Sains Malaysiana 51(1)(2022): 1-13

http://doi.org/10.17576/jsm-2022-5101-01

Molecular Identification and Ochratoxigenic Potential of Black Aspergillus from Various Substrates and Indoor Environment

(Pengenalpastian Molekul dan Potensi Okratoksigen Aspergillus Hitam daripada Pelbagai Substrat dan Persekitaran Dalaman)

WARDAH ABDUL RAHMAN & LATIFAH ZAKARIA*

School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

Received: 28 July 2020/Accepted: 17 May 2021

ABSTRACT

Aspergillus section Nigri or black Aspergillus is characterized by dark brown to black colonies. Several species of this group showcase the ability to produce ochratoxin A (OTA), contaminating various types of food and feed. This study was conducted to identify black Aspergillus isolated from rice, groundnuts, spices, corn grains, soil, and indoor environment and determine the ability of the isolates to produce OTA. Based on ITS, β-tubulin and calmodulin sequences, 177 isolates of black Aspergillus from various substrates and indoor environment were identified as A. niger, A. aculeatus, and A. tubingensis. By using two sets of primers to amplify two ochratoxin biosynthesis genes (PKS15KS and PKS15C-MeT genes), the OTA genes were detected in only six A. niger isolates from rice and indoor environment. These six A. niger isolates produced 8 to 308 µg/g of OTA as quantified using UPLC analysis. No OTA gene was detected in any of the A. tubingensis and A. aculeatus isolates. In conclusion, three black Aspergillus species, A. niger, A. aculeatus, and A. tubingensis, were identified from rice, groundnuts, spices, corn grains, soil, and indoor environment. Only six isolates of A. niger produced OTA indicating most of the A. niger isolates were non-OTA producers. These results could thus portray the occurrence of OTA in the field. Since both the number of isolates producing OTA and the levels of OTA production were low, it could be possibly assumed that the occurrence as well as the levels of OTA in the field are also low.

Keywords: Aspergillus aculeatus; A. niger; A. tubingensis; black Aspergillus; ochratoxin A

ABSTRAK

Aspergillus seksyen Nigri atau Aspergillus hitam dicirikan oleh koloni perang gelap ke hitam. Beberapa spesies kumpulan ini mempamerkan keupayaan menghasilkan okratoxin A (OTA) yang dapat dikesan dalam pelbagai jenis makanan dan makanan ternakan. Pengenalpastian ke tahap spesies Aspergillus hitam agak sukar kerana persamaan ciri makroskopik dan ciri mikroskopik. Dalam kajian ini, Aspergillus hitam dipencilkan daripada bahan makanan (beras, kacang tanah rempah dan bijirin jagung,), tanah dan persekitaran dalaman. Sebanyak 177 pencilan ini dikenal pasti sebagai A. niger, A. aculeatus dan A. tubingensis berdasarkan jujukan kawasan transkripsi penjarak dalaman (ITS), β-tubulin dan kalmodulin. Gen OTA hanya dikesan dalam enam pencilan A. niger yang diperoleh daripada beras dan persekitaran dalaman. Pencilan A. niger ini menghasilkan 8 hingga 308 µg/g OTA yang dikuantifikasi menggunakan analisis UPLC. Tiada gen OTA yang dikesan dalam pencilan A. tubingensis dan A. aculeatus. Kesimpulannya, tiga spesies Aspergillus hitam, A. niger, A. aculeatus dan A. tubingensis dikenal pasti daripada beras, kacang tanah, rempah, bijirin jagung, tanah dan persekitaran dalaman. Hanya 4.2% daripada pencilan A. niger menghasilkan OTA dan ini menunjukkan kebanyakan pencilan A. niger bukan pengeluar OTA. Keputusan kajian dapat menggambarkan kejadian OTA di lapangan. Oleh kerana bilangan pencilan yang menghasilkan OTA dan tahap penghasilan OTA adalah rendah, kemungkinan kejadian dan tahap OTA di lapangan juga adalah rendah.

Kata kunci: Aspergillus aculeatus; Aspergillus hitam; A. niger; A. tubingensis; okratoksin A.

