Isolation and identification of Bacillus thuringiensis strains native of the Eastern Province of Saudi Arabia

Bacillus thuringiensis (Bt) produces a group of δ-endotoxin proteins designated as cry toxins. No doubt that Bt isolates are excellent potential candidates for biological control strategies. The present study showed that 12 Bt strains were isolated and characterized at morphological, biochemical, and molecular levels. All the tested 12 Bt strains were gram-positive, endospore-forming, and possessing typical Bt crystal structures under the scanning electron microscopy (SEM). Universal primers direct and reverse of five pairs were used to detect five Cry-type genes (Cry1, Cry2, Cry3, Cry4, Cry7, and Cry8) by the PCR sizes produced from the studied Bt strains. The 16S rRNA PCR technique, 16S gene primer, DNA template, dNTPs, and Taq polymerase produced unique and distinguishable restriction patterns used for the molecular characterization of the studied Bt strains. Based on the PCR products, the frequency of Cry-gene distribution among the tested strains was Cry1 100%, Cry4 85%, and Cry3 62%, and Cry2 and Cry7 frequency was 54%. Based on the activity of insecticidal of the tested Bt strains, Bt1, Bt9, Bt10, and Bt11 were extremely pathogenic; their pathogenicity ranged from 93 to 100% against dipteran and lepidopteran larvae, compared to the other Bt isolates. The nucleotide sequences of amplified 1500 bp conserved region of 16S rRNA genes of four strains blasted using NCBI database compared to NCBI database sequences, and they were reported as native strains of Bt showing high homology with the known Bt strains (99–100%). The nucleotide sequences of Bt1, Bt9, Bt10, and Bt11 were placed in the GenBank database under accession numbers MN860017, MN843958, MN843959, and MN843960, respectively. The strategies of enhancing the sustainability of crops and vegetables that are targeted by a large number of pathogenic insects require a great effort of exploring novel species and strains of Bt. Herein, native strains of Bt were documented from the eastern province of Saudi Arabia that displayed bio-insecticidal action on larvae of Diptera and Lepidoptera.


Background
Enhancing the resistance of crop and vegetable plants against pathogenic organism diseases is an excellent strategy to increase their productivity and achieve sustainable development. Several microorganisms induce toxins that can be used to control the pathogenicity of a wide range of plant pathogens. Bt is a soil organism that produces crystal inclusions during sporulation. The inclusions are toxic proteins encoded by Cry genes and shown to have toxic effects against different groups of insects, nematodes, and protozoa (Abo-Bakr et al. 2020). Bt toxic proteins neither affect human health nor nontarget organisms. Several research articles proved that Bt synthesizes Cry toxins terminating the growth of pathogenic insect larvae.
Controlling crop and disease insects, Bt secretes parasporal crystal protein known as Cry toxins that have high toxicity towards specific orders of pathogenic insects. Taxonomic classification of these toxins placed themes in the delta category of insect toxins because of their intracellular position (Schnepf et al. 1998). Crystalline toxins or δ-endotoxins are considered the main factor conferring entomopathogenic properties to Bt (Bouslama et al. 2020). δ-endotoxins are composed of 74 types of toxins and holotypes of 295 that belong to the 3D protein family synthesized by Bt (Leopoldo et al. 2014). Bt delta toxins commercialized have been used as pesticides over decades as they have constitutive blocks of amino acids and different specificities against different orders of insects (Van Frankenhuyzen 2009). δ-endotoxins of Bt attack the insects' membrane pores and forming channels (Melo et al. 2016). The three-domain proteins have various complementary aspects of insect toxification. The first domain is responsible for the formation of pores; the second domain is specific in binding to the receptors in the epithelial cells of the insects' midgut; the third domain is functioning in stabilizing the bond between toxin and receptor that results in osmatic discrepancy and finally death of insect (Melo et al. 2016). Therefore, it is clear that the three domains of toxin protein have correlations and their activity can be described as complementary action. This complementary action does not affect the phylogeny or phyletic lineage among groups. It is important to mention that various Bt strains produce various toxins, each toxin specific for a certain category of insects. Recently, Cry genes have been expressed in transgenic crops and vegetables to resist pests. Using transgenic crops is a protocol for increasing the yields and minimizing the use of chemical insecticides (Yutao and Kongming 2019).
Bt Cry toxins are environmentally safe and effective pest control tools. The only negative point of using Bt to control pests is the development of new species of targeted insects that are able to resist these toxins, for example, Plutella xylostella (diamondback moth) has developed strains that resist Bt pesticides in different locations worldwide. It is documented that B. thuringiensis strain AB1 (Sri Lanka) has high toxicity against P. xylostella larvae that are resistant to the commercial Bt available in the market. Exploring new species of Bt will provide another way to cope up with the population of insects that are resistant to the known Bt biopesticides (Baragamaarachchi et al. 2019).
Polymerase chain reaction (PCR) is the most used method to characterize Bt genes. PCR protocol is precise and requires minor amounts of DNA and fast detection of the DNA sequences in a given organism. This protocol allows quick screening of large numbers of Bt species to identify novel Cry-type genes to determine their distribution (Yilmaz et al. 2017). The use of universal primers is a common method to detect the presence of Cry genes (Reinoso-Pozo et al. 2016).
In the current research, we aimed to isolate, identify, and screen for novel Bt that might be highly potent pathogenic from the eastern province of the Kingdom of Saudi Arabia.

