Safety of the fungal workhorses of industrial biotechnology: update on the mycotoxin and secondary metabolite potential of Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei

This review presents an update on the current knowledge of the secondary metabolite potential of the major fungal species used in industrial biotechnology, i.e., Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei. These species have a long history of safe use for enzyme production. Like most microorganisms that exist in a challenging environment in nature, these fungi can produce a large variety and number of secondary metabolites. Many of these compounds present several properties that make them attractive for different industrial and medical applications. A description of all known secondary metabolites produced by these species is presented here. Mycotoxins are a very limited group of secondary metabolites that can be produced by fungi and that pose health hazards in humans and other vertebrates when ingested in small amounts. Some mycotoxins are species-specific. Here, we present scientific basis for (1) the definition of mycotoxins including an update on their toxicity and (2) the clarity on misclassification of species and their mycotoxin potential reported in literature, e.g., A. oryzae has been wrongly reported as an aflatoxin producer, due to misclassification of Aspergillus flavus strains. It is therefore of paramount importance to accurately describe the mycotoxins that can potentially be produced by a fungal species that is to be used as a production organism and to ensure that production strains are not capable of producing mycotoxins during enzyme production. This review is intended as a reference paper for authorities, companies, and researchers dealing with secondary metabolite assessment, risk evaluation for food or feed enzyme production, or considerations on the use of these species as production hosts.


Introduction
Earlier reviews on the safety of Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei have been published (Schuster et al. 2002;Tanaka et al. 2002;Barbesgaard et al. 1992;Jørgensen 2007;Blumenthal 2004), but since these reviews were written, much progress has been made in the taxonomy, toxicology, natural product chemistry, genomics, genetics, and molecular biology of these fungi.
There is a clear distinction between mycotoxins and other secondary metabolites with attractive properties for diverse applications. Fungal species containing industrial strains have the potential to produce a rather limited number of compounds that are toxic to vertebrates (mycotoxins) and a large variety of other compounds that can display anticarcinogenic or antimicrobial activity, antioxidant activity, be pigments, etc. (Mushtaq et al. 2018). A clear definition of mycotoxin and secondary metabolite is presented here to provide a clear basis for the consideration of safety. The fungal strains that represent the workhorses of industrial biotechnology have a long and extensively documented history of safe use for food and feed applications. Strains belonging to the Aspergillus species A. niger and A. oryzae have been used for fermentation of food for more than 2 millennia and to manufacture food enzymes for over 50 years, while strains of Trichoderma reesei have been used safely for decades in enzyme production. Hundreds of enzymes produced in these species are considered as safe by regulatory authorities. Furthermore, mycotoxins and other secondary metabolites are not produced during the controlled, industrially relevant growth conditions where nutrients are not limited and where there is no growth challenge by any other microorganism.
This report includes a comprehensive update of the current knowledge about the mycotoxin and the promising secondary metabolite potential of these industry relevant fungal species. We have considered all published work and have critically evaluated the validity of the data and the accuracy of the taxonomic identification in each case. Consequently, not all publications have been included herein. The report is divided into three sections (taxonomy, mycotoxins, and secondary metabolite potential) for each species.
Taxonomy of Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei Traditional identification of fungal species relied on microscopic and macroscopic morphological traits, e.g., sporulation structures and other phenotypic features like growth and colony features (see Fig. 1 for examples of A. niger, A. oryzae, and its close relative Aspergillus flavus, together with T. reesei). In the last decades, taxonomical classification aided by secondary metabolite profiles has also proven successful (Frisvad and Larsen 2015;Samson et al. 2014). More recently, the use of diagnostic gene sequences like rRNA and, later, the availability of whole genome sequences, have enabled direct comparison of different species at the nucleotide level, throughout the genome. In fact, rDNA-derived ITS sequences are recommended as one of the main "barcodes" for species identification (Samson et al. 2014). However, at least in some Aspergillus clades, there is limited variation in, e.g., ITS sequences, requiring the use of additional barcodes like calmodulin or β-tubulin (Samson et al. 2014). The level of resolution of these molecular techniques provides new ways to investigate what defines the species boundaries . Still, differences in DNA sequences alone cannot always provide a biological understanding. Also, the profiles of secondary metabolites are species-specific (Frisvad and Larsen 2015) and thereby consistent with phylogenetic relationships in fungi (Larsen et al. 2005;Kocsubé et al. 2016). When taxonomical identification is required, it is therefore advantageous to combine the accumulated knowledge on morphological, physiological, and molecular characteristics. Taxonomical classification of A. niger, A. oryzae, and T. reesei together with relevant related species is described in the following texts.

Aspergillus niger
Aspergillus niger is placed within the Aspergillus niger clade in the Aspergillus section Nigri (Varga et al. 2011a). The species is well-circumscribed, but it has a sibling species, with the same properties, called Aspergillus welwitschiae (Hong et al. 2013a). The latter species shares all morphological, physiological, and chemical characters with A. niger (Fig. 1), and the two species can only be distinguished by sequencing preferably one of the secondary bar-coding genes (Hong et al. 2013b). The DNA barcodes of Aspergillus welwitschiae are as follows: ITS (internally transcribed spacer regions and the 5.8 S of the ribosomal gene): FJ629340; BenA (β-tubulin): FJ629291; CaM (calmodulin): KC480196, while A. niger has the following barcodes: ITS: EF 661186; BenA (β-tubulin): EF661089; CaM (calmodulin): EF661154; RPB2 (RNA polymerase B2: EF661058). Different strains of Aspergillus niger have been genome-sequenced (see Baker 2006;Pel et al. 2007;Andersen et al. 2011).
Other species closely related to A. niger are Aspergillus neoniger, Aspergillus tubingensis, Aspergillus vadensis, Aspergillus luchuensis, Aspergillus eucalypticola, Aspergillus costaricaensis, and Aspergillus piperis, but it is mostly A. luchuensis (formerly Aspergillus acidus or Aspergillus foetidus var. acidus), A. vadensis, and A. tubingensis that are used in the industry. In some cases, the latter have been misidentified as A. niger, and A. niger is by far most commonly used species in the industry . A. luchuensis is found in fermented Puerh tea ) and is used often for koji production (also under the names Aspergillus kawachii and Aspergillus awamori) (Fujimoto et al. 1993;Hong et al. 2013a;Fujii et al. 2016). A. niger sensu stricto is the most commonly used species in biotechnology (Andersen et al. 2011;). An often examined typical strain of A. niger is ATCC 1015.
Unlike the situation in A. flavus, which has a taxonomically accepted domesticated form A. oryzae, the domesticated form of A. niger, A. awamori (Nakazawa 1907, Sakaguchi et al. 1951Raper and Fennell 1965;Murakami 1979;Al-Musallam 1980), has not been accepted as a valid name, probably because of a mistaken neotypification. Perrone et al. (2011) used the name A. awamori for a taxon that was isolated from Welwitschia mirabilis, but since the ex-type isolate (CBS 557.65) was not from a koji environment, that species was renamed A. welwitschiae by Hong et al. (2013a). Other names such as Aspergillus usamii and A. kawachii have also been used for domesticated forms of A. niger or A. luchuensis (Hong et al. 2013b). However, none of these names have been officially taken up for the domesticated form of A. niger. The names Aspergillus phoenicis and Aspergillus ficuum predate A. niger and have therefore been rejected, and the name A. niger officially conserved because of the economical importance of the latter species (Kozakiewicz et al. 1992).
Average nucleotide identity (ANI) has become the gold standard for taxonomic confirmation of prokaryotes. Two species having > 95% ANI are considered the same species (Rodriguez and Konstantinidis 2014). Although ANI is not widely used in eukaryotes and there are no studies done to layout an ANI-based species framework in fungi, ANI values can still be used to determine the relatedness of two strains or species and can give a better resolution of phylogenetic tree-based inferences (Goris et al. 2007). ANI can also discriminate between closely related populations, and it provides a higher resolution than other sequence analyses, at least in bacteria (Rodriguez and Konstantinidis 2014).
We performed comparative genomics within species of the Nigri clade for which the genome sequence is available using ANI that showed a relatively high identity (85% or higher) between different species in this clade, while a lower level (7 6%) was obtained when comparing to species outside the clade like A. oryzae or Aspergillus nidulans (Table 1). Remarkably, a higher ANI was obtained when comparing A. tubingensis and A. luchuensis (~93%) and a slightly lower ANI when comparing A. tubingensis with A. vadensis or A. luchuensis with A. vadensis (~92%). These three species appear to be more closely related (Table 1), and they all produce asperazines ). A phylogenetic tree based on the above-mentioned genome comparison displays the closer relationship between these three species and the clustering of A. niger and Aspergillus brasiliensis (Fig. 2).
Recently, wild-type A. niger has been considered as a class 2 microorganism by the German authorities (BAUA see previous texts) because of its potential mycotoxin production and pathogenicity to humans and animals. It is important to discriminate between (1) mycotoxin production as a health hazard during food manufacture and spoilage and corn silage and (2) the growing number of reports of opportunistic pathogens that have resulted in disease, normally in immunocompromised patients. In fact, the baker's yeast (Saccharomyces cerevisiae) can also be considered as a pathogen since it has been associated with disease in severely immunocompromised patients. Perhaps the concept of what constitutes a "pathogen" needs a comprehensive revision and it is not solely related to the taxonomy of the microbe (Casadevall and Pirofski 2003).

Aspergillus oryzae
A. oryzae is regarded by most taxonomists as the domesticated form of A. flavus (Blochwitz 1929;Wicklow 1984;Klich and Pitt 1988;Georgianna et al. 2009;Rokas 2009;Varga et al. 2011b;Gibbons et al. 2012;Houbraken et al. 2014;Frisvad et al. 2019). Wicklow (1984) claims that domestication (in rice fermentations) has resulted in the following phenotypic  Andersen) differences: conidia in A. oryzae are smoother and slightly larger (to adapt to the rice habitat), amylase production is higher, the conidiophore stipes are longer, the mycelium is more floccose, and the conidium color en masse is light brownish green rather than yellow grass green as compared to A. flavus (Fig. 1). While there are no genotypic differences between A. oryzae and A. flavus (Thom and Church 1921;Raper and Fennell 1965;Murakami 1971;Christensen 1981;Pitt et al. 1983;Wicklow 1984;Pitt 1985, 1988;Geiser et al. 1998Geiser et al. , 2000Gibbons et al. 2012;Powell et al. 2008;Varga et al. 2011;Gilbert et al. 2018;Frisvad et al. 2019), there are several morphological and physiological differences between the two species as listed previously. Furthermore Aspergillus oryzae cannot produce aflatoxins, aspergillic acid, and flavimine, that are otherwise present in most strains of Aspergillus flavus (Thom and Church 1921;Raper and Fennell 1965;Murakami 1971;Christensen 1981;Wicklow 1984;Pitt 1985, 1988;Pitt et al. 1983;Varga et al. 2011;; Fig. 1). Klich and Mullaney (1987) were able to distinguish between strains of A. oryzae and A. flavus by DNA restriction enzyme fragment polymorphisms. Nearly all strains of A. flavus produce a bright orange reverse on the medium AFPA (Aspergillus flavus parasiticus agar), while A. oryzae strains produce a cream-colored reverse (Bothast and Fennell 1974;Hamsa and Ayres 1977;Pitt et al. 1983).
In accordance with this, genome sequencing of A. flavus (Nierman et al. 2015;Faustinelli et al. 2016) and strains of A. oryzae Galagan et al. 2005;Umemura et al. 2012Umemura et al. , 2013aZhao et al. 2012Zhao et al. , 2013aZhao et al. ,b, 2014a have shown that these two species are very similar. Interestingly, the first sequenced strain of A. oryzae may be an A. flavus Fig. 2 Phylogram based on whole genome sequences of available Aspergillus species within the Nigri and Flavi clades. The phylogram was made using kSNP (version 3.1; Gardner et al. 2015) which computes a core SNP matrix from all the genomes and then executed FastTree (Price et al. 2009) with the maximum likelihood option to compute the tree. The tree was then midpoint-rooted and rendered as a Phylogram using Dendroscope (Huson and Scornavacca 2012) "sensu stricto." The isolate RIB40 produces large globose sclerotia (Rank et al. 2012;Fig. 1) and was isolated from a broad bean, Kuriyamacho, Kyoto, Japan, in a field, not from a fermentation factory. Based on the first identification as A. oryzae var. brunneus, it has brownish conidia and, therefore, resemble A. oryzae. RIB40 does not produce aflatoxin as it contains disabling mutations in the gene cluster (Tominaga et al. 2006). It has been shown that A. flavus isolates gradually lose their ability to produce spores, sclerotia, and aflatoxinproducing capability after several serial transfers (Torres et al. 1980;Horn and Dorner 2001;Chang et al. 2007). The production of large globose sclerotia is characteristic for A. flavus sensu stricto (Geiser et al. 2000), and only few strains of A. flavus (for example NRRL 3251) produce small sclerotia (Hesseltine et al. 1970;Saito and Tsuruta 1993), while other strains with small sclerotia belong to the species Aspergillus minisclerotigenes, Aspergillus aflatoxiformans, Aspergillus austwickii, and Aspergillus cerealis (Varga et al. 2011;. Overall, none of the characterized true A. oryzae isolates produce aflatoxins. For A. flavus, the situation is more complex since some isolates, including the extype strain (NRRL1957), do not produce aflatoxin. However, aflatoxin production has been shown for a large number of A. flavus including NRRL3357.
Genome sequencing has allowed several comparative studies to be carried out (Abe et al. 2006;Payne et al. 2006;Kobayashi et al. 2007;Rokas et al. 2007;Machida et al. 2008). A. oryzae is used extensively in enzyme production at industrial scale (Barbesgaard et al. 1992;Jørgensen 2007) and as a successful expression host for production of secondary metabolites (Sakai et al. 2008;Liu et al. 2015;Minami et al. 2016;He et al. 2018). In practice, sequence barcodes for A. oryzae include the following: (1) ITS (accession no. EF661560); (2) BenA (β-tubulin, accession no. EF661483); (3) CaM (calmodulin accession no. EF661506); and (4) RPB2 (RNA polymerase B2, accession no. EF661438) and for A. flavus: (1) ITS: (AF027863); (2) BenA (EF661485); (3) CaM (EF661508); and RPB2 (EF661440). Remarkably, the barcodes are not sufficient to effectively separate A. flavus and A. oryzae. More elaborate molecular techniques are required to distinguish these species (Godet and Munaut 2010). ANI analysis showed a very high degree of sequence homology, well above 99%, between RIB40 and other A. oryzae strains used in industrial enzyme production like A1560 (synonym IFO 4177), while a slightly lower percentage is observed when comparing A. flavus and A. oryzae (Table 1). The use of the % identity between A. oryzae RIB40, A1560 or A. flavus NRRL3357 (99.9% versus 99.1%) does not allow a direct species discrimination based on ANI. Furthermore, ANI between A. oryzae and species from the Nigri section display an ANI value below 80%. Members of the Nigri section display an ANI of 85% or higher. Lower ANI (approx. 75%) is obtained when comparing either A. oryzae/A. flavus to A. nidulans or species from the Nigri section to A. nidulans. Overall, as in the case of A. niger, the above-mentioned data demonstrate that genome homology data alone cannot be used for taxonomical purposes and need to be complemented by phenotypic properties.

Trichoderma reesei
Trichoderma reesei (anamorph) has also been named Hypocrea jecorina (teleomorph and holomorph), but with the new nomenclatural system used after 2011, Trichoderma reesei is considered the correct name for this fungus (Samuels et al. 1998;Samuels et al. 2012, Fig. 1). Most of the industrial strains have a single common ancestor, RUT-C30, which displays a blue-green color on solid medium (Fig. 1). The genome sequence has also been reported for this species (Martinez et al. 2008). The T. reesei type strain is QM6a.

Mycotoxins are a very limited group of fungal secondary metabolites
Fungal secondary metabolites can be defined as outward-directed, small differentiational molecules of restricted taxonomic distribution that are genetically encoded by clustered genes and accumulated and normally secreted. Secondary metabolites are a very heterogeneous chemical group of low molecular weight compounds that include antimicrobials, antioxidants, pigments, hormones, and metal chelators. A great number of these compounds have therefore a very significant potential application.
In general, any competition-selected fungal species has the potential to produce hundreds of individual secondary metabolites coded by up to 90 biosynthetic gene clusters (Clevenger et al. 2017;Lind et al. 2017). The major biosynthetic classes of secondary metabolites are polyketides, non-ribosomal peptides, terpenes, and shikimic acid-derived compounds, but many compounds are hybrids of these classes. The genes coding for the enzymes involved in the biosynthesis of these compounds are associated in gene clusters. The genomes of A. niger, A. oryzae, and T. reesei include 78, 75, and 27 gene clusters for secondary metabolite biosynthesis, respectively (Lind et al. 2015;Zeilinger et al. 2016;Wasil et al. 2018), although these numbers may vary depending on the strain and the software package used. Furthermore, each biosynthetic gene cluster may be responsible for the production of a large number of precursors, shunt products, and final products. For example Aspergillus oryzae was reported to produce many members (26) of the cyclopiazonic acid biosynthetic family of compounds (Liu et al. 2018), including cyclopazonic acids, speradines, cyclopiamides, and asporydines. With the development of new genome mining approaches (Kjaerbølling et al. 2018) and algorithms such as antiSMASH (Blin et al. 2017), the prediction of secondary metabolite encoding gene clusters has become easier. On the other hand, the chemical modifications based on important accessory tailoring genes on the core structure of secondary metabolites may be more difficult to predict from sequences (Bertrand et al. 2018) and often require full structure elucidation. In this context, it is important to note that majority of gene clusters are not expressed under standard cultivation and that no fungal species synthesizes all potential secondary metabolites at any given time. As mentioned previously, production of secondary metabolites does not normally occur under production-relevant growth conditions where no species competition or nutrient starvation threat is used.
Mycotoxins are a very limited group of fungal secondary metabolites. Regarding biotechnology, mycotoxins are important if they pose a safety concern in the industrial application of fungi for enzyme or bulk metabolite production as well as in other areas like food spoilage and in building environments. There have been numerous definitions of the word mycotoxin (Bennett and Inamdar 2015;Taevernier et al. 2016), but a strict consensus definition that we endorse is the following: Mycotoxins are secondary metabolites genetically encoded by clustered genes and produced by fungi. These mycotoxins are acutely or chronically toxic and pose health hazards or death in humans and other vertebrates when acquired in small amounts via a natural route (orally, by inhalation, or via the skin). This definition is a combination of that of Jarvis and Miller (2005), Frisvad (2011), Bennett and Inamdar (2015), and Taevernier et al. (2016). Taevernier et al. (2016) suggested that a quantitative level of cell cytotoxicity on preferably human cell cultures with an IC 50 (the concentration required for 50% of cell viability) of less than 1000 μM could be used to determine whether a fungal secondary metabolite was considered a mycotoxin or not. We cannot accept this definition as such molecules may be cytotoxic, while not necessarily being toxic when acquired via a natural route. Earlier claims of mycotoxicity were based on other toxicity data, such as toxicity including cancerogenicity after intraperitoneal or subcutaneous injection (Dickens and Jones 1961;Cole and Cox 1981;Lu et al. 2017), but this too is not a natural route of intake. For example, patulin and penicillic acid were originally claimed to be cancerogenic based on subcutaneous injection (Dickens and Jones 1961), but Enomoto and Saito (1972) rightly mention that experimental production of cancer should be confirmed in animals by oral administration of mycotoxin.
The safe use of fungal strains is recognized in official classifications of biological agents into risk groups; e.g., BAUA (German Federal Institute for Occupational Safety and Health) classifies A. niger and A. oryzae as risk group 2 biological agents. Importantly, BAUA recognizes that strains belonging to these species may still be classified as risk group 1 biological agents if documentation of safety and/or history of safe use is provided.
In the following sections, we describe the mycotoxins that are potentially produced by the three industrial organisms and relevant related species. Only mycotoxins with a documented effect are described. All other secondary metabolites are described in the section on secondary metabolite potential and are not considered mycotoxins according to the definition herein.

Mycotoxins potentially produced by Aspergillus niger
Aspergillus niger has been claimed to produce a very large number of mycotoxins and other secondary metabolites (Table 2; Nielsen et al. 2009). Apart from a large number of volatiles and small organic acids (Wani et al. 2010;Priegnitz et al. 2015;Costa et al. 2016), A. niger sensu stricto can produce very few mycotoxins but a large number of other secondary metabolites. In many cases, fungi identified as A. niger were indeed A. tubingensis or other closely related species (Table 2).

Fumonisins
Fumonisins are strongly reduced polyketides with two added tricaballyllic acid groups and an amino group added from a non-ribosomal peptide. They are mycotoxins associated with multiple human and animal diseases, as they are produced in large amounts in cereals by common Fusarium species (Braun and Wink 2018;Cendoya et al. 2018). Fumonisins induce leukoencephalomalacia in horses, nephro-and hepatotoxicity in rodents, and pulmonary toxicity in pigs, and they have been classified as International Agency for Research on Cancer (IARC) type 2B carcinogens in humans (esophageal cancer) (Cendoya et al. 2018). However, Aspergillus niger and its sibling species A. welwitschiae (originally named A. awamori) also produce fumonisins of the B 2 , B 4 , and B 6 types  and may produce fumonisins in cereals and grapes (Logrieco et al. 2009;Mogensen et al. 2010;Munkvold et al. 2018). Several industrial strains have the capability to produce fumonisins Han et al. 2017), so it is important to use strains that do not produce these mycotoxins. Current A. niger production strains have been developed that either have been selected due to the lack of fumonisin production or contain a deletion of the fumonisin gene cluster (unpublished results).
The impact of fumonisins on human health remains poorly understood (Voss and Riley 2013). It has been known for long that fumonisins are hepatotoxic, nephrotoxic, atherogenic (induces formation of plaque in arteries), immunosuppressive, and embryotoxic in experimental animal systems (Nair 1998). Structurally, fumonisin B1 shows similarity to the cellular sphingolipids, and this similarity has been shown to disturb the metabolism of sphingolipids leading to accumulation of sphinganine in cells and tissues. The cellular mechanisms behind fumonisin B 1 -induced toxicity include the induction of oxidative stress, apoptosis, and cytotoxicity, as well as alterations in cytokine expression (Stockmann-Juvala and Savolainen 2008). Mechanistically, the toxicity of fumonisin B 2 and B 3 is relatively poorly understood, but a comparison of the toxicities of fumonisin B 1 , B 2 , and B 3 individually and in combination has shown that all three are toxic, but with fumonisin B 1 being the most toxic of the three (Henry and Wyatt 2001).

Ochratoxin
Ochratoxin A (OTA) is a mycotoxin that is a common contaminant of a wide variety of food products. The molecular s t r u c t u r e c o m p r i s e s a c h l o r i n a t e d p o l y k e t i d e dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a major part of the biosynthetic pathway Massi et al. 2016;Gallo et al. 2017;Gill-Serna et al. 2018). OTA inhibits protein synthesis and energy production, induces oxidative stress, cell apoptosis and necrosis, and DNA adduct formation, and is mostly recognized as a nephrotoxin (Heussner and Bingle 2015; Közégi and Poór 2016). It is classified as an IARC type B2 carcinogen in human beings.

Oxalic acid
Oxalic acid is a strong dicarboxylic acid. Oxalic acid is a reducing agent and its conjugate base, known as oxalate, is a chelating agent for metal cations. Typically, oxalic acid occurs as the dihydrate. Excessive ingestion of oxalic acid or prolonged skin contact can be dangerous. Oxalic acid is hepatotoxic, but it will only have a negative effect in quite high doses (Jahn 1977). Aspergillus niger infections are often accompanied with oxalosis (Kredics et al. 2008;Oda et al. 2013), and in one case, calcium oxalate produced by A. niger in the lungs caused hyperoxaluria in the kidneys (Vaideeswar and Sakhdeo 2009), so both kidneys and the liver can be affected. However, such cases are rare and will only happen in severely immunocompromised patients. Otomycoses are often caused by Aspergillus tubingensis rather than A. niger (Kredics et al. 2008).

Improved safety of A. niger industrial strains
As mentioned previously, A. niger has the potential to produce ochratoxin, fumonisin, and oxalic acid. Industrial strains have been developed by classical mutagenesis and by deletion of the genes involved in the biosynthesis ).

Mycotoxins from Aspergillus oryzae and A. flavus
A. oryzae and its closely related species A. flavus can produce a very limited number of mycotoxins (Table 3). Their macroscopic similarity has contributed to a disparity of reports on the potential production of mycotoxins from either species. Mycotoxins produced by these species are described in the following texts with attention to knowledge about the potential production of these compounds by either species and reports that describe production in wrongly assigned species.
A. oryzae is a domesticated species originating probably from Aspergillus flavus, and the two species can not be distinguished by DNA sequence differences. Since A. oryzae is domesticated, it can only be expected to be found in fermentation environments. Any A. oryzae recovered in nature can only be found there if it has escaped such a fermentation plant, and based on its adaptation to the fermentation environment, it must be expected to be a poor competitor in cereals, oilseeds, and nuts, where A. flavus is a very competitive species (Wicklow 1984).

Aflatoxins
The aflatoxins (B 1 and B 2 primarily) are polyketides that have been found in many strains of Aspergillus flavus, albeit not the culture ex type of A. flavus ). Aflatoxin (AFL) has been reported from strains of Aspergillus oryzae, but these data are based on misidentified strains or misidentified mycotoxins or contaminated cultures ). It has been shown that strains of Aspergillus oryzae Ochratoxin A Abarca et al. (1994) This mycotoxins has been found in less than 10% of the strains of A. niger examined ) Oxalic acid Raistrick and Clark (1919); Yassin et al.
Nearly all strains of A. niger produce oxalic acid sensu stricto cannot produce AFL, as a result of the lack of essential parts of the gene cluster, e.g., deletion of the aflR gene involved in induction of biosynthesis (Cary and Ehrlich 2006;Chang et al. 2007;Lee et al. 2006a,b;Tominaga et al. 2006;Takahashi et al. 2008Takahashi et al. , 2012Kiyota et al. 2011;Hong et al. 2013b;Lee et al. 2014;Tao and Chung 2014). Therefore, AFL production can be excluded in A. oryzae sensu stricto. Furthermore, current industrial strains contain a deletion of the whole AFL gene cluster, providing additional safety in enzyme production. Type G aflatoxins (aflatoxin G 1 and G 2 ) have rarely been reported from Aspergillus flavus. In some cases, the G type aflatoxins were produced by A. parasiticus and other species from section Flavi (Varga et al. 2011), rather than isolates that confidently can be allocated to A. flavus sensu stricto. Saldan et al. (2018) reported on aflatoxin G 1 production by A. flavus ATCC 9643, but this strain may not be an A. flavus sensu stricto. Five Korean strains of A. flavus sensu stricto were reported to produce the G-type aflatoxins (Frisvad et al. 2019).

Cyclopiazonic acids
Cyclopiazonic acid (= α-cyclopiazonic acid) (CPA) is an indol tetrameric acid, hybrid polyketide/non-ribosomal peptide/ DMAT (dimethylallyl terpene unit) compound that was isolated from A. flavus originally by Luk et al. (1977) and Gallagher et al. (1978) but has since been found repeatedly in A. flavus (Varga et al. 2011b). It was originally isolated from a fungus identified as Penicillium cyclopium, but the strains of Penicillium-producing cyclopiazonic acid were Penicillium griseofulvum and Penicillium commune (Frisvad 1989;Frisvad et al. 2004). CPA has also been isolated repeatedly from Aspergillus oryzae (Orth 1977;Ohmomo et al. 1973, erroneously reported as A. versicolor;see Domsch et al. 2007;Frisvad 1989;Tokuoda et al. 2008;Shaaban et al. 2014). It is possible to remove the CPA gene cluster and thus avoid CPA production in biotechnological processes (Kato et al. 2011). A. oryzae and A. flavus can produce a large number of secondary metabolites related to CPA including iso-α-cyclopiazonic acid, β-cyclopiazonic acid (= bissecodehydrocyclopiazonic acid), α-cyclopiazonic acid imine, 2-oxocyclopiazonic acid, cyclopiamide (A), cyclopiamide E & H, speradine A, B, C, D, E, F, H, I, 3-hydroxy-speradine A, cAATrp, and asperorydine A-M (Ohmomo et al. 1973;Holzapfel et al. 1990;Hu et al. 2014a,b;Ma et al. 2015;Tokuoka et al. 2015;Xu et al. 2015;Uka et al. 2017;Liu et al. 2018) from A. oryzae and A. flavus, but speradine A is also produced by Aspergillus tamarii (Tsuda et al. 2003). Some of the strains reported as A. oryzae producing these tetramic acids have been isolated from marine sources, so they may in fact be A. flavus. However, the speradines are related to CPA, produced by many strains of both A. flavus and A. oryzae, and so speradines are not unlikely secondary metabolites in A. oryzae. There have been some problems with the naming of speradine B that is a different speradine in Penicillium dipodomyicola  than that from A. flavus, so some of the speradines need to be renamed.
β-nitropropionic acid β-nitropropionic acid (BNP) is one of the real mycotoxins reported from authentic Aspergillus oryzae strains, but also from A. flavus strains (Bush et al. 1951;Nakamura and Shimoda 1954;Iwasaki and Kosikowski 1973;Orth 1977).  (1969) It has caused sugarcane disease in children eating sugarcane infected with Nigrospora spp. that produce β-nitropropionic acid also (Liu et al. 1989;Ming 1995;Fu et al. 1995;Johnson et al. 2000;Fernagut et al. 2002;He et al. 1995). The genetic basis for production of BNP is not completely understood. Therefore, BNP levels are monitored in industrial enzyme productions.

Mycotoxins from Trichoderma reesei
It seems that chemotaxonomy is working excellently at the species level in Trichoderma ). In the latter paper, T. reesei was not included, and it is only few mycotoxins that are ascribed to T. reesei ) (Table 4). Reported mycotoxins from T. reesei (claimed to be a mutant of QM 9414 and called P-12) include trichodermin (Watts et al. 1988), but this ability to produce trichodermin by T. reesei has been rejected by Nielsen et al. (2005). The latter authors claimed that only Trichoderma brevicompactum can produce trichodermin, and possibly also Trichoderma arundinaceum ). There are also some trichothecene genes in Trichoderma gamsii and Trichoderma asperellum, but such genes have not been observed in T. reesei . Also, the mycotoxin gliotoxin has been mentioned as a potential secondary metabolite in Trichoderma, because a gene cluster seems to be present in the genome of this fungus ). However, gliotoxin has never been detected in any culture of T. reesei (Martinez et al. 2008;Kubicek and Druzhinina 2016). T. reesei thus seems to be unable to produce mycotoxins.
Toxicity of fungal mycotoxins relevant for A. niger, A. oryzae, and T. reesei Mycotoxins often affect different vertebrate species very differently. However, to enable a comparison of the relative toxicity of the mycotoxins potentially produced by A. niger, A. oryzae, and T. reesei, an overview of acute oral toxicity is provided (Table 5). In the enzyme industry, it is ensured that production strains based on A. niger, A. oryzae, and T. reesei do not produce mycotoxins when grown at large scale. As shown previously, A. niger can produce the mycotoxins ochratoxin A, fumonisins B 2 , B 4 , and B 6 , and oxalic acid, and A. oryzae can produce the mycotoxins cyclopiazonic acid and β-nitropropionic acid, and T. reesei has not been convincingly shown to produce any mycotoxins.

Improved safety of A. oryzae industrial strains
As mentioned previously, A. oryzae strains are not able to produce aflatoxins due to the presence of disabling mutations in the gene cluster. Modern industrial strains have been developed that contain a large DNA deletion. This region includes the aflatoxin gene cluster and genes involved in the biosynthesis of cyclopiazonic acid (CPA, Christensen et al. 2000). Thus, during industrial enzyme production using strains derived from A1560 containing the chromosomal deletion, the presence of neither aflatoxin nor CPA is a concern.
Secondary metabolite potential Fungal secondary metabolites are very diverse and include compounds with a wide range of applications (e.g., antibiotics, cancer treatment, immunosuppressing drugs, pigments, antioxidants).
Like many other fungi, Aspergillus species are capable of producing a very large number of drugs and drug-lead compounds. Among the best known for medical applications are the antibiotic penicillin to combat bacterial infections, the cholesterol-lowering mevinolin from Aspergillus terreus, the anticancer compound fumagillin from Aspergillus fumigatus, the antifungal echinocandin from Aspergillus pachycristatus and mulundocandin from Aspergillus mulundensis (Baltz et al. 2010;Houbraken et al. 2014;Zeiliger et al. 2015;Bills et al. 2016;Park et al. 2017a).
Fungi produce a large number of other secondary metabolites. Among them, fungal pigments such as polyketidederived azaphilones are used to add color and as antioxidants in food. Aspergillus species are used to produce yellow and brown pigments like fumigatin (Hanson 2008). Additionally, red pigments have been reported in, e.g., an A. flavus strain (Gurupavithra et al. 2017). Carotenes are important terpenoid pigments and antioxidants that are produced in many bacteria, fungi, algae, and plants. Interestingly, carotene is produced by few Aspergillus species and not by Trichoderma reesei (Avalos and Limon 2015). Culture could have been contaminated, but claimed to be derived from QM 9414 as strain P-12; the claim that T. reesei produces this mycotoxin may also be based on insufficient analytical chemical methods
A strain identified as A. niger was reported to produce asperiamide B and C (Wu et al. 2008), but it also produces the aflatoxin precursors averufin and nidurufin, so this strain was probably an A. flavus.

Small acids of Aspergillus niger
Oxalic acid, gluconic acid, and citric acid are small chelating organic acids derived from the citric acid cycle, but since they are secreted and accumulated may be characterized as secondary metabolites (Poulsen et al. 2012;Niu et al. 2016). These are by far the small organic acids produced in the highest amounts, but other acids can be produced by A. niger (Table 5).   Jefferson (1967a,b); Yu et al. (1967); Pattenden (1969Pattenden ( , 1970; Rabache et al. (1974); Rao et al. (2002) Also referred to as asperyellone and asperrubrol

Aflavinines
Aflavinines are indoloterpenes biosynthesized from tryptophan and dimethylallyl units. They are only produced in sclerotia of A. niger (Frisvad et al. 2014). Such sclerotia are not produced on ordinary laboratory media, except if they are induced by the presence of small dried fruits, such as raisins (Frisvad et al. 2014). Most aflavinines are antiinsectan, but are not known to be toxic towards vertebrates (Gloer et al. 1988).

Asperamides
Asperamides are sphingolipids and unusual cerebrosides (Zhang et al. 2007a,b,c,d). Such sphingolipids appear to be pretty widespread in fungi, but their function in fungi is often unknown. The similar flavusides from A. flavus are antibacterial (Yang et al. 2011).

Asperenones
The terpenes asperenones, asperyellones, and asperrubrols are carotenoid-like secondary metabolites. Asperenone is a human platelet aggregation inhibitor, and a strain of A. niger has been optimized for higher production of this bioactive compound (Chidananda et al. 2008).

Aspergitides
Aspergitides are NRP-derived tetrapeptides which are potentially anti-inflammatory (Lee et al. 2015). These hydrophobic tetrapeptides have some similarity with fungisporins and nidulanins which appear to be generally present in Aspergillus and Penicillium species (Ali et al. 2014;Klitgaard et al. 2015;Hautbergue et al. 2017).

Aspergillin
Aspergillin is a green polyketide (Ray and Eakin 1975) that may be connected with the production of the black pigment in the spores of A. niger. Other (yellow) pigments, such as funalenone and naphtho-γ-pyrones, are also connected with black melanin (Jørgensen et al. 2011).

Azanigerones
The azanigerones A-F needed chromatin remodeling in order to be produced by Aspergillus niger (Zabala et al. 2012). These compounds are polyketides, and little is known of their activity. However, like other azaphilones, they can probably bind amino acids, but no nitrogen-containing derivatives have been found yet.

Cycloleucomelone
Cycloleucomelone, leucomelone, and atromentin are shikimic acid-derived secondary metabolites that have been found in basidiomycetes (Turner 1971;Turner and Aldridge 1983) but also species in Aspergillus section Nigri (Hiort et al. 2004; Nielsen et al. 2009). These types of compounds may have radiation-protective characteristics, and they are widespread in Aspergillus (Frisvad and Larsen 2015). The analogous (heteroisoextrolites) terphenyllins are for example produced by members of Aspergillus section Candidi and aspulvinones by Aspergillus section Terrei (Turner 1971;Turner and Aldridge 1983;Frisvad and Larsen 2015).

Funalenone and naphtho-γ-pyrones
These polyketides have some genes in common with the pksA gene for production of the black pigment in Aspergillus niger (Jørgensen et al. 2011). Some naphtho-γ-pyrones have been claimed to be toxic (Ghosal et al. 1979), but they are not mycotoxins according to the definition accepted here. In fact, they can be exploited industrially as they have anti-oxidant, anti-cancer, anti-microbial, anti-HIV, anti-hyperuricuric, and anti-tubercular effects (Choque et al. 2015).

Malformins
Malformins are NRP cyclic peptides that originally were cited as toxic (Anderegg et al. 1976;Kobbe et al. 1977;Cole and Cox 1981), but they are not within the definition of mycotoxins in a strict sense, as malformin A has an oral LD 50 of more than 50 mg/kg body weight in male mice. The toxicity data of Anderegg et al. (1976) and Kobbe et al. (1977) were based on malformin injection, which is not a natural route of intake. Furthermore, malformins have never been detected after mycotoxicosis caused by A. niger. Malformins are very promising anti-cancer agents, however (Park et al. 2017b).

Nafuredin
Nafuredin is a polyketide terpene-derived secondary metabolite and is an inhibitor of anaerobic electron transport in pig roundworm, but it has very low effect on mammalian enzymes (Ui et al. 2001). It is a promising antihelminthic drug lead candidate.

Nigerasterols
Nigerasterols are terpene-derived sterols that display potent activity against tumor cell lines ). There are as yet no data on vertebrate toxicity. The fungus (MA-132) was identified only by using ITS sequences, so it may be another species in Aspergillus section Nigri than A. niger that produces nigerasterols.

Nigerazines, aspernigerin, and nigragillins
The nigerazines, nigragillins, and aspernigerin are all related NRP-derived secondary metabolites. They are weakly insecticidal, and nigerazine B inhibits the root growth of lettuce seedlings (Caesar et al. 1969;Iwamoto et al. 1983). They have not been reported as mycotoxins.

Nigerloxin
Nigerloxin is derived from an inhibitor of soy bean lipoxygenase and rat lens aldose reductase (Rao et al. 2002a,b). It is a polyketide NRP hybrid. It is a strong antioxidant and is anti-diabetic and of low toxicity (Rao et al. 2005;Suresha and Srinivasan 2013;Vasantha et al. 2018).

Pseurotins
Pseurotins are NRP polyketide hybrid secondary metabolites that have neuritogenic (Komagata et al. 1996) However, psurotins have not been reported from any other isolate of A. niger ), so the two metabolites may be produced by another species in Aspergillus section Nigri.

Yanuthones
The yanuthones are meroterpenoids with a 6-methyl salicylic acid precursor and terpene units attached (Holm et al. 2014;Petersen et al. 2015;Nielsen et al. 2017). There are no toxicity data for yanuthones, but they have antifungal activity (Petersen et al. 2015).

Secondary metabolites described in A. oryzae and A. flavus
A. flavus and A. oryzae can produce many secondary metabolites (Table 7). These can be subdivided into biosynthetic families. It is very interesting to note that, e.g., ustiloxin B and ustilaginoidin C, have both been isolated from the rice false smut pathogen Villosiclava virens (= Ustilaginoidea virens) even though they are not biosynthetically related. However, these two types of secondary metabolites have also been found in Aspergillus flavus (Umemura et al. 2014;Tsukui et al. 2015;Yoshimi et al. 2016). This is remarkable as both unrelated fungi occur on rice. One speculation could be that the gene clusters for both ustiloxins and ustilaginoidins were horizontally transferred from one fungus to the other during evolution. Ustilaginoidins are bis-naphtho-γ-pyrones (even called "mycotoxins" in the paper of Meng et al. 2015 and ustiloxins for toxic cyclic peptides by Tsukui et al. 2015).
Like the heteroisoextrolite (Frisvad and Larsen 2015) analogues in Aspergillus section Nigri (normally also called naphtho-γ-pyrones, Nielsen et al. 2009;Lu et al. 2014;Choque et al. 2015), the ustilaginoidins are probably also involved in the formation of the green conidium color of Aspergillus section Flavi as it is known for the involvement of naphtho-γ-pyrones in black pigmentation in Aspergillus section Nigri isolates (Chiang et al. 2011;Jørgensen et al. 2011;Frisvad et al. 2014;Niu et al. 2016). Other important secondary metabolites are described subsequently. Additionally, secondary metabolites that have been erroneously assigned to A. flavus or A. oryzae are also listed (Table 8).

Aflavarins
Aflavarins are polyketides found in the sclerotia of Aspergillus flavus (TePaske et al. 1992). These polyketides have not yet been found in any A. oryzae strain. Leporins, also found in A. leporis (TePaske et al. 1991), have been found in A. flavus (Cary et al. 2015), but they are not expected to be produced by A. oryzae.

Aspergillic acids
These iron-chelating compounds have been used for discrimination between A. flavus and A. oryzae, in that Aspergillus oryzae sensu stricto has been claimed not to produce any of these pyrazine compounds. Testing Aspergillus flavus sensu stricto and Aspergillus oryzae sensu stricto has shown that it is only the former that can produce aspergillic acids (Bothast and Fennell 1974;Hamsa and Ayres 1977;Pitt et al. 1983;Assante et al. 1981;Liljegren et al. 1988;Varga et al. 2011b). However, compounds in this class have been reported from A. oryzae, including mutaaspergillic acid (Nakamura and Shiro 1959a,b;Nakamura 1961;Sugiyama et al. 1967;Ohta and Ohta 1983), hydroxyaspergillic acid (Nakamura and Shiro 1959a,b;Dutcher 1958 (as A. flavus);MacDonald 1962;Ohta and Ohta 1983;Sano et al. 2007), VI-2 (Ueno et al. 1977), A-2 (Sano et al. 2007), and aspergillic acid (Nishimura et al. 1991). Aspergillic acids have been evaluated for toxicity (Sasaki et al. 1968;MacDonald 1973;Perry et al. 1984), but Sano et al. (2007) suggest that the toxicity of aspergillic acids is so low that it can be present in fermented foods used for consumption. The strains producing aspergillic acid, indicated by the medium AFPA (Aspergillus flavus parasiticus agar), are probably representing Aspergillus flavus sensu stricto, but because of issues with potential aflatoxin production, they are called A. oryzae "short stipes." The indicative red-orange color is caused by reaction of ferric ions with aspergillic acids (Assante et al. 1981) with none of these strains have been reported to produce aflatoxins (Sano et al. 2007).

Aspergillomarasmins
Aspergillomarasmin A, anhydroaspergillomarasmin A, and anhydromarasmic acid are polyamino acid compounds/ phytotoxins related to lycomarasmin from Fusarium (Plattner and Clauson-Kaas 1945;Hardegger et al. 1963), but they have also been found in Aspergillus oryzae or A. flavus (A. "flavus oryzae") (Haenni et al. 1962;Haenni et al. 1965;Robert et al. 1962). Aspergillomarasmin A is very interesting as it inhibits metallo-beta-lactamases and could thus help in overcoming bacterial resistance to penicillin (King et al. 2014;Koteva et al. 2016). There are no data of toxicity of these compounds yet.

Aspirochlorines
Aspirochlorine is a halogenated diketopiperazine with a central disulfide bridge that was first isolated from Aspergillus oryzae IAM-2613 under the name oryzachlorin (Kato et al. 1969). The compound has also been chemically synthesized (Miknis and Williams 1993;Wu et al. 2000). However, oryzachlorin was later shown to be the same as aspirochlorine and A30641 (Sakata et al. 1982(Sakata et al. , 1983(Sakata et al. , 1987a(Sakata et al. , 1987b. It was isolated under the name A30641 from Aspergillus tamarii NRRL 8101, where it was co-occurring with canadensolide (Berg et al. 1976), as was also the case of a strain identified as A. flavus (Sakata et al. 1982). A strain of the latter was not available for more detailed studies. Another strain identified as A. flavus ("MDH-1420") was shown to produce aspirochlorin and the related compound tetrathioaspirochlorine, and evidence for presence of the trithio analogue also (Klausmeyer et al. 2005). Furthermore, a bromoaspirochlorin, dechloroaspirochlorine, and O,Odimethylaspirochlorine have been reported (Sakata et al. 1987). Aspirochlorin has been shown to be a highly selective and potent inhibitor of protein synthesis (Monti et al. 1999) and an effective inhibitor of fungi, bacteria, viruses, and murine tumor cells (Monti et al. 1999;Chankhamjon et al. 2014). For these reasons and because epipolythiodiketopiperazines are generally toxic, the latter authors called aspirochlorin for a mycotoxin. The related mycotoxin gliotoxin was reported from 4 and 13% of clinical Aspergillus flavus strains (Lewis et al. 2005;Kupfahl et al. 2008), but there is some doubt whether these data are correct (Patron et al. 2007;Manzanares-Miralles et al. 2016;Vidal-Garcia et al. 2018). On the other hand, Shaaban et al. (2014) isolated the reduced form of gliotoxin from A. "oryzae" MMAO1, and this latter isolate could be Aspergillus flavus sensu stricto. Gliotoxin-producing isolates have not been available for the scientific community (Varga et al. 2011b).

Asporyergosterol
Asporyergosterol and several other sterols were isolated from isolated from Aspergillus oryzae "cf-2" = CCTCC M 2010045, isolated from a marine alga (Qiao et al. 2010b). The strain isolated could equally well be another Aspergillus, as A. oryzae in principle cannot be isolated from natural sources. An oxylipin and several sterols were isolated from A. flavus, isolated from an alga by the same authors (Qiao et al. 2011). The isolate also produced emeniveol and similar compounds and could probably in reality be A. cejpii, A. niveus, or A. striatus.  Atalla et al. (2003) Apparently both fungus and mycotoxin were misidentified in this work (3S,6S)-Terramide A and B Garson et al. (1986) Listed as being produced also by A. flavus, in addition to A. terreus in AntiBase, no references could be found to the possible fact that A. flavus can produce terramides Terrein Saldan et al. (2018) Identity of strain and compound dubious Taxol El-Sayed et al. (2018) Both producer and secondary metabolite production needs to be verified Violacetin Kobayashi (1966); Aiso et al. (1955) Violacetin is a Streptomyces secondary metabolite, not of fungal origin Zearalenone Atalla et al. (2003) Apparently both fungus and mycotoxin were misidentified in this work

Avenaciolides and canadensolides
Canadensolides are formed via condensation of an acetate derived chain with a TCA cycle intermediate (Brookes et al. 1963;Turner 1971;Tanabe et al. 1973). It was first isolated from "Penicillium" canadense (McCorkindale et al. 1968), but it has been reported once from A. flavus (Sakata et al. 1982), and the related avenaciolide has been reported from A. avenaceus in Aspergillus section Flavi (Brookes et al. 1963;Tanabem et al. 1973;Varga et al. 2011b). The avenaciolides are also present in Aspergillus glaber and A. stramenius from Aspergillus section Fumigati (Ellis et al. 1964;Samson et al. 2007), and the avenaciolides are active against methicillin-resistant Staphylococci (Chang et al. 2015). Avenaciolide is also a specific inhibitor of glutamate transport in rat liver mitochondria (McGivan and Chappell 1970). Isoavenaciolide has been reported as an anti-cancer agent (Al-Tel et al. 2009). However, there are no indications that avenaciolide is a mycotoxin.

Kojic acids
Kojic acid was the first compound to be isolated from Aspergillus oryzae (Yabuta 1912(Yabuta , 1922Birkinshaw et al. 1931;Jennings and Williams 1945;Parrish et al. 1966;Morton et al. 1945;Marston 1949;Bentley 2006). A dimer of kojic acid has been structure-elucidated as the bright greenish yellow flourescence pigment from Aspergillus flavus (Zeringue et al. 1999). Koji acid is common for nearly all species in Aspergillus section Flavi (Varga et al. 2011b). However, kojic acid is not regarded as a mycotoxin (Bentley 2006). The gene cluster coding for kojic acid production is known (Terabayashi et al. 2010). 7-O-acetylkojic acid has also been isolated from A. flavus ).

Kojistatins
An isolate of an industrial strain of Aspergillus oryzae (ATCC 20386 and FERM-15834) produced kojistatin A = CPI-4 and related cystein protease inhibitors, called CPI 1-5 (Sato et al. 1996;Yamada et al. 1998). The kojistatins are nonribosomal peptide-polyketide hybride molecules. There are no data on the toxicity of these compounds.

Maltoryzin
The polyketide maltoryzin was reported from a strain of A. oryzae var. "microsporis" isolated from malting barley (Iizuka and Iida 1962). However, the fungus could also be an Aspergillus clavatus, which is very common in malting barley (Lopez Diaz and Flannigan 1997). A. oryzae or A. flavus has not been reported from malting barley. On the other hand, Bakhali et al. (2015) reported on maltoryzin production by A. flavus from walnuts.

Miyakamides
Miyakamides A 1 , A 2 , B 1 , and B 2 (Shiomi et al. 2002), and oryzamide A 1-2 (Rank et al. 2012) have been reported from both Aspergillus flavus "var. columnaris" FKI-0739 and A. oryzae RIB40 and are NRPs. The miyakamides are antimicrobial compounds, but there are no toxicity data on these compounds. Since the A. flavus strain FKI-0739 produced hydroxyaspergillic acid also (Shiomi et al. 2002), it was probably an A. flavus sensu stricto. As discussed earlier, RIB40 may also in reality be an A. flavus sensu stricto.

Oryzaeins
The polyketides oryzaein A-D, tabaisocoumarin A, caudacoumarin C, versicolol B, and exserolide D and F are antiviral and cytotoxic isocoumarin derivatives isolated from a fungus identified as A. oryzae isolated from the rhizome of the marine Paris polyphylla var. yunnanensis (Zhou et al. 2016), and thus, the producing strain is probably an A. flavus sensu stricto. However, compounds with isochroman chromophores have been found in extracts of some A. oryzae (Frisvad JC, "personal data"). The four oryzaeins had moderate to weak inhibitory effect against some human tumor cell lines (Zhou et al. 2016), but their actual toxicity is unknown.

Oryzines
Oryzines are maleidrides biosynthetically produced from acyl CoA thiolester and from oxaloacetic acid (Wasil et al. 2018). RIB 203, the producing strain, is from sake-koji and thus represents a real A. oryzae. There are no data on the bioactivity of these compounds as yet.

