Identification and Characterization of Non-aflatoxin Producing Aspergillus Species
DOI:
https://doi.org/10.55627/pbulletin.004.01.1773Keywords:
Aspergillus flavus, Aspergillus niger, Disease incidenc, UV light screening, AFPA media, Ammonia vapor testAbstract
Aflatoxins are secondary metabolites formed by Aspergillus species, mostly Aspergillus flavus and Aspergillus parasiticus. Among the mycotoxins that contaminate agricultural products, AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 are the most toxic and carcinogenic. Not all species of Aspergillus produce aflatoxins; some strains are non-toxin. It is estimated that about 1.6 billion dollars of the global food crops are thought to be mycotoxin contaminated. AFs contamination in maize grains is 10.0 µg/kg in central Punjab, Pakistan. It is a major global challenge to manage Aspergillus infection, biocontrol methods are most effective and innovative by applying non-aflatoxigenic spores onto the field prior to harvest. The study aimed to estimate the incidence of aflatoxin and to identify non-aflatoxin Aspergillus species in four Tehsils of Multan district. The survey in Multan district revealed varying incidence percentage across tehsils, the highest occurrence of A. flavus (26.21%) was recorded in Tehsil Multan while lowest (13.45%) incidence was found in Tehsil Multan Saddar. The use of CMA, AFPA and PDA media in this study enabled sufficient growth, sporulation and pigmentation, allowing for a thorough identification of aflatoxin and non-aflatoxin producing Aspergillus isolates. A. flavus and A. niger isolates on CMA media under UV light at 365 nm were screened, 19 out of 20 isolates showed blue fluorescence while non-aflatoxigenic A. niger showed none. On AFPA media, A. flavus isolates indicated orange coloration underside their colonies which were aflatoxin producing and A. niger isolate did not show any coloration and considered as non-aflatoxin Aspergillus isolate. While on PDA media, the 7 days old isolates of A. flavus were detected through Ammonia Vapor Test (AVT) with a drop of concentrated ammonia hydroxide turned plum red indicating aflatoxin and A. niger showed no color change and confirmed as non-aflatoxin producing Aspergillus isolate. The use of selective media like CMA and AFPA were effective for morphological identification by distinguishing between aflatoxigenic and non-aflatoxigenic isolates when advance tools were accessible. Incorporating non-aflatoxin producing A. niger strain into crop management can reduce the risk of aflatoxin contamination, improving food safety and economic stability.
References
Ajmal, M., Bedale, W., Akram, A., & Yu, J.-H. (2022). Comprehensive review of aflatoxin contamination, impact on health and food security, and management strategies in Pakistan. Toxins, 14(11), 845.
Al-Masoodi, I. H., Al-Rubaye, A. F. M., & Hussein, H. J. (2023). Isolation and diagnosis of the fungi associated with maize seeds collected from local markets in Karbala, Iraq. Caspian Journal of Environmental Sciences, 21(3), 665–672.
Ashraf, W., Rehman, A., Rabbani, M., Shaukat, W., & Wang, J.-S. (2023). Aflatoxins posing threat to food safety and security in Pakistan: Call for a one health approach. Food and Chemical Toxicology, 180, 114006. https://doi.org/10.1016/j.fct.2023.114006
Bhatnagar-Mathur, P., Sunkara, S., Bhatnagar-Panwar, M., Waliyar, F., & Sharma, K. K. (2015). Biotechnological advances for combating Aspergillus flavus and aflatoxin contamination in crops. Plant Science, 234, 119–132. https://doi.org/10.1016/j.plantsci.2015.02.009
Bandyopadhyay, R., Ortega-Beltran, A., Akande, A., Mutegi, C., Atehnkeng, J., Kaptoge, L., Senghor, A. L., Adhikari, B. N., & Cotty, P. J. (2016). Biological control of aflatoxins in Africa: Current status and potential challenges in the face of climate change. World Mycotoxin Journal, 9(5), 771–790.