Methods
Isolation and identification of native B. thuringiensis strains Soil sampling was performed at six different regions at Al-Ahsa province in the eastern region of Saudi Arabia. Four samples are from cultivated soil (Al-Buhairia, Al-Batalieh, Ghubaiba Villages) and two from non-cultivated soil (El-Qaarh and El-Shoabah mountains). The top layer of soil was removed to avoid the destructive effect of atmospheric UV radiation on the viability of Bt spores. The samples were placed into zip-lock bags and stored in a refrigerator at 4°C until isolation (Palma 2015). Soil samples were suspended in saline 0.85% solution (1 g soil/saline solution); all samples were shacked at 250 rpm for 1 h. All soil samples were exposed to 75°C for 20 min to kill most of the non-spore-forming cells. About 0.2 ml of each soil suspension was seeded onto nutrient agar (NA) medium, and incubation time was 24 h at 30°C (Daniel et al. 2018). Bacterial colonies exhibiting Bt like phenotype flat, matte white color, dry, and uneven borders were picked up carefully and subcultured on nutrient agar medium for single-colony isolation (Palma 2015). The selected colonies were grown on T3 agar medium (per liter: 3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate [pH 6.8], and 0.005 g MnCl (Travers et al. 1987)) and incubated at 30°C for 72 h. The cells of Bt potential isolates grown on T3 agar medium were stained with Gram staining as well as spore-staining reagents. All Bt strains were examined for the presence of vegetative cells, spores, and parasporal inclusion (Padole et al. 2017). Bt isolates were further characterized by examining the enzymatic activities for catalase, urease, caseinase, gelatinase, amylase, and lecithinase according to Cinar et al. (2008). All Bt strains were given code names for recognition.

Preparation and capturing of insecticidal crystal proteins (ICPs)
The cells of Bt potential isolates were grown on T3 medium at 30°C and 200 rpm for 7 days for spore induction. Spore suspension of Bt isolate was centrifuged at 15,000 rpm and 4°C for 10 min to harvest spore-crystal mixtures. Pure spore crystals were fixed and sputter-coated with 10 nm Au/Pd using an SC7620 Mini-sputter coater; parasporal crystal protein were examined and captured using a LEO440 scanning electron microscope at 20 kV beam ( Fig. 1) (Yilmaz et al. 2017).

Analysis of insecticidal crystal proteins (ICPs)
Proteins of parasporal crystal were solubilized, characterized, and accessed using 10% SDS-PAGE according to Laemmli (1970) with minor modification as follows: Aliquots of an overnight LB culture of Bt strains and Bt-HD1 reference strain were injected on to liquid T3 medium (Bozlagan et al. 2010) at 30°C for more than 3 days. Ten milliliters from Bt broth was harvested by centrifugation at 7500 rpm for 20 min, 4°C. The pelleted protein crystals were then suspended in 3 ml of sterilized distilled water (SDW) at 4°C. The pelleted proteins were washed twice by the SDW at the same conditions; later, the pelleted crystal proteins were dissolved in 100 μl sterile distilled water and 100 μl of 2X breaking buffer composed of 125 mM Tris-HCl, pH 6.8, of sodium dodecyl sulfate (SDS) 4%, β-mercaptophenol 0.2%, glycerol 50%, bromophenol blue 0.02%. The Bt pellet was mixed well, boiled for 10 min, and allowed to cool down for 1 min, finally centrifuged for 15 min at 10,000 rpm, and loaded on to 12% SDS polyacrylamide gel page. Scanning of B. thuringiensis strains for cry genes DNA isolation The tests were conducted as follows: 15 μl of Bt pure single colony was picked from nutrient agar culture and suspended in 150 μl SDW, the suspension was subjected to boiling for 5 min, followed by cooling at room temperature, and centrifuged at 10,000 rpm at 4°C over 10 min. This supernatant comprising crude DNA was used in PCR amplification according to Carozzi et al. (1991).

