Insecticidal activity of Bacillus thuringiensis strains isolated from tropical greenhouses towards Cydia pomonella and Spodoptera exigua larvae

Three Bacillus thuringiensis strains were isolated from a specific habitat of tropical greenhouses located in the Botanical Garden of Adam Mickiewicz University in Poznań, Poland. The BG11, BG12 and BG15 strains harbour genes encoding a specific set of insecticidal proteins ( cry1Ba , cry1Ia , cry2Ab , vip3Aa )—entirely different from those found in commercial isolates, currently used as bioinsecticides. Despite high genetic similarity of the new strains, each of them produces unique Cry1Ba toxin as a main component of the parasporal crystals. Moreover, the tested entomopathogens contain genetic determinants encoding two types of chitinolytic enzymes ChiA and ChiB. The tested strains display insecticidal activity against two distinct, economically important pest insects, Cydia pomonella L. (Lepidoptera: Tortricidae) and Spodoptera exigua Hübner (Lepidoptera: Noctuidae). However, BG12 and BG15 strains are significantly more active than BG11 towards both pests. The BG12 and BG15 strains can be considered as candidates for the pro-duction of new lepidopteran-active bioinsecticides with high potential to augment the existing biocontrol strategies.


Introduction
Bioinsecticides based on Bacillus thuringiensis (Bt) insecticidal proteins are currently the best-selling biological plant protection products (Olson 2015;Fernández-Chapa et al. 2019). The main advantages of these agents, which enabled their worldwide implementation, include high activity against certain economically important pests and low cost of their use (Sanahuja et al. 2011;Sansinenea 2012;George and Crickmore 2012). Moreover, bioinsecticides are an alternative for detrimental synthetic pesticides, known for their negative impacts on the environment and human health (Kumar et al. 2021). Bt-based products are used in a form of microbial formulations or plant incorporated protectants (PIPs) called Btcrops. The most active ingredients of these bioinsecticides include insecticidal proteins such as Cry, Cyt and Vip but the entomopathogenic arsenal of B. thuringiensis comprises much wider variety of toxins (Crickmore et al. 2020). Microbial formulations usually contain spore-crystal mixtures derived from certain B. thuringiensis variants, such as: (1) B. thuringiensis subsp. kurstaki (e.g., HD-1 strain; synthesizing Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa insecticidal proteins) or B. thuringiensis subsp. aizawai (synthesizing Cry1Aa, Cry1Ba, Cry1Ca, Cry1Da), used for controlling lepidopterans; (2) B. thuringiensis subsp. tenebrionis (synthesizing Cry3Aa), used for controlling coleopterans; or (3) B. thuringiensis subsp. israelensis (synthesizing Cry4A, Cry4B, Cry11A and Cyt1Aa) used for controlling dipterans (Sansinenea 2012;Fernández-Chapa et al. 2019). Btcrops are genetically modified plants, usually synthesizing Cry1, Cry2 and/or Vip3 toxins for the control of lepidopterans or Cry3 and/or Gpp34/Tpp34 toxins (the latter proteins were originally designated Cry34/ Cry35), for the control of coleopterans (Castagnola and Jurat-Fuentes 2012;Carrière et al. 2016). Btcrops are planted in most regions of the world. Top ten growers include USA, Brazil, Argentina, Canada, India, Paraguay, China, Pakistan, South Africa and Uruguay. For many Bt-based cultivars of crops such as soybean, maize or cotton the adoption rates exceeded 90% (ISAAA Brief 54 2018). Notably, in some regions (i.e., in European Union) the biotech crops are not allowed for commercial use. However, the use of Bt microbial formulations is generally accepted around the world and even strongly recommended as one of the possible Integrated Pest Management (IPM) strategies (Barzman et al. 2015).
The future perspectives and efficiency of Bt-based plant protection products (regardless of the form of PIPs or microbial formulations) is endangered by limited content of Bt insecticidal proteins (mainly Cry1-type, Cry2-type or Cry3-type, as referenced above) present in currently used agents. The first consequence of this fact is the relatively narrow spectrum of target pests (comparing, e.g., to synthetic pesticides) of Bt-based bioinsecticides (George and Crickmore 2012). Thus, many of the economically important pest species are not susceptible and cannot be controlled using these products. Such examples are Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae), Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae) or S. exigua Hübner (Lepidoptera: Noctuidae)-lepidopteran pest species, which larvae cause tremendous losses in agriculture, but feature low susceptibility to most of the Cry proteins (Palma et al. 2014;Chakrabarty et al. 2020). Another threat is the emergence of secondary pests, associated with broad usage of Bt-crops in some regions, and in consequence the necessity to increase the usage of synthetic pesticides. Such cases have been reported in China (Xia et al. 2000;Li et al. 2007;Su et al. 2010Su et al. , 2015 or USA (Frisvold and Reeves 2014). The direct effect of low diversity of insecticidal proteins in Bt-based bioinsecticides is high selective pressure exerted by these products, and in consequence constantly growing number of reports, showing the emergence of resistant insect populations (Jurat-Fuentes et al. 2021).
Taking the above into consideration, there is a clear need for new approaches to sustain the efficient use of Bt-based bioinsecticides in the future. One of the possible strategies is the search for new isolates of entomopathogenic B. thuringiensis. These strains should produce new sets of toxins, capable of high activity against insect pest, but simultaneously featuring low amino acid sequence similarity to currently used insecticidal proteins. The strains may be used to control insect populations, which are naturally not susceptible to currently used Bt-products or insect populations that developed resistance to these agents. Moreover, genes encoding the insecticidal proteins found in novel isolates may be used to generate new generation of Bt-crops. The aim of this study was to search for novel Bt isolates in a specific habitat found in greenhouses of a botanical garden, and to comprehensively characterize the bacteria for their genetic and molecular properties, as well as insecticidal activity towards insect pest species. Upon the gathered results, the potential use of the obtained entomopathogens in biological pest management is discussed.

Materials and methods
Sample collection and isolation of bacteria Samples were collected from diverse materials obtained from two tropical greenhouses of the Botanical Garden of Adam Mickiewicz University in Poznań, Poland (52° 25′ 12′′ N, 16° 52′ 57′′ E). The greenhouses (No. 9 and No. 10) are used to grow plants from equatorial climate (e.g., Coffea arabica L., Hibiscus rosa-sinensis L., Vanilla planifolia Jacks. ex Andrews) in high humidity and stable temperatures of 18 °C during the night and 21-22 °C during the day all year round. These sites have no history of use of B. thuringiensis or its products. Soil samples were obtained by scraping off the surface layer with a sterile spatula and subsequently transfering ~ 50 ml of the material into sterile plastic containers. Water samples were collected by submerging sterile 50 ml Falcon tubes in ponds or running water. Plant material was collected by breaking off leaves/flowers/small stems, and placing them in sterile containers. All the above procedures were done using aseptic technique to avoid contamination of samples. Each sample was subjected to acetate selection (Travers et al. 1987), diluted, spread on BHI medium and incubated for 24 h. Next, ~ 20 representative colonies from each sample were re-streaked on Sporulation Medium (SM) agar plates (0.75% peptone; 50 mM KH 2 PO 4 ; 0.5 mM MgSO 4 × 7H 2 O; 0.01 mM MnSO 4 × 1H 2 O; 0.05 mM ZnSO 4 × 7H 2 O; 0.05 mM Fe 2 (SO 4 ) 3 ; 0.3 mM H 2 SO 4 ; 1 mM CaCl 2 × 2H 2 O; 1.5% agar; pH 7.2), which is a slightly modified BP medium described by Lecadet et al. (1980). After five days of incubation at 30 °C, the microbiological material from each colony was subjected to crystal-spore staining, as described earlier (Smirnoff 1962), and examined under a light microscope. Strains with apparent parasporal crystals visible as dark objects were plated on Mannitol Yolk Polymyxin (MYP) medium (BIOCORP, Poland) for confirmation and subjected to further analysis. B. thuringiensis subsp. kurstaki HD-1 (BGSC 4D1) and/or B. thuringiensis subsp. thuringiensis HD-2 (BGSC 4A3), kindly provided by Bacillus Genetic Stock Center (Columbus, Ohio, USA), were used as reference strains in clonal analysis and 16S rRNA gene sequencing.

Clonal analysis
To eliminate identical B. thuringiensis isolates from further study, the clonal analysis was performed using three combined approaches: ERIC-PCR, BOX-PCR and REP-PCR. Genomic DNA was isolated as described below. ERIC-PCR was done according to da Silva and Valicente (2013), using primers ERIC1 and ERIC2. BOX-PCR was performed as described by Subbanna et al. (2018) with primer BOX1. REP-PCR was performed according to Katara et al. (2012) with oligonucleotides Bc-REP-1 and Bc-REP-2. Primer sequences were summarized in Supplementary Table S1. Comparison of the combined DNA profiles obtained by ERIC-PCR, BOX-PCR and REP-PCR methods was done with GelCompar II 3.0 software (Applied Maths, Belgium), using the Dice factor and grouping by UPGMA. The same software was used for inferring dendrograms presented in this study. Isolates showing more than 95% relative similarity were considered clones.

16S rRNA gene sequencing
Genomic DNAs from the B. thuringiensis strains were isolated using GeneMATRIX Bacterial & Yeast Genomic DNA Purification kit (EURx) according to the manufacturer's instructions. To determine the sequence of the 16S rRNA gene, this fragment was amplified using primer pair including: 16S_27F and 16S_1391R (Supplementary Table S1). Amplification was performed with a C1000 thermal cycler (BioRad) in the following mixture: 60 ng of template DNA; 0.2 mM dNTPs (EURx); 5 μl of 10×PCR buffer supplemented with 25 mM MgCl 2 (Novazym); 0.45 μM of each primer (Oligo.pl); 1.5 U HiFiTaq polymerase (Novazym); sterile, demineralized water up to 50 μl. The parameters of the PCR included initial denaturation (94 °C for 3 min), followed by 30 cycles consisting of: (1) denaturation (94 °C for 30 s); (2) annealing (59 °C for 30 s); (3) elongation (72 °C for 50 s). Final extension was carried out at 72 °C for 7 min. Amplicons were sequenced using the Genetic Analyzer 3130 × 1 sequencer (Applied Biosystems) at the Laboratory of Molecular Biology Techniques at the Faculty of Biology, Adam Mickiewicz University, Poland. The obtained sequences were analyzed using FINCHTV (Genospiza, USA) and MEGA (Kumar et al. 2016).

Protein content of parasporal crystals
Bacterial strains were spread onto sporulation agar medium (as described above) and incubated at 30 °C for five days. After incubation, each spore-crystal mixture was collected, transferred to a 50 ml Falcon tube, suspended in 40 ml of deionized water and centrifuged at 3000 rpm for 15 min at 4 °C. The washing was repeated two more times, and finally the supernatant was removed. The pellet was weighed and resuspended in MiliQ water to a concentration of 40 mg ml −1 . The samples were checked by Zeiss Evo 40 scanning electron microscope (SEM) and stored at − 80 °C until further use.
For mass spectrometry analysis, 225 µl of each spore-crystal mixture was mixed with 150 µl of solubilization buffer (200 mM Tris/HCl, pH 6.8; 32% glycerol; 4.8% SDS; 0.032% bromophenol blue; 20% 2-mercaptoethanol) and incubated 15 min at 30 °C. Next, 225 µl of water was added to each sample and 5 min incubation was carried out at 100 °C, followed by centrifugation at 10,000 rpm for 5 min. Samples in a volume of 15 µl were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) carried out in 10% gels stained with Coomassie brilliant blue dye. PageRuler Unstained Protein Ladder (Thermo Scientific; Cat. 26614) was used as a molecular weight marker. Main bands, corresponding with insecticidal crystal proteins, were excised from the gel and analyzed by mass spectrometry in the laboratory of mass spectrometry (Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland). Mascot software (http:// www. matri xscie nce. com) was used to detect peptide fragments match and assess the fit, exponentially modified protein abundance index (emPAI) and score.
