Identification of Aspergillus westerdijkiae and its potential risk of Ochratoxin A synthesis in Cannabis inflorescences

Emily Aguirre-Ortega; Mónica J. Henao-López; Jolián A.  Vargas-Alzate; Katherin Castro-Ríos; Claudia N.  Montoya-Estrada

doi:10.17268/sci.agropecu.2024.004

Autores

Emily Aguirre-Ortega Universidad de Caldas, Manizales, Colombia. https://orcid.org/0000-0003-1446-209X
Mónica J. Henao-López Universidad Católica de Manizales, Manizales, Colombia. https://orcid.org/0009-0000-0708-8234
Jolián A. Vargas-Alzate Cubikan Group, Colombia. https://orcid.org/0009-0000-7657-4092
Katherin Castro-Ríos Universidad Católica de Manizales, Manizales, Colombia. https://orcid.org/0000-0003-2520-1696
Claudia N. Montoya-Estrada Universidad Católica de Manizales, Manizales, Colombia. https://orcid.org/0000-0003-3802-1125

DOI:

https://doi.org/10.17268/sci.agropecu.2024.004

Palavras-chave:

biosynthesis, risk analysis, Cannabis sativa, fungal contamination, genes, mycotoxins

Resumo

Nowadays, fungal contamination of medical Cannabis inflorescences during postharvest has become an increasingly frequent and worrisome problem for consumers and the industry in general. This is because some of these microorganisms can produce secondary metabolites, such as mycotoxins, which can be toxic to humans. To assess the risk posed by fungal contamination and evaluate the potential for fungal isolates to produce mycotoxins, samples of medicinal Cannabis were tested for the presence of mycotoxin-forming fungi. Inflorescences were isolated on PDA agar at 23 ± 2 °C for ten days, and the microorganisms were identified. The strain with morphological characteristics compatible with the genus Aspergillus spp. was selected as the fungus with the highest risk of forming hazardous mycotoxins. This isolate was characterized conventionally and by molecular identification using primers for the internal transcribed spacer region (ITS) of ribosomal DNA and different coding genes and was identified as Aspergillus westerdijkiae. To determine mycotoxin formation, the genome of A. westerdijkiae was sequenced using the Illumina Novaseq platform in South Korea. The antiSMASH tool was used to search for gene clusters associated with producing secondary metabolites, and genes related to toxins were manually curated. Regions where the cluster of genes directly involved in OTA biosynthesis (otaA, otaB, otaC, otaR and otaD) were found, suggesting a potential risk of synthesis of this toxin.

Referências

Amaradasa, B. S., & Amundsen, K. (2014). First Report of Curvularia inaequalis and Bipolaris spicifera Causing Leaf Blight of Buffalograss in Nebraska. Plant Disease, 98(2), 279-279. https://doi.org/10.1094/PDIS-05-13-0487-PDN

Amézqueta, S., Schorr-Galindo, S., Murillo-Arbizu, M., González-Peñas, E., López de Cerain, A., & Guiraud, J. P. (2012). OTA-producing fungi in foodstuffs: A review. Food Control, 26(2), 259-268. https://doi.org/10.1016/j.foodcont.2012.01.042

Ámon, J., Keisham, K., Bokor, E., Kelemen, E., Vágvölgyi, C., & Hamari, Z. (2018). Sterigmatocystin production is restricted to hyphae located in the proximity of hülle cells. Journal of Basic Microbiology, 58(7), 590-596. https://doi.org/10.1002/jobm.201800020

Ángeles López, G. E., Brindis, F., Cristians Niizawa, S., & Ventura Martínez, R. (2014). Cannabis sativa L., una planta singular. Revista mexicana de ciencias farmacéuticas, 45(4), 1-6.

Barnett, H. L., & Hunter, B. B. (1998). Illustrated Genera of Imperfect Fungi (Fourth Edition).

