STUDY OF Bacillus licheniformis BACTERIA AS A BIO-CONTROL AGENT IN REDUCING AFLATOXIN B1 TOXICITY IN DRIED GRAPE SAMPLES
DOI:
https://doi.org/10.36103/zdgppj34Keywords:
fungal toxins, Aspergillus flavus, ammonia, secondary metabolites, hplcAbstract
This study was aimed to isolate and identify fungi contaminating raisin samples and evaluate the effectiveness of bacteria in inhibiting fungal growth and reducing toxin levels. The isolation results the fungus A. flavus was the most frequent and appeared in the samples, reaching 54.5 and 44.4%, respectively. The results showed that all isolates of A. flavus had the ability to produce aflatoxin B1, as confirmed by the ammonia vapor test. The isolate with the highest toxin production was identified through nucleotide sequences deposited in the World Genomic Organization's Gene Bank under accession number OR192858. The results demonstrated the ability of the live bacteria Bacillus licheniformis to inhibit fungal growth, reaching 94.4%, compared to 72.56% for the killed bacterial filtrate. The toxin concentration was 62.11 ppb in the toxin-producing isolate treated with bacteria, while it was 285.866 ppb in the toxin-producing isolate without bacteria. An 85.65% reduction in toxin levels was observed compared to the control, which registered 0%, with a toxin concentration of 41.08 ppb in the bacteria-treated toxin-producing isolate, while it was 285.866 ppb in the untreated toxin-producing isolate.
References
1. Albarrán-de la Luz, L., M. A. Rodríguez-Barrera, G. Hernández-Floreset et al. , 2022. Antagonismo de Bacillus licheniformis M2–7 contra hongos fitopatógenos de Mangifera indica. Revista Internacional de Contaminacion,38 (Ambientey Bioenergía), pp:1-10. https://doi.org/10.20937/RICA.54217
2. Abdul-Karim, E. K. 2023. The Role of secondary metabolites in the Conocarpus spp. in controlling plant diseases: A Review. International Journal of Biological Engineering and Agriculture, 2(8), 18-26. https://doi.org/10.51699/ijbea.v2i8.2382
3. Abdul-Karim, E. K. 2021. The efficiency of magnesium oxide, nano magnesium oxide, and cinnamon alcoholic extract in controlling Fusarium oxysporum f. sp. lycopersici causing Fusarium wilt on tomato. International Journal of Agricultural and Statistical Sciences, 17(1), 1611-1618. https://doi.org/connectjournals.com/03899.2021.17.1611
4. Abdul-Karim, E. K. and N. S. Aljarah. 2023. Morphological and molecular identification of the Neoscytalidium dimidiatum, and evaluation of kaolin and magnesium oxide nano-particles efficacy to control in vitro . IOP Conf. Series: Earth and Environmental Science 1252, 012011 doi:10.1088/1755-1315/1252/1/012011
5. Abdul-Karim , E.K. , N. S. Aljarah and H. A.Ali .2023. Molecular characterization of neoscytalidium spp. the cause of wilting of branches and blackening of the stem.IOP Conf. Series: Earth and Environmental Science. 1262,032032.doi:10.1088/17551315/1262/3/03203 .
6. Abdul-Karim, E. K. and H.Z. Hussein . 2022. The biosynthesis of nanoparticles by fungi and the role of nanoparticles in resisting pathogenic fungi to plants: A Review. Basrah Journal of Agricultural Science, 35, 243-256. https://doi.org/10.37077/25200860.2022.35.1.18
7. Abdul-Karim, E. K., S.N. Hawar and H.Z. Hussein. 2025. Antagonistic activity of plant growth-promoting rhizobacteria to control Rhizoctonia solani fungus causing black scurf disease in potato. Iraqi Journal of Agricultural Sciences.56(4).in press.
8. Ajanal, M., M. B. Gundkalle and S. U. Nayak. 2012. Estimation of total alkaloid in chitrakadivati by UV spectrophotometer. Ancient Science Life,31(4),198-201. https://doi.org/10.4103%2F02577941.107361
9. Al-Barazanchi, T. A., Z. H. Abood and R. S. Al-Rawi. 2022. Molecular investigation of heat shock protein 70 (hsp70) expression levels in aspergillosis patients. Iraqi Journal of Agricultural Sciences,53(3),534-541. https://doi.org/10.36103/ijas.v53i3.1561
10. Aljbori, N. M. and H. Z. Hussein. 2023. Testing the efficiency of silver nanoparticles manufactured locally by the alga Spirogyra in inhibiting the fungus Aspergillus flavus and reduction of aflatoxin B1. IOP Conf. Series: Earth and Environmental Science, 1225, 012062.
doi:10.1088/17551315/1225/1/012062.