The amplicons were resolved in 1% agarose gel electrophoresis and sequenced using the same approach as described above.

Insect bioassays
Bioassays were performed with Cydia pomonella L. (Lepidoptera: Tortricidae) and Spodoptera exigua Hübner (Lepidoptera: Noctuidae) as target organisms. The laboratory lines of these insects were routinely kept at 26 °C with 40-60% RH and a L:D 16:8 photoperiod. Larvae were routinely fed with semi-artificial diet according to Poitout and Bues (1970).
The insecticidal activity of spore-crystal mixtures derived from tested strains BG11, BG12 and BG15 (prepared as described above) was assessed towards first instar larvae: one-day-old Cydia pomonella and 2-3-days-old S. exigua. Diet surface contamination bioassays were done using 12-well plates, and lepidopteran-dedicated diet, as described previously (Baranek et al. 2021a). The spore-crystal mixtures were tested at six concentrations (5, 50, 500, 5000, 50 000 and 500 000 ng cm −2 ) on C. pomonella and at four concentrations (100, 5000, 50 000 and 500 000 ng cm −2 ) against S. exigua. MilliQ water was used as negative control. For each concentration, 12 test larvae were assigned, and all tests were done in triplicate. Mortality was scored ten days after treatment. The mortality in the control was below 5% in case of both species, and therefore the mortality correction of the spore-crystal mixture treatments was omitted. The LC 50 values, as well as the slopes of the regression lines, were calculated in BioStat Professional ver. 6.9.1.0 (AnalystSoft). The differences between LC 50 values were recognized as statistically significantly different, when 95% fiducial limits did not overlap.

Isolation of bacteria and clonal analysis
Soil, water and phylloplane samples were obtained from two tropical greenhouses of Adam Mickiewicz University (AMU) Botanical Garden in Poznań, Poland. In total, 370 isolates of spore-forming bacteria were obtained from the collected material. Bacillus thuringiensis strains were in a minority (~ 4%).
Five, randomly chosen isolates (BG11, BG12, BG13, BG15 and BG18) were confirmed on MYP agar plates as Bacillus cereus group (isolates did not ferment mannitol but formed diglyceride precipitation surrounding the colonies; Supplementary Fig. S1). These isolates were then subjected to next parts of this work.
To eliminate duplicate, genetically identical isolates from further studies the clonal analysis has been performed using combination of three methods: ERIC-PCR, BOX-PCR and REP-PCR. The individual results obtained for these methods are presented in Supplementary Figs. S2-S7. The combined DNA profiles (Fig. 1), obtained for tested isolates, show that BG12 and BG13 as well as BG15 and BG18 share more than 95% mutual similarity. Therefore, the isolates BG13 and BG18 were eliminated, whereas isolates BG11, BG12 and BG15 were selected for further studies.
16S rRNA gene sequencing 16S rRNA gene fragments of BG11, BG12 and BG15 strains were amplified and sequenced. For comparison, the same procedures were applied to B. thuringiensis subsp. kurstaki reference strain HD-1. For all tested strains, the amplicons of expected size (~ 1400 bp) were obtained ( Supplementary Fig. S8). Sequencing of the amplified fragments showed no differences in 16S rRNA gene fragment among BG11 (GenBank accession number ON506251), BG12 (GenBank accession number ON506252), BG15 (GenBank accession number ON506253) and the reference strain HD-1 (GenBank accession number ON506250). The data obtained for HD-1 in this study is in line with 16S rRNA gene sequence established for this strain earlier (GenBank accession number ASM71753v1).

