Identification of a native Bacillus thuringiensis strain from Sri Lanka active against Dipel-resistant Plutella xylostella

Bacterial strains and Plasmids

Reference strains B. thuringiensis subsp. kurstaki HD-1 (ATCC 33679) and B. thuringiensis subsp. israelensis (ATCC 35646) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Strains, B. thuringiensis subsp. aizawai HD-133 (BGSC 4J1, 4J3) and B. thuringiensis subsp. tenebrionis (BGSC 4AA1) were purchased from the Bacillus Genetic Stock Center (BGSC, Ohio State University, Columbus, OH, USA). The TA cloning vector pGEM-T Easy used in this study was purchased from Promega (Madison, WI, USA).

Isolation and identification of Bacillus thuringiensis strains

Previously, B. thuringiensis strains were isolated from 63 soil samples from natural habitats in Sri Lanka (Travers, Martin & Reichelderfer, 1987). Isolates were designated with given numbers and are stored at the Industrial Technology Institute B. thuringiensis collection (Colombo, Sri Lanka). Isolated spore-forming Bacilli were grown on chromogenic Bacillus agar (HiMedia, Mumbai, India), to identify putative Bt colonies based on their blue–green color and irregular margins.

Putative Bt isolates were sub-cultured on Luria-Bertani (LB) agar plates and incubated at 30 ± 2 °C for 16 h to obtain pure cultures, which were used for genomic DNA extraction using the PureLink Genomic DNA Mini Kit (Invitrogen), following manufacturer’s instructions. The gyrase B (gyrB) gene was amplified from genomic DNA using nested PCR primers gyrB-F1 (5′ATGGAACAAAAGCAAATGCA-3′) and gyrB-R1 (5′TTAAATATCAAGGTTTTTCA-3′), and gyrB-F2 (5′-CCTTGYTTTGCWGAWCCDCC-3′) and gyrB-R2 (5′ACWCGTATGCGTGARYTRGC-3′) as second primer pair (Soufiane & Côté, 2009). The first PCR was carried out in 25 µL containing template genomic DNA (250 ng), 1× reaction buffer, 200 µM dNTPs, 1 mM MgCl₂, 0.8 µM (each) primer and 1.25 U of Taq DNA polymerase (Promega). Amplification included initial denaturation at 95 °C for 3 min followed by 30 amplification cycles of denaturation at 95 °C for 30 s, annealing at 45 °C for 30 s and extension at 72 °C for 30 s in a Veriti thermocycler (Applied Biosystems, USA). A final extension step of 7 min at 72 °C was added after completion of 30 cycles. The second PCR was performed in a 50 µL reaction including 2 µL of the amplicon from the first PCR as template DNA, 1× reaction buffer, 200 µM dNTPs, 1 mM MgCl_2, 0.8 µM (each) primer and 2.5 U of Taq DNA polymerase (Promega). Amplification included initial denaturation at 95 °C for 3 min followed by 30 amplification cycles of denaturation at 95 °C for 45 s, annealing at 50 °C for 30 s and extension at 72 °C for 1 min. A final extension step of 7 min at 72 °C was added after completion of 30 cycles. Amplified products were purified from 1% low-melting agarose using PureLink Quick Gel Extraction Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Purified amplicons of gyrB were bi-directionally sequenced using the Big Dye Terminator Cycle sequencing kit in an Applied Biosystems 3500 dx genetic analyzer (Applied Biosystems). Forward and reverse sequences were aligned and edited using the BioEdit sequence alignment editor (version 7.2.5). The sequences were used as query over the GenBank database at NCBI using BLAST searches.

