CC BY
0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
GENETIC DIVERSITY AND INSECTICIDAL POTENTIAL OF
BACILLUS THURINGIENSIS STRAINS ISOLATED FROM UZBEKISTAN: PREVALENCE OF CRY1, CRY2, AND VIP3A
GENES
1Kobilov Fazliddin Bozorovich, 2Khalilov Ilkhom Mamatqulovich, 3Nazirov Muhammad Latif Maruf o'g'li, 4Aliyev Zafar Zokirovich, 5Imomova Muqaddas Khasnovna, 6Azimova
Nodira Shoyim qizi
1PhD (student), IMUAS, 2DSc, IMUAS, Junior researcher, IMUAS, 4Junior researcher, IMUAS, 5Junior researcher, IPQP, 6PhD, IMUAS https://doi.org/10.5281/zenodo.13827586
Abstract. In this study, we investigated the presence and diversity of insecticidal cry-type genes in native 68 Bacillus thuringiensis (Bt) strains isolatedfrom Uzbekistan. To determine the presence of lepidopteran-active insecticide genes, cryl, cry2, and vip3A gene families were selected and analyzed through PCR-based screening. The total genomic DNA was extracted and used as a template for PCR amplification. The results demonstrated that cryl-type genes were the most prevalent, present in 88.23% of the native 68 Bt strains, followed by cry2 genes in 61.76% and vip3A genes in 17.64% of the strains. These findings were consistent with other studies, indicating a strong presence of cryl genes across different Bt strains. This research contributes to the understanding of the genetic diversity of Bt strains in Uzbekistan and highlights the importance of further investigations into the cloning, characterization, and expression of these unique insecticidal genes. Such efforts could provide new opportunities for integrated pest management in sustainable agriculture, enhancing the development of novel biopesticides for effective pest control.
Keywords: Bacillus thuringiensis, cry-vip3 genes, genetic diversity, biopesticides, PCR, primers.
Introduction
The commonplace Gram-positive, rod-shaped, and sporulating bacterium Bacillus thuringiensis (Bt) has been isolated globally from a wide range of environments, including soil, water, dead insects, silt, dust from silos, leaves from deciduous trees, various conifers, and insectivorous mammals, as well as from severely necrotic human tissues [1-4]. Each strain may be particularly active against pests that are lepidopteran, dipteran, coleopteran, or hymenopteran, as well as other invertebrates like mites and nematodes, depending on the levels of Cry, Cyt, and Vip proteins [5-7]. There are 14 subfamilies (cry1A-N) within the cryl family, and between them are 275 cry genes. Most of the cryl genes are functional in preventing lepidopteran pests from spreading. This family's crylb and crylI genes are effective against coleopteran pests as well [8]. With roughly 82 genes, the cry2 family is ranked second and mostly targets lepidopteran or dipteran pests. During the vegetative growth phase, species of B. thuringiensis also produce vegetative insecticidal proteins, or VIPs [9]. Binary toxins Vipl and Vip2 exhibit insecticidal action against pests of the Hemiptera and Coleoptera [l0], while Vip3 proteins exhibit insecticidal activity against pests of the Lepidoptera [ll, l2]. The toxicity potential of these Vip3 proteins against vulnerable insects is comparable to that of Cry proteins.
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
To improve resistance to lepidopteran genes, seven anti-lepidopteran cry and vip genes-crylAb, cry1A105, crylAc, crylF, cry2Ab, cry2Ae, and vip3A-have been employed. The most utilized genes to create lepidopteran-resistant crops are crylAb, crylF, and crylAc; these genes have been included into 6l, 5l, and 32 genetically modified varieties, respectively [l3].
In Uzbekistan, the cotton bollworm mostly affects the Fergana, Andijan, Namangan, and Tashkent regions, causing significant harm to cotton farms. comparatively less harm in other areas. However, it seriously damaged the Kashkadarya region's Nishon and Miri shkor districts in 2020. It seriously damages tomatoes, corn, eggplant, peas, and legumes in addition to cotton. Should no control measures be implemented, 20-30% of the crop will sustain damage. 60-80% damage if neglected. Damage from average hard impact years can reach 30%.