REFERENCES

Abbott, S.P. 2002. Mycotoxins and indoor molds. Indoor Environment Connections 3(4): 14-24.

Abdullah, S.K. & Muhammed, A.A.H. 2011. Prevalence of black aspergilli in soil at vineyards in Duhok, Kurdistan Region of Iraq. The 4th International Scientific Conference of Salahaddin University-Erbil, Iraq. pp. 966-969.

Accensi, F., Abarca, M.L. & Cabañes, F.J. 2004. Occurrence of Aspergillus species in mixed feeds and component raw materials and their ability to produce ochratoxin A. Food Microbiology 21(5): 623-627.

Ali, N., Hashim, N.H. & Shuib, N.S. 2015. Natural occurrence of aflatoxins and ochratoxin A in processed spices marketed in Malaysia. Food Additives & Contaminants: Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment 32(4): 518-532.

Almeida, M.I., Almeida, N.G., Carvalho, K.L., Gonçalves, G.A.A., Silva, C.N., Santos, E.A. & Vargas, E.A. 2012. Ochratoxin A, zearalenone, deoxynivalenol, and citreoviridin in rice in Brazil. Food Additives & Contaminants: Part A 29(4): 694-703.

Belli, N., Pardo, E., Mar, S., Farr, G., Ramos, A.J. & Sanchis, V. 2004. Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes. Journal of the Science of Food and Agriculture 84(6): 541-546.

Benford, D., Boyle, C., Dekant, W., Fuchs, E., Gaylor, D.W., Hard, G. & Walker, R. 2001. Ochratoxin A Safety Evaluation of Certain Mycotoxins in Food. WHO Food Additives Series 47. FAO Food and Nutrition Paper 74, Geneva: World Health Organization. pp. 281-415.

Beuchat, L.R. 1979. Survival of conidia of Aspergillus flavus in dried foods. Journal of Stored Products Research 15(1): 25-31.

Bisbal, F., Vicente, J., Daniel, G. & Martínez-culebras, P.V. 2009. ITS-RFLP characterization of black Aspergillus isolates responsible for ochratoxin A contamination in cocoa beans. European Food Research and Technology 229: 751-755.

Bragulat, M.R., Abarca, M.L. & Cabanes, F.J. 2001. An easy screening method for fungi producing ochratoxin A in pure culture. International Journal of Food Microbiology 71(2-3): 139-144.

Dalcero, A., Magnoli, C., Hallak, C., Chiacchiera, S.M., Palacio, G. & Rosa, C.A. 2002. Detection of ochratoxin A in animal feeds and capacity to produce this mycotoxin by Aspergillus section Nigri in Argentina. Food Additives and Contaminants 19(11): 1065-1072.

Di-Paolo, N., Guarnieri, A. & Garosi, G. 1994. Inhaled mycotoxins lead to acute renal failure. Nephrology Dialysis Transplantation 9(Suppl. 4): 116-120.

Esteban, A., Abarca, M.L., Bragulat, M.R. & Cabañes, F.J. 2004. Effects of temperature and incubation time on production of ochratoxin A by black aspergilli. Research in Microbiology 155(10): 861-866.

Ferracin, L., Beatriz, C., Lucia, M., Vieira, C., Monteiro-Vitorello, C.B., Mello, A. & Fungaro, P.D. 2012. Specific polyketide synthase genes of Aspergillus niger. International Journal of Food Microbiology 155(3): 137-145.

Fredj, S.M.B., Chebil, S. & Mliki, A. 2009. Isolation and characterization of ochratoxin A and aflatoxin B1 producing fungi infecting grapevines cultivated in Tunisia. African Journal of Microbiology Research 3(9): 523-527.

Frisvad, J.C., Larsen, T.O., De Vries, R., Meijer, M., Houbraken, J., Cabañes, F.J. & Samson, R.A. 2007. Secondary metabolite profiling, growth profiles and other tools for species recognition and important Aspergillus mycotoxins. Studies in Mycology 59: 31-37.