Protein content of parasporal crystals
The sporulated cultures of BG11, BG12 and BG15 strains were examined under scanning electron microscope, and it was established that the bacteria produce bipyramidal parasporal crystals ( Supplementary Fig.  S9). Next, the composition of parasporal crystals produced by strains from AMU Botanical Garden has been determined. SDS-PAGE analysis showed similar banding patterns for all three tested strains (Fig. 2,  lanes 2, 3, 4). The SDS-PAGE bands, corresponding with insecticidal crystal proteins of BG11, BG12 and BG15, were excised from the gel and analyzed by mass spectrometry. The MASCOT analysis determined that the main proteins present in parasporal crystals of BG isolates are Cry1Ba (fit: > 79%; score > 32 000; emPAI: > 10.55). However, the used method also indicated a low probability of the presence of other insecticidal proteins such as Cry1Be, Cry1Bc, Cry1Jb, Cry1Aa, Cry1Ac and Cry9Ea. The full results of the MASCOT analysis are presented in Supplementary Table S2.
Detection and sequence analysis of insecticidal protein genes The PCR method was used to detect insecticidal protein genes present in genomes of the strains isolated in AMU Botanical Garden. The detection was done for genes encoding: (1) insecticidal proteins with probable presence in parasporal crystals, as suggested by MASCOT analysis (cry1B, cry1J, cry1A and cry9E); (2) B. thuringiensis insecticidal proteins known to be synthesized in a soluble form, thus typically not present in parasporal crystals (cry1I, vip3A); (3) other abundant lepidopteran-active proteins (cry2A-type); and (4) chitinolytic enzymes known to augment insecticidal activity (chi1 and chi2). It was shown that B. thuringiensis BG11, BG12 and BG15 strains harbour genes encoding: (1) four Cry proteins (Cry1B-type, Cry1I-type, Cry2A-type and Cry9E-type); (2) Vip3Aa protein; and (3) two distinct chitinolytic enzymes, ChiA and ChiB, belonging to glycoside hydrolase GH18 family as reviewed by Drewnowska et al. (2020). Insecticidal protein gene content of the BG strains was summarized in Supplementary Table S2, along with results from mass spectrometry showing protein content of parasporal crystals. The full results of PCR reactions are presented in Supplementary Figs. S10 and S11. It was shown that despite the presence of genes encoding Cry1I, Cry2A, Vip3Aa, ChiA and ChiB in genomes of BG11, BG12 and BG15 strains, the proteins are not detected in parasporal crystals. On the other hand, proteins Cry1Be, Cry1Bc, Cry1Jb, Cry1Aa and Cry1Ac, predicted in parasporal crystals with low probability, were not confirmed to have genetic determinants in genomes of BG strains.
The cry genes characteristic for BG strains were sequenced. Next, the amino acid sequences of the encoded proteins were deduced from nucleotide arrangement and compared to known Cry insecticidal protein sequences deposited in Bacterial Pesticidal Protein Resource Center (BPPRC) database (Crickmore et al. 2020), showing that BG strains harbour genes encoding Cry1Ba, Cry1Ia, Cry2Ab and Cry9Ea toxins. The sequences have been deposited in GenBank database under the following accession numbers: cry1Ba (BG11-ON527016; BG12-ON527017; BG15-ON527018), cry1Ia (BG11-ON527019; BG12-ON527020; BG15-ON527021), cry2Ab (BG11-ON527022; BG12-ON527023; BG15-ON527024), cry9Ea (BG11-ON527025; BG12-ON527026; BG15-ON527027). The mutual comparison of nucleotide sequences showed that cry1Ia, cry2Ab and cry9Ea genes are identical in all three tested strains. However, significant differences were detected in the case of cry1Ba gene: 12 nucleotide substitutions were noted between BG11 and BG12 strains; 16 substitutions between BG11 and BG15, and 18 substitutions between BG12 and BG15 (Supplementary Table S3). Nucleotide substitutions resulted in ten amino acid substitutions between BG11 and BG12 strains, 12 substitutions between BG11 and BG15, and 12 substitutions between the proteins of the BG12 and BG15 strains (Fig. 3). Four of these differences (at positions 178, 487, 891 and 900) occur within the conserved blocks, characteristic for three-domain Cry proteins. It is suggested that these eight blocks are crucial for the mode of action of Cry proteins, being responsible for important steps such as pore formation or receptor binding (Schnepf et al. 1998). However, the exact effect of the amino acid substitutions in Cry1Ba from BG11, BG12 and BG15 on insecticidal activity was not yet determined.
