جداسازی و شناسایی قارچ‌های مرتبط با بذر پنبه و بررسی قارچ آفلاتوکسین‌زای Aspergillus flavus

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشکده فناوری کشاورزی، دانشگاه فنی شمالی، موصل، عراق.

2 دانشکده پلی‌تکنیک، دانشگاه فنی شمالی، موصل، عراق

3 بخش زیست‌شناسی، دانشکده علوم و سلامت، و مرکز تحقیقات علوم و سلامت، دانشگاه کویه، کویه ۴۴۰۲۳، اقلیم کردستان، عراق

چکیده

هدف: این پژوهش با هدف جداسازی و شناسایی قارچ‌های آلوده‌کننده‌ی بذرهای دو رقم پنبه که به مدت طولانی در انبار نگهداری شده بودند، و همچنین تعیین جدایه‌ای که بیشترین میزان آفلاتوکسین را تولید می‌کند، انجام شد تا خطرات بالقوه برای ایمنی بذر ارزیابی شود.
مواد و روش‌ها: دو رقم پنبه، عاشورا و لاشتا، که حدود سه سال نگهداری شده بودند، در کیسه‌های استریل جمع‌آوری و به آزمایشگاه منتقل شدند و تا زمان آزمایش در دمای ۴ درجه سانتی‌گراد ذخیره گردیدند. جداسازی قارچ‌ها از طریق کشت سطحی بر روی محیط‌های Potato Dextrose Agar و Malt Extract Agar با انکوباسیون در دمای ۲۵ تا ۲۸ درجه سانتی‌گراد انجام شد. کلنی‌ها بر اساس ویژگی‌های ماکروسکوپی توصیف و سپس با استفاده از رنگ‌آمیزی Lactophenol Cotton Blue از نظر میکروسکوپی بررسی شدند. آزمون قرارگیری در معرض بخار آمونیاک به‌عنوان آزمایشی سریع برای شناسایی کیفی سویه‌های آفلاتوکسین‌زای Aspergillus به کار رفت. میزان تولید آفلاتوکسین توسط جدایه‌ها با استفاده از روش ELISA اندازه‌گیری شد. شناسایی مولکولی جدایه‌ی دارای بیشترین تولید آفلاتوکسین از طریق تکثیر ناحیه‌ی ITS با روش PCR و به‌کارگیری آغازگرهای استاندارد و DNA ژنومی استخراج‌شده بر اساس دستورالعمل تولیدکننده انجام گرفت.
نتایج: در مجموع ۲۸۵ جدایه‌ی قارچی از دو نمونه‌ی بذر پنبه به‌دست آمد که متعلق به چندین جنس مختلف بودند. نمونه‌ی رقم عاشورا ۱۵۰ جدایه و رقم لاشتا ۱۳۵ جدایه تولید کرد. توزیع گونه‌های شناسایی‌شده شامل Aspergillus niger (48 درصد)، Rhizopus spp. (56/44 درصد)، Aspergillus flavus (01/7 درصد)، و Penicillium spp. (35/0 درصد) بود. سه جدایه‌ی A. flavus در شرایط کشت آزمایشگاهی مقادیر قابل اندازه‌گیری آفلاتوکسین تولید کردند که غلظت آن‌ها بین 27 تا 42 نانوگرم بر میلی‌لیتر متغیر بود. جدایه‌ای که بیشترین میزان آفلاتوکسین (۴۲ نانوگرم بر میلی‌لیتر) را تولید کرد، از طریق PCR به عنوان Aspergillus flavus تأیید شد و با شناسایی مورفولوژیک نیز مطابقت داشت؛ این جدایه با شماره‌ی دسترسی منحصربه‌فرد در پایگاه GenBank ثبت گردید.
نتیجه‌گیری: نگهداری طولانی‌مدت بذر پنبه می‌تواند موجب کلونیزه شدن آن توسط اجتماعات قارچی متنوع، شامل سویه‌های آفلاتوکسین‌زای A. flavus شود. پایش مستمر دسته‌های بذر ذخیره‌شده، بهبود بهداشت شرایط انبار و اجرای راهبردهای کاهنده‌ی هدفمند برای تضمین ایمنی بذر و کاهش خطرات بهداشتی و اقتصادی مرتبط توصیه می‌شود.