Materials and methods
Bacterial strains, chemicals and oligonucleotide primers
The Bt were isolated from Uzbekistan. "GenPak® PCR Core" was used for PCR amplifications, oligonucleotide primers were synthesized by IDT (Integrated DNA Technologies). To determine active lepidopteran active insecticide genes, cryl, cry2, and vip3A gene families were selected.
PCR-based cry gene screening in local Bt strains
The total genomic DNA of 68 Bt strains were extracted according to standard protocols "Marmur procedure" [14]. The total DNA pattern of all Bt strains were analyzed on agarose gel. Genomic DNA isolated from Bt strains were used as template for the PCR amplification. Each 20 ul reaction mixture contained 50 ng of genomic DNA of Bt strain, l0 ul dilution, 0.4 ul of forward and reverse primers each, 8.2 ul double distilled water. The amplifications were conducted with the thermal cycler (BioRad, USA). The PCR was performed for 35 cycles as follows: 94°C for 3 min, primer annealing for 40 s and 72°C for 1 min., the final extension was performed for 10 min at 72°C. Electrophoresis was carried out at 140 V for 25 min in TAE electrophoresis buffer. Data analysis was done using GelDoc (BioRad).
Results and discussion
Cry gene detection through PCR in regional Bt strains
The amplification of PCR fragments of the expected size using different primer pairs (Table l) confirmed the presence of the mentioned insecticide-type genes in the 68 Bt strains. Since all the strains carried cryl-type genes, they were the most prevalent (88.23%) among native 68 Bt strains, followed by cry2 (6l.76%) and vip3A (l7.64%) (Fig.l). Using the same screening primers, agarose gel electrophoresis revealed both nonspecific amplification and a particular partial cry gene amplicon, which were also seen in numerous published research findings.
Table 1 Oligonucleotide primers utilized for the screening of partial insecticide-type genes for Lepidopteran insects
№ Primers name Sequence (5' 3') —► Gene name Product size bp References
1 LeplA LeplB CCGGTGCTGGATTTGTGTTA AATCCCGTATTGTACCAGCG crylAa, crylAb, crylAc 490 l5]
2 cry2F cry2R GTTATTCTTAATGCAGATGAAT GGG CGGATAAAATAATCTGGGAAATAGT cry2 70l l6]
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
3 vip3AF ATGAACAAGAATAATACTAAA vip3A 2300 [17]
vip3AR GCGGCCGCTTACTTAATAGAGAC
b
Fig. 1 Agarose gel electrophoresis of a. PCR amplification of partial cry 1A gene from 68 Bt strains. b. PCR amplification of partial cry2 gene from Bt strains. c. PCR amplification of Vip3A gene from Bt strains. M-100 bp DNA ladder (a and b figures); M-1kb DNA ladder (c figure).
