Bulletin "Veterinary biotechnology"

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Veterynarna biotekhnolohiia– Veterinary biotechnology, 2023, 43, 44-52 [in Ukrainian]. https://doi.org/10.31073/vet_biotech43-05

ZHAKHAROVA O., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., YANGOL Y., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., TARASOV O., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine of the NAAS

 

DEVELOPMENT OF A PCR TEST KIT FOR THE IDENTIFICATION OF MICROSCOPIC FUNGI OF THE GENUS FUSARIUM IN CORN GRAIN

Introduction. The mainly used methods for detecting of microscopic fungi are mycological, which are aimed at cultivating the pathogen on nutrient media and typing by studying morphological (microscopy) and fermentative properties. Another important method is molecular biological diagnostics, namely the use of polymerase chain reaction (PCR), which allows to identify individual species or genera of the pathogen, including the presence of toxin-producing genes.

Establishing the species composition of the predominant species is an important approach to preventing contamination of grain products and to assess the risks of mycotoxin accumulation in grain, since certain species produce only certain types of mycotoxins.

The use of new approaches to the species identification of microscopic fungi – contaminants of grain of the genus Fusarium, is an important measure to prevent grain contamination with mycotoxins during storage and is an important measure to preserve animal and human health.

The goal of the work was to develop the polymerase chain reaction (PCR) test kit for genus and species identification of Fusarium fungi and to study the species composition of corn grain samples from some regions of Ukraine.

Materials and methods. 57 isolates of fungi of the genus Fuzarium, isolated from maize grain samples from the northern regions of Ukraine (Kyiv, Chernihiv, Zhytomyr) were used for the study. Samples were obtained for research from farms where signs of chronic animal toxicosis were observed or grain damage by microscopic fungi was visually recorded.The isolation of microscopic fungi and confirmation of the species was carried out with conventional microbiological methods using with the use of Chapek and Sabouraud nutrient media, which were prepared according to the manufacturer's recommendations (HiMedia, India).

DNA extraction from mycelial culture samples was perormed using PureLink™ Microbiome DNA Purification Kit, A29790 (Invitrogen, USA) according to the manufacturer’s instructions.

Primer sets detecting individual Fusarium species were used from the data on recommended sequences published by Wolny-Koładka et al. 2015, and genus-specific primers were calculated by the authors. The primers were synthesized for us by local vendor, Ukraine.

The data obtained were analyzed using descriptive statistical methods.

Results of research and discussion. The Fusarium genus-specific primer pair (FU_gen_f and FU_gen_r), which we calculated based on the TEF gene sequences of different Fusarium species, was specific exclusively for the genus Fusarium in both in silico and in vitro tests using the fungal strains we isolated. As a result of the study, it was found that the use of genus-specific primers allows the successful detection of all the target species studied, which are the most common contaminants of maize grain, while in the study of 57 species-unspecific isolates that were assigned to Fusarium on the basis of morphological properties, only 55 were identified as representatives of the genus Fusarium by PCR and two isolates were stated as non-fusarium species of microscopic fungi similar in morphological properties.

The identity of all isolates was also confirmed using a pair of primers to determine the genus Fusarium. 55 isolates studied were classified by species: F. oxysporum (9%), F. proliferatum (13%), F. sporotrichioides (33%), F. graminearum (25%), F. verticillioides (9%) and F. culmorum (11%).

This approach allowed us to identify the predominant Fusarium species associated with the contamination of maize grain from farms in the northern region of Ukraine. Two isolates tentatively assigned to the genus Fusarium were not identified at the species and genus level.

Conclusions and prospects for further research:

1. The calculated genus-specific primer pair (FU_gen_f and FU_gen_r) based on the TEF gene sequences of different Fusarium species was specific exclusively for the genus Fusarium in both in silico and in vitro tests using isolated fungal isolates.

2. As a result of the study, it was found that the use of genus-specific primers allows the successful detection of all target species that are the most common contaminants of corn grain. The identity of all isolates was also confirmed as the genus Fusarium, and the species distribution consisted of the predominant species: F. oxysporum (9%), F. proliferatum (13%), F. sporotrichioides (33%), F. graminearum (25%), F. verticillioides (9%) and F. culmorum (11%).

As a next step, new approaches will be developed to detect toxin-producing species of microscopic fungi in grain and grain products.

Keywords: Fusarium, microscopic fungi, toxigenecity, corn feed, contamination.

REFERENCES

  1. Dvorskaya, U. (2016). Mycotoksini v kormah vliianie na givotnih [Mycotoxins in feed: impact on animals]. Efectivny kormy ta godivlia – Effective feeds and feeding, 2, 34-38 [in Russian].
  2. Trufanov, O.V. (2011). Monitoring zagriaznennosti mycotoksinamy zerna i kormov v Ukraine v 2005-2010 godah [Monitoring of cereals and feed with mycotoxines contamination in Ukraine in 2005-2010]. Suchasni problemy toksikologii – Contemporary problems of toxicology,
  3. 1-2, 55-59 [in Ukrainian].
  4. Ji, F., He, D., Olaniran, A.O., et al. (2019). Occurrence, toxicity, production and detection of Fusarium mycotoxin: a review. Food Prod Process and Nutr., 1, 6. https://doi.org/10.1186/s43014-019-0007-2.
  5. Kępińska-Pacelik, J., & Biel, W. (2021). Alimentary Risk of Mycotoxins for Humans and Animals. Toxins, 13(11), 822. https://doi.org/10.3390/toxins13110822.
  6. Anfossi, L., Giovannoli, C., & Baggiani, C. (2016). Mycotoxin detection. Current Opinion in Biotechnology, 37, 120-126. https://doi.org/10.1016/j.copbio.2015.11.005.
  7. Nan, M., Xue, H., & Bi, Y. (2022). Contamination, Detection and Control of Mycotoxins in Fruits and Vegetables. Toxins, 14(5), 309. https://doi.org/10.3390/toxins14050309.
  8. Locatelli, S., Scarpino, V., Lanzanova, C., Romano, E., & Reyneri, A. (2022). Multi-Mycotoxin Long-Term Monitoring Survey on North-Italian Maize over an 11-Year Period (2011-2021): The Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Fungal Metabolites. Toxins, 14(8), 520. https://doi.org/10.3390/toxins14080520.
  9. Yu, J., & Pedroso, I.R. (2023). Mycotoxins in Cereal-Based Products and Their Impacts on the Health of Humans, Livestock Animals and Pets. Toxins, 15(8), 480. https://doi.org/10.3390/toxins15080480.
  10. Penagos-Tabares, F., Khiaosa-Ard, R., Schmidt, M., Pacífico, C., Faas, J., Jenkins, T., Nagl, V., Sulyok, M., Labuda, R., & Zebeli, Q. (2022). Fungal species and mycotoxins in mouldy spots of grass and maize silages in Austria. Mycotoxin research, 38(2), 117-136. https://doi.org/10.1007/s12550-022-00453-3.
  11. Zhang, D., Zhao, L., Chen, Y., Gao, H., Hua, Y., Yuan, X., & Yang, H. (2022). Mycotoxins in Maize Silage from China in 2019. Toxins, 14(4), 241. https://doi.org/10.3390/toxins140402419.
  12. Chauhan, R., Singh, J., Sachdev, T., Basu, T., & Malhotra, B. D. (2016). Recent advances in mycotoxins detection. Biosensors & Bioelectronics, 81, 532-545. https://doi.org/10.1016/j.bios.2016.03.004.
  13. Awuchi, C.G., Ondari, E.N., Nwozo, S., Odongo, G.A., Eseoghene, I.J., Twinomuhwezi, H., Ogbonna, C.U., Upadhyay, A.K., Adeleye, A.O., & Okpala, C.O.R. (2022). Mycotoxins’ Toxicological Mechanisms Involving Humans, Livestock and Their Associated Health Concerns: A Review. Toxins (Basel), 14(3), 167. doi: 10.3390/toxins14030167.
  14. Arif, M., Chawla, S., Zaidi, M.W., Rayar, J.K., Variar, M., Singh, U.S. (2012). Development of specific primers for genus Fusarium and F. solani using rDNA sub-unit and transcription elongation factor (TEF-1α) gene. African Journal of Biotechnology, 11, 444-447. https://doi.org/10.5897/AJB10.489.
  15. Bayraktar, H., and Dolar, F.S. (2011). Molecular identification and genetic diversity of Fusarium species associated with onion fields in Turkey. Journal of Phytopathology, 159, 28-34.
  16. Geiser, D.M., Aoki, T., Bacon, C.W., Baker, S.E., Bhattacharyya, M.K., Brandt, M.E., Brown, D.W. et al (2013). One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology, 103(5), 400-408.
  17. Aoki, T., O’Donnell, K., & Geiser, D.M. (2014). Systematics of key phytopathogenic Fusarium species: current status and future challenges. J Gen Plant Pathol., 80(3), 189-201.
  18. Woloshuk, C.P., & Shim, W.B. (2013). Aflatoxins, fumonisins, and trichothecenes: a convergence of knowledge. FEMS Microbiol Rev., 37(1), 94-109.
  19. Schatzmayr, G., & Streit, E. (2013). Global occurrence of mycotoxins in the food and feed chain: facts and figures. World Mycotoxin J., 6(3), 213-222.
  20. Lattanzio, V.M.T., von Holst, C., Visconti, A. (2013). Experimental design for in-house validation of a screening immunoassay kit. The case of a multiplex dipstick for Fusarium mycotoxins in cereals. Anal Bioanal Chem., 405(24), 7773-7782.
  21. Wolny-Koładka, K., Lenart-Boroń, A., & Boroń, P. (2015). Species composition and molecular assessment of the toxigenic potential in the population of Fusarium spp. isolated from ears of winter wheat in southern Poland. Journal of Applied Botany and Food Quality, 88, 139-144. https://doi.org/10.5073/JABFQ.2015.088.020.

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