Blin, K., Shaw, S., Kloosterman, A. M., Charlop-Powers, Z., van Wezel, G. P., Medema, M. H., & Weber, T. (2021). antiSMASH 6.0: Improving cluster detection and comparison capabilities. Nucleic Acids Research, 49(W1), W29-W35. https://doi.org/10.1093/nar/gkab335

Boggs, D. L., Nguyen, J. D., Morgenson, D., Taffe, M. A., & Ranganathan, M. (2018). Clinical and Preclinical Evidence for Functional Interactions of Cannabidiol and Δ9-Tetrahydrocannabinol. Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology, 43(1), 142-154. https://doi.org/10.1038/npp.2017.209

Buchicchio, L., Asselborn, L., Schneider, S., van Nieuwenhuyse, A., Moris, G., & Schummer, C. (2022). Investigation of aflatoxin and ochratoxin A contamination of seized cannabis and cannabis resin samples. Mycotoxin Research, 38(1), 71-78. https://doi.org/10.1007/s12550-022-00449-z

Carver, T., Harris, S. R., Berriman, M., Parkhill, J., & McQuillan, J. A. (2012). Artemis: An integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics, 28(4), 464-469. https://doi.org/10.1093/bioinformatics/btr703

Castaño, J. (2015). Principios básicos de hongos fitopatógenos (1.a ed.). Universidad de Caldas.

Castaño Zapata, J., & Salazar Pineda, H. (1998). Ilustrated Guide for Identification of Plant Pathogens. Universidad de Caldas.

Chakrabortti, A., Li, J., & Liang, Z.-X. (2016). Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A. Genome Announcements, 4(5), e00982-16. https://doi.org/10.1128/genomeA.00982-16

Chakraborty, P. (2022). Gene cluster from plant to microbes: Their role in genome architecture, organism’s development, specialized metabolism and drug discovery. Biochimie, 193, 1-15. https://doi.org/10.1016/j.biochi.2021.12.001

Copetti, M. V., Pereira, J. L., Iamanaka, B. T., Pitt, J. I., & Taniwaki, M. H. (2010). Ochratoxigenic fungi and ochratoxin A in cocoa during farm processing. International Journal of Food Microbiology, 143(1), 67-70. https://doi.org/10.1016/j.ijfoodmicro.2010.07.031

Coton, M., & Dantigny, P. (2019). Mycotoxin migration in moldy foods. Current Opinion in Food Science, 29, 88-93. https://doi.org/10.1016/j.cofs.2019.08.007

Departamento Administrativo de la Función Pública. (2016). Ley 1787 de 2016. https://www.funcionpublica.gov.co/eva/gestornormativo/norma.php?i=80394

Ding, L., Han, M., Wang, X., & Guo, Y. (2023). Ochratoxin A: Overview of Prevention, Removal, and Detoxification Methods. Toxins, 15(9), 565. https://doi.org/10.3390/toxins15090565

Dryburgh, L. M., Bolan, N. S., Grof, C. P. L., Galettis, P., Schneider, J., Lucas, C. J., & Martin, J. H. (2018). Cannabis contaminants: Sources, distribution, human toxicity and pharmacologic effects. British Journal of Clinical Pharmacology, 84(11), 2468-2476. https://doi.org/10.1111/bcp.13695

Estrada, G., & Ramirez, M. (2019). Micología General (1.a ed.). Universidad Católica de Manizales.

Fordjour, E., Manful, C. F., Sey, A. A., Javed, R., Pham, T. H., Thomas, R., & Cheema, M. (2023). Cannabis: A multifaceted plant with endless potentials. Frontiers in Pharmacology, 14. https://doi.org/10.3389/fphar.2023.1200269

Gallo, A., Ferrara, M., & Perrone, G. (2017). Recent advances on the molecular aspects of ochratoxin A biosynthesis. Current Opinion in Food Science, 17, 49-56. https://doi.org/10.1016/j.cofs.2017.09.011

Gil, J., García-Díaz, M., González-Jaén, M. T., Vázquez, C., & Patiño, B. (2018). Description of an orthologous cluster of ochratoxin A biosynthetic genes in Aspergillus and Penicillium species. A comparative analysis. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2017.12.028

