A predominant genus of SGM-associated fungi is Fusarium, which contains numerous mycotoxigenic species and is among the most common genera in the disease complex (Williams and Rao, 1981). The most common species associated with sorghum grain mold are F. verticillioides (synonymous with F. moniliforme in the literature for the purposes of this review), F. thapsinum, and F. proliferatum, although many others have also been isolated from sorghum grain (Pena et al., 2019). F. verticillioides and related taxa produce the mycotoxin fumonisin, which has been implicated in human and animal diseases such as esophageal cancer, equine leukoencephalomalacia, and impaired growth (Chen et al., 2018). In more temperate growing environments, DON, a mycotoxin produced by F. graminearum, is a major concern across susceptible cereal crops (Das et al., 2011; Dweba et al., 2017). Sorghum contamination with DON has been occasionally reported (Das et al., 2011), but F. graminearum is demonstrably less virulent in the disease complex than other fusaria, such as F. thapsinum (Mpofu and Mclaren, 2014).

The Aspergillus genus contains among the most prolific producers of the mycotoxins aflatoxin (AF) and ochratoxin. The Aspergillus species common in the SGM disease complex include soil-borne saprophytes that can opportunistically infect the developing caryopsis following events such as insect damage (Pfliegler et al., 2020). The Aspergilli commonly found in sorghum grain molds are: A. flavus, A. niger, A. parasiticus, A. fumigatus, and A. glaucus (Little et al., 2011; Gemede and University, 2016). A. flavus and A. parasiticus are the most notable producers of aflatoxins, and while A. fumigatus produces the immunosuppressive mycotoxin gliotoxin to increase virulence to plants, animals and humans, it is not known to produce aflatoxin (Kamei and Watanabe, 2005). The risks associated with aflatoxin exposure are most pronounced in tropical and subtropical environments, where Aspergillus spp. proliferate abundantly, and the risk is less in regions with both cooler and drier off seasons (Cotty et al., 1994; Amaike and Keller, 2011). Additionally, growing regions with the inability to utilize modern grain storing technologies, which boast increased aeration and drying of stored grain, are at risk of enhanced fungal growth during storage.

The consumption of aflatoxin-contaminated grain can lead to aflatoxicoses (Marin et al., 2013; Mahato et al., 2019), which are some of the most widespread mold-associated diseases globally (Latgeì, 1999). Aflatoxin exposure can result in cancer, lack of immune suppression, liver damage, and mortality (Bennett and Klich, 2003; Sarma et al., 2017). From a public health view and in regard to the SGM complex, A. flavus and its close relative within the Flavi section, A. parasiticus, are of the highest concern within the Aspergillus genus (Sarma et al., 2017). Compared to A. parasiticus, A. flavus has a wide host range, and is much more prevalent in the SGM complex across locations (Amaike and Keller, 2011; Little et al., 2011; Gemede and University, 2016; del Palacio and Pan, 2020). Out of the more than 20 known aflatoxins there are four major aflatoxins which are a focus of studies for their abundance in foods and toxicity: B1, B2, G1, G2, and M1 (Iqbal et al., 2015; Kumar et al., 2017). AFB1 is the most toxic of these four, being a potent genotoxic and hepatoxic agent as well as being classified as a group 2A carcinogen by the International Agency for Research on Cancer (IARC) (Khoury et al., 2011). The A. flavus S and L strains are prolific producers of aflatoxins B1 and B2, and in addition, the L strains produce aflatoxins G1 and G2 (Amaike and Keller, 2011).

