Seven hundred Bt isolates were collected from soil samples in Qatar. Spore-forming isolates were obtained using the acetate selection method from Travers et al. (1987) with slight modifications. One gram of each soil sample was suspended in 10 ml of Luria Bertini (LB) broth, buffered with 250 mM Sodium Acetate (pH 6.8). The mixture was then incubated in a shaker incubator at 30°C for 4 h. After incubation, the samples were heated at 80°C for 15 min. From each of these samples, 100 μl was spread on T3 agar plates and incubated for 72 h at 30°C. Each pure isolate was subjected to microscopic observation to confirm the presence of spores and crystals. Each parasporal crystal forming Bt isolate was named QBT followed by its serial number. Bt isolates were then grown on T3 sporulation media (Travers et al., 1987) for 96 h and the spores-crystal mixture was stored in 30% glycerol at −80°C.

Each sample was grown on T3 agar plates and incubated at 30°C for 96 h. After sporulation, the spore crystal mixture was checked under light microscope to identify the shape of the crystals and group the collection based on their crystal shapes: Bipyramidal, Cuboidal and Spherical. The crystals were then studied in detail under FEI Nova NanoSem 450 Scanning Electron Microscope (SEM), USA to identify intrinsic details and differences between crystals of different isolates. Bacillus thuringiensis israelensis (Bti) and Bacillus thuringiensis kurstaki (Btk) were used as reference strains.

The plasmid DNA of of Bt isolates representing the 4 classes forming different spherical crystals was used as template for amplification of 16s rRNA gene using the primer sets Rib73 (5′-AGAGTTTGATCCTGGCTCAG-3′) and Rib74 (5′-AGGAGGTGATCCAGCCGCA-3′). The amplification was carried out using polymerase chain reaction (PCR) in Applied Biosystems 96 wells Veriti Thermal Cycler from Thermo Fisher, USA. The amplified products were run on a 1.2% agarose gel. Expected band (1.5 kb) was gel purified using QIAquick gel extraction kit from Qiagen by following the instruction manual. The purified product was sequenced by Sangers sequencing using 3500 Series Genetic Analyzer by Thermo Fisher Scientific, USA. The 16s ribosomal DNA sequences were submitted to NCBI (National Center for Biotechnology Information) database. The sequences obtained were compared to published sequences on NCBI database.

After complete sporulation, T3 culture with spore crystal mixture of each isolate was centrifuged and the pellet was washed thrice with 1 N NaCl. The pellet was then washed 3x with distilled water. The spore-crystal pellet was re-suspended in 50 mM NaOH and incubated at room temperature for 1 h to solubilize the crystal proteins.

The purified crystal proteins were combined with 2x boiling buffer (containing 0.1% ß-mercaptoethanol, 1% SDS, 0.025% Bromophenol blue and 10% glycerol) in the ratio 2:1, respectively. The samples were then boiled for 5 min along with a GelPilot broad range protein marker. Samples were loaded on a SDS-PAGE gel with a 10% separating gel and 3% stacking gel. The electrophoresis was run at 100 V for 2 h. The gels were stained with a staining solution containing 0.025% Coomassie Brilliant Blue R250. Destaining was performed overnight with a solution containing Ethanol, Glacial Acetic acid and Water in the ratio 5:7:88, respectively.

Total plasmid DNA was isolated by alkaline lysis method combined with lysozyme treatment and purified using alcohol precipitation as per Sambrook et al. (1989) with slight modifications. The plasmid profiles were obtained by running on 1% agarose gels with 0.5 μg/ml Ethidium bromide. The gels were loaded with 40 μl of each sample, and run at 10 V overnight in 1% TAE tank buffer. The plasmid patterns of the Bti and Btk were used as references.

Blood Nutrient agar plates were prepared by adding 5 ml fresh sheep's blood to the 100 ml autoclaved Nutrient agar medium. The plates were divided into grids and each isolate was inoculated onto the Blood agar plates using sterile toothpicks. The plates were then incubated at 30°C overnight. The zone of clearance around the colony of each isolate were checked and the results were interpreted accordingly.

Plasmid DNA was used as template for the PCR amplification of genes encoding Cry and Cyt proteins that are insecticidal to Dipteran, Lepidopteran and Coleopteran insects (Table 1). The PCR amplifications were carried out as per Jaoua et al. (1996) using Applied Biosystems 96 wells Veriti Thermal Cycler from Thermo Fisher. Two groups of primers were used for amplifications. First group consisted of Dipteran specific genes including cry4A/4B, cry11, cry10, cyt1A, cyt1C, cyt2A, p19, and p20. Second group consisted of Lepidopteran and Coleopteran specific genes including cry1A, cry1IA, cry1B, cry1D, vip3a, and cry2.

A single colony from 24 h incubated LB agar plates was re-suspended in 50 ml LB broth in 250 ml flask and incubated at 30°C in a shaker incubator for 24 h. The O.D of the culture was checked after 24 h and 50 ml T3 broth was inoculated with this pre-culture such that the starting O.D of all the isolates were 0.1. The T3 broth cultures were incubated at 30°C in shaker incubator for 96 h, ensuring complete sporulation. The broth was centrifuged at 10000 rpm for 10 min and the pellet was re-suspended in 10 ml distilled water. The spore crystal mixture was then diluted 5 times and was used as testing solution for the bioassay.

