with Coomassie brilliant blue stain (G250), and examined under a bright-field microscope (Iscope, Euromex, The Netherlands) at 100× magnification. These cultures were established by inoculating 250 µl of a 12-h-old culture into 25 ml of T3 broth and then subjected to incubation at 30°C for 48 h with constant agitation at 200 rpm.

Spore crystal mixture isolation

After confirming the lysis of 90% of the cells, the broth culture was centrifuged at 4°C for 10 min at 7,000 rpm. The pellet obtained was solubilized in 25 ml of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, and 1 mM phenylmethanesulfony fluoride [PMSF], pH 8.0). Subsequently, the pellet was subjected to a single wash with 25 ml of 0.5 M NaCl, followed by another two additional rinses with Tris-EDTA buffer. The resulting pellet of spore crystal was resuspended in 500 µl of nuclease-free water (Qiagen, Hilden, Germany) with the addition of 1 mM PMSF and stored at −20°C for further analysis.

Scanning electron microscopy (SEM)

The spore crystal image was obtained using a Quanta 250 scanning electron microscope (FEI, Brno, Praha, Czech Republic) located at the Centre for Agricultural Nanotechnology, TNAU, Coimbatore, Tamil Nadu, India. For SEM, the spore crystal mixture was subjected to two washes with sterile nuclease-free water (Qiagen, Tegelen, The Netherlands). Subsequently, 100 µl of spore crystal mixture was added to 1,900 µl of sterile distilled water and subjected to sonication at 37 kHz to disperse aggregates and then vortexed. A volume of 30 µl from the resulting solution was applied to the SEM sample holder and left to dry. The sample was then coated with gold nanoparticles by using a sputter coating process, enabling the crystals and spores to acquire conductivity. The samples were placed in the vacuum chamber of SEM, and the imaging process was conducted at a voltage of 15 kV, with the sample positioned 4 mm away from the target.

Protein profiling with SDS-PAGE

The spore crystal mixture was resuspended in 1× sample solubilizing buffer (0.5 M Tris, pH 6.8; 0.1% bromophenol blue; 2% SDS; 5% β-mercaptoethanol; and 10% sucrose) before subjecting to incubation at 95°C for 10 min to facilitate protein denaturation. The denatured samples were analyzed on 12% SDS-PAGE following the protocol outlined by Laemmli (1970) by using 0.133% Coomassie brilliant blue R250 staining and destaining. The protein bands in the gel were compared with the prestained protein marker PGPMT2962 (Puregene, Genetix Biotech Asia Pvt Ltd., New Delhi, India) that spans a molecular weight range of 10–315 kDa.

Genomic DNA isolation and PCR sequence analysis

The genomic DNA was isolated from overnight-grown cultures following the methodology of Sambrook and Russell (2001). For PCR amplification, a quantity of 30–50 ng of the extracted DNA was used as a template. The PCR was performed using Nexus GX2 Eppendorf PCR master cycler. The reaction mixture consisted of 30–50 ng of total genomic DNA, forward and reverse primer at a 1 µM concentration each, 2× master mix (EmeraldAmp, Takara, Takara Bio India Pvt. Ltd., New Delhi, India), which contained deoxynucleoside triphosphates (dNTPs), Taq polymerase, and reaction buffer. The Vip1 primers were designed based on the conserved regions of vip1Aa (GU992203), vip1Ad (JQ855505), vip1Bb (HM485584; KR065727), vip1Bc (HM485583), and vip1Ca (KR065725) to characterize the vip1 genes. The primers listed in Table 1 were used to perform PCR targeting cry3, vip1, and vip2 genes. The PCR products obtained were examined through a 1.2% agarose gel. The PCR products were Sanger sequenced with corresponding gene-specific primers at Biokart India Pvt. Ltd., Bangalore, India. The gene sequences were analyzed using the BioEdit program (Hall 1999) and subjected to BLAST analysis against the nucleotide and protein databases. The resulting aligned sequences were submitted to GenBank to obtain accession numbers.

Table 1.Primer sequences used for amplification of cry and vip genes encoding coleopteran toxic Cry proteins.^a

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Insecticidal activity of Bt isolate T121 against H. vigintioctopunctata

A laboratory population of H. vigintioctopunctata was maintained from the field-collected adults and was consistently maintained in the laboratory (25 ± 1°C, 75 ± 5% relative humidity, and a photoperiod of 16:8 [light:dark] h). The bioassay was conducted using a spore crystal mixture of the Bt isolate T121 against first-instar grubs by using the leaf disc method. Freshly cut eggplant (Solanum melongena L.) leaves were used to cut circular discs 40 mm in diameter. Forty microliters of diluted spore crystal mixtures with concentrations ranging from 6.5 to 0.5 µg were spread on both sides of the leaf discs (20 µl/side) and air dried. Sterile distilled water was spread on leaf discs to serve as controls along with spore crystal suspensions from the standard strains Btt (positive control) and 4Q7 (negative control). Three replications were conducted per treatment, with 10 first-instar grubs per replicate. Mortality of the grubs was monitored at 24–72 h. Data were subjected to probit analysis (Finney 1971) using POLO-PC LeOra 2.0 software.

Colony and crystal morphology characterization

Bt has been documented to exhibit variations in color (white, off-white, and yellowish white) and type (resembling a fried egg or a glossy appearance). The texture may vary and can be described as circular, flat, or raised elevation, with complete or undulated margins (Abo-Bakr et al. 2020, Ma et al. 2023, Mukhija and Khanna 2018, Navya et al. 2021). The morphological characteristics of the Bt isolate T121, as well as the standard strains Btt and 4Q7, were examined by assessing various attributes such as color, elevation, margin, shape, and surface characteristics. All three Bt isolates exhibited a creamy white hue. The surface resembled a fried egg, circular with a raised elevation, and a complete margin.