Comparison of the amino acid sequences of Cry1Ba proteins characteristic for BG11, BG12 and BG15 strains with National Center for Biotechnology Information (NCBI) and BPPRC databases has shown that they are unique. The closest known proteins are Cry1Ba1 (GenBank accession number CAA29898.1), Cry1Ba5 (GenBank accession number ABO20894.1) and Cry1Ba8 (GenBank accession number AIA96503.1), sharing 99.1-99.8% similarity with the corresponding toxins of BG11/ BG12/BG15 strains.

Insecticidal activity of B. thuringiensis strains
The insecticidal activity of B. thuringiensis BG11, BG12 and BG15 was assessed against two economically important pest species: C. pomonella and S. exigua, representing families Tortricidae and Noctuidae, respectively. The tested strains are active against both distinct pest insects. However the calculated LC 50 values of the spore-crystal mixtures vary, depending on strain (Table 1). BG12 and BG15 are equally active against C. pomonella and S. exigua and they are significantly more potent than BG11 against both insect pests.

Discussion
To date, many different B. thuringiensis strains were isolated from soil, insects, stored-product dusts, insectivorous mammals and phylloplane samples obtained from various geographical sites and different habitats (Fernández-Chapa et al. 2019). In this work, B. thuringiensis strains were isolated for the first time from a specific, semi-contained environment of two tropical greenhouses located in the AMU Botanical Garden. These greenhouses are used to grow plants from the equatorial climate. The ratio of B. thuringiensis isolates comparing to all grown isolates noted in this work (~ 4%) was comparable to other reports, describing B. thuringiensis isolations from different geographic locations and various samples, where this value reached 1.5-5.0% (Berón and Salerno 2006;Ramalakshmi and Udayasuriyan 2010;Aramideh et al. 2016;Cerqueira et al. 2016;Lone et al. 2017), but it was lower than in some other studies, showing that B. thuringiensis accounted for 10-60% of the isolates (Kim 2000;Apaydin et al. 2005;Rabha et al. 2017). Noteworthy is the fact that despite evident activity of BG11/BG12/BG15 strains against lepidopterans (they harbour genes encoding lepidopteran-active Cry1Ba/1Ia/2Ab/9Ea and Vip3Aa insecticidal proteins and were proven to be active against C. pomonella and S. exigua larvae), insects representing Lepidoptera order are not noted in the tropical greenhouses No. 9 and No. 10 (Adam Mickiewicz Botanical Garden data).