Jain et al. [18] examined the frequency of insecticide-type genes in eight Bt strains (IS1-IS8) and found that, as determined by PCR, cry1 type genes were the most prevalent in the native isolates because all the strains carried them. These were followed by vip3A (87.5%), cry2 (75%), cry9 (62%), cry3 (50%), cry11 (37.5%), cry7-8 (37.5%), cry5, 12, 14, 21 (25%), cyt1 (25%), cry4 (12.5%), and cyt2 (12.5%). The diversity of cry genes from various soil types and climatic environments was reported by Patel et al. [19]. They also found that while cry3 and cry13 genes were absent from isolates of non-agricultural samples, cry1, cry2, cry3, 7, 8, cry4, cry5, 12, 14, 21, cry11, cry13, and cyt1 genes were found to be present in Bt. Parallel to this, Salekjalali et al. [20] used PCR to identify isolates carrying distinct cry-type genes and discovered that 47% of the
a
c
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
strains amplified using the cryl primer, 29% with the cry3 primer, and 13% with the cry4 primer. The cryl gene is the most prevalent of the investigated cry-type genes in these isolates (83.33%), according to Salama et al. [21]. Cryl gene subfamilies (cryl B and crylC) had percentages of 38.88 and 77.77%, respectively, behind the cryl gene. The cry2A gene was present in the examined isolates, however not all the isolates (6l.76%) tested positive for the cry2 gene. Of the examined isolates, only 6l.76% and l7.64%, respectively, had cry2 and vip3A genes. A presence of all three genes family in Btlfo, Btl0, Btl3, Btl6, Btl9, Bt24, Bt99, Bt ambl; and cryl and cry2 genes family in Btl, Bt2fo, Bt3, Bt3fo, Bt4, Bt6, Bt7, Bt7fo, Btl2, Btl9fo, Btx20, Bt2l, Btx24, Bt27, Btx27, Btx28, Bt28, Bt29, Bt3l, Bt32, Btx33, Bt38, Bt39, Bt77, Btl00, Btl06, Bt mth, Bt var.thur, Bt26 strains; cry2 and vip3A genes family in Bt35, Bt98 strains, and only vip3A gene in Btl05 strain; only cry2 gene family in Bt43, Bt80, Btl03 strains; in addition to these, only cryl family genes in Bt6fo, Btl0fo, Btl2fo, Btl3fo, Btl7fo, Btxl8, Btl8fo, Bt20fo, Btx30, Btx45, Bt45, Bt57, Bt76, Bt84, Bt93, Bt94, Btl0l, Btl02, Btl04, Bt300, Btl-4, Bt2-3, Bt amd strains and absence of all three genes family in Bt30 and Bt72 strains were determined by PCR results. Cry gene detection has been substantially enhanced using PCR; nevertheless, this technique is primarily restricted to members of already identified gene families and necessitates many primers. The current study's findings imply that the native Bt isolates are diverse. Additional research on the cloning and characterization and expression of those unique insecticide genes from these fresh isolates of Bt will be beneficial and present fresh prospects for integrated pest management in sustainable agriculture in Uzbekistan.
Conclusion
This study highlights the predominance of cryl-type genes in native 68 Bt strains, with a significant majority (88.23%) of the strains carrying these genes. The detection of cry2 and vip3A genes, though less frequent, indicates additional insecticidal potential within the strains. Overall, these findings enhance our understanding of the genetic diversity of Bt strains and their potential utility in developing effective biopesticides.
LITERATURE
1. Höfte, H.; Whiteley, H.R. Insecticidal Crystal Proteins of Bacillus thuringiensis. Microbiol. Rev. l989, 53, 242-255. 2.
2. Knowles, B.H.; Dow, J.A.T. The crystal delta-endotoxins of Bacillus thuringiensis— Models for their mechanism of action on the insect gut. Bioessays l993, l5, 469-476.
3. Raymond, B.; Johnston, P.R.; Nielsen-LeRoux, C.; Lereclus, D.; Crickmore, N. Bacillus thuringiensis: An impotent pathogen? Trends Microbiol. 20l0, l8, l89-l94.
4. Roh, J.Y.; Choi, J.Y.; Li, M.S.; Jin, B.R.; Je, Y.H. Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control. J. Mol. Biol. 2007, l7, 547-559.
5. Abdelmalek N, Sellami S, Ben Kridis A, Tounsi S, Rouis S (20l5) Molecular characterisation of Bacillus thuringiensis strain MEB4 highly toxic to the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Pest Manag Sci. doi:l0.l002/ps.4066.
6. Salehi Jouzani G, Abad AP, Seifinejad A, Marzban R, Kariman K, Maleki B (2008a) Distribution and diversity of dipteran-specific cry and cyt genes in native Bacillus thuringiensis strains obtained from different ecosystems of Iran. J Ind Microbiol Biotechnol 35:83-94. doi:l0. l007/s l0295-007-0269-6.
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
7. Salehi Jouzani G, Seifinejad A, Saeedizadeh A, Nazarian A, Yousefloo M, Soheilivand S, Mousivand M, Jahangiri R, Yazdani M, Amiri RM, Akbari S (2GG8b) Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes. Canad J Microbiol 54:812-822. doi:1G.1139/WG8-G74.
8. Nazarian A, Jahangiri R, Salehi Jouzani G, Seifinejad A, Soheilivand S, Bagheri O, Keshavarzi M, Alamisaeid K (2GG9) Coleopteranspecific and putative novel cry genes in Iranian native Bacillus thuringiensis collection. J Invertebr Pathol 1G2:1G1-1G9. doi:1G. 1G16/j.jip.2GG9.G7.GG9.
9. Mamta Gupta, Harish Kumar and Sarvjeet Kaur. Vegetative Insecticidal Protein (Vip): A potential contender from Bacillus thuringiensis for Efficient Management of Various Detrimental
Agricultural Pests. Front_Microbiol. 2G21;12:
659736.PMCID: PMC815894G.PMID: 34G54756.doi:1G.3389/fmicb.2G21.659736.
1G. Warren G. W. (1997). "Vegetative insecticidal proteins: novel proteins for control
of corn pests," in Advances in Insect Control: The Role of Transgenic Plants, eds Carozzi N., Koziol M. (London: Taylor and Francis), 1G9-121. [Google Scholar] [Ref list].
11. Palma L., Muñoz D., Berry C., Murillo J., Caballero P. (2014). Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 6 3296-3325. 1G.339G/toxins6123296 [PMC free article] [PubMed] [CrossRef] [Google Scholar] [Ref list].
12. Chakroun M., Banyuls N., Bel Y., Escriche B., Ferré J. (2016). Bacterial vegetative insecticidal proteins (Vip) from entomopathogenic bacteria. Microbiol. Mol. Biol. Rev. 8G 329-35G. 1G.1128/MMBR.GGG6G-15 [PMC free article] [PubMed] [CrossRef] [Google Scholar] [Ref list].
13. Jouzani, G. S., Valijanian, E., & Sharafi, R. (2G17). Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Applied Microbiology and Biotechnology, 1G1(7), 2691-2711. doi:1G.1GG7/sGG253-G17-8175-y (https://doi.org/1G.1GG7/sGG253-G17-8175-y.
14. Francisco Salvà-Serra, Liselott Svensson-Stadler, Antonio Busquets, Daniel Jaén-Luchoro, Roger Karlsson, Edward R. B. Moore & Margarita Gomila Culture Collection University of Gothenburg (CCUG). A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: a modified version of the Marmur procedure. Method in Protocol Exchange • August 2018. DOI: 10.1038/protex.2018.084.
15. Carozzi NB, Kramer VC, Warren GW, Evola S, Koziel MC (1991) Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles. Appl Environ Microbiol 57:3G57-3G61.
16. Isolation and identification of Bacillus thuringiensis strains native of the Eastern Province of Saudi Arabia. Amina A. Hassan, Mohamed A. Youssef, M. M. A. Elashtokhy, I. M. Ismail, Munirah Aldayel and Eman Afkar. Egyptian Journal of Biological Pest Control (2G21) 31:6 https://doi.org/1G.1186/s41938-G2G-GG352-8.
17. Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (1996) vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum activity against lepidopteran insects. Proc Natl Acad Sci USA 93:5389-5394.
18. Jain D, Kachhwaha S, Jain R, Kothari S (2G12) PCR based detection of cry genes in indigenous strains of Bacillus thuringiensis isolated from the soils of Rajasthan. Indian J Biotechol 11:491-494.
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
19. Patel KD, Purani S, Ingle SS (2012) Distribution and diversity analysis of Bacillus thuringiensis cry genes in different soil types and geographical regions of India. J Invertebr Pathol 112:116-121.
20. Salekjalali M, Barzegari A, Jafari B (2012) Isolation, PCR detection and diversity of native Bacillus thuringiensis strains collection isolated from diverse Arasbaran Natural Ecosystems. World App Sci J 18:1133-1138.
21. Salama HS, El-Ghany NM, Saker MM (2015) Diversity of Bacillus thuringiensis isolates from Egyptian soils as shown by molecular characterization. J Genet Eng Biotechnol 13:101-109.