Oligonucleotide PCR primers
Five pairs of universal primers (direct and reverse) were used to explore 5 Cry-encoding genes by sizes from PCR products. The sequences, gene recognized, and the expected Cry-gene sizes of PCR products are presented in Table 1.

DNA amplification cycles
Perkin-Elmer GeneAmp PCR system (model 2400), used with temperature cycling program, was applied as described by Temnykh et al. (2000). The amplification steps were carried out as follows: a single cycle runs at 94°C for 5 min, then 30 cycles composed of denaturation at 94°C for 1 min, a single step of annealing at 55°C for 1 min, next single step of synthesis at 72°C for 2 min, followed by one step of extension at 72°C for 7 min, and a final 4°C infinitive. Each experiment was associated with negative and positive controls. The negative control was run in absence of a DNA template, and the positive control was run by a standard template. The PCR products were assessed by electrophoresis, using 1.4% agarose gels, after finishing the electrophoresis the gels stained with ethidium bromide, and UV light was used to capture the photographs. One hundred base pair DNA ladder purchased from Pharmacia was applied to access the PCR product band sizes.

Insects
Insects used in this study are the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), and the fruit fly, Drosophila melanogaster Meigen (Diptera: Drosophilidae). The required instars from both insects were kindly provided by Mr. Ibrahim Hamed and Ms. Marwa El-Saleh, Plant Protection Department, Faculty of Agriculture, Egypt.

Bioassay
Spore-crystal mixtures of Bt isolates were prepared according to Ammouneh et al. (2011). A single colony from preliminary identified Bt strains was cultured in 100 ml of T3 liquid medium and incubated on a shaker (150 rpm) at 30°C for 5 days. Crystal proteins and Bt spores were collected and suspended in 1000 mM NaCl, centrifuged at 10,000 rpm at 4°C for 10 min, washed with SDW two times, later suspended in SDW, and subjected to freeze-drying. Both Bt crystals and spore powders are used in further experimental work. Bio-insecticidal potentials of the native Bt strain studies in this work were tested against the cotton leafworm larvae (S. littoralis) and the fruit fly (D. melanogaster). Ten of 3rd to 4th instar tested larvae were put in 30-ml plastic cups in 3 replicates, with a total number of 30 larvae for each concentration which was maintained at 27 ± 2°C, 60-65% RH, and 14:10 h (light:dark). Five milliliters (ug/ml −1 ) of each native Bt strain lyophilized powders of the mixture of crystals and spores were introduced to the larvae and left at room temperature (Aramideh et al. 2010 performed using a spore-crystal mixture which was collected from each Bt isolate individually, and prepared serial concentrations ranged from 25 to 50 μg/ ml −1 in sterile distilled water (dH 2 O). The leafdipping technique was used for the tested cotton leafworm larvae. Fresh and clean leaves were dipped for 30 s in each original isolate concentration of Bt spore-crystal mixture separately and offered into instar larvae of S. littoralis larvae. For control, the same number of larvae was exposed to clean leaves dipped in dH 2 O only. Also, 20 g from the fresh prepared artificial fruit fly feed was mixed well with each original isolate of Bt concentrations.