Third and fourth instar larvae of Culex pipiens complex were used for bioassay. For each test, five larvae were transferred to the test solution. And incubated overnight at room temperature. All the 19 Bti like isolates and the representatives of other classes with spherical crystals were tested for insecticidal activity. The number of larvae that survived was calculated for each Bt isolate δ-endotoxin sample.

The observation of crystal forms of the 700 isolates carried out by light microscope allowed the collection to be classified into two main classes.:441 isolates producing spherical crystals and 259 isolates producing bipyramidal and cuboidal crystals. The crystals were observed by SEM in order to evidence any further differences among the same crystal forms of each class. The results showed that the bipyramidal and cuboidal crystals resembled the standard crystals of the reference Bt kurstaki HD1. However, the spherical crystals showed four different shapes when studied at higher magnifications of SEM. In fact, among the collection of 441 spherical crystal producing isolates four groups were identified: smooth spherical (like the reference Bti), spherical with undulated surface, spherical but deflated balloon shape, spherical with one concave side and pointy edged shape (Figure 1).

The plasmid DNA of Bt isolates representing the 4 classes forming different spherical crystals was used for the amplification of the 16s ribosomal DNA using the primers Rib73 and Rib74. The PCR products obtained were purified from the gel using the QIAquick gel extraction kit, following the manual instructions. The purified PCR products were then sequenced by Sangers sequencing. The sequences were submitted to NCBI GeneBank for 16s rRNA and the accession numbers for the same were obtained as MG995012, MG995013, MG995014, MG995015. The in-silico analyses of these sequences showed that they had very high (up to 99%) similarity to the published sequences of Bt 16s rRNA genes in the NCBI database. This confirms that the isolates that were identified and characterized in this collection definitely belong to the Bt family.

The protein patterns of the isolates producing bipyramidal and cuboidal crystals were similar to that of the reference subspecies kurstaki. They have one protein of about 130 kD, two proteins of about 65 kD and a protein of about 40 kD. However, the spherical crystals harbor different protein patterns when compared to the reference subspecies israelensis. In fact, 15 different protein patterns (Figure 2) were evidenced. One protein pattern matched that of the reference Bt israelensis H14. These profiles showed mainly the presence of one protein of 130 kD, two proteins of about 65 kD, three proteins of about 45 kD and two proteins of about 27 kD. But 14 other types of protein patterns were observed among the collection. The main proteins as per their sizes are listed in Table 2. Hence, as per the protein patterns, 16 groups (Prot 1 to Prot 16) were evidenced in the collection: 256 Btk like isolates with one protein pattern (Type 11), 19 Bti like isolates with one protein pattern (Type 1) and 14 protein patterns shown by 422 spherical crystals producing isolates (bifurcations shown in Table 2).

The investigation of plasmid patterns of the Bt isolates showed that all bipyramidal and cuboidal crystal producing isolates had the same plasmid pattern; similar to that of the reference Bt kurstaki HD1. Additionally, the 19 Bti like spherical crystal producing isolates have the same plasmid pattern as that of the Bt israelensis H14. However, among the other 422 spherical crystal producing isolates, only 5 types of plasmid patterns were observed. Contrary to the 16 groups of protein patterns in our collection, we have only 7 types (Plas 1 to Plas 7) of plasmid patterns: one Btk like, one Bti like and 5 Non-Bti like (Figure 3). These findings showed that the 700 isolates harbor a total of seven plasmid patterns and 16 different protein patterns (Table 2).

The 19 Bti like isolates with smooth spherical crystals, all showed positive hemolysis around their colonies after overnight incubation. The others showed different degrees of hemolysis. Among the crystal forms, the isolates with spherical deflated balloon shaped crystals did not show any hemolytic activity. On the other hand, the isolates with spherical undulated surface crystals showed different hemolytic activities. Some had no activity while some had good or slight activities. The isolates with spherical concave crystals and spherical pointy edged crystals showed good hemolytic activity (Figure 4, Table 2). The isolates with bipyramidal crystals showed hemolytic activity among them like the reference Bt kurstaki.

The 19 isolates that resembled the profile of the reference Bt israelensis H14 gave the expected PCR product for amplifications with primers designed for exploring genes encoding Dipteran specific endotoxins; except for cry10 and cyt1C genes. Hence, these isolates were tested for their insecticidal activity against Culex pipiens complex. The 259 isolates that resembled the profile of the reference Bt kurstaki HD1gave all the expected PCR products with primers designed for identifying the presence of genes encoding Lepidopteran and Coleopteran specific endotoxins. The rest of the isolates with spherical crystals did not give expected PCR products with the primers that we tested.

As the 19 Bti like isolates that showed the possible absence of the two important Dipteran specific endotoxin coding genes, the insecticidal bioassay was conducted to check the effect, if any, on the activity. First group consisted of 19 Bti like isolates which were able to kill all the larvae as expected. The second group consisted of 14 representatives that had different protein patterns; they did not show insecticidal activity (Table 2).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.