کلیدواژه‌ها

عنوان مقاله [English]

Isolation and identification of fungi associated with cotton seeds and investigation of aflatoxigenic Aspergillus flavus

نویسندگان [English]

  • Janan K. Al-Tarjuman 1
  • Maha M. Taha Al-Nuaimy 2
  • Yaseen N.M. Al-Shekhany 3
1 College of Agricultural Technology, Northern Technical University, Mosul, Iraq.
2 Polytechnic College, Northern Technical University, Mosul, Iraq.
3 Department of Biology, Faculty of Science and Health, and Department of Science and Health Research Center, Koya University, Koya 44023, Kurdistan Region, Iraq.
چکیده [English]


Objective
This investigation targeted to isolate and identify fungi contaminating seeds of two long-term stored cotton cultivars and to identify which isolate generated the greatest level of aflatoxin, thereby evaluating potential risks to seed safety.

Materials and methods
Two cotton cultivars, Ashura and Lashta, stored for almost three years, were gathered in sterile bags, transported to the laboratory, and stored at 4 °C until analysis. Fungal isolation was carried out by surface plating on potato dextrose agar and malt extract agar with incubation at 25-28 °C. Colonies were described by macroscopic morphology and microscopic examination applying lactophenol cotton blue staining. Ammonia vapor exposure acted as a fast qualitative experiment for aflatoxigenic Aspergillus strains. Aflatoxin making by isolates was quantified applying an enzyme-linked immunosorbent assay (ELISA). Molecular identifying of the greatest aflatoxin-generating isolate was carried out by PCR amplification of the ITS region applying standard primers and genomic DNA extracted based on producer protocols.

Results
A total of 285 fungal isolates demonstrating multiple genera were regained from the two cottonseed samples. The Ashura sample produced 150 isolates, while Lashta produced 135. The dispensation of recognized taxa was Aspergillus niger (48.00%), Rhizopus spp. (44.56%), Aspergillus flavus (7.01%), and Penicillium spp. (0.35%). Three A. flavus isolates generated measurable aflatoxin under culture situations, with concentrations ranging from 27.0 ng·mL⁻¹ to 42.0 ng·mL⁻¹. The isolate with the greatest aflatoxin product (42.0 ng·mL⁻¹) was affirmed as Aspergillus flavus by PCR and was in line with morphological identifying; this isolate has been deposited with a unique accession in GenBank.

Conclusions
Extended storage of cotton seeds can favor colonization by diverse fungal communities, containing aflatoxigenic A. flavus strains. Routine surveillance of stored seed lots, progressed storage hygiene, and aimed mitigation strategies are recommended to ensure seed safety and to reduce related health and economic risks.

کلیدواژه‌ها [English]