Detection of insecticidal protein genes

Polymerase chain reaction (PCR) with specific primers was used to detect insecticidal genes (cry, vip and cyt) in isolated Bt strains. Strains were cultured in 10 mL of LB broth enriched with salts (0.2% NH₄SO₄, 1.4% K₂HPO₄, 0.6% KH₂PO₄, 0.1% sodium citrate, 0.02% MgSO₄.7H₂O) and 0.5% glucose at 30 °C, shaking at 200 rpm for 17 h, and then plasmid DNA was extracted (Reyes-Ramırez & Ibarra, 2008). Reference strains; B. thuringiensis subsp. kurstaki HD-1 (ATCC 33679), B. thuringiensis subsp. israelensis (ATCC 35646), B. thuringiensis subsp. aizawai HD-133 (BGSC 4J1, 4J3) and B. thuringiensis subsp. tenebrionis (BGSC 4AA1) served as controls in PCR reactions. Bacillus thuringiensis strains were screened for lepidopteran-specific cry and vip genes (cry1, cry2, cry9 and vip3A), Coleopteran-specific cry genes (cry3, cry7 and cry8), and Dipteran-specific cry and cyt genes (cry4, cry10, cry11, cyt1 and cyt2), using universal and gene specific primers (Ben-Dov et al., 1997; Ben-Dov et al., 1999; Bravo et al., 1998; Ejiofor & Johnson, 2002; Porcar & Juárez-Pérez, 2003). Each amplification reaction (25 µL) contained 200 µM dNTP, 0.5 µM of each primer, 2.5 mM MgCl₂, and 2.5 U of Taq DNA polymerase. Amplifications were carried out in thermocycler following a program of initial denaturation at 94 °C for 3 min, followed by 35-cycles of denaturation at 94 °C for 45 s, annealing at corresponding annealing temperatures of the primer (Table S1) for 45 s and an extension at 72 °C for 1 min. A final extension step of 7 min at 72 °C was included after completion of 35 cycles (Ben-Dov et al., 1997; Bravo et al., 1998). Reaction products were resolved in 1% agarose gels and examined for the presence of the expected amplicon size.

Observation and purification of insecticidal crystal proteins (ICPs)

Bacillus thuringiensis strains were grown on 1/3 Tryptic Soy Broth (TSB) agar for 3 days at 28 °C until sporulation. Individual colonies were suspended in a drop of sterile distilled water and observed in a Differential Interference Contrast (DIC) microscope to document the presence of crystals.

For production of ICPs from B. thuringiensis strain AB1, three colonies of a sporulated culture were inoculated separately into 1 mL of sterile distilled H₂O, vortexed to homogenize and heated at 70 °C for 45 min to kill vegetative cells and synchronize the culture. This pre-inoculum (500 µL) was used to inoculate 500 mL of 1/3 TSB, which were then incubated at 28 °C with shaking at 160 rpm for 3 days until 90% of cells appeared sporulated under microscopic inspection. Parasporal crystal proteins were isolated from spores using solubilization in 50 mM Na₂CO₃ containing 0.1% 2-mercaptoethanol, as described elsewhere (Perera et al., 2009). Solubilized crystal proteins were dialyzed overnight against 2 L Na₂CO₃ buffer (50 mM) with two changes of buffer, to eliminate 2-mercaptoethanol, and then stored at 4 °C until used (within 1 week).

Proteins present in the isolated parasporal crystal protein mixtures from strain AB1 were analyzed using electrophoresis. Aliquots (10 µL) of each protein sample were mixed with equal volume of 2% electrophoresis buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 0.2% bromophenol blue, 2% β-mercaptoethanol), and heat-denatured for 10 min before loading on lanes of an SDS-10% PAGE gel. After the electrophoresis run, proteins were detected by staining with ProtoBlue Coomassie stain (National Diagnostics, Atlanta, GA, USA).

Insects and bioassays

Eggs of Dipel susceptible and resistant (NO-QAGE) strains of P. xylostella were purchased from Benzon Research (Carlisle, PA). The NO-QAGE strain was derived from a strain with field-evolved resistance to Dipel from Hawaii (Tabashnik et al., 1990), and it displays 500-1,000-fold resistance to B. thuringiensis subsp. kurstaki (https://www.benzonresearch.com/species-detail). Eggs were placed in an incubator (27 °C, 21% relative humidity, and 16-h light/8-h dark photoperiod) until hatched.