Gallo, A., Bruno, K.S., Solfrizzo, M., Perrone, G, Mulè, G., Visconti, A. & Baker, S.E. 2012. New insight into the ochratoxin A biosynthetic pathway through deletion of a nonribosomal peptide synthetase gene in Aspergillus carbonarius. Applied and Environmental Microbiology 78(23): 8208-8218.

Garcia-Cela, E., Crespo-Sempere, A., Ramos, A.J., Sanchis, V. & Marin, S. 2014. Ecophysiological characterization of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger isolated from grapes in Spanish vineyards. International Journal of Food Microbiology 173: 89-98.

Ghosh, D., Dhar, P., Chakraborty, T., Uddin, N., Das, A.K., North, P.G.S. & Bengal, W. 2014. Study of aeromycoflora in indoor and outdoor environment of national library, Kolkata. International Journal of Plant, Animal and Environmental Sciences 4: 663-672.

Glass, N.L. & Donaldson, G.C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Applied and Environmental Microbiology 61(4): 1323-1330.

Hayer, K., Stratford, M. & Archer, D.B. 2014. Germination of Aspergillus niger conidia is triggered by nitrogen compounds related to L-amino acids. Applied and Environmental Microbiology 80(19): 6046-6053.

Hong, S., Lee, M., Kim, D., Varga, J., Frisvad, J.C., Perrone, G. & Samson, R.A. 2013. Aspergillus luchuensis, an industrially important black Aspergillus in East Asia. PLoS ONE 8: e63769.

Hong, S., Cho, H., Shin, H., Frisvad, J.C. & Samson, R.A. 2006. Novel Neosartorya species isolated from soil in Korea. International Journal of Systematic and Evolutionary Microbiology 56(2): 477-486.

Jalili, S., Jinap, S. & Radu, S. 2010. Natural occurrence of ochratoxin A contamination in commercial black and white pepper products. Mycopathologia 170(4): 251-258.

Kim, D.H., Kim, S.H., Kwon, S.W., Lee, J.K. & Hong, S.B. 2013. Fungal diversity of rice straw for meju fermentation. Journal of Microbiology and Biotechnology 23(12): 1654-1663.

Kim, N.Y., Lee, I. & Ji, G.E. 2014. Reliable and simple detection of ochratoxin and fumonisin production in black Aspergillus. Journal of Food Protection 77(4): 653-658.

Kittikamhaeng, N. & Dachoupakan, C. 2011. Contamination of aflatoxigenic and ochratoxigenic Aspergillus in Thai rice. Proceeding of the 23rd Annual Meeting of the Thai Society for Biotechnology “Systems Biotechnology: Quality & Success”. pp. 144-145.

Lamboni, Y., Nielsen, K.F., Linnemann, A.R., Gezgin, Y.K., Hell, K., Nout, M.J.R. & Frisvad, J.C. 2016. Diversity in secondary metabolites including mycotoxins from strains of aspergillus section Nigri isolated from raw cashew nuts from Benin, West Africa. PLoS ONE 11(10): e0164310.

Lasram, S., Oueslati, S., Jouira, H.B., Chebil, S., Mliki, A. & Ghorbel, A. 2013. Identification of ochratoxigenic Aspergillus section Nigri isolated from grapes by ITS-5.8S rDNA sequencing analysis and in silico RFLP. Journal of Phytopathology 161(4): 280-283.

Leong, S.L., Hocking, A.D. & Scott, E.S. 2007. Aspergillus species producing ochratoxin A: Isolation from vineyard soils and infection of Semillon bunches in Australia. Journal of Applied Microbiology 102(1): 124-133.

Lopez-Mendoza, M.C., Crespo-Sempere, A. & Martinez-Culebras, P.V. 2009. Identification of Aspergillus tubingensis strains responsible for OTA contamination in grapes and wine based on the acyl transferase domain of a polyketide synthase gene. International Journal of Food Science & Technology 44(11): 2147-2152.

Magan, N. & Aldred, D. 2005. Conditions of formation of ochratoxin A in drying, transport and in different commodities. Food Additives & Contaminants 22(Suppl. 1): 10-16.

