The present study determined the characterization of some Bt strains collected from various biomes and sites of Iran. The results showed that 73.33 and 53.33% of the isolates produced spherical and bipyramidal crystals, respectively, compared to those of Seifinejad et al. (2008) who reported that most of Iranian isolates created bipyramidal ones. Similar to the obtained results, extraordinary variations of crystals (like spherical, irregular, bipyramidal) were observed among native Bt isolates collected from soil and other materials by El-kersh et al. (2016).
Among different characteristics of Bt strains, the plasmid profiles could help to identify strains (Saadaoui et al., 2010). Our result was in accordance with that of BenFarhat-Touzri et al. (2016), who demonstrated that BLB250 strain was identical to Bt strain aizawai and concluded that the strain might be a serotype of the reference isolate. Present study indicated also that some isolates from various regions and provinces had different plasmid profile even in comparison with the reference strains.
Likewise, Yilmaz et al. (2012) found several forms of plasmid patterns in the SY49.1 strain compared with Btk like Seifinejad et al. (2008) for Iranian isolates. Although, the 1013 and 1019 isolates were collected from different provinces, they showed the same pattern and produced one band.
Due to genetic diversity observed in the Bt strains and containing more than one cry gene located on the plasmids or chromosomes, polymerase chain reaction (PCR) can be a precise procedure to discover and characterize the genes (Salama et al., 2015). In the present study, all isolates exhibited bands of the expected size for cry1A, and few ones showed the expected bands for cry1Aa. Similar bands of the expected size for cry1A have been reported for new strains isolated from Tunisian soil samples with the primer pairs (BenFarhat-Touzri et al., 2016). On the contrary, Seifinejad et al. (2008) manifested that cry1Aa was the most abundant gene belonging to the strains isolated from different regions of Iran. Most of the strains harbored cry1C that was consistent with Seifinejad et al. (2008). However, previous study showed that cry genes isolated from Iran exhibited unexpected size bands for cry1C (Nazarian et al., 2009).
Totally, the findings of the present study are similar to those presented by Bravo et al. (1998) who reported that the most abundant gene was cry1 among the Mexican strains collected from soil samples. Compatible to our results, characterization of the strains isolated from Egyptian soil samples clarified that 77.77% of them contained the cry1C genes (Salama et al., 2015). Similar to the present study, Yilmaz et al. (2012) found that cry1A along with cry1C from the most pathogenic Bt strain (SY49.1) collected from Turkey. By comparison, Salehi Jouzani et al. (2008) elucidated that the most abundant gene detected from Iranian isolates was cry2-type such as cry2Ab (55%) and cry2Aa1 (37.5%). Compatible to the obtained results, all Iranian Bt strains isolated from soil and larvae harbored the cry2 gene, and some of them showed amplification products for cry1Ac, cry1Aa, and cry1C (Khorramnejad et al., 2018).
The results of the sequence analyzing of the highest pathogenic strain, 1019, showed more than (99%) identity to all Bt strains used. Yilmaz et al. (2012) analyzed the 16S ITS rDNA gene of the SY49.1 strain and indicated that the similarity of the tested strain was (98%) with Bt subspecies andalousiensis BGSC 4AW1 and Bt subspecies monterrey BGSC 4AJ1. Moreover, the 16S rRNA gene analysis of native Bt isolates reported by El-kersh et al. (2016) showed that the high pathogenic isolate, Bt63, had a strict relationship to Bt subspecies israelensis, and all isolates were homologous together. Consistent with our output, analysis of an Indian strain by the gene sequencing revealed high identity with B. cereus and Bt (Banik et al., 2019).
A previous investigation revealed that molecular mass of Cry1Aa, Cry1Ac, and Cry1C proteins were 133.2, 133.3, and 134.8 kDa analyzed through the SDS–PAGE, respectively (Kalman et al., 1995). All strains studied by Alper et al. (2016), harboring the cry1 and cry2 genes, and the reference strain, Btk, almost revealed similar crystal protein contents. Similar results have been achieved for the most Iranian strains studied in the present research. It was shown that all protein profiles of Iranian strains were almost similar to the reference strains, Btk and Btt, except those of 1013, 1022, 1032, and 1046 that were almost similar to the Btg protein pattern.
According to the present results, the 1013 and 1032 strains had different protein profiles. Both of them carried cry1Ac and cry1C; however, 1032 only showed an unexpected band for cry1Aa. Both of the 1022 and 1046 strains, with similar protein patterns, had cry1Ac; however, the 1022 strain harbored 2 extra genes including cry1C and cry2Ab2. Khorramnejad et al. (2018) clarified that the protein patterns of tested strains were similar to Btk with molecular mass between 60 and 130 kDa comprising Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da, and Cry2Aa proteins. Also, these profiles were observed for the tested crystals in the present study.
Based on previous study, more than 50% of Iranian isolates structured proteins of 130–140 kDa, and there were also strains that produced proteins with a molecular mass between 28 and 140 kDa or even lower than 28 kDa (Seifinejad et al., 2008), in which the first range was active against lepidopteran pests (Herandeza et al., 2005) that was in accordance with our findings. Additionally, the most of Iranian strains which harbored cry2-type genes showed protein bands of 21–140 kDa (Salehi Jouzani et al., 2008). Boukedi et al. (2016) reported that Bt HD1 and BUPM95 carried Cry proteins of 65–70 and 130–135 kDa related to the cry2 and cry1 genes, respectively. All investigations mentioned above for protein profiles of parasporal inclusions were in accordance with our results and confirmed that the Iranian strains were able to produce effective crystal protein toxin against lepidopteran pests, and the toxins were associated to their PCR profiles.
It was obvious that the cry1 and cry2A genes are active against lepidopteran insects (Park et al., 2011). Although, all tested strains especially 1019 were analyzed for the cry1A and cry1C genes and presented expected bands, they manifested different median lethal concentrations. Similarly, the high toxic Turkish strain, SY49.1, against E. kuehniella harbored the two latter genes (Yilmaz et al., 2012). Furthermore, E. kuehniella was so sensitive to the novel strain, BLB250, as it contained cry1A (BenFarhat-Touzri et al., 2016). Jalapathi et al. (2020) indicated that Cry1Ac protein toxin from Bt was highly pathogenic to the larvae of Tuta absoluta (Meyrick) (Gelechiidae: Lepidoptera) which was in accordance with our results. As it was obvious, our findings were consistent with those of previous outcomes, and the important role of cry1, as a determinant of pathogenicity against lepidopteran pests, was supported by the current results.
Consistent with this research, 2 Iranian strains, KON4 and YD5, harboring cry1A, cry1C, and cry2A, were more pathogenic against Helicoverpa armigera (Hübner) larvae than Btk (Seifinejad et al., 2008). The highest mortality for E. kuehniella happened by the Tunisia isolate, BLB1, harboring cry1 and cry2 in comparison with Btk HD1 (Saadaoui et al., 2010). In comparison with the current bioassay result, some Turkish Bt strains harboring cry2Ab and cry2Aa1 led to 42% mortality in E. kuehniella (higher than Btk) (Alper et al., 2016).
The investigated Iranian strains had the lepidopteran-active cry genes; however, their toxicity potential against the Mediterranean flour moth varied. This finding was in close agreement with Ferrandis et al. (1999) that exhibited the inactivation possibility of the specific genes.