Insecticidal activity of isolated solubilized crystal proteins was evaluated first against Dipel-susceptible P. xylostella larvae using surface-diet contamination bioassays. Freshly prepared meridic diet (General Lepidopteran Diet; Frontier Agricultural Sciences, Newark, DE, USA) was dispensed into 128 well bioassay trays (CD International, Pitman, NJ) and allowed to solidify. Toxin (crystal protein) concentrations were measured using a Qubit 4 Fluorometer (Thermo Fisher, Waltham, MA, USA). Selected concentrations of solubilized crystal protein solutions (75 µL) were applied to each well ensuring uniform distribution. Crystal solubilization buffer (50 mM Na₂CO₃) without 2-mercaptoethanol was used as control treatment. After drying, five neonates were introduced into each well before sealing with adhesive tray lids (CD International, Pitman, NJ). Trays were incubated at 27 °C, 21% relative humidity, and 16-h light/8-h dark photoperiod and mortality recorded after 7 days. Bioassays were replicated three times with 20 larvae tested per each concentration. Further bioassays were performed with larvae from susceptible and Dipel-resistant (NO-QAGE) strains of P. xylostella to estimate median lethal concentration (LC₅₀) of solubilized crystal proteins from the most active B. thuringiensis strains in bioassays above. These bioassays were conducted as above, except for testing eleven crystal protein concentrations against 50 larvae from each strain per concentration, and bioassays replicated three times with larvae from different cohorts. Mortality data were analyzed by probit analysis using PoloPlus 1.0 (PoloPlus, 1987) to estimate the LC₅₀s and 95% fiducial limits. The ratio between LC₅₀ of AB1 against the susceptible and Dipel-resistant P. xyslotella strains and approximate 95% confidential boundaries were estimated as described elsewhere (Robertson et al., 2007).

Genome sequencing and assembly

Genomic DNA of B. thuringiensis strain AB1 was purified as described above. DNA was quantified using Qubit 4 fluorometer (Thermo Fischer Scientific, Waltham, MA, USA) and randomly sheared using Covaris M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA) to sizes between 200 and 500 bp per the manufacturer’s instructions (Barchenger et al., 2017). PCR free Illumina libraries were constructed using KAPA Hyper Prep Kit (Kapa Biosystems, Roche, Basel, Switzerland) followed by library quantification by qPCR KAPA Library Quantification Kit according to manufacturer’s instructions. The library was sequenced on Illumina Hiseq X at Admera Health (Illumina, San Diego, CA, USA). High throughput sequencing generated 38,737,140 high quality reads with an average length of 151 bp. The raw reads were deposited into Sequence Read Archive (SRA) database under NCBI accession PRJNA514414. Raw reads were quality trimmed and assembled (de novo) using CLC Genomic Workbench 11.0.1 software (Qiagen Bioinformatics, Redwood City, CA). De novo assembly of paired reads resulted in 213 contigs (N50 = 67 kbp). The assembled genome was submitted to the Prokaryotic GeneFinding tool in Blast2Go ver. 5.2.1 (Conesa et al., 2005) to obtain a FASTA file listing all possible Open Reading Frames (ORFs) in the contigs, which was used for proteomic analysis (see below). Homologous sequences to the predicted ORFs were searched using BLASTX in CloudBlast of Blast2Go software. GO terms were then assigned to each BLAST hit sequence by performing Mapping function followed by Annotation function, where the sequences were annotated using an annotation rule to the assigned GO’s using Blast2Go.

Typing of Bacillus thuringiensis strain AB1

Multilocus sequence typing (MLST) analysis was performed using the PubMLST Bacillus cereus database (https://pubmlst.org/bcereus/) to identify the sequence type (genetically similar subspecies) of strain AB1. The AB1 genome was queried to identify the seven sequence type loci (glpF, gmk, ilvD, pta, pur, pycA and tpi) used for MLST with B. thuringiensis strains (Wang et al., 2018). The PubMLST database was then queried to determine the allele pattern for the seven loci, which matched the profile of sequence type 15 (ST15).

The full-length GyrB gene sequence from strain AB1 was identified as contig 122 in the AB1 genome using a BLAST in CLC Genomics Workbench ver. 11 (Qiagen Bioinformatics, Redwood City, CA, USA). Contig 122 was used as query in a BLAST search against NCBInr, and the 60 best matching coding sequences were used for sequence alignment in CLC Genomics Workbench. A neighbor joining phylogenetic tree was constructed using the aligned sequences. Stability of each node was assessed by bootstrap analysis with 1,000 replicates, and only bootstrap values >90% were considered for branches in the graphical representation of the tree.