Gil, J., Vázquez, C., & Patiño, B. (2020). The Genomic Regions That Contain Ochratoxin A Biosynthetic Genes Widely Differ in Aspergillus Section Circumdati Species. Toxins, 12(12), 754. https://doi.org/10.3390/toxins12120754

Gonçalves, J., Rosado, T., Soares, S., Simão, A. Y., Caramelo, D., Luís, Â., Fernández, N., Barroso, M., Gallardo, E., & Duarte, A. P. (2019). Cannabis and Its Secondary Metabolites: Their Use as Therapeutic Drugs, Toxicological Aspects, and Analytical Determination. Medicines, 6(1), Article 1. https://doi.org/10.3390/medicines6010031

Gwinn, K. D., Leung, M. C. K., Stephens, A. B., & Punja, Z. K. (2023). Fungal and mycotoxin contaminants in cannabis and hemp flowers: Implications for consumer health and directions for further research. Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1278189

Han, X., Chakrabortti, A., Zhu, J., Liang, Z.-X., & Li, J. (2016). Sequencing and functional annotation of the whole genome of the filamentous fungus Aspergillus westerdijkiae. BMC Genomics, 17(1), 633. https://doi.org/10.1186/s12864-016-2974-x

Inglis, D. O., Binkley, J., Skrzypek, M. S., Arnaud, M. B., Cerqueira, G. C., Shah, P., Wymore, F., Wortman, J. R., & Sherlock, G. (2013). Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiology, 13(1), 91. https://doi.org/10.1186/1471-2180-13-91

International Agency For Research On Cancer. (2012). Agents Classified by the IARC Monographs, Volumes 1–133 – IARC Monographs on the Identification of Carcinogenic Hazards to Humans. https://monographs.iarc.who.int/agents-classified-by-the-iarc/

Jameson, L. E., Conrow, K. D., Pinkhasova, D. V., Boulanger, H. L., Ha, H., Jourabchian, N., Johnson, S. A., Simeone, M. P., Afia, I. A., Cahill, T. M., Orser, C. S., & Leung, M. C. K. (2022). Comparison of State-Level Regulations for Cannabis Contaminants and Implications for Public Health. Environmental Health Perspectives, 130(9), 097001. https://doi.org/10.1289/EHP11206

Jerushalmi, S., Maymon, M., Dombrovsky, A., & Freeman, S. (2020). Fungal Pathogens Affecting the Production and Quality of Medical Cannabis in Israel. Plants, 9(7), Article 7. https://doi.org/10.3390/plants9070882

Liu, W.-C., Pushparaj, K., Meyyazhagan, A., Arumugam, V. A., Pappuswamy, M., Bhotla, H. K., Baskaran, R., Issara, U., Balasubramanian, B., & Mousavi Khaneghah, A. (2022). Ochratoxin A as an alarming health threat for livestock and human: A review on molecular interactions, mechanism of toxicity, detection, detoxification, and dietary prophylaxis. Toxicon, 213, 59-75. https://doi.org/10.1016/j.toxicon.2022.04.012

Malir, F., Ostry, V., Pfohl-Leszkowicz, A., Malir, J., & Toman, J. (2016). Ochratoxin A: 50 Years of Research. Toxins, 8(7), E191. https://doi.org/10.3390/toxins8070191

Maman, M., Sangchote, S., Piasai, O., Leesutthiphonchai, W., Sukorini, H., & Khewkhom, N. (2021). Storage fungi and ochratoxin A associated with arabica coffee bean in postharvest processes in Northern Thailand. Food Control, 130, 108351. https://doi.org/10.1016/j.foodcont.2021.108351

Ministerio de Salud y Protección Social. (2017). Resolución N° 02892 de 2017. https://www.funcionpublica.gov.co/eva/gestornormativo/norma.php?i=80394

Ministerio de Salud y Protección Social. (2021). Decreto N°811 de 2021. https://www.funcionpublica.gov.co/eva/gestornormativo/norma.php?i=80394

Montero-Oleas, N., Arevalo-Rodriguez, I., Nuñez-González, S., Viteri-García, A., & Simancas-Racines, D. (2020). Therapeutic use of cannabis and cannabinoids: An evidence mapping and appraisal of systematic reviews. BMC Complementary Medicine and Therapies, 20(12), 1-15. https://doi.org/10.1186/s12906-019-2803-2

Moretti, A., Logrieco, A. F., & Susca, A. (2017). Mycotoxins: An Underhand Food Problem. En A. Moretti & A. Susca (Eds.), Mycotoxigenic Fungi: Methods and Protocols (pp. 3-12). Springer New York. https://doi.org/10.1007/978-1-4939-6707-0_1

Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology and Evolution, 32(1), 268-274. https://doi.org/10.1093/molbev/msu300

Paterson, R. R. M., Lima, N., & Taniwaki, M. H. (2014). Coffee, mycotoxins and climate change. Food Research International, 61, 1-15. https://doi.org/10.1016/j.foodres.2014.03.037

Pauli, C. S., Conroy, M., Vanden Heuvel, B. D., & Park, S.-H. (2020). Cannabidiol Drugs Clinical Trial Outcomes and Adverse Effects. Frontiers in Pharmacology, 11, 63. https://doi.org/10.3389/fphar.2020.00063

Pérez-Moreno, M., Pérez-Lloret, P., González-Soriano, J., & Santos-Álvarez, I. (2019). Cannabis resin in the region of Madrid: Adulteration and contamination. Forensic Science International, 298, 34-38. https://doi.org/10.1016/j.forsciint.2019.02.049

Pintori, N., Caria, F., De Luca, M. A., & Miliano, C. (2023). THC and CBD: Villain versus Hero? Insights into Adolescent Exposure. International Journal of Molecular Sciences, 24(6), 5251. https://doi.org/10.3390/ijms24065251

Pitt, J. I., & Hocking, A. D. (2009). Fungi and Food Spoilage. En Fungi and Food Spoilage. Springer.

Prjibelski, A., Antipov, D., Meleshko, D., Lapidus, A., & Korobeynikov, A. (2020). Using SPAdes De Novo Assembler. Current Protocols in Bioinformatics, 70(1), e102. https://doi.org/10.1002/cpbi.102

Punja, Z. K. (2021a). Emerging diseases of Cannabis sativa and sustainable management. Pest Management Science, 77(9), 3857-3870. https://doi.org/10.1002/ps.6307

Punja, Z. K. (2021b). The diverse mycoflora present on dried cannabis (Cannabis sativa L., marijuana) inflorescences in commercial production. Canadian Journal of Plant Pathology, 43(1), 88-100. https://doi.org/10.1080/07060661.2020.1758959

Punja, Z. K., Collyer, D., Scott, C., Lung, S., Holmes, J., & Sutton, D. (2019). Pathogens and Molds Affecting Production and Quality of Cannabis sativa L. Frontiers in Plant Science, 10. https://doi.org/10.3389/fpls.2019.01120

Punja, Z. K., Ni, L., Lung, S., & Buirs, L. (2023). Total yeast and mold levels in high THC-containing cannabis (Cannabis sativa L.) inflorescences are influenced by genotype, environment, and pre-and post-harvest handling practices. Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1192035

Russo, E. B. (2018). Cannabis Therapeutics and the Future of Neurology. Frontiers in Integrative Neuroscience, 12(51), 1-11. https://doi.org/10.3389/fnint.2018.00051

Sartori, D., Massi, F. P., Ferranti, L. S., & Fungaro, M. H. P. (2014). Identification of Genes Differentially Expressed Between Ochratoxin Producing and Non Producing Strains of Aspergillus westerdijkiae. Indian Journal of Microbiology, 54(1), 41-45. https://doi.org/10.1007/s12088-013-0408-x