  • aflatoxins
  • aspergillus flavus
  • cotton seeds
  • fungal pollution

مراجع

Abd-Elsalam, K. A., Habeeb, M. M., & Asran, A. A. (2023). Efeitos de cultivar, período de armazenamento e fungos transmitidos por sementes no teor de aflatoxina em sementes de algodão. Summa Phytopathologica, 49, Article e257388. https://doi.org/10.1590/0100-5405/257388
Abdullah, A. A., Al-Tarjuman, J. K., & Al-Mamar, H. A. S. (2024). Detection of seed-borne fungal pathogens associated with barley (Hordeum vulgare) seeds and their bio-control under laboratory conditions. IOP Conference Series: Earth and Environmental Science, 1371(3), Article 032042. https://doi.org/10.1088/1755-1315/1371/3/032042
Abdullah, A. A., Al-Tarjuman, J. K., & Al-Mamar, H. A. S. (2025). Biological and chemical control of Fusarium graminearum contaminating barley seeds under greenhouse conditions. NTU Journal of Agricultural and Veterinary Sciences, 5(2), 144-150. https://doi.org/10.56286/ep9ey944
Adhikari, B. N., Bandyopadhyay, R., & Cotty, P. J. (2016). Degeneration of aflatoxin gene clusters in Aspergillus flavus from Africa and North America. AMB Express, 6(1), Article 62. https://doi.org/10.1186/s13568-016-0228-6
Ahsani, M. R., Mohammadabadi, M. R., & Shamsaddini, M. B. (2010). Clostridium perfringens isolate typing by multiplex PCR. Journal of Venomous Animals and Toxins Including Tropical Diseases, 16(4), 573-578. https://doi.org/10.1590/S1678-91992010000400006
Al-Abdalall, A. H. A. (2008). Pathological studies of fungi associated with pulse seed during storage in Dammam Province, Kingdom of Saudi Arabia. Middle Eastern and Russian Journal of Plant Science and Biotechnology, 2(2), 71-77. http://www.globalsciencebooks.info/Online/GSBOnline/OnlineMERJPSB_2_1_2.html
Al-Nuaimy, M. M., & Abed, F. N. (2021). Isolation and identification of Phytophthora infestans from some citrus trees by using polymerase chain reaction (PCR) and DNA sequencing technique in Iraq. Biochemia Medica, 21(1), 769-776. https://www.connectjournals.com/toc/CJ-033216/21/2021/01/April
Al-Nuaimy, M. M., & Abed, F. N. (2024). Zearalenone toxins produced by some species of Fusarium. NTU Journal of Agriculture and Veterinary Science, 4(4). https://doi.org/10.56286/bb44fh80
Al-Rejaboo, M. A., Al-Tarjuman, J. K., Al-Dulaimi, F. K. Y., & Al-Sumaidaiea, B. Z. (2025). Fungi isolated from soils adjacent to olive trees in Nineveh Governorate. Regulatory Mechanisms in Biosystems, 16(1), Article e25017. https://doi.org/10.15421/0225017
Al-Tamimi, M. J., & Al-Zobaidy, B. M. (2022). Phenotypic and molecular diagnosis using polymerase chain reaction (PCR) technology for some aflatoxin-producing fungi isolated from food. International Journal of Health Sciences, 6(S5), 11228-11237. https://doi.org/10.53730/ijhs.v6nS5.10986
Aly, A., Omar, M., El-Abbasi, I., & El-Samawaty, A. (2008). Effect of seed mycoflora on incidence of Fusarium wilt disease in cotton genotypes. Journal of Plant Production, 33(10), 7243-7251. https://doi.org/10.21608/jpp.2008.171237
Al-Zubaidi, A. S. M. A. (2022). The effect of some elements of climate and its relationship to the cultivation of the cotton crop in the central governorates of Iraq. Journal of the College of Basic Education, 3(SI), 94-112. https://cbej.uomustansiriyah.edu.iq/index.php/cbej/article/view/5592
Amaike, S., & Keller, N. P. (2011). Aspergillus flavus. Annual Review of Phytopathology, 49(1), 107-133. https://doi.org/10.1146/annurev-phyto-072910-095221
Areo, O. M., Abafe, O. A., Gbashi, S., & Njobeh, P. B. (2023). Detection of multi-mycotoxins in rooibos and other consumed teas in South Africa by a modified QuEChERS method and ultra-high performance liquid chromatography tandem mass spectrometry. Food Control, 143, Article 109255. https://doi.org/10.1016/j.foodcont.2022.109255
Demirev, A. V., Lee, C. H., Jaishy, B. P., Nam, D. H., & Ryu, D. D. Y. (2006). Substrate specificity of nonribosomal peptide synthetase modules liable for the biosynthesis of the oligopeptide moiety of cephabacin in Lysobacter lactamgenus. FEMS Microbiology Letters, 255(1), 121-128. https://doi.org/10.1111/j.1574-6968.2005.00067.x
El-Naghy, M. A., Mazen, M. B., & Fadl-Allah, E. M. (1991). Studies on the fungus flora and aflatoxin production of cotton seeds in Egypt. Medical Journal of Islamic World Academy of Sciences, 4(2), 141-145. https://medicaljournal-ias.org/jvi.aspx?pdir=ias&plng=eng&volume=4&issue=2
Eskola, M., Kos, G., Elliott, C. T., Hajšlová, J., Mayar, S., & Krska, R. (2020). Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited ‘FAO estimate’ of 25%. Critical Reviews in Food Science and Nutrition, 60(16), 2773-2789. https://doi.org/10.1080/10408398.2019.1658570
Gbashi, S., Njobeh, P. B., Madala, N. E., De Boevre, M., Kagot, V., & De Saeger, S. (2020). Parallel validation of a green-solvent extraction method and quantitative estimation of multi-mycotoxins in staple cereals using LC-MS/MS. Scientific Reports, 10(1), Article 10334. https://doi.org/10.1038/s41598-020-66787-z
