Ethylenediaminetetraacetic Acid Disodium Salt Acts as an Antifungal Candidate Molecule against Fusarium graminearum by Inhibiting DON Biosynthesis and Chitin Synthase Activity

Fusarium fungi are the cause of an array of devastating diseases affecting yield losses and accumulating mycotoxins. Fungicides can be exploited against Fusarium and deoxynivalenol (DON) production. However, Fusarium resistance to common chemicals has become a therapeutic challenge worldwide, which indicates that new control agents carrying different mechanisms of action are desperately needed. Here, we found that a nonantibiotic drug, ethylenediaminetetraacetic acid disodium salt (EDTANa2), exhibited various antifungal activities against Fusarium species and DON biosynthesis. The infection of wheat seeding caused by F. graminearum was suppressed over 90% at 4 mM EDTANa2. A similar control effect was observed in field tests. Mycotoxin production assays showed DON production was significantly inhibited, 47% lower than the control, by 0.4 mM EDTANa2. In vitro experiments revealed a timely inhibition of H2O2 production as quickly as 4 h after amending cultures with EDTANa2 and the expression of several TRI genes significantly decreased. Chitin synthases of Fusarium were Mn2+-containing enzymes that were strongly inhibited by Mn2+ deficiency. EDTANa2 inhibited chitin synthesis and destroyed the cell wall and cytomembrane integrity of Fusarium, mainly via the chelation of Mn2+ by EDTANa2, and thus led to Mn deficiency in Fusarium cells. Taken together, these findings uncover the potential of EDTANa2 as a fungicide candidate to manage Fusarium head blight (FHB) and DON in agricultural production.


Introduction
Fusarium is a globally important genus of fungal pathogens, responsible for many devastating diseases of plants and various serious diseases of humans [1,2]. Fusarium species are widely present in soil, plants and other organic substrates and have widespread distributions [3]. Species such as Fusarium graminearum, Fusarium oxysporum and Fusarium verticillioides can infect many crop plants, vegetables and flowers [2,[4][5][6][7][8]. One of the major diseases caused by Fusarium is Fusarium head blight (FHB), which is becoming more and more serious recently and causing concern. FHB results in yield loss and damaging of cereal grains [9,10]. Additionally, Fusarium spp. produce various types of mycotoxins, including deoxynivalenol (DON) and acetyl-deoxy-nivalenol (3-ADON and 15-ADON), that suppress humoral and cellular immunity and are thus highly detrimental to human and animal health [11,12]. DON is a mycotoxin virulence factor that promotes growth of the F. graminearum fungus in wheat floral tissues [13]. Practices used to control FHB and DON include rotation with nonhost crops and tillage [14], planting of resistant cultivars [15], and application of fungicides [16]. Essentially, current protective measures against Fusarium species mainly rely on fungicides, such as benzimidazole, triazole, demethylation inhibitor and quinone outside inhibitor. However, some of these can lead to the enhancement of DON biosynthesis in the infected wheat [17,18]. Therefore, new molecules are needed to control FHB and inhibit DON biosynthesis.
In addition, Fusarium isolates are susceptible to mutations that lead to phenotypes of tolerance towards common antifungal drugs [19,20]. Resistance to fungicides allows pathogens to survive fungicide treatment. The time taken for a new resistant mutant to reach a population size that is unlikely to die out by chance is called "emergence time". Prolonging emergence time would delay loss of control [21]. To date, the drawbacks of both scientific control strategies and the use of effective fungicides need to be addressed [8,22,23]. Undoubtedly, the processes of discovery and development of new antifungal drugs in the pharmaceutical industry are not only laborious and time consuming but also costly [24]. This is particularly true, as fungicides with novel modes of action are only rarely found, and resistance to single-target fungicides may occur within few years [25]. Some nonantibiotic drugs were recently reported to exhibit some antimicrobial activity against bacteria and Candida albicