bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190306; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Wild rice (O. latifolia) from natural ecosystems in the Pantanal region of Brazil: host to Fusarium 2 incarnatum-equiseti species complex and highly contaminated by zearalenone. 3 4 Sabina Moser Tralamazza1*, Karim Cristina Piacentini1, Geovana Dagostim Savi2, Lorena Carnielli- 5 Queiroz1, Lívia de Carvalho Fontes1, Camila Siedlarczyk Martins3, Benedito Corrêa1, Liliana Oliveira 6 Rocha3* 7 8 Affiliation 9 1 Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, 10 Brazil. 11 2 University of Southern Santa Catarina (UNESC), Scientific and Technological Park, Santa Catarina, 12 Brazil 13 3 Department of Food Science, Food Engineering Faculty, University of Campinas, Campinas, Brazil. 14 15 *Corresponding authors 16 Liliana O. Rocha 17 [email protected] 18 Department of Food Science, Food Engineering Faculty, University of Campinas, Campinas, Brazil. 19 20 Sabina M. Tralamazza 21 [email protected] 22 Present address: Laboratory of Evolutionary Genetics, Institute of Biology, University of Neuchatel, 23 Neuchâtel, Switzerland. 24 25 26 27 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190306; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 28 Abstract 29 30 We assessed the mycobiota diversity and mycotoxin levels present in wild rice (Oryza latifolia) from 31 the Pantanal region of Brazil; fundamental aspects of which are severely understudied as an edible 32 plant from a natural ecosystem. We found a variety of fungal species contaminating the rice samples; 33 the most frequent genera being Fusarium, Nigrospora and Cladosporium (35.9%, 26.1% and 15%, 34 respectively). Within the Fusarium genus, the wild rice samples were mostly contaminated by the 35 Fusarium incarnatum-equiseti species complex (FIESC) (80%) along with Fusarium fujikuroi species 36 complex (20%). Phylogenetic analysis supported multiple FIESC species and gave strong support to 37 the presence of two previously uncharacterized lineages within the complex (LN1 and LN2). 38 Deoxynivalenol (DON) and zearalenone (ZEA) chemical analysis showed that most of the isolates 39 were DON/ZEA producers and some were defined as high ZEA producers, displaying abundant ZEA 40 levels over DON (over 19 times more). Suggesting that ZEA likely has a key adaptive role for FIESC in 41 wild rice (O. latifolia). Mycotoxin determination in the rice samples revealed high frequency of ZEA, 42 and 85% of rice samples had levels >100 µg/kg; the recommended limit set by regulatory agencies. 43 DON was only detected in 5.2% of the samples. Our data shows that FIESC species are the main 44 source of ZEA contamination in wild rice and the excessive levels of ZEA found in the rice samples 45 raises considerable safety concerns regarding wild rice consumption by humans and animals. 46 47 Keywords 48 native rice, fungi, mycotoxin, deoxynivalenol, FIESC 49 50 1 Introduction 51 52 The Pantanal region is a 140,000 km2 sedimentary floodplain in western Brazil and one of the 53 largest wetlands in the world (Pott and Silva, 2015); which experiences months-long floods every year 54 during the rainy season from October to April (Bergier and Assine, 2016). The region harbors more bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190306; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 55 than 200 wild grass species (Pott and Pott, 2000) that are commonly used for cattle grazing and are 56 also a food source for native wildlife (Pott and Pott, 2004). 57 Oryza latifolia is a tetraploid wild species of rice, with a distribution ranging from Mexico to Brazil 58 and the Caribbean Islands (Tateoka, 1962). The species is characterized as drought resistant, aquatic 59 emergent and is largely found in the Pantanal wetland of Brazil (Bertazonni and Alves Damasceno- 60 Júnior, 2011). 61 O. latifolia has been employed as a genetic resource to improve resistance to biotic and abiotic 62 stress in conventional rice crops (O. sativa). Notable examples include resistance to bacterial blight, 63 the brown planthopper (Nilaparvata lugens) and white-backed planthopper (Sogatella furcifera) 64 (Multani et al., 2003, Angeles-Shim et al., 2020). More importantly, wild rice is also a source of 65 nutrition for local communities (Bertazonni and Alves Damasceno-Júnior, 2011, Bortolloto et al., 2017), 66 forage for livestock (Pott & Pott, 2000) and a component of wild animal diets, like jaguars, pumas and 67 ocelots (Montalvo et al., 2020). 