11. Al-Hamiri, K. A. A. and H. Z. Hussein . 2022. The efficiency of aloe vera gel extract in inhibiting the growth of Aspergillus flavus fungus associated with imported and domestic rice grains in Iraq and its ability to reduce aflatoxin B1 production. Arab Society for Plant Protection,40(2),164-168. https://doi.org/10.22268/AJPP040.2.164168
12. AlNuaimy, M.A.A. and S.N.Hawar. 2024. Antagonistic activity of endophytic fungi isolated from aloevera leaves against some plant pathogenic fungi. Iraqi Journal of Agricultural Sciences, 55(Special Issue):63-79. https://doi.org/10.36103/ijas.v55iSpecial.1886
13. Benyan, L. A. and A. A. Alhaddad . 2019. Short communication: scanning of processed food contaminating fungi and determine the potential aflatoxigenic type. Basrah Journal of Agricultural Science, 32(Spec Issue), 315-322. DOI:10.21276/basjas.
14. Bibi, F., M. I. Naseer and E. I. Azhar. 2021. Assessing the diversity of bacterial communities from marine sponges and their bioactive compounds. Saudi Journal Biological Science, 28, 2747–2754. https://doi.org/10.1016/j.sjbs.2021.03.042
15. Del Palacio, A., L. Bettucci and D. Pan . 2016. Fusarium and Aspergillus mycotoxins contaminating wheat silage for dairy cattle feeding in Uruguay. Brazilian Journal of Microbiology, 47(4): 1000-1005. https://doi.org/10.1016%2Fj.bjm.2016.06.004
16. El-Feky, A. M. and W. M. Aboulthana.2016. Phytochemical and Biochemical Studies of Sage (Salvia officinalis L.). UK Journal of Pharmaceutical and Biosciences, 4(5), 56-62. http://dx.doi.org/10.20510/ukjpb/4/i5/118037
17. Effmert, U., J. Kalderas, R. Warnke and B. Piechulla .2012. Volatile-mediated interactions between bacteria and fungi in the Soil. Journal Chemical Ecology,38,665−703.https://doi.org/10.1007/s10886-012-0135-5
18. Faria, C. B.; F. C. D. Santos , F. F. D.Castro, L. M. Sergio, M. V. Silva and I. P. Barbosa-Tessmann. 2017. Occurrence of toxigenic Aspergillus flavus in commercial Bulgur wheat. Food Science and Technology , 37(1): 103-111. https://doi.org/10.1590/1678457x.09316
19. Habibatni, S., A. F. Zohra, H. Khalida, S. Anwar, I. Mansi and N. A. A., Ali 2017. In-vitro antioxidant, xanthine oxidase-inhibitory and in-vivo anti-inflammatory, analgesic, antipyretic activity of Onopordum acanthium. International Journal of Phytomedicine, 9(1),92-100. http://dx.doi.org/10.5138/09750185.2030
20. Haider, A. A. and H. Z. Hussein. 2022. Efficiency of biologically and locally manufactured silver nanoparticles from Aspergillus niger in preventing Aspergillus flavus to produce aflatoxin B1 on the stored maize grains. Caspian Journal of Environmental Sciences, 20(4),765-773. https://doi.org/10.22124/cjes.2022.5760
21. Harwood, C. R., M. Mouillon, S. Pohl and J. Arnau. 2018. Secondary metabolite production and the safety of industrially important members of the Bacillus subtilis group. FEMS. Microbiology Reviews,42,721–738. https://doi.org/10.1093/femsre/fuy028
22. Hassan, K .I. 2023.Molecular techniques for detection of aflatoxigenic of Aspergillus flavus and determine their aflatoxin in pistachios. Iraqi Journal of Agricultural Sciences ,54(6):1773-1783. https://doi.org/10.36103/ijas.v54i6.1876
23. Hassan, Z. U., R. Al Thani, H. Alnaimi, , Q. Migheli and S Jaoua . 2019. Investigation and application of Bacillus licheniformis volatile compounds for the biological control of toxigenic Aspergillus and Penicillium spp. ACS Omega,4,17186−17193. http://dx.doi.org/10.1021/acsomega.9b01638