Under Aspergillus infection, sorghum has been shown to produce varying levels of antifungal proteins including sormatin, glucanases, and chitinases (Seetharaman et al., 1997; Ratnavathi and Sashidhar, 2004). Ratnavathi and Sashidhar (2004) showed chitinase as a response mechanism to Aspergillus from relatively strong positive correlations between chitinase activity and aflatoxin levels for both white-pericarp (r² = 0.482) and red-pericarp (r² = 0.600) sorghums. The authors added that the lower rate of chitinase activity in red to white-seeded sorghums is likely due to red-seeded sorghums reliance on a constitutively higher presence of polyphenols. Chitinase and glucanase production in an effort to fragment a pathogen cell wall is a common defense mechanism in host plants (de Ferreira and da Monteiro, 2017), and while further studies in sorghum are limited, chitinase production has been shown to be an effective defense response to Aspergillus in maize and peanut (Fountain et al., 2015; Hawkins et al., 2015).

In addition to their roles as pathogens in the SGM disease complex, Aspergilli also constitute a serious post-harvest spoilage threat for sorghum and other crops when grain is stored under sub-optimal conditions conducive for fungal growth and mycotoxin accumulation. Post-harvest genetic resistance is especially important in ensuring the prevention of aflatoxin infection throughout storage, as Aspergillus remains a top threat to storage safety. Sorghum is a staple crop in many developing countries which lack proper storage facilities capable of limiting contamination, and post-harvest genetic resistance is an important defense measure which prevents further expenses such as fumigation from occurring (Gemede and University, 2016; Meseka et al., 2018; Soni et al., 2020).

Compared to the Aspergilli associated with maize and groundnuts, the crops most vulnerable to aflatoxin accumulation in many environments, there is relatively little understanding of the mycotoxigenic characteristics of sorghum-associated Aspergillus strains. In parts of the world where sorghum is a major staple, notably India and sub-Saharan Africa, some evidence has accrued enabling characterization of aflatoxin biosynthetic potential of sorghum-derived isolates. In Egypt, Abdel-Sater et al. (2017) found that local sorghum-derived isolates of Aspergillus flavus produced an average of 254 μg/kg AFB1 when incubated on potato dextrose agar at 28°C for 10 days. Tunisian sorghum-derived isolates, on the other hand, produced just 1.15 μg/kg AFB1 on whole sorghum grains incubated at 37°C for 7 days (Lahouar et al., 2016). This discrepancy is consistent with earlier evidence that the thermal optimum for aflatoxin production is ∼30°C (Mousa et al., 2011), but could also be indicative of differential mycotoxigenicity of fungal isolates even from the same geographic region.

In India, Aspergillus flavus isolates from sorghum grain produced on average 2567 μg/kg AFB1 in yeast extract broth when incubated at 27°C for 7 days (Usha et al., 1994). Other studies in India also report aflatoxin biosynthetic potential in this ballpark in culture media (Somashekar et al., 2004; Reddy et al., 2011). Incubation on sorghum grain as a substrate has yielded much lower levels of aflatoxin production by sorghum-derived Aspergillus isolates in India (Mukherjee and Lakshminarasimham, 1995). This is illustrative of the importance of substrate characteristics in aflatoxigenesis; Winn and Lane (1978), for example, observed marked differences in aflatoxin deposition across sorghum substrates in different forms (whole, cracked, and ground) (Winn and Lane, 1978).

Another ascomycete genus commonly associated with SGM is Curvularia, particularly C. lunata, which has been shown to play an increased role in the disease complex in drier environments and to have greater negative impact on germination in early season (Prom et al., 2016). Similarly, to F. verticillioides, Curvularia is typically associated with early-stage infection of the developing sorghum caryopsis and is known to elicit defense response genes in the host plant (Little and Magill, 2003). There is some evidence that Curvularia may be more competitive in the disease complex than Fusarium spp. in moist environments and that infection by this fungus substantially reduces seed viability even without presenting severe symptoms of infection (Prom et al., 2003). While certainly regarded as an important disease agent in SGM, there is no confirmed evidence of mycotoxin deposition by Curvularia in sorghum and it is unlikely that this genus plays and active role in sorghum mycotoxin contamination.