Parasiticolides
Parasiticolide A = astellolide A is a sesquiterpene that was first found in Aspergillus parasiticus from section Flavi (Hamasaki et al. 1975) and Aspergillus stellatus from section Nidulantes (Gould et al. 1981), and later parasiticolide A, dideacetylparasiticolide A, and 14-deacetyl parasiticolide Awere isolated from A. oryzae RIB40 (Rank et al. 2012). Ren et al. (2015) found astellolides A, B, C-E, and F-I in Aspergillus oryzae QXPV-4 isolated from the insect Coccinella septempunctata. The origin of QXPV-4 indicates that this was also an A. flavus sensu stricto, rather than an A. flavus. Shinohara et al. (2016) also found parasiticolides = astellolides in A.oryzae RIB40: 14-deacetyl astellolide A = 14-deacetyl parasiticolide A (already found by Rank et al. 2012), and 14-deacetyl astellolide B. Depending on the opinion of the taxonomic status of A. flavus and A. oryzae, parasiticolides are secondary metabolites of one of these species or both.

Penicillins
Penicillins are non-ribosomally synthesized tripeptides (NRP) that have been reported from A. oryzae (Waksman and Bugie 1943;Foster and Karow 1945;Marui et al. 2010) and A. flavus originally as flavicidin (Bush and Goth 1943;McKee and MacPhillamy 1943;McKee et al. 1944;Waksman and Bugie 1943;RG Benedict, unpublished in Raper 1946). This important antibiotic is not regarded as a mycotoxin, but it is unwanted in industrial fermentations due to its wide use to treat microbial infections.

Sporogens
Sporogen AO1 (=13-desoxyphomenone) is a sesquiterpenoid that was isolated from Aspergillus oryzae NOY-2, but the strain is not available to the scientific community. This compound induces conidiation in a less sporulating strain (Tanaka et al. 1984,b). Sporogen AO1 has later been found in strains of A. flavus (Frisvad and Larsen, unpublished). Phomenone, related to sporogen AO1, is a potent inhibitor of protein synthesis (Moule et al. 1977) and has moderate toxicity to shrimps (Capasso et al. 1984). Phomenone was recently shown to stimulate pro-inflammatory responses in murine cells and thus may exacerbate allergic reactions if inhaled (Rand et al. 2017). There are no direct data showing that these compounds are mycotoxins, but they are not unlike the mycotoxin PR-toxin in structure (Cole and Cox 1981; Moule et al. 1977;Capasso et al. 1984

TMC-2A, -2B and -2C
The NRP-derived peptide-like compounds TMC-2A, -2B, and 2C were isolated from a strain identified as A. oryzae A374 = FERM P-14934 Asai et al. 1997). From the description of the strain, and as it was isolated from soil, it appears that the strain is rather an Aspergillus tamarii, as the conidia were large, distinctly roughened, and brown. These peptide-like compounds may be used as lead compounds to find better rheumatoid arthritis inhibitors, but toxicity data have not been presented.

Ustilaginoidins
The polyketide ustilaginoidin C was isolated as a suggested conidium pigment from A. parasiticus (Brown et al. 2003), and a compound with the same chromophore has been isolated from A. flavus (Frisvad, JC, unpublished data), so it could be representing the general naphtho-γ-pyrone pigment type produced in Aspergillus section Flavi. There are no toxicity data on these compounds.

Ustiloxin B
This ribosomally produced cyclic peptide (RIPS, ribosomally produced peptides) compound was isolated from Aspergillus flavus and A. oryzae (Umemura et al. 2013(Umemura et al. , b, 2014Ye et al. 2016;Yoshimi et al. 2016). The ustiloxins are phytotoxins first isolated from Villosiclava virens (= Ustilaginoidea virens), and they exibit potent antimitotic activity and inhibit microtubule assembly (Koiso et al. 1994), and they have also been called mycotoxins (Koiso et al. 1992). The ustiloxins are not established as mycotoxins. A. oryzae RIB40 does not produce ustiloxin B, probably because of the large deletion of the ustR gene encoding a transcriptional regulation that regulates ustiloxin B production (Umemura et al. 2014).
Secondary metabolites that are not produced by A. flavus or A. oryzae Due to the close relatedness between A. flavus and A. oryzae as well as their similarity to other species, some reports have misleadingly described production of secondary metabolites in A. flavus or A. oryzae that they do not produce (Table 8).

Secondary metabolites described in T. reesei
Peptaibol non-ribosomal peptides (peptaibiotics) and similar peptides are produced by many Trichoderma species ), but it is only paracelsin A, C, and D in this class that have been reported from T. reesei (Brückner and Graf 1983;Brückner et al. 1984;Pócsfalvi et al. 1997;Przybylski et al. 1984 Paracelsins are linear peptides containing a high level of uncommon amino acids, alphaaminoisobutyric acid (Aib), and isovaline (Iva), together with an acetylated N-terminal amino acid and a Cterminal amino alcohol (Pócsfalvi et al. 1997). These compounds have shown antimicrobial activity. There are no data on the toxicity of the paracelsins. The sorbicillin biosynthetic family compounds have been reported from Trichoderma sp. USF 2690 (Abe et al. 2001) (strain not available in any culture collection), and it is only mentioned to be a product of T. reesei in the Antibase secondary metabolite database. The Trichodermatides (A-D) are produced by a fungus claimed to be a marine T. reesei (Sun et al. 2008;Shigehisa et al. 2015), but the culture is unavailable in culture collections, and may be one of the many other known Trichoderma species. T. reesei may also produce some other non-ribosomal peptides, including intracellular and extracellular siderophores ). Siderophores such as ferricrocin have not been claimed to be toxic. Among the polyketides, the genes for a conidium pigment related to aurofusarin and bikaverin have been reported ). This polyketide compound (not structure elucidated) is probably a precursor for the green pigment (melanin) in the conidia of T. reesei, and generally, these conidium pigments have not been claimed to be toxic. T. reesei have PKS gene clusters for production of other polyketides, which are not unlike those for citrinin and fumonisins (Baker et al. 2012), but neither citrinin nor fumonisins have been detected in T. reesei.
In conclusion, the only secondary metabolites that appear to be naturally produced by T. reesei are the paracelsins.
Based on genome sequencing data (Schmoll et al. 2016), several potential toxic secondary metabolites may be produced under special conditions. Such secondary metabolites have not been detected yet in T. reesei, however. Genome sequencing showed that there are 8 NRKS, 11 PKS, 2 NRPS-PKS hybrid, and 12 terpenoid synthase encoding genes (Schmoll et al. 2016;Zeilinger et al. 2016). The LaeA and VELVET regulatory genes are important for the expression of secondary metabolites in T. reesei, but nevertheless, only few of the putative gene clusters for secondary metabolites seem to be actually expressed.

Conclusions
Aspergillus oryzae produce few recognized mycotoxins, and they are only produced by few strains. If they are produced, there are genetic means of inactivating the biosynthetic pathways, so isolates of the species can be exploited for production of enzymes and as a transformation host for industrially relevant secondary metabolites or enzymes. Some isolates of Aspergillus niger can produce three types of mycotoxins, ochratoxin A, fumonisin B 2 (B 4 and B 6 ), and oxalic acid. Again, genetic means have been employed to inactivate the gene clusters for ochratoxins and fumonisins, while accumulation of the less toxic oxalic acid can be avoided by chosing an optimal substrate or use optimal procedures for the industrial products. Trichoderma reesei cannot produce any recognized mycotoxins and is one of the most important enzyme producers in the industry. All three species can produce interesting secondary metabolites, of which some are drug lead candidates and others, such as citric acid, are important bulk chemicals that are produced by fermentation.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.   JC (1989) The connection between the penicillia and aspergilli and mycotoxins with special emphasis on misidentified isolates.