24. Jangala, S., K. Sowjanya, N. Kumara , K. V. N. R. Reddi, , M. J. Sravanthi, , P. Chitta and A. K. Satya. 2014. Identification and assessment of frequency distribution in fungi isolated from coastal Andhra Pradesh. Malaysian Journal of Microbiology, 10(3), 215-218. https://doi.org/10.21161/MJM.56513
25. Kizhakkekalam, V. K., K. Chakraborty and M. Joy. 2020. Oxygenated elansolid type of polyketide spanned macrolides from a marine heterotrophic Bacillus as prospective antimicrobial agents against multidrug resistant pathogens. International Journal of Antimicrobial Agents,55,105892. https://doi.org/10.1016/j.ijantimicag.2020.105892
26. Kareem, T. A., S. S. Mutar, E., K. A Karim and N. A. S. A.Kuwaiti . 2020. Protective effect of olive polyphenols on watermelonagainst Fusarium oxysporum sp. niveum infection. Pakistan Journal of Phytopathology,32(1),27-31. https://doi.org/10.33866/phytopathol.032.01.0542
27. Klich, M. A. 2002 . Identification of common Aspergillus species. Centraalbureau voor Schimmel culture, UTRECHT, The Netherlands. pp. 116.
28. Kříţová, L., K. Dadáková, M. Dvořáčková and T. Kašparovský . 2021. Feedborne mycotoxins beauvericin and enniatins and livestock animals. Toxins, 13(1),32. https://doi.org/10.3390/toxins13010032
29. Kyei, N., B. Cramer, H. U. Humpf, G. H. Degen, N. Ali and S. Gabrysch. 2022. Assessment of multiple mycotoxin exposure and its association with food consumption: a human biomonitoring study in a pregnant cohort in rural Bangladesh. Archives of Toxicology, 96(7),2123–2138. https://doi.org/10.1007/s00204-022-03288-0
30. Lasram, S., Z. Hamdi, , S. Chenenaoui, A. Mliki and A. Ghorbel. 2016. Comparative study of toxigenic potential of Aspergillus flavus and Aspergillus niger isolated from barley as affected by temperature, water activity, and carbon source. Journal of Stored Products Research,69,58-64. https://doi.org/10.1016/j.jspr.2016.06.002
31. Liu, J., L. Sun, N. Zhang, J. Zhang, J. Guo, C. Li, S. A. Rajput and D. Qi . 2016. Effects of nutrients in substrates of different grains on aflatoxin B1 production by Aspergillus flavus. BioMed Research International,. https://doi.org/10.1155/2016/7232858
32. Lusine, H., K. Grigoryan and A. Kirakosyan . 2010. Contamination of raisin by filamentous fungi-potential producers of Ochratoxin A. Potravinarstvo,4,28-33. https://doi.org/10.5219/95
33. Mohamed A. M. and E. I. Al – Shamary .2022. Isolation and identification of aflatoxin B1 productng fungi from stored wheat in some silos of Baghdad. Iraqi Journal of Agricultural, 53(6):1427-1436. https://doi.org/10.36103/ijas.v53i6.1659
34. Marchese, S., A. Polo, A. Ariano, S. Velotto, S. Constantine and L. Severino. 2018. Aflatoxin B1 and M1: biological properties and their involvement in cancer development. Toxins, 10(6), 214. https://doi.org/10.3390%2Ftoxins10060214
35. Maung, C. E. H., T. G. Choi, H. H. Nam and K. Y. Kim .2017. Role of Bacillus amyloliquefaciens Y1 in the control of fusarium wilt disease and growth promotion of tomato. Biocontrol Science Technology, 27(12), 1400–1415. https://doi.org/10.1080/09583157.2017.1406064
36. Mondol, M. A., H. J. Shin and M. T. Islam . 2013. Diversity of secondary metabolites from marine Bacillus species: chemistry and biological activity. Mar Drugs, 11(8),2846-72. https://doi.org/10.3390%2Fmd11082846