Schlösser, I., & Prange, A. (2019). Antifungal Activity of Selected Natural Preservatives against Aspergillus westerdijkiae and Penicillium verrucosum and the Interactions of These Preservatives with Food Components. Journal of Food Protection, 82(10), 1751-1760. https://doi.org/10.4315/0362-028X.JFP-19-082

Seltenrich, N. (2019). Cannabis Contaminants: Regulating Solvents, Microbes, and Metals in Legal Weed. Environmental Health Perspectives, 127(8), 082001. https://doi.org/10.1289/EHP5785

Serafimovska, T., Stefanovski, S., Erler, J., Keskovski, Z., Stefkov, G., Mitevska, M., Darkovska Serafimovska, M., Balkanov, T., & Tonic Ribarska, J. (2021). Does Cannabis Extract Obtained From Cannabis Flowers With Maximum Allowed Residual Level of Aflatoxins and Ochratoxin a Have an Impact on Human Safety and Health? Frontiers in Medicine, 8. https://doi.org/10.3389/fmed.2021.759856

Sopovski, D. S., Han, J., Stevens-Riley, M., Wang, Q., Erickson, B. D., Oktem, B., Vanlandingham, M., Taylor, C. L., & Foley, S. L. (2023). Investigation of microorganisms in cannabis after heating in a commercial vaporizer. Frontiers in Cellular and Infection Microbiology, 12, 1051272. https://doi.org/10.3389/fcimb.2022.1051272

Steenwyk, J. L., Shen, X.-X., Lind, A. L., Goldman, G. H., & Rokas, A. (2019). A Robust Phylogenomic Time Tree for Biotechnologically and Medically Important Fungi in the Genera Aspergillus and Penicillium. mBio, 10(4), e00925-19. https://doi.org/10.1128/mBio.00925-19

Susca, A., Anelli, P., Haidukowski, M., Probyn, C. E., Epifani, F., Logrieco, A. F., Moretti, A., & Proctor, R. H. (2021). A PCR method to identify ochratoxin A-producing Aspergillus westerdijkiae strains on dried and aged foods. International Journal of Food Microbiology, 344, 109113. https://doi.org/10.1016/j.ijfoodmicro.2021.109113

Villa, Y. V. M., Correa, L. A. M., & López, A. (2020). Estandarización de técnicas moleculares basadas en amplificación y análisis de restricción para la identificación de especies de Aspergillus spp. Universidad de Antioquia, 14.

Vujanovic, V., Korber, D. R., Vujanovic, S., Vujanovic, J., & Jabaji, S. (2020). Scientific Prospects for Cannabis - Microbiome Research to Ensure Quality and Safety of Products. Microorganisms, 8(2), Article 2. https://doi.org/10.3390/microorganisms8020290

Wang, G., Li, E., Gallo, A., Perrone, G., Varga, E., Ma, J., Yang, B., Tai, B., & Xing, F. (2023). Impact of environmental factors on ochratoxin A: From natural occurrence to control strategy. Environmental Pollution, 317, 120767. https://doi.org/10.1016/j.envpol.2022.120767

Wang, L., Hua, X., Shi, J., Jing, N., Ji, T., Lv, B., Liu, L., & Chen, Y. (2022). Ochratoxin A: Occurrence and recent advances in detoxification. Toxicon, 210, 11-18. https://doi.org/10.1016/j.toxicon.2022.02.010

Wang, Q., Garrity, G. M., Tiedje, J. M., & Cole, J. R. (2007). Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy. Applied and Environmental Microbiology, 73(16), 5261-5267. https://doi.org/10.1128/AEM.00062-07

Yin, G., Zhang, Y., Pennerman, K. K., Wu, G., Hua, S. S. T., Yu, J., Jurick, W. M., Guo, A., & Bennett, J. W. (2017). Characterization of Blue Mold Penicillium Species Isolated from Stored Fruits Using Multiple Highly Conserved Loci. Journal of Fungi, 3(1), Article 1. https://doi.org/10.3390/jof3010012