The Colletotrichum genus, also the causal agent of another economically important sorghum disease, anthracnose, is known to play a role in the SGM disease complex. The predominant species implicated in both pathosystems is C. graminicola, which is abundant in warm, humid sorghum production contexts and has high levels of genetic diversity (Cuevas et al., 2016). While Colletotrichum spp. can occasionally predominate over other taxa in the SGM disease complex (Bandyopadhyay et al., 2000), the relative importance of this species is likely highly dependent on climatic conditions in the growing environment. In Texas, a low-humidity environment, for example, there is evidence that Colletotrichum spp. is a far less prominent member of the fungal assemblage than Fusarium, Curvularia, or Alternaria taxa (Prom et al., 2011). Like Curvularia spp., Colletotrichum is likely not implicated directly in mycotoxigenesis in the SGM disease complex. However, it remains unknown the extent to which atoxigenic taxa involved in SGM symptom manifestation modulate or enable toxin deposition by toxigenic fungi via disruption of physical, biochemical, or immune defenses of the host – this should be the subject of future investigation into the host-pathogen and pathogen-pathogen interactions within the disease complex.

While not known to infect early flowering tissue of sorghum, A. alternata and A. solani are common contributors to SGM disease symptoms and have also been implicated in mycotoxin contamination (Bandyopadhyay et al., 2000). In certain environments, such as Texas and Turkey, this genus can be the most prominent member of the SGM fungal assemblage (Prom et al., 2020a). Alternaria is a diverse genus of ascomycete fungi, which is a prolific mycotoxin producer and is implicated in a range of plant diseases, notably in high-value crops (Logrieco et al., 2009). Like Fusarium spp., the mode of infection of Alternaria spp. in the SGM disease complex is largely via airborne spores, which are present in the growing environment throughout caryopsis development (Bandyopadhyay et al., 1991). Sorghum-associated Alternaria can be prolific producers of key mycotoxins such as tenuazonic acid, alternariol, alternariol methyl, altenuene, and altertoxin. The aforementioned Alternaria mycotoxins can be mutagenic and cytotoxic and, and while less toxic than aflatoxins or fumonisins, are believed to have synergistic effects (Patriarca et al., 2007). At present, tenzuazonic acid contamination in sorghum- or millet-based infant foods is the only governmentally regulated Alternaria toxin (Tralamazza et al., 2018).

Sorghum contamination with Alternaria mycotoxins has been observed in many production contexts globally. Despite the known role of this genus in the disease complex, relatively little attention has been given to the toxigenicity of Alternaria associated with SGM, compared to higher-profile toxins in public health dialog. In India, isolates of sorghum-derived Alternaria tenuissima produced 32.6, 22.1, 9.2, 2.7, and 1.8 μg/g of these toxins, respectively, on rice medium incubated at 25°C for 28 days (Bilgrami et al., 1995). Epicoccum sorghinum (formerly Phoma sorghinum) is another important contributor to SGM symptoms in some production systems and has also been implicated in the production of tenuazonic acid. A recent study from Brazil documented substantial tenuazonic acid production by sorghum-derived isolates between 0.0986 and 148 μg/g incubated on rice substrate at 25°C for 21 days (Oliveira et al., 2019).

The phenylpropanoid pathway begins with the enzymatic conversion of phenylalanine by phenylalanine ammonia lysase (PAL) (Tohge et al., 2013), which then undergoes additional enzymatic conversions resulting in naringenin chalcone, the intermediate that chalcone isomerase (CHI) ultimately converts to the flavanone naringenin and marks the beginning of the flavonoid biosynthesis pathway (Figure 2). Many of the phenolic acids contributing to SGM disease resistance are derivatives of phenylalanine, and while part of the phenylpropanoid pathway, are not part of the downstream flavonoid pathway.