Chi, M.-H., & Craven, K. D. (2016). RacA-mediated ROS signaling is required for polarized cell differentiation in conidiogenesis of Aspergillus fumigatus. PLoS ONE, 11(2), e0149548. https://doi.org/10.1371/journal.pone.0149548
Compaore, H., Samandoulougou, S., Tapsoba, F. W., Bambara, A., Ratongue, H., Sawadogo, I., Kabore, D., Ouattara-Sourabie, P. B., & Sawadogo-Lingani, H. (2021). Aflatoxigenic potential of Aspergillus section Flavi isolated from maize seeds, in Burkina Faso. African Journal of Microbiology Research, 15(8), 420–428.
Criseo, G., Bagnara, A., & Bisignano, G. (2001). Differentiation of aflatoxin-producing and non-producing strains of Aspergillus flavus group. Letters in Applied Microbiology, 33(4), 291–295. https://doi.org/10.1046/j.1472-765X.2001.00998.x
Damann, K. E., Jr. (2015). Atoxigenic Aspergillus flavus biological control of aflatoxin contamination: What is the mechanism? World Mycotoxin Journal, 8(2), 235–244. https://doi.org/10.3920/WMJ2014.1719
Dorner, J. W. (2009). Biological control of aflatoxin contamination in corn using a nontoxigenic strain of Aspergillus flavus. Journal of Food Protection, 72(4), 801–804. https://doi.org/10.4315/0362-028X-72.4.801
Fente, C. A., Ordaz, J. J., Vázquez, B. I., Franco, C. M., & Cepeda, A. (2001). New additive for culture media for rapid identification of aflatoxin-producing Aspergillus strains. Applied and Environmental Microbiology, 67(10), 4858–4862. https://doi.org/10.1128/AEM.67.10.4858-4862.2001
Frisvad, J. C., Hubka, V., Ezekiel, C. N., Hong, S.-B., Nováková, A., Chen, A. J., Arzanlou, M., Larsen, T. O., Sklenář, F., Mahakarnchanakul, W., & Samson, R. A. (2019). Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins. Studies in Mycology, 93(1), 1–63.
Frisvad, J. C., Skouboe, P., & Samson, R. A. (2005). Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov. Systematic and Applied Microbiology, 28(5), 442–453. https://doi.org/10.1016/j.syapm.2005.02.012
Giray, B., Girgin, G., Engin, A. B., Aydın, S., & Sahin, G. (2007). Aflatoxin levels in wheat samples consumed in some regions of Turkey. Food Control, 18(1), 23–29. https://doi.org/10.1016/j.foodcont.2005.08.002
Houbraken, J., Kocsubé, S., Visagie, C. M., Yilmaz, N., Wang, X.-C., Meijer, M., Kraak, B., Hubka, V., Bensch, K., Samson, R. A., & Frisvad, J. C. (2020). Classification of Aspergillus, Penicillium, Talaromyces and related genera (Eurotiales): An overview of families, genera, subgenera, sections, series and species. Studies in Mycology, 95, 5–169. https://doi.org/10.1016/j.simyco.2020.05.002
Khan, R., Ghazali, F. M., Mahyudin, N. A., & Samsudin, N. I. P. (2020). Morphological characterization and determination of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolated from sweet corn kernels and soil in Malaysia. Agriculture, 10(10), 450. https://doi.org/10.3390/agriculture10100450
Khan, W. A., Khan, M. Z., Khan, A., & Hussain, I. (2010). Pathological effects of aflatoxin and their amelioration by vitamin E in white leghorn layers. Pakistan Veterinary Journal, 30(3), 155–162.