Molecular typing of native strains
Based on the insecticidal bioassay results, the strains of Bt that expressed the highest insecticidal activities against Diptera and Lepidoptera were selected for molecular typing 16S rRNA according to the methods of Rochelle et al. (1995). Universal primers for 16S rRNA, forward primer 8F 5′AGT TGA TCC TGG CTC AG 3′ and reverse primer 1492R 5′TAC CTT GTT ACG ACT T3′ were used to analyze the 16S rRNA. DNA templates were isolated from the 4 native (Bt) strains used in this experiment. The reaction was carried out as follows: 50 μl of the reaction mixture includes DNA template buffer, 16S gene primer, Taq polymerase, dNTPs, and MgCl 2 . The program of PCR was performed in Bio-RAD i-cycler with these steps: (a) 94°C for 40 s set for denaturation, (b) 55°C for 50 s set for annealing, (c) 72°C for 1.5 min set for extension; all running over 35 cycles, the initial temperature of denaturation was 94°C for 3 min and 72°C for 7 min for the final extension. A 1.5% agarose gel electrophoresis was used to analyze the PCR outcome fragments. The agarose gels were purified according to the catalog of purification kit purchased from (Promega Wizard SV Gel and PCR Clean Upsystem Kit cat. #A9282). Purified PCR products were sent for sequencing at Macrogen (South Korea). The blast algorithm at NCBI database was used to set the comparison among the native Bt sequences, and the documented Bt sequences (Bt1, Bt9, Bt10, and Bt11) were stored in NCBI (Altschul et al. 1990).  Phylogeny of native strains Bt1, Bt9, Bt10, and Bt11 16S rRNA gene sequences were compared and submitted to the GeneBank database based on the nucleotidenucleotide standard BLAST algorithm. The MEGA5 software was used to align the sequences, and the phyletic lineage was analyzed, and the tree was constructed. The neighbor-joining method inferred the history of evolution among the taxa. One thousand replicates were taken to represent the evolutionary history of the analyzed taxa, and the bootstrap consensus tree may be inferred (Saitou and Nei 1987).

Results
Isolation, identification, and characterizations of 12 Bt isolate from cultivated and non-cultivated soils, collected from 6 different regions of Al-Ahsa in the eastern province of Saudi Arabia, were reported. Four out of the 12 isolates were novel and highly toxic against S. littoralis and D. melanogaster. The isolated Bt strains were primarily identified based on their morphological characteristics (flat, matte white color, dry, and uneven borders) and their positivity towards gram-staining reagents. Moreover, the 12 Bt isolates showed positive results when examined for the activity of catalase, urease, caseinase, gelatinase, amylase, and lecithinase. Spore-staining reagents declared the presence of parasporal crystal proteins (PCP), and pure spore suspensions of Bt isolates showed clear parasporal protein crystals (PCP) when examined under scanning electron microscopy (SEM) (Fig. 1).
Electrophoretic patterns of the 12 Bt strains along with a single type strain Bt subsp. kurstaki HD1 showed several bands of protein that had different molecular weights in a range of 130-20 kDa. Although each strain had its unique protein profile, there were common bands among different strains and the reference strain HD1 (Fig. 2a). Besides, the SDS-PAGE protein profile of the parasporal crystal protein was isolated after 72 h from sporulated Bt culture were also represented (Fig. 2b).
Direct and reverse universal primers consisted of 5 pairs that were used to detect PCR products by sizes of five Cry-type genes from the 12 isolates of Bt; the results revealed the presence of fragments for Cry1, Cry2, Cry3,  Cry4, Cry7, and Cry8 genes (Table 1 and Fig. 3). Cry1 gene was frequently distributed among Bt isolate strains (100%), followed by Cry4 (85%) and Cry3 (62%), and Cry2, Cry7, and Cry8 are equally distributed (54%) among all Bt isolates ( Table 2). Bioassay of the 12 Bt isolates for larvicidal activity against 3rd instar larvae of the D. melanogaster as dipteran demonstrated that some strains were highly effective in killing the insect larvae than the reference strain (HD1). Depending on the values of LC 50 and slope of the tested Bt, Bt5, Bt6, Bt9, and Bt10 isolates showed the highest mortality percentage for dipteran larvae after 24 h of exposure which ranged from 70 to 86% with LC 50 values ranged from 48.1 to 66.6 μg/ ml −1 , respectively. While the exposure after 48 h, the mortality percentage ranged from 75 to 93% with LC 50 values ranged from 32.5 to 54.4 μg/ml −1 , respectively as shown in Table 3.
Regarding the larvicidal activity of Bt against S. littoralis as lipedopteran, the highest larva mortality percentage appeared with isolates Bt1, Bt8, Bt9, Bt10, and Bt11, which ranged between 87 and 94%, after 24 h of exposure with LC 50 values between 34.0 and 61.4 μg/ml −1 , respectively, while after 48 h the mortality percentage increased to 100% with LC 50 values ranged from 45.5 to 46.4 μg/ml −1 as shown in Table 4.
Referencing to these results, the highest effectiveness against both dipteran and lepidopteran insects (Bt1, Bt9, Bt10, and Bt11) were assigned to further molecular typing. The amplified sequences of these 4 strains were blasted against sequences from the NCBI database, using the blast algorithm (Altschul et al. 1990). The nucleotide sequencing of the amplified 1500 bp conserved region of 16SrRNA genes of all the Bt strains Bt1, Bt9, Bt10, and Bt11 were blasted using NCBI database showing high homology with Bt strains (99-100%).
The 4 native Bt (Bt1, Bt9, Bt10, and Bt11) were given the names SA1, SA9, SA10, and SA11 reflecting the site of isolation and reported in the GenBank under accession numbers MN860017, MN843958, MN843959, and MN843960, respectively (Table 5 and Fig. 4).  The aligned sequences were challenged for neighbor-joining analysis to build the phylogenetic relationship. Nine clusters were produced with different percentages of similarities. Bt9 isolate showed a similarity of 15% with the two clusters of eight isolates with an internal similarity of 16%. The Bt10 isolate showed a similarity of 63% with the cluster containing four isolates with an internal similarity of 100% (Fig. 5).

Discussion
Bt toxins are highly selective and covering more than 50% of the market share (Lacey et al. 2015). The morphology of the isolated bacteria along with the results obtained from the biochemical tests was confirming that the 12 isolated bacterial strains belong to Bt. Similar results were obtained by Cinar et al. (2008); besides, the presence of parasporal crystal proteins (PCP) in the SDS-PAGE profile and the pure spore suspensions of the bacterial isolates is further confirming that the isolated bacteria belong to Bt. This result agreed with other studies on Bt protein crystal (Yilmaz et al. 2017).
Also, the diverse profiles of proteins on SDS-PAGE were consistent with the diverse insecticidal toxins due to the different chemical properties of insecticidal proteins. The presence of very highly crystal proteins soluble in high basic pH is aligned with similar findings by Bukhari and Shakoori (2010) while studying the molecular characterization of Bt cry genes and their mosquitocidal activity. The severe pathogenicity levels of Bt parasporal crystal proteins against larvae of S. littoralis and D. melanogaster were explained by the presence of various insecticidal parasporal crystal proteins and by the possibility of different binding sites on the larvae midguts, similar findings reported by Ammouneh et al. (2011) and Padole et al. (2017).  Cry-gene specific and competition-binding studies were carried out with lectins, and specific sugars confirmed that Cry1Ac and Cry1Fa share similar numbers of the midgut binding sites of both Anticarsia gemmatalis and Chrysodeixis includes (Bel et al. 2017). Also, 5 pairs of universal primers have been used to detect the highly conserved Cry-gene sequences and differentiate 34 out of about 60 genes known in the following groups: 20 Cry1, 3 Cry2, 4 Cry3, 2 Cry4, 2 Cry7, and 3 Cry8 genes (Ben-Dov et al. 1997); PCR screening using universal primers of two pairs to detect Cry1 and Cry2 gene families in their collection is performed by Lone et al. (2017). Therefore, Cry1 protein and other Cry proteins found in the 12 Bt isolates could have similar binding sites on the midguts of S. littoralis and D. melanogaster. Also, it was obvious that Cry1 genes were highly distributed in all Bt isolates (frequency of distribution was 100%). Recent proteomic data supported that Cry1C and Cry1D parasporal crystal proteins make up approximately 92% of the insecticidal crystal proteins in AB1 (Baragamaarachchi et al. 2019).
Although the 4 native Bt strains Bt1, Bt9, Bt10, and Bt11 exhibited high levels of pathogenicity against lepidopteran and dipteran, the percentage of mortality was 93% for lepidopteran, whereas it was 100% for dipteran. Besides, the Bt11 isolate showed a similarity of 100% with Bt9 isolate, while clustered with 25% of similarity with Bt1 isolate. These results strongly support the typing of Bt1, Bt9, Bt10, and Bt11 as Bt novel stains. It remains to match and compare the peptides in the parasporal crystal proteome of Bt1, Bt9, Bt10, and Bt11 to describe precisely their mode of action; this work is ongoing in our laboratories.

Conclusion
In this study, 12 strains of Bt were isolated, identified, and screened for novel Bt that might have highly potent pathogenic as the result of variable sequences that can be used as pest control. Here, we used universal primers, five pairs (direct and reverse) to explore five Cry-genes on different isolates of Bt from the eastern province of the Kingdom of Saudi Arabia. The 4 native strains of Bt types, Bt1, Bt9, Bt10, and Bt11, producing mostly Cry1, Cry2, Cry3, Cry4, Cry7, and Cry8 genes that express insecticidal parasporal crystal proteins, were isolated and identified from the eastern province of Saudi Arabia. Continuous experimental work is needed to evaluate the possibility of culturing Bt: Bt1, Bt9, Bt10, and Bt11 for commercialization.