Massi, F.P., Sartori, D., Ferranti, L.S., Iamanaka, B.T., Taniwaki, M.H., Vieira, M.L.C. & Fungaro, M.H.P. 2016. Data on the presence or absence of genes encoding essential proteins for ochratoxin and fumonisin biosynthesis in Aspergillus niger and Aspergillus welwitschiae. Data in Brief 7: 704-708.

Medina, A., Mateo, R., López-ocaña, L., Valle-algarra, M. & Jiménez, M. 2005. Study of spanish grape mycobiota and ochratoxin a production by isolates of Aspergillus tubingensis and other members of Aspergillus Section Nigri. Applied and Environmental Microbiology 71(8): 4696-4702.

Nielsen, K.F., Mogensen, J.M., Johansen, M., Larsen, T.O. & Frisvad, J.C. 2009. Review of secondary metabolites and mycotoxins from the Aspergillus niger group. Analytical and Bioanalytical Chemistry 395(5): 1225-1242.

Nugroho, A.D., Setyabudi, F.M.C., Salleh, B. & Rahayu, E.S. 2013. Ochratoxigenic black aspergilli isolated from dried agricultural products in Yogyakarta, Indonesia. Journal of Food Science and Engineering 3: 472-480.

Nyongesa, B.W., Okoth, S. & Ayugi, V. 2015. Identification key for Aspergillus species isolated from maize and soil of Nandi Country, Kenya. Advances in Microbiology 5(4): 205-229.

Palumbo, J.D., O’Keeffe, T.L. & McGarvey, J.A. 2011. Incidence of fumonisin B2 production within Aspergillus section Nigri populations isolated from California raisins. Journal of Food Protection 74(4): 672-675.

Parenicová, L., Skouboe, P., Frisvad, J., Samson, R.A., Rossen, L., ten Hoor-Suykerbuyk, M. & Visser, J. 2001. Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species. Applied and Environmental Microbiology 67(2): 521-527.

Perrone, G., Mulè, G., Susca, A., Battilani, P., Pietri, A. & Logrieco, A. 2006. Ochratoxin A production and amplified fragment length polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger strains isolated from grapes in Italy. Applied and Environmental Microbiology 72(1): 680-685.

Pettersson, O.V. & Leong, S.L. 2011. Fungal xerophiles (osmophiles). eLS. Chichester: John Wiley & Sons Ltd.

Pitt, J.I. & Hocking, A.D. 2009. Fungi and Food Spoilage. New York: Springer.

Priyanka, S.R., Venkataramana, M., Kumar, G.P., Rao, V.K., Murali, H.C.S. & Batra, H.V. 2014. Occurrence and molecular detection of toxigenic Aspergillus species in food grain samples from India. Journal of the Science of Food and Agriculture 94(3): 537-543.

Rahmani, A., Jinap, S. & Soleimany, F. 2010. Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin A and zearalenone in cereals using HPLC-FLD. Food Additives & Contaminants. Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment 27(12): 1683-1693.

Richard, J.L., Plattner, R.D., May, J. & Liska, S.L. 1999. The occurrence of ochratoxin A in dust collected from a problem household. Mycopathologia 146(2): 99-103.

Saadabi, A.M. 2011. Prevalence and assessment of air-borne fungi at vegetable central market of Khartoum State, Sudan. Journal of Applied Sciences Research 7(5): 550-553.

Saadullah, A.A.M. 2013. Identification and determination of aflatoxin G1 and aflatoxigenic Aspergillus isolates from dried vine in Duhok by LC/MS-MS technique. Journal of Basrah Researches (Sciences) 39(1): 66-74.

Samson, R.A., Noonim, P., Meijer, M., Houbraken, J., Frisvad, J.C. & Varga, J. 2007. Diagnostic tools to identify black Aspergilli. Studies in Mycology 59: 129-145.

Samson, R.A., Visagie, C.M., Houbraken, J., Hong, S.B., Hubka, V., Klaassen, C.H.W. & Frisvad, J.C. 2014. Phylogeny, identification and nomenclature of the genus Aspergillus. Studies in Mycology 78: 141-173.