Proteomic identification of crystal proteins

Proteins in solubilized isolated parasporal crystal samples were identified and relatively quantified using SDS-10% PAGE and nano liquid chromatography followed by tandem mass spectrometry (nano LC/MS/MS). Solubilized crystal proteins (10 µg) were resolved by Bis-Tris SDS-10% PAGE, the gel stained with Coommassie blue, and the entire mobility region excised. The gel piece was then submitted to MS Bioworks LLC (Ann Arbor, MI) for nano LC/MS/MS. Trypsin digestion was performed by washing the gel piece with 25 mM ammonium bicarbonate followed by acetonitrile. Gel was reduced with 10 mM dithiothreitol at 60 °C followed by alkylation with 50 mM iodoacetamide at room temperature and digested with trypsin for 4 h at 37 °C. Trypsin activity was quenched with formic acid. The processed protein sample (supernatant) was analyzed by nano LC/MS/MS in a Waters NanoAcquity HPLC system interfaced to a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Peptides were loaded on a trapping column and eluted over a 75 µM analytical column at 350 nL/min; both columns were packed with Luna C18 resin (Phenomenex, Torrance, CA, USA). The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 70,000 FWHM resolution and 17,500 FWHM resolution, respectively. The fifteen most abundant ions were selected for MS/MS. Data were queried against a database including all the translated protein sequences from predicted open reading frames identified in the B. thuringiensis  AB1 strain genome through the Prokaryotic GeneFinding tool in Blast2Go, as described above. Querying was performed using the Mascot software (Matrix Science, London) with carbamidomethyl (C) as fixed modification, and oxidation (M), acetyl (protein N-terminus), and deamidation (NQ) as variable modifications. Additional parameters included 10 ppm peptide mass tolerance, 0.02 Da fragment mass tolerance and two maximum missed cleavages. Mascot DAT files were parsed into the Scaffold ver. 4.8.8 (Proteome Software Inc., Portland, OR, USA) for validation based on peptide identification, filtering and to create a non-redundant list. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. The list of identified proteins was organized based on protein abundance considering the Normalized Spectral Abundance Factor (NASF) quantitative method (Zhang et al., 2010) which takes into account normalized spectra and protein length.

Isolation and identification of Bacillus thuringiensis strains from Sri Lanka

Twenty-one B. thuringiensis strains were isolated from sixty-three soil samples collected from natural habitats in Sri Lanka (Table 1). For rapid and efficient isolation of Bt, we combined the conventional Travers method (Travers, Martin & Reichelderfer, 1987) with chromogenic identification of Bt as blue/green color colonies in Bacillus agar (HiChrome) (Fig. 1), making the identification of Bt from other spore-forming Bacilli much faster and easier.

Sequencing of the gyrB gene further identified the strains as Bt. Amplified gyrB sequences aligned with the gyrB gene sequences of reported B. thuringiensis strains available in the GenBank database with 99–100% sequence similarity. Sequence data have been deposited in GenBank (Table 1). The gyrB sequence data showed that the most common subspecies among the 21 tested Bt strains were graciosiensis (eight strains), kurstaki (five), konkukian (two) and israelensis (two).

Detection of insecticidal genes by PCR was used to predict insecticidal activity of native B. thuringiensis strains (Carozzi et al., 1991). Analysis of the 21 B. thuringiensis strains revealed the presence of amplified fragments characteristic of cry1, cry2, cry4, cry8, cry9, cry10, cry11, vip3A, cyt1 and cyt2 genes (Table 1). The most abundant (76% of strains) insecticidal gene among the screened B. thuringiensis strains was the dipteran-specific cry10. Among the lepidopteran-specific genes, cry1, cry2 and cry9 genes were present in 52%, 28% and 71% of the tested strains, respectively, while vip3A gene was present in 42% of the strains. The frequency of dipteran-specific cry4/cry11, cyt1 and cyt2 genes was 19%, 23% and 4%, respectively. Nineteen percent of strains harbored the coleopteran-specific cry8 gene.

In contrast to detection of ICP genes in all tested strains, not all B. thuringiensis strains produced observable parasporal crystal proteins (Table 1). In fact, only 5 (AB1, AB2, AB8, AB125, AB142) out of the 21 strains showed high amounts of clear crystal inclusions. Lower levels of crystals inclusions were observed in strains AB21, AB22 and AB24, while crystals were not detected in the remaining strains. Different morphologies, including rhomboidal, spherical and spore-attached crystals were observed (Fig. 2).