Phenolic acids represent the largest and simplest group of non-flavonoid phenolics in sorghum grain. Having been subject to an array of anti-mycotoxin research, phenolic acids have been shown to both inhibit and activate mycotoxin production (Atanasova-Penichon et al., 2016; Stuper-Szablewska and Perkowski, 2017). The main phenolic acids connected to SGM resistance reported in sorghum consist of the (1) cinnamic acids: ferulic, chlorogenic, caffeic, p-coumaric, and sinapic acids (2) benzoic acids: gallic, protocatechuic, vanillic, and syringic acids (Xiong et al., 2019b). In cereals, cinnamic acids such as ferulic and p-coumaric acid have been shown to have inhibitory effects for Fusarium growth and mycotoxin production (Ferruz et al., 2016b), and additionally shown to directly inhibit F. verticillioides and F. proliferatum mycelial proliferation and fumonisin production on maize-based media (Ferrochio et al., 2013). Studies have shown caffeic acid and vanillic acid to drastically reduce FB1 production and mycelial growth of Fusarium (Beekrum et al., 2003; Schöneberg et al., 2018). In contrast to cinnamic acids, benzoic acids with the exception of syringic acid have generally been shown to have activating effects, even providing slight stimulation to mycelial growth (Boutigny et al., 2009; Atanasova-Penichon et al., 2016; Schöneberg et al., 2018).

Phenolic acids exist in both bound and free forms. Bound forms represent 70–95% of phenolic acid in sorghum grain (Xiong et al., 2019b) and present a multifaceted grain mold resistance source. Alongside of contributing to antioxidant potential and limiting mycotoxins on a cellular level, bound forms of phenolic acids strengthen grain hardness (Chiremba et al., 2012). Alongside ferulic acid, p-coumaric acid has also been shown to positively correlate with sorghum grain hardness (Chiremba et al., 2012). Grain hardiness contributes to grain mold resistance by reducing weathering of the pericarp and ultimately limits the ability of grain mold pathogens to degrade the endosperm. This connection of phenolic acids to grain hardness suggests an interesting contribution to SGM-resistance (Jambunathan et al., 1992; Waniska et al., 2001).

Compared to bound phenolic acids, free phenolic acids represent a smaller portion of total phenolic acids within sorghum, maize and wheat. Additionally, biosynthesis levels undergo more drastic fluctuations than their bound counterparts (Atanasova-Penichon et al., 2016). In many cereals, free chlorogenic acid is the most common free phenolic acid. Gauthier et al. (2016) demonstrated in maize that chlorogenic acid is a valuable source of resistance to F. graminearum proliferation and limiter of type B trichothecene mycotoxin production. While chlorogenic acid exhibits antifungal properties of its own, the authors showed that F. graminearum transforms host chlorogenic acid into caffeic acid, which exhibits antifungal properties of increased potency over the former. The authors concluded this phenomenon regarding chlorogenic acid exhibits “pro-drug” qualities, demonstrating its highest toxicity levels when degraded into caffeic acid (Gauthier et al., 2016). Chlorogenic acid is shown to be a major phenolic acid present in sorghum (Pasha et al., 2015), shown to play roles in essential physiological processes such as photosynthesis (Turner et al., 2016); however, the role of chlorogenic acid as a fungal inhibitor in sorghum has not been explored to the extent that it has in maize or wheat.

Both the strain of pathogen and host are critical determinants in the overall efficiencies of phenolic acids as they interact with pathogens in inhibitory, neutral or activating roles (Ferruz et al., 2016a; Gauthier et al., 2016). Research has helped create a solid understanding of the roles phenolic acids play in diseases such as wheat Fusarium head blight and maize ear rot. Even though sorghum boasts a diverse and abundant phenolic profile well suited for further exploration in relation to biotic resistance, phenolic acid activity has not been as extensively studied with sorghum-based substrates using grain mold pathogens and related mycotoxins. Presence of common phenolic acids across crops and studies showing functional properties of phenolic acids on mycotoxins demonstrates a potential for increased contribution to grain mold resistance in sorghum. To understand the nuances of phenolic acid based-sorghum grain mold resistances, interactions of phenolic acids with grain mold pathogens must undergo further research using sorghum-based media or in vivo studies to understand these relationships in planta.