68 Despite being a food source for humans and animals, fundamental aspects of food-safety, such as 69 the microbial diversity, and the presence of hazardous toxins, are severely understudied in wild rice 70 from natural ecosystems. The lack of information is worrisome as a multitude of studies have shown 71 that rice can be heavily afflicted by fungal pathogens in the field, particularly mycotoxigenic species of 72 the Fusarium genus (Petrovic et al., 2013, Gonçalves et al., 2019). Their presence can cause 73 significant economic losses through crop diseases and production of hazardous toxins (mycotoxins) 74 that hinders cereal commercialization as food and feedstuff (Brown and Proctor, 2013). 75 The Fusarium fujikuroi species complex (FFSC) is one of the most prominent Fusarium complexes 76 in rice crops. The group which includes the species F. fujikuroi, F. proliferatum and F. verticillioides are 77 reported as the causal agent of the fast-emerging Bakanae disease. This disease can cause seedling 78 blight, root and crown rot, etiolation, and the excessive elongation of infected rice plants. (Gupta et al., 79 2015). The FFSC members are also prolific producers of fumonisin, a mycotoxin which can have 80 carcinogenic, hepatotoxic, nephrotoxic and embryotoxic effects in laboratory animals. In humans 81 fumonisin is associated with esophageal cancer and neural tube defects (Scott, 2012). bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190306; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 82 Species within the Fusarium graminearum species complex (FGSC) became important plant 83 pathogens in major rice producing regions, such as China (Qiu and Shi, 2014, Yang et al., 2018) and 84 Brazil (Gomes et al., 2015, Moreira et al., 2020). The FGSC includes distinct species capable of 85 causing Fusarium head blight in cereals and producing the sesquiterpene trichothecenes and the non- 86 steroidal estrogenic mycotoxin zearalenone. (O’Donnel et al., 2004, Aoki et al., 2012). 87 Recently, the Fusarium incarnatum-equiseti species complex (FIESC) gained attention as a 88 relevant mycotoxigenic contaminant of crops worldwide (Goswami et al., 2005, Castellá and Cabañes, 89 2014, Avila, et al., 2019). This complex has an intricate taxonomy (O’Donnel et al., 2012), and ongoing 90 studies (Villani et al., 2016) are trying to resolve the species complex phylogeny. The complex was 91 divided in two large clades, named incarnatum and equiseti (O’Donnell et al., 2009), which currently 92 comprise more than 31 phylogenetically distinct species (O’Donnel et al., 2012, Villani et al., 2016). 93 Like, the FGSC, the species of this group are known to produce significant amounts of trichothecenes 94 and zearalenone and other mycotoxins such as equisetin, butenolide and fusarohromanone (Thrane, 95 1989, Kosiak et al., 2005, Goswami et al., 2008). 96 Deoxynivalenol (DON), the most prevalent variant of trichothecene is reported to inhibit protein 97 synthesis by binding to the ribosome and causing anorexia, immune dysregulation as well as growth, 98 reproductive, and teratogenic effects in mammals (Chen, Kistler and Ma, 2019). Zearalenone (ZEA) 99 has been highly associated with significant changes in reproductive organs and fertility loss in animals 100 (Kowalska et al., 2016). Also, the toxin has been found to induce the production of progesterone, 101 estradiol, testosterone in the cell line H295R, indicating its potential as an endocrine-disruptive agent 102 in humans (Frizzell, et al., 2011). 103 The presence of fungi and mycotoxins in wild rice is still poorly understood. Yet, the use of 104 edible wild plants from natural ecosystems is a relevant ecological alternative resource to 105 deforestation and monocultures (Bartollo et al., 2017). Moreover, the consumption of O. latifolia has 106 been gaining more traction in recent years because of its higher nutritional value in comparison to O. 107 sativa (Bertazzoni and Damasceno-Júnior, 2011). Due to the increasing relevance of wild cereal bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190306; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 108 consumption, including O. latifolia, it is essential to investigate the safety concerns regarding the 109 introduction of these novel sources of nutrition for human and animal use.