Canadian Journal of Microbiology Risk assessment of secondary metabolites produced by fungi in the genus Stemphylium Journal: Canadian Journal of Microbiology Manuscript ID cjm-2020-0351.R1 Manuscript Type: Review Date Submitted by the 29-Oct-2020 Author: Complete List of Authors: Stricker, Sara; University of Guelph, Plant Agriculture Gossen, Bruce D.; Agriculture and Agri-Food Canada, Saskatoon Research and Development Centre; McDonald,Draft Mary Ruth; University of Guelph, Plant Agriculture Keyword: Stemphylium, toxins, allergen, Pleospora Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : © The Author(s) or their Institution(s) Page 1 of 19 Canadian Journal of Microbiology 1 Risk assessment of secondary metabolites 2 produced by fungi in the genus Stemphylium 3 4 Sara M. Stricker1, Bruce D. Gossen2, Mary Ruth McDonald1* 5 6 1Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada 7 8 2Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, Saskatchewan. 9 Canada. 10 11 * [email protected] (MRM) 12 Draft 1 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 2 of 19 13 Abstract 14 The fungal genus Stemphylium (phylum Ascomycota, teleomorph Pleospora) includes plant 15 pathogenic, endophytic, and saprophytic species with worldwide distributions. Stemphylium spp. 16 produce prodigious numbers of air-borne spores, so are a human health concern as allergens. 17 Some species also produce secondary metabolites such as glucosides, ferric chelates, aromatic 18 polyketides, and others that function as toxins that damage plants and other fungal species. Some 19 of these compounds also exhibit a low level of mammalian toxicity. The high production of air- 20 borne spores by this genus can result in a high incidence of human exposure. Concern about 21 toxin production appears to be the reason that S. vesicarium, which is a pathogen of several 22 vegetable crops, was classified in Canada as a potential risk of harm to humans for many years. 23 A detailed assessment of the risk of exposureDraft was provided to the relevant regulatory body, 24 Public Health Agency of Canada, which then determined that Stemphylium spp. in nature or 25 under laboratory conditions posed little to no risk to humans or animals, and the species was re- 26 assigned as a basic (level 1) risk agent. 27 28 Keywords: Stemphylium, toxins, allergen, Pleospora 29 2 © The Author(s) or their Institution(s) Page 3 of 19 Canadian Journal of Microbiology 30 Introduction 31 Until recently, the Public Health Agency of Canada (PHAC) of the Government of Canada listed 32 Stemphylium spp. in Risk Group 2 under the authority of the Human Pathogens and Toxins Act 33 (Government of Canada 2009). This risk group contains human or animal pathogens that are 34 judged to represent a moderate risk to individuals but have a low risk of spread in communities 35 (Government of Canada 2018). Canadian regulators had listed Stemphylium vesicarium Wallr 36 (Simmons) as belonging in Risk Group 2 in previous versions of this legislation. This review was 37 undertaken to summarize the literature on the major classes of metabolites produced by 38 Stemphylium species and assess if the precaution to treat them as potential human and animal 39 pathogens was warranted based on theirDraft potential impact on human and animal health. Based on 40 this review, PHAC agreed to reduce the risk classification of Stemphylium spp. 41 Several of the more than 50 species in the fungal genus Stemphylium (phylum 42 Ascomycota, class Dothideomycetes, order Pleosporales, family Pleosporaceae) are important 43 pathogens of vegetable and fruit crops. The nomenclature for this genus has changed 44 considerably over time (see a list of the names of S. vesicarium, S1 Table). Use of the anamorph 45 name, Stemphylium, instead of the teleomorph name, Pleospora, is recommended by the 46 International Committee on the Taxonomy of Fungi (McNeill et al. 2012; Rossman et al. 2015). 47 The focus of this paper was on S. vesicarium, since it is an economically important pathogen of 48 Pyrus and Allium spp. worldwide (Aveling 1993; Llorente and Montesinos 2002). The systemic 49 review was broadened to include the entire genus due to a paucity of literature about 50 S. vesicarium alone. 51 3 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 4 of 19 52 Toxins in S. vesicarium 53 Stemphylium vesicarium was the initial Stemphylium species identified by PHAC as a Risk 54 Group 2 pathogen. It has a wide host range, from onion and asparagus to pears, and an equally 55 wide geographic distribution, including South Africa, Egypt, Turkey, India, Australia, Brazil, the 56 USA, and Canada (S2 Table) (Aveling and Naude 1992; Boiteux et al. 1994; Gupta et al. 1994; 57 Suheri and Price 2000; Hassan et al. 2007; Polat et al. 2012; Hoepting and Pethybridge 2016; 58 McDonald et al. 2016). Isolates of S. vesicarium from pear produce phytotoxic metabolites 59 (Singh et al. 1999) and the leaf blight symptoms on onion and garlic may be caused by a toxin 60 (Basallote-Ureba et al. 1999). 