Examination of the spore crystal mixture with bright-field microscopy and SEM revealed the presence of cuboidal and spherically shaped crystals in T121 (Fig. 1), whereas cuboidal-shaped crystals were observed with Btt. These results agree with descriptions by Naveenarani et al. (2022) and Soltani-Nezhad et al. (2022). Various shape types of crystals, including bipyramidal, rectangular, cuboidal, and spherical, are reportedly produced by Bt (Gothandaraman et al. 2022, Ramalakshmi and Udayasuriyan 2010).

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PCR screening and DNA sequencing

PCR analysis confirmed the presence of binary toxin gene vip1 and vip2 in addition to cry3 gene in the Bt isolate T121. Validation of the PCR products was achieved through Sanger sequencing using gene-specific primers. The acquired sequences were aligned, followed by Blastn analysis against National Center for Biotechnology Information (NCBI) nucleotide databases. This revealed 100% homology with the previously identified cry and vip genes available in the NCBI database. The nucleotide sequences of cry3, vip1, and vip2 from the T121 Bt isolate were submitted to NCBI, and their GenBank accession numbers are OM777031, OR921178, and OR947062, respectively.

Protein profiling through SDS-PAGE

Examining the molecular weights of crystal proteins through SDS-PAGE profiling provides a valuable method for anticipating the existence of cry genes. The SDS-PAGE analysis of Bt isolate T121 revealed four prominent bands, with molecular weights of 74, 65, 48, and 31 kDa, as did the positive standard Btt (Fig. 2). The varied patterns of protein bands detected in Bt isolates suggest differences in biological activity and specificity against different insect pests. Nazarian et al. (2009) and Asokan et al. (2014) observed a range of electrophoretic patterns encompassing molecular weights ranging from 20 to 135 kDa. Our findings are consistent with the results reported by Rupar et al. (1991): they observed a prominent 74-kDa protein band in the EG2838 isolate and a 73-kDa protein in the EG2158 that also generated minor crystal proteins of approximately 31 and 29 kDa. Notably, these proteins exhibit significant toxicity toward Colorado potato beetle (Leptinotarsa decemlineata (Say)) larvae.

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Specifically, Cry proteins exhibit toxicity against lepidopteran larvae (Cry1, 130–140 kDa), both lepidopteran and dipteran larvae (Cry2, 71 kDa), coleopteran larvae (Cry3, 66–77 kDa), and dipteran larvae (Cry4, 125–145 and 68 kDa), as documented by Höfte and Whiteley (1989). Besides the cry3 gene found in isolate T121, binary toxins vip1 and vip2 also were identified and confirmed through Sanger sequencing with gene-specific primers. However, in SDS-PAGE, a prominent wide band around 74 kDa, encoding the Cry3Aa protein, was observed. No protein associated with the Vip1/Vip2 could be detected. Bi et al. (2014) also reported that Bt strain HBF-18 contains vip1Ad1 and vip2Ag1 genes in addition to cry8Ga, which were undetected in SDS-PAGE. However, cloning and expression of vip1Ad1 and vip2Ag1 revealed binary toxin activity against Scarabaeoidea larvae. The DNA sequences of cry3, vip1, and vip2 genes from T121 exhibit a high degree of similarity to the previously documented sequences.

Insecticidal activity against H. vigintioctopunctata

The Bt isolate T121 had a median lethal concentration (LC₅₀) of 4.675 µg/ml (95% fiducial limit [95% FL] = 5.118–4.271 µg/ml) against first-instar larvae of H. vigintioctopunctata, whereas the reference strain Btt had an LC₅₀ of 3.636 µg/ml (95% FL = 3.978–3.324 µg/ml) (Table 2; Fig. 3). According to Park et al. (2009), the LC₅₀ for the Colorado potato beetle was 3.56 µg/ml (95% FL = 2.41–4.97 µg/ml) for the Cry3Aa crystal suspension. In bioassays of the Cry7Ab3 protoxin against H. vigintioctopunctata, Song et al. (2012) determined an LC₅₀ of 0.209 mg/ml for second-instar larvae and 1.711 mg/ml for third-instar larvae. The engineered Bt strain HD7AB exhibited significant toxicity against second-instar H. vigintioctomaculata larvae, with an LC₅₀ of 0.779 μg/μl (Wang et al. 2010). Cry3Aa has been documented for its toxicity against various pests, including L. decemlineata, Alphitobius diaperinus (Panzer), Diabrotica virgifera virgifera Leconte, Acanthoscelides obtectus (Say), Cylas puncticollis Boheman, and Cylas brunneus (F.) (Ekobu et al. 2010, Li et al. 2013, Park et al. 2009, Rodríguez-Gonzalez et al. 2020). Our results demonstrate toxicity of Cry3Aa against H. vigintioctopunctata first instars, thereby expanding the list of target insect species for this toxin. The characterization of native Bt isolate T121, encompassing morphological, genetic, and functional aspects, underscores its potential as a biopesticide with enhanced insecticidal efficacy against coleopteran pests. Confirming the presence of essential toxin genes, protein expression, and significant mortality rates against H. vigintioctopunctata highlights Bt isolate T121 as a promising candidate for integrated pest management strategies.

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Table 2.Toxicity of spore crystal mixture from Bacillus thuringiensis isolate T121 against first-instar larvae of Henosepilachna vigintioctopunctata.

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