Naringenin marks the beginning of the flavonoid biosynthesis pathway, acting as a substrate that is subject to competition from an array of enzymes: flavonoid 3′ hydroxylase (F3′H), flavanone-3-hydroxylase (F3H) as well as dihydroflavonol 4-reductase (DFR) and the DFR-like enzyme flavanone 4-reductase (FNR) (Xiong et al., 2019a; Figure 2). The ultimate fate of naringenin is heavily influenced by genotype by environment interactions, with studies such as (Taleon et al., 2012) demonstrating 48% of total variation in naringenin abundance being a consequence of environmental variation, and separate studies having reported major influence on naringenin by biotic factors such as fungal ingression (Boddu et al., 2005). The fate of naringenin is also tissue-dependent, as downstream products such as 3-deoxyanthocyanidins (3-DAs) are maintained constitutively in unchallenged grain while synthesis is only induced in leaf tissue upon fungal ingress (Boddu et al., 2005; Ibraheem et al., 2015; Kawahigashi et al., 2016).

Sorghum demonstrates a high level of host sensitivity to fungal ingression, as spatial accuracy in the induction of phytoalexins critical to SGM resistance occur through site-specific synthesis at place of infection. Naringenin is a precursor to many of these phytoalexins and marks an important junction in the flavonoid pathway. A resistant response to fungal ingression is dependent on the availability of naringenin to enzymes that synthesize SGM resistance-related compounds. As described in detail in later sections, synthesis of 3-DA phytoalexins is an inducible resistance-related host response to SGM, and a lack of activity by F3H can be a consequence of inducible synthesis of 3-DAs resulting from increased channeling of DFR3/FNR on naringenin (Figure 2; Liu et al., 2010). As will be explored throughout this text, the availability of naringenin at optimal time and point of infection is crucial to host response. At its core, a large portion of host biochemical responses to grain mold may in many ways a consequence of the management of the enzymatic competition over naringenin.

Condensed tannins (proanthocyanidins) are well understood for their roles in increased antioxidant capabilities and significant correlations with SGM resistance (Harris and Burns, 1973; Forbes, 1986; Melake-Berhan et al., 1996; Dicko et al., 2005; Cuevas et al., 2019b). The presence of condensed tannins throughout SGM resistant germplasm is interesting, as there is little knowledge to suggest condensed tannins demonstrate direct toxicity to fungal pathogens as do products of other branches of the flavonoid biosynthesis pathway such as 3-DAs and flavones (Melake-Berhan et al., 1996; Du et al., 2009; Nida et al., 2021). Regardless, the use of condensed tannins is limited in sorghum germplasms for their reduction in protein digestibility of animals. However, the toxicity of condensed tannins to humans has been misunderstood, and have been shown to provide antioxidants, fiber and reduce obesity as a food source (Dykes et al., 2005). Sorghum tannin types have been divided amongst three categories: type I) no tannins; type II) tannins in pigmented testa; type III) tannins in pigmented testa and pericarp (presence of spreader gene) (Waniska et al., 2001).

SbF3H1 of sorghum codes the flavanone-3-hydroxylase (SbF3H) enzyme that is capable of converting naringenin to condensed tannins (Figure 3). As another competitive enzyme for naringenin, host management of SbF3H activity could play an important role in managing enzymatic competition for naringenin (Mizuno et al., 2014). SbF3H converts flavanones to dihydroflavonols from which SbDFR1 synthesizes flavan-3,4,-diols (leucoanthocyanidins). Leucoanthocyanidins represents a junction in this branch of flavonoid synthesis from which anthocyanidins or flavan-3-ols can be synthesized. Condensed tannins are the result of oligomerization and polymerization of flavan-3-ol compounds (He et al., 2008; Wu et al., 2012).