Samsudin, N.I.P. & Abdullah, N. 2013. A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia. Mycotoxin Research 29(2): 89-96.

Sánchez-Hervás, M., Gil, J.V., Bisbal, F., Ramón, D. & Martínez-Culebras, P.V. 2008. Mycobiota and mycotoxin producing fungi from cocoa beans. International Journal of Food Microbiology 125(3): 336-340.

Schatzmayr, G. & Streit, E. 2013. Global occurrence of mycotoxins in the food and feed chain: Facts and figures. World Mycotoxin Journal 6(3): 213-222.

Serra, R., Caban, F.J., Perrone, G. & Castella, G. 2006. Aspergillus ibericus: A new species of section Nigri isolated from grapes. Mycologia 98(2): 295-306.

Shehu, K. & Muhammad, S. 2012. Fungi associated with storage rots of onion bulbs in Sokoto, Nigeria. International Journal of Modern Botany 1(1): 1-3.

Soleimany, F., Jinap, S. & Abas, F. 2012. Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry. Food Chemistry 130(4): 1055-1060.

Spadaro, D., Patharajan, S., Lorè, A., Garibaldi, A. & Gullino, M.L. 2012. Ochratoxigenic black species of aspergilli in grape fruits of northern Italy identified by an improved PCR-RFLP procedure. Toxins 4(2): 42-54.

Storari, M., Bigler, L., Gessler, C. & Broggini, G.A.L. 2012. Assessment of the ochratoxin A production ability of Aspergillus tubingensis. Food Additives & Contaminants. Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment 29(9): 1450-1454.

Susca, A., Proctor, R.H., Morelli, M., Haidukowski, M., Gallo, A., Logrieco, A.F. & Moretti, A. 2016. Variation in fumonisin and ochratoxin production associated with differences in biosynthetic gene content in Aspergillus niger and A. welwitschiae isolates from multiple crop and geographic origins. Frontiers in Microbiology 7(1412): 1-15.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28(10): 2731-2739.

Teh, L.Y. & Latiffah, Z. 2015. Occurrence and molecular characterization of Aspergillus species in beach sand. Malaysian Applied Biology 44(2): 119-127.

Varga, J., Kocsube, S., Szigeti, G., Baranyi, N., Vagvlogyi, C., Despot, D.J. & Klaric, M.S. 2014. Occurrence of black Aspergilli in indoor environments of six countries. Archives of Industrial Hygiene and Toxicology 65: 219-223.

Varga, J., Frisvad, J.C., Kocsubé, S., Brankovics, B., Tóth, B., Szigeti, G. & Samson, R.A. 2011. New and revisited species in Aspergillus section Nigri. Studies in Mycology 69(1): 1-17.

Vitale, A., Cirvilleri, G., Panebianco, A., Epifani, F., Perrone, G. & Polizzi, G. 2012. Molecular characterisation and pathogenicity of Aspergillus section Nigri causing Aspergillus vine canker of table grapes in Italy. European Journal of Plant Pathology 132: 483-487.

Wardah, A.R., Hafizi, R., Baharuddin, S.N. & Salleh, B. 2012. Incidence and remediation of fungi in a sick building in Malaysia: A case study. Aerobiologia 28: 275-283.

White, T.J., Bruns, T. & Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications, edited by Innis, M.A., Gelfand, D.H., Sninsky, J.J. & White, T.J. New York: Academic Press. pp. 315-322.

Zarei, F., Mirhendi, H., Motamedi, M., Ahmadi, B., Nouripour-Sisakht, S., Zarrinfar, H. & Hashemi, J. 2016. Black Aspergillus species isolated from clinical and environmental samples in Iran. Journal of Medical Microbiology 64(11): 1454-1456.

Zheng, M.Z., Richard, J.L. & Binder, J. 2006. A review of rapid methods for the analysis of mycotoxins. Mycopathologia 161: 261-273.

*Corresponding author; email: Lfah@usm.my