International Agency for Research on Cancer. (2012). Aflatoxins (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 100F). World Health Organization. https://publications.iarc.fr/120 (Original work published 2012)
Khabiri, A., Toroghi, R., Mohammadabadi, M., & Tabatabaeizadeh, S. E. (2023). Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran. Veterinary Research Forum, 14(4), 221-227. https://doi.org/10.30466/vrf.2022.548152.3373
Khabiri, A., Toroghi, R., Mohammadabadi, M., & Tabatabaeizadeh, S. E. (2025). Whole genome sequencing and phylogenetic relative of a pure virulent Newcastle disease virus isolated from an outbreak in northeast Iran. Letters in Applied Microbiology, 78(4), Article ovaf049. https://doi.org/10.1093/lambio/ovaf049
Kumar, A., Pathak, H., Bhadauria, S., & Sudan, J. (2021). Aflatoxin contamination in food crops: Causes, detection, and management: A review. Food Production, Processing and Nutrition, 3(1), Article 17. https://doi.org/10.1186/s43014-021-00064-y
Li, P., Zhou, Q., Wang, T., Zhou, H., Zhang, W., Ding, X., Zhang, Z., Chang, P.-K., & Zhang, Q. (2016). Development of an enzyme-linked immunosorbent assay method specific for the detection of G-group aflatoxins. Toxins, 8(1), Article 5. https://doi.org/10.3390/toxins8010005
Magnussen, A., & Parsi, M. A. (2013). Aflatoxins, hepatocellular carcinoma and public health. World Journal of Gastroenterology, 19(10), 1508-1513. https://doi.org/10.3748/wjg.v19.i10.1508
Matsushima, K., Chang, P. K., Yu, J., Abe, K., Bhatnagar, D., & Cleveland, T. E. (2001). Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis. Applied Microbiology and Biotechnology, 55(5), 585-589. https://doi.org/10.1007/s002530100607
Mohammadabadi, M. R., Shaikhaev, G. O., Sulimova, G. E., & Rahman, O. (2004). Detection of bovine leukemia virus proviral DNA in Yaroslavl, Mongolian, and Black Pied cattle by PCR. Cellular and Molecular Biology Letters, 9(4A), 766-768.
Mohammadabadi, M. R., Soflaei, M., Mostafavi, H., & Honarmand, M. (2011). Using PCR for early diagnosis of bovine leukemia virus infection in some native cattle. Genetics and Molecular Research, 10(4), 2658-2663. https://doi.org/10.4238/2011.October.27.2
Mohammadabadi, M., Afsharmanesh, M., Khezri, A., Kheyrodin, H., Babenko, O. I., Borshch, O., Kalashnyk, O., Nechyporenko, O., Afanasenko, V., Slynko, V., & Usenko, S. (2025). Effect of mealworm on GBP4L gene expression in the spleen tissue of Ross broiler chickens. Agricultural Biotechnology Journal, 17(2), 343-360. https://doi.org/10.22103/jab.2025.25277.1714
Mohammadabadi, M., Babenko, O. I., Borshch, O., Kalashnyk, O., Ievstafiieva, Y., & Buchkovska, V. (2024). Measuring the relative expression pattern of the UCP2 gene in different tissues of the Raini Cashmere goat. Agricultural Biotechnology Journal, 16(3), 317-332. https://doi.org/10.22103/jab.2024.24337.1627
Mohana, D., Prasad, P., Vijaykumar, V., & Raveesha, K. (2011). Plant extract effect on seed-borne pathogenic fungi from seeds of paddy grown in Southern India. Journal of Plant Protection Research, 51(2), 136-143. https://doi.org/10.2478/v10045-011-0018-8
Mohankumar, M. M., Vijayasamundeeswari, A. V., Karthikeyan, M. K., Mathiyazhagan, S. M., Paranidharan, V. P., & Velazhahan, R. V. (2010). Analysis of molecular variability among isolates of Aspergillus flavus by PCR-RFLP of the ITS regions of rDNA. Journal of Plant Protection Research, 50(4), 425-431. https://doi.org/10.2478/v10045-010-0075-4
Nazhand, A., Durazzo, A., Lucarini, M., Souto, E. B., & Santini, A. (2020). Characteristics, occurrence, detection and detoxification of aflatoxins in foods and feeds. Foods, 9(5), Article 644. https://doi.org/10.3390/foods9050644
Saadallah, H. A., Al-Tarjuman, J. K., & Sultan, F. I. (2023). Isolation and identification of phenolic compounds from Eugenia caryophyllus and study on its biological effect against Macrophomina phaseolina. Journal of Applied & Natural Science, 15(4), 1432-1440. https://doi.org/10.31018/jans.v15i4.4880
Salih, R. F., Osman, G. A., & Aziz, L. H. (2019). Growth and yield response of flax (Linum usitatissimum L.) to different rates of charcoal and potassium fertilizer in Erbil, Kurdistan region—Iraq. Journal of Duhok University, 22(2), 71-80. https://doi.org/10.26682/AJUOD.2019.22.2.6
Shahdadnejad, N., Mohammadabadi, M. R., & Shamsadini, M. (2016). Typing of Clostridium perfringens isolated from broiler chickens using multiplex PCR. Genetics in the 3rd Millennium, 14(4), 4368-4374.
Zhang, W., He, H., Zang, M., Wu, Q., Zhao, H., Lu, L. L., & Jiao, Y. (2017). Genetic features of aflatoxin-associated hepatocellular carcinoma. Gastroenterology, 153(1), 249-262.e2. https://doi.org/10.1053/j.gastro.2017.03.024
مجله بیوتکنولوژی کشاورزی
دوره 17، شماره 4
آبان 1404
صفحه 347-362

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