Early understandings of SGM resistances recognized condensed tannin content as a lone dominant influence on SGM resistance (Harris and Burns, 1973). While this outlook was justified at the time, it was chemical studies such as Menkir et al. (1996) that began to expand this perspective to consider a more complete host phenolic profile by showing inconsistencies in condensed tannin content of SGM resistant phenotypes. Recent studies have assisted in the accurate understanding of the contributions of condensed tannin content to SGM resistance as the expansive genetic underpinnings of grain mold resistance continue to be elucidated. Biosynthesis of condensed tannins is controlled by the Tannin-1 (TAN1) transcription factor (Wu et al., 2012). TAN1 demonstrates influence over enzymatic competition for naringenin by regulating expression of enzyme-coding genes in the pericarp responsible for the transcription of CHI, F3H, F3′H, DFR and ANS (Wu et al., 2012; Mizuno et al., 2014), and consequently influencing the synthesis of 3-DAs, flavones, and anthocyanins in addition to condensed tannins (Liu et al., 2010).

Wu et al. (2012) found the TAN1 allele was present throughout 78% of a diverse collection of sorghum germplasm consisting of 161 accessions. Similar to the 161 accessions used by Wu et al. (2012), Cuevas et al. (2019a) found that a functional TAN1 is present in 79% of the sorghum association panel (SAP) (Casa et al., 2008), an interesting finding considering that a majority of the accessions within the SAP were also found be highly susceptible to SGM. The study by Cuevas et al. (2019a) demonstrated that genotypes harboring a non-functional TAN1 allele (tan1-a) were more susceptible to grain mold on average compared to accessions with the functional allele. Interestingly, the Genome-Wide Association Studies (GWAS) of the SAP did not associate TAN1 with grain mold resistance, illustrating the complexity of the SGM resistance phenotype that supports the author’s conclusion that resistance is a culmination of many additional factors in addition to the concentration of tannins (Cuevas et al., 2019a).

Another recent SGM association study employing 635 Ethiopian genotypes by Nida et al. (2021) identified a collection of protein-coding loci, and consistently detected SNPs within the TAN1 coding region. The authors suggested TAN1’s role in the flavonoid pathway affecting biosynthesis of 3-DAs and tannins could contribute to TAN1’s role in grain mold resistance. While widespread presence of TAN1 across mapping populations is a factor of both aforementioned studies, a key difference is the differing levels of resistance between the two populations, with the former (SAP) demonstrating increased susceptibility overall than the latter (Ethiopian). This notable difference in SGM resistance between mapping populations may be a consequence of varying levels of TAN1’s role in transcriptional regulation of the enzyme-coding genes in the flavonoid pathway, and could be factoring into why TAN1 was not associated with SGM resistance in the SAP, but consistently identified in the Ethiopian panel.

Additional GWAS such as those by Prom et al. (2020a) and Ahn et al. (2019) further highlight the genetic complexities of SGM resistance, as many identified genes show minor rather than major effects, such as a variety of zinc finger proteins, resistance genes (R-genes), and the underlying genetics associated with systemic acquired resistance (SAR) mechanisms (Ahn et al., 2019; Prom et al., 2020a). While condensed tannin content remains an important component of host resistance, it only represents a portion of the phenolic profile that contributes to a complex host resistance mechanism. Many additional components of host resistance such as sorghum SAR mechanisms, R-genes and the phenolic profile in its entirety should be explored in order to provide a more complete understanding of mold resistance in sorghum germplasms (Cuevas et al., 2019a). Additionally, at the core of the grain mold resistance phenotype in sorghum is host management of enzymatic competition over naringenin and the ability to ensure naringenin availability to the correct branch of the flavonoid pathway at time of grain mold infection.

Flavone synthase II (FNSII) enzymes are responsible for the synthesis of the flavones apigenin and luteolin (Figure 5), and like F3′H, are part of the cytochrome P450 superfamily (Yonekura-Sakakibara et al., 2019). Flavones in sorghum represent a separate branch of the flavonoid biosynthesis pathway that begins with the hydroxylation of naringenin or eriodyctiol by SbFNSII resulting in the formation of 2-hydroxyflavone intermediates (Du et al., 2009). Subsequent conversion of 2-hydroxyflavones by an unknown ANS results in the formation of the flavones apigenin and luteolin (Figure 5). Flavones are one of the largest subgroups of flavonoids and are well understood for their ability to affect color by complexing with anthocyanins as well as antioxidant potential and nutrient value amongst other benefits (Martens and Mithöfer, 2005). Additionally, flavones such as luteolin and apigenin have been shown to demonstrate levels of fungal toxicity of their own, with increased fungal toxicity and higher presence in resistant germplasm demonstrated by the former (Du et al., 2009; Aboody and Mickymaray, 2020). Research in sorghum has suggested flavones are a phytoalexin due to a combination of the aforementioned fungal toxicity and pathogen-related induction (Du et al., 2009). Functional characterization of flavone synthase genes in other crops however, has reported an interesting mechanism in balancing the competition between FNSII and other enzymes and even pathogen-manipulation of flavone production potentially being a component of host susceptibility (Ferreyra et al., 2015).

Darker pericarp color and purple plant sorghum lines have been generally shown to be more resistant to disease (Melake-Berhan et al., 1996; Menkir et al., 1996; Kawahigashi et al., 2016). Kawahigashi et al. (2016) reported that during tissue wounding, red pericarp, purple plant sorghum types favor conversion of naringenin and eriodyctiol by SbFNR (3-DA synthesis), while seemingly suppressing conversion by SbFNSII - suggesting increased reliance on flavan-4-ols and 3-DA phytoalexins pathways over flavones (Dykes et al., 2005; Kawahigashi et al., 2016). On the contrary, tan plants favor conversion of naringenin and eriodyctiol by SbFNSII while seemingly blocking the production of flavan-4-ols, resulting in leaf and glume tissue showing higher levels of apigenin and luteolin over 3-DAs (Kawahigashi et al., 2016; Mizuno et al., 2016). This phenomenon could potentially be a factor contributing to the higher antioxidant capabilities and disease resistance of purple plant/dark pericarp sorghum genotypes over tan plants.

The findings described above from Kawahigashi et al. (2016) suggest potential mechanisms of sorghum in managing enzymatic activity on naringenin to balance compound synthesis between separate flavonoid pathway branches. Research in maize and Arabidopsis has begun to show this as an important trait of SA-mediated resistance, a phytohormone commonly associated with SAR responses. Flavone synthase enzymes responsible for flavone synthesis such as FNSII are widely present throughout the flavonoid biosynthesis pathways of plants. FNSI and FNSII enzymes of maize are functionally similar to SbFNSII, as both play roles in the synthesis of flavones from naringenin and both have been shown to play an important role in the SA – flavone cross talk affecting maize salicylic acid-mediated resistance (Ferreyra et al., 2015; Righini et al., 2019). It is important to note that FNSI distinctly differs from FNSII in that it is part of the 2-oxoglutarate-dependent dioxygenase family and shows higher sequence identity to F3H than to FNSII (Du et al., 2009; Yonekura-Sakakibara et al., 2019). While maize has been shown to rely on both ZmFNSI and ZmFNSII, sorghum has thus far been shown to be reliant on SbFNSII for flavone synthesis (Righini et al., 2019). An important dissimilarity in the roles of FNSI and FNSII exists: FNSI converts flavanones directly to flavones, while most FNSII enzymes convert flavanones to 2-hydroxyflavanones prior to conversion to flavones by an unknown protein (Figure 5; Du et al., 2009; Kawahigashi et al., 2016).