Klich, M. A. (2007). Aspergillus flavus: The major producer of aflatoxin. Molecular Plant Pathology, 8(6), 713–722. https://doi.org/10.1111/j.1364-3703.2007.00436.x
Kowalska, A., Walkiewicz, K., Kozieł, P., & Muc-Wierzgoń, M. (2017). Aflatoxins: Characteristics and impact on human health. Postępy Higieny i Medycyny Doświadczalnej, 71, 315–327. https://doi.org/10.5604/01.3001.0010.3816
Luttfullah, G., & Hussain, A. (2011). Studies on contamination level of aflatoxins in some dried fruits and nuts of Pakistan. Food Control, 22(3–4), 426–429. https://doi.org/10.1016/j.foodcont.2010.09.015
Mamo, F. T., Shang, B., Selvaraj, J. N., Wang, Y., & Liu, Y. (2018). Isolation and characterization of Aspergillus flavus strains in China. Journal of Microbiology, 56(2), 119–127. https://doi.org/10.1007/s12275-018-7144-1
Nasir, U., Naeem, I., Asif, M., Ismail, A., Gong, Y. Y., Routledge, M. N., Amjad, A., Fazal, A., & Ismail, Z. (2021). Assessment of aflatoxins exposure through urinary biomarker approach and the evaluation of the impacts of aflatoxins exposure on the selected health parameters of the children of Multan city of Pakistan. Food Control, 123, 107863. https://doi.org/10.1016/j.foodcont.2020.107863
Paulussen, C., Hallsworth, J. E., Álvarez-Pérez, S., Nierman, W. C., Hamill, P. G., Blain, D., Rediers, H., & Lievens, B. (2017). Ecology of aspergillosis: Insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species. Microbial Biotechnology, 10(2), 296–322. https://doi.org/10.1111/1751-7915.12367
Perrone, G., Susca, A., Cozzi, G., Ehrlich, K., Varga, J., Frisvad, J. C., Meijer, M., Noonim, P., Mahakarnchanakul, W., & Samson, R. A. (2007). Biodiversity of Aspergillus species in some important agricultural products. Studies in Mycology, 59, 53–66. https://doi.org/10.3114/sim.2007.59.07
Pitt, J. I., & Hocking, A. D. (2009). Fungi and food spoilage (3rd ed.). Springer, New York.
Reis, T. A., Baquião, A. C., Atayde, D. D., Grabarz, F., & Corrêa, B. (2014). Characterization of Aspergillus section Flavi isolated from organic Brazil nuts using a polyphasic approach. Food Microbiology, 42, 34–39. https://doi.org/10.1016/j.fm.2014.02.013
Ribeiro, J. M. M., Cavaglieri, L. R., Fraga, M. E., Direito, G. M., Dalcero, A. M., & Rosa, C. A. R. (2006). Influence of water activity, temperature and time on mycotoxins production on barley rootlets. Letters in Applied Microbiology, 42(2), 179–184. https://doi.org/10.1111/j.1472-765X.2005.01830.x
Samson, R. A., Visagie, C. M., Houbraken, J., Hong, S.-B., Hubka, V., Klaassen, C. H. W., Perrone, G., Seifert, K. A., Susca, A., Tanney, J. B., Varga, J., Kocsubé, S., Szigeti, G., Yaguchi, T., & Frisvad, J. C. (2014). Phylogeny, identification and nomenclature of the genus Aspergillus. Studies in Mycology, 78, 141–173. https://doi.org/10.1016/j.simyco.2014.07.004
Santos, L., Kasper, R., Sardiñas, N., Marín, S., Sanchis, V., & Ramos, A. J. (2010). Effect of Capsicum carotenoids on growth and aflatoxins production by Aspergillus flavus isolated from paprika and chilli. Food Microbiology, 27(8), 1064–1070. https://doi.org/10.1016/j.fm.2010.07.009
Thathana, M. G., Murage, H., Abia, A. L. K., & Pillay, M. (2017). Morphological characterization and determination of aflatoxin-production potentials of Aspergillus flavus isolated from maize and soil in Kenya. Agriculture, 7(10), 80. https://doi.org/10.3390/agriculture7100080
Wyatt, T. T., Golovina, E. A., van Leeuwen, R., Hallsworth, J. E., Wösten, H. A. B., & Dijksterhuis, J. (2015). A decrease in bulk water and mannitol and accumulation of trehalose and trehalose-based oligosaccharides define a two-stage maturation process towards extreme stress resistance in ascospores of Neosartorya fischeri (Aspergillus fischeri). Environmental Microbiology, 17(2), 383–394. https://doi.org/10.1111/1462-2920.12557
Yu, J. (2012). Current understanding on aflatoxin biosynthesis and future perspective in reducing aflatoxin contamination. Toxins, 4(11), 1024–1057. https://doi.org/10.3390/toxins4111024
Downloads
Published
Issue
Section
License
Copyright (c) 2025 Hassan Riaz, Shehbaz Sabir, Amna Ikram

This work is licensed under a Creative Commons Attribution 4.0 International License.