Toxicity of Bacillus thuringiensis strains against Plutella xylostella

Due to limited resources, we focused toxicity assays on B. thuringiensis strains classified as producing high amounts of crystals (AB1, AB2, AB8, AB125 and AB142). The composition of the solubilized crystal protein fractions was assessed by electrophoresis (Fig. 3). Diversity in protein profiles were observed among the tested strains, in agreement with the diverse toxin genes detected by PCR in each strain.

Toxicity of the solubilized crystal protein fractions was assessed using a range of protein concentrations (from 0.1 µg to 10 µg /cm²) against larvae from Dipel susceptible and resistant populations of P. xylostella (Table 2). Percentage mortality data from this preliminary screen identified solubilized crystal proteins from B. thuringiensis strain AB1 as the most active against both susceptible and Dipel-resistant (NO-QAGE) strains of P. xylostella. Solubilized crystal proteins from strains AB2, AB8 and AB125 displayed similar toxicity against susceptible P. xylostella compared to AB1, but they did not present comparable toxicity against larvae from the NO-QAGE strain. B. thuringiensis strain AB142 displayed the lowest levels of activity against both strains of P. xylostella tested (Fig. 4).

Characterization of Bacillus thuringiensis strain AB1

The AB1 strain of B. thuringiensis was selected for further characterization based on its relatively higher toxicity against larvae from the susceptible and NO-QAGE strains of P. xylostella. This strain was deposited and is archived in the B. thuringiensis strain collection at the Industrial Technology Institute in Colombo (Sri Lanka), with accession number ITI-SL-AB1-2013. The genome of the strain was sequenced using Hiseq Illumina to identify insecticidal protein genes present and to generate a custom database for proteomic querying in the identification of insecticidal proteins among the solubilized crystal proteins.

Concentration-mortality assays with the solubilized crystal proteins from B. thuringiensis strain AB1 were performed to estimate toxicity parameters (Table 2). While not statistically significant due to overlapping fiducial limits values (Gonzalez-Cabrera et al., 2001; Mushtaq, Shakoori & Jurat-Fuentes, 2018), results from these bioassays identified a lower LC₅₀ for these proteins against larvae from the NO-QAGE (0.091 µg/cm²) compared to the susceptible (0.313 µg/cm²) strain. The ratio of LC₅₀ values and the corresponding 95% confidence intervals calculated as described by Robertson et al. (2007) suggested a 3.4-fold difference in susceptibility between the strains with 2.0 and 5.8 as lower and upper limits.

We used high throughput sequencing to further identify genes expressed in B. thuringiensis strain AB1. Assembly of reads resulted in 213 contigs with and N50 of 67 kb. Genome annotation results showed that B. thuringiensis strain AB1 contained pesticidal genes cry1Aa, cry1Ca, cry1Da, cry1Ia, cry2Ab, cry9Ea and vip3A.

Nano LC/MS/MS was performed on the solubilized crystal proteins of strain AB1 to identify the insecticidal genes expressed in the strain. Queries of MS data against the database of predicted ORFs from the genome confirmed the presence of 5 insecticidal Cry proteins in the AB1 sample (Table 3). Among these detected Cry toxins, Cry1Ca and Cry1Da were the most abundant, representing 61.2% and 31.2% of the total normalized spectra. The other three toxins detected, Cry1Aa, Cry1Ia, and Cry2Ab, represented <3% of the normalized spectra (Table 3).

Typing of Bacillus thuringiensis strain AB1

Multilocus sequence typing using the AB1 genome in the PubMLST database identified the allele pattern for the seven loci used for classification: nine (glp), eight (gmk), 16 (ilv), 13 (pta), two (pur), 16 (pyc A) and nine (tpi), which matched the profile of sequence type 15 (ST15). This ST15 group comprises 12 bacterial isolates, of which eight are Bt. Seven out of the eight Bt isolates belong to serovar aizawai, supporting that strain AB1 belongs to this serovar.

Further evidence for serotyping of strain AB1 as aizawai was obtained through analysis of the full length gyrB gene sequence. A neighbor-joining tree constructed using the gyrB sequence from the strain AB1 genome (contig 122) and the 60 most similar gyrB sequences in the NCBInr database (Fig. 5), identified different clades (only considering branches with bootstrap values >90%). Strain AB1 was clustered in a clade together with strains of Bacillus thuringiensis serovar aizawai.