37. Muras, A., M. Romero, C. Mayer and A. Otero . 2021. Biotechnological applications of Bacillus licheniformis. Critical Reviews in Biotechnology, 41, 609–627. https://doi.org/10.1080/07388551.2021.1873239
38. Pitt, J. I. and A. D. Hocking . 2009. Fungi and Food Spoilage. 3rd Edition. Springer US. New York: pp.520
39. Ravindran, P., J. M. Lajapathy, S. G. Lalithakumari, A. K., Mohan, T. Cyriac and S. S. Usha. 2023. Efficacy of Bacillus licheniformis: a biocontrol agent against Colletotrichum gloeosporioides Penz. (Penz. & Sacc.) causing anthracnose in greater yam (Dioscorea alata L.) Amrutha. Egyptian Journal of Biological Pest Control ,33,112.00755-3 https://doi.org/10.1186/s41938-023-00755-3
40. Sharma, D., S. S. Singh, P. Baindara, S. Sharma, N. Khatri, V. Grover, P. B. Patil and S. Korpole. 2020. Surfactin like broad spectrum antimicrobial lipopeptide co-produced with sublancin from Bacillus subtilis strain A52: Dual Reservoir of Bioactives. Frontiers in Microbiology, 11,1167. https://doi.org/10.3389/fmicb.2020.01167
41. Shekhar, M., N. Singh, R. Dutta, S. Kumar and V. Mahajan . 2017. Comparative study of qualitative and quantitative methods to determine toxicity level of Aspergillus flavus isolates in maize. PLoS One, 12(12), e0189760. https://doi.org/10.1371/journal.pone.0189760
42. Shleeva, M. O., D. A. Kondratieva and A. S. Kaprelyants .2023. Bacillus licheniformis: a producer of antimicrobial substances, including antimycobacterials, which are feasible for medical applications. Pharmaceutics, 15,1893.https://doi.org/10.3390/pharmaceutics15071893
43. Sidharthan, V.K., R. Aggarwa, N. Surenthi and V. Shanm .2018. Selection and characterization of the virulent Fusarium oxysporum f.sp. lycopersici isolate inciting vascular wilt of tomato. International Journal of Current Microbiology and Applied Sciences. 7(2):1749-1756. https://doi.org/10.20546/ijcmas.2018.702.212
44. Sun, F., D. Yu, H. Zhou, H. Lin, Z. Yan and A. Wu . 2023. CotA laccase from Bacillus licheniformis ZOM-1 effectively degrades zearalenone, aflatoxin B1 and alternariol. Food Control, 145, 109472. https://doi.org/10.1016/j.foodcont.2022.109472
45. Uppala, S. S., K. L. Bowen and F. M. Woods . 2013. Pre-harvest aflatoxin contamination and soluble sugars of peanut. Peanut Science, 40(1), 40-51. https://doi.org/10.3146/PS12-9.1
46. Wang, Y., H. Zhang, H. Yan, C. Yin, Y. Liu, Q. Xu, et al. 2018. Effective biodegradation of aflatoxin B1 using the Bacillus licheniformis (BL010) strain. Toxins, 10, 497. https://doi.org/10.3390%2Ftoxins1012049
47. Zhang, D., R. Qiang, Z. Zhou, Y Pan, S. Yu, W. Yuan, J. Cheng, J. Wang, Zhao, D., J. Zhu and Z. Yang . 2022. Biocontrol and action mechanism of Bacillus subtilis lipopeptides’ fengycins against Alternaria solani in potato as assessed by a transcriptome analysis. Frontiers in Microbiology, 13, 861113.
https://doi.org/10.3389/fmicb. 2022.861113
48. Zhang, L. and C. Sun .2018. Fengycins, cyclic lipopeptides from marine Bacillus subtilis strains, kill the plant-pathogenic fungus Magnaporthe grisea by inducing reactive oxygen species production and chromatin condensation. Applied and Environmental Microbiology, 84(18). https://doi.org/10.1128/aem.00445
Downloads
Published
Issue
Section
License
Copyright (c) 2025 IRAQI JOURNAL OF AGRICULTURAL SCIENCES
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
2.jpg)
https://orcid.org/0000-0002-5774-5906 2.jpg)