61 Four secondary metabolites that Draftappear to be host-specific toxins are produced by 62 S. vesicarium: stemphylin, stemphyperylenol, stemphyloxin, and stemphol (Andersen et al. 63 1995). Host-specific toxins are compounds produced by plant pathogens that promote pathogen 64 colonization and symptom development by disrupting or killing host cells or tissues, but the 65 reaction only develops in susceptible host species (Tsuge et al. 2013). However, the four 66 compounds from S. vesicarium are not listed on the U. S. National Center for Computational 67 Toxicology database (U.S. Environmental Protection Agency 2020). Also, Stemphylium species 68 are not listed as threats by the Center for Disease Control, the Animal and Plant Health 69 Inspection Service or the Agriculture Select Agent Services (Federal Select Agent Program 70 2017) of the USA, or the European Union (European Parliament and of the Council, 2019). In 71 fact, several of the plant toxins are being examined as potential treatments for human cancer (see 72 below). 4 © The Author(s) or their Institution(s) Page 5 of 19 Canadian Journal of Microbiology 73 Stemphylin 74 Stemphylin (3-hydroxy-2,2-dimethyl-5-α-d-glycopryranoside-2,3-dihydrochrome, C17H22O9) is a 75 chromone glucoside that has been isolated from Stemphylium botryosum Wallr., a pathogen of 76 lettuce (Lactuca sativa) (Barash et al. 1975). The structure of stemphylin is identical to 77 altersolanol A, an anthraquinone-derivative mycotoxin produced by Alternaria and Phomopsis 78 spp. (Assante and Nasini 1987; Mishra et al. 2015). Stemphylin produced necrotic lesions on 79 lettuce, vetch (Vicia sativa), alfalfa (Medicago sativa), and tobacco (Nicotiana tabacum), but not 80 on tomato (Solanum lycopersicum) (Barash et al. 1975). Interestingly, low doses of stemphylin 81 have anti-cancer properties and have been used to treat mouse leukemia cells (Assante and 82 Nasini 1987). Conversely, stemphylin had no cytotoxic effects against five human cancer types 83 at 40 μM but exhibited toxicity to bacteriaDraft at <10 ug mL-1(Zhou et al. 2015). 84 Stemphyloxins (I and II) 85 Stemphyloxin I (C21H34O6) and stemphyloxin II (C21H32O5) are phytotoxic ferric ion chelates 86 that are trans-decalin derivatives (Li et al. 2014) identified from S. botryosum (Barash et al. 87 1983; Manulis et al. 1984). Stemphyloxin I produced lesions on tomato but not on barley 88 (Hordeum vulgare) (Barash et al. 1982). Their mammalian toxicity is not known. 89 Stemphyperylenol 90 Stemphyperylenol (C20H16O6) was isolated from S. botryosum (Arnone et al. 1986), and has 91 since been isolated from several other fungal species. Stemphyperylenol is a perylenequinone, a 92 class of aromatic polyketides with a pentacyclic core that function as pigments and have potent 93 light-induced bioactivity (Hu et al. 2019). 5 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 6 of 19 94 Inoculation with stemphyperylenol extracted from Alternaria cassiae induced necrosis on 95 crabgrass, but not on sicklepod (Senna obtusifolia), corn (Zea mays), timothy (Phleum pretense), 96 or soybean (Glycine max) (Hradil et al. 1989). It exhibited antibacterial activity at 3 μg mL-1 (Liu 97 et al. 2010). Stemphyperylenol extracted from Botryosphaeria dothidea, an endophyte, exhibited 98 antimicrobial activity against bacteria and fungi, and cytotoxicity against human colon cancer 99 cells (Xiao et al. 2014). Stemphyperylenol from another endophyte, Talaromyces sp., inhibited 100 bacterial growth at 3 μg mL-1, which is lower than that of ampicillin (12.5 μg mL-1) (Liu et al. 101 2010). However, stemphyperylenol from A. alternata did not exhibit cytotoxic effects against 102 human cancer cells (Zhao et al. 2019). 103 Stemphol Draft 104 Stemphol (2-butyl-5-pentylbenzene-1,3-diol, C15H24O2) is a crystalline dialkyl resorcinol that was 105 first isolated from Stemphylum majusculum E.G. Simmons (Stodola et al. 1973). Subsequent 106 studies have isolated stemphol from Stemphylium herbarum E.G. Simmons in vitro (Marumo et 107 al. 1985), S. botryosum from oilseed rape (Brassica napus) (Solfrizzo et al. 1994) and 108 Stemphylium lycopersici (Enjoji) W. Yamam on tomato (Andersen and Frisvad 2004). An 109 endophytic fungus, Gaeumannomyces amomi, also produces stemphol (Jumpathong et al. 2009). 110 Stemphol has antimicrobial activity against fungi, yeasts, and bacteria (Marumo et al. 1985). 111 The amount of stemphol produced differs with fungal species, media, and length of time 112 the culture has been in storage (Solfrizzo et al. 1994). Stemphol induces apoptosis (programmed 113 cell death) in human leukemia cells by increasing cytosolic calcium levels, and cancer cells were 114 more sensitive compared to healthy cells (Ji et al. 2018). An endophytic Stemphylium isolate 115 produced stemphol A (C15H23O5SNa), stemphol B (C17H25O6SNa), and stemphol, which all 116 exhibited antibacterial properties but were not toxic to human cancer cells (Zhou et al.