Clonal analysis has shown that BG11, BG12 and BG15 isolates are genetically distinct. Therefore, certain diversity of B. thuringiensis exists despite rather low and closed sampling area (the total surface of greenhouses No. 9 and No. 10 is approximately 250 m 2 ). On the other hand, further analysis proved Fig. 3 Differences in the deduced amino acid sequence of the Cry1Ba protein among B. thuringiensis BG strains. Boxes indicate consecutive (1-8) conserved blocks, characteristic for three-domain Cry proteins, as summarized by Schnepf et al. (1998). Amino acid positions with differences among BG11, BG12 and BG15 strains are indicated and the exact amino acids, characteristic for each B. thuringiensis strain, are shown below in standard one-letter code that all three strains produce insecticidal parasporal crystals composed mainly of Cry1Ba protein, and they harbour similar set of insecticidal protein genes. Despite this obvious similarity among the strains, the sequence of cry1Ba gene in each of them is slightly different. It is probable, that BG11, BG12 and BG15 strains are descendants of a recent common ancestor and some differences among the offspring are caused by mutations in certain genome fragments, e.g., in the above-mentioned cry1Ba gene. Insecticidal protein gene content of BG11, BG12 and BG15 strains is distinctive. These strains harbour sequences encoding Cry1Ba, Cry1Ia, Cry2Ab, Cry9Ea and Vip3Aa toxins. This set is entirely different, than in commercial B. thuringiensis strains currently used in biological control of lepidopteran pests i.e., B. thuringiensis subsp. kurstaki or B. thuringiensis subsp. aizawai (Bravo et al. 2011). B. thuringiensis subsp. kurstaki is an active ingredient of microbial formulations such as Biobit, Dipel, Javelin, etc., while B. thuringiensis subsp. aizawai is used to produce microbial formulations such as Agree, Selectgyn, Xentari, and others (complete list of bioinsecticides was provided by Sansinenea 2012). Moreover, the deduced amino acid sequences of Cry1Ba characteristic for BG11, BG12 and BG15 are unique-no identical sequences were deposited in databases so far. Such dissimilarity between the commercially used B. thuringiensis strains and BG isolates is an obvious asset, depicting the latter as good candidates for development of new lepidopteran-active bioinsecticides, featuring low risk of cross-resistance with currently used Bt-based bioinsecticides.
It was shown that BG strains harbour genes encoding two types of chitinolytic enzymes: ChiA and ChiB. Both enzymes belong to glycoside hydrolase GH18 family and exert endochitinase and possibly also chitobiosidase activity (Drewnowska et al. 2020). Chitinases play an important role in pathogenesis process of B. thuringiensis in insects, by destructing the chitin-made peritrophic membrane, which protects insects' gut from harmful molecules such as toxins. Moreover, chitinases are used to enhance protection of Bt-crops against pests (Berini et al. 2018). Therefore, the presence of genetic determinants encoding chitinolytic enzymes further increases insecticidal potential of BG strains.
To assess the actual insecticidal properties of the isolates obtained during this study, a series of bioassays has been performed on two economically important pest species. The results show that spore-crystal mixtures derived from B. thuringiensis strains BG11, BG12 and BG15 are active against larvae of C. pomonella and S. exigua. It was previously established that these two insect species feature entirely different susceptibility to various B. thuringiensis Cry/Vip insecticidal proteins (Boncheva et al. 2006;Hernández-Martínez et al. 2008;Baranek et al. 2021a, b). Thus, the biocidal activity towards both lepidopteran species suggests a broad specificity range of three tested B. thuringiensis strains. However, significant differences in LC 50 values were observed between BG11 and BG12/BG15 isolates. One of the possible reasons for the dissimilar activity of the strains may include differences in Cry1Ba protein sequences (Fig. 3). It was previously shown that even single amino acid replacements in Cry toxin structure can dramatically alter its insecticidal activity (Schnepf et al. 1998). Functional analysis of amino-acid substitutions in Cry1Ba will be performed in future studies to elucidate this matter. Nevertheless, strains BG12 and BG15 (rather than BG11) are recommended for future attempts to develop new biological insecticides for the control of lepidopteran pests.
In conclusion, three distinct B. thuringiensis strains (BG11, BG12 and BG15) were obtained from tropical greenhouses of a botanical garden. The collected strains harbour genetic determinants encoding lepidopteran-active insecticidal proteinsan entirely different set than in commercial B. thuringiensis strains currently used to control caterpillars. The tested strains are active against larvae of two economically important insect pests C. pomonella and S. exigua. However, BG12 and BG15 are more active than BG11. B. thuringiensis BG12 and BG15 have the potential to be used as new bioinsecticides featuring high activity and low risk of cross-resistance with currently used microbial formulations, based on B. thuringiensis spore-crystal mixtures.