ZmFNSI-1 is commonly coexpressed with maize R2R3-MYB transcription regulator P1 (Ferreyra et al., 2015), an ortholog of the critical SGM resistance gene Y1 (Boddu et al., 2005; Nida et al., 2019). Transcriptional studies show ZmFNSI-1 is highly active in maize pericarps and silk - tissues commonly under pressure from ear rot-related pathogens (Righini et al., 2019). In maize and Arabidopsis, a SA – flavone cross talk that fine-tunes homeostasis between the phytohormones and flavones has been well described. Studies have characterized an Arabidopsis SA 5-hydroxylase DMR6 (Downy mildew resistance), a recently reported homolog of ZmFNSI-1 (Zhang et al., 2017b). In Arabidopsis, the ZmFNSI-1 homolog DMR6 was shown to be responsible for controlling levels of salicylic acid (SA) and naringenin through use of either compound as a substrate. However, studies show that DMR6 prefers SA as a substrate over naringenin, showing that increased availability of SA over naringenin to DMR6/ZmFNSI-1 will consequently result in higher availability of naringenin. The authors conclude that while enhanced SA levels are responsible for pathogen resistance, lowered levels of apigenin and luteolin are likely not directly connected to resistance. Rather, the resistant mechanism is a result of the preoccupation of ZmFNSI-1/DMR6 with the hydroxylation of SA rather than naringenin, increasing the availability of naringenin as a substrate to other resistance-related enzymes to increase phytoalexin production (Zhang et al., 2017b).

Metabolic studies of sorghum under Burkholderia andropogonis (leaf blight) infection point toward induction of flavone synthase as a defense response (Mareya et al., 2019), while studies in Arabidopsis and maize have suggested the potential of flavones in active immune suppression through metabolic manipulation by the pathogen resulting in host susceptibility (Ferreyra et al., 2015). Studies regarding pigmented sorghums suggest a role in limiting SbFNSII activity to support resistance related enzymes such as SbFNR (Dykes et al., 2005; Kawahigashi et al., 2016). Additionally, recent SGM association studies identified BTB/POZ domains within SAR response pathways that involve phytohormone induction (Cuevas et al., 2019a). Rather than SA-induced resistance as described above, Liu et al. (2010) found that expression of SbDFR3 and 3-DA pathways were induced by methyl jasmonate treatment of sorghum roots and antagonized by SA. The enzymatic competition for naringenin in sorghum and coexpression of Y1 ortholog P1 with ZmFNSI-1 in maize suggest flavones may be carefully managed in SGM interactions; however, host mechanisms must be further characterized in reproductive tissue using grain mold pathogens to elucidate this relationship.

With confirmation of the role of Y1 in SGM resistance, the combined increased expression of SbDFR3 and elucidation of implicated SAR mechanisms identified in SGM resistant sorghum suggests there is potential for heightened enzymatic competition for naringenin and a phytohormone-mediated mechanism for management of enzymatic activity on naringenin (Nida et al., 2019; Prom et al., 2020a). The presence of flavones in SGM resistant varieties has been demonstrated, but further functional characterization of SbFNSII in SGM pathosystems is required to understand to which extent that host reliance on flavone synthesis branches of the flavonoid pathway over SbDFR3/SbFNR and F3H-mediated branches contributes to a SGM resistant phenotype.

Part of the cytochrome P450 superfamily, flavonoid 3′-hydroxylase enzyme (F3′H) plays a potentially important role in the SGM response of sorghum as it is responsible for the conversion of naringenin to the flavanone intermediate eriodyctiol (Figure 2; Poloni et al., 2014; Yonekura-Sakakibara et al., 2019). Through hydroxylation of the 3′ position of the B-ring of naringenin, F3′H synthesizes eriodyctiol, the precursor to luteolinidin and luteolin (Figures 4, 5; Mizuno et al., 2016). Luteolinidin has been shown to be present at a higher rate than apigenidin throughout red pericarp sorghum, a pigment trait that correlates with SGM resistance (Dykes et al., 2005). Additionally, luteolinidin exhibited increased fungal toxicity over apigenidin (Nicholson et al., 1987). Luteolin has also been described to exhibit higher presence in disease resistant sorghum lines as well (Du et al., 2009). This phenomenon is suggestive that the extra enzymatic step in F3′H hydroxylation to produce eriodyctiol as an intermediate may play a role in enhancing resistance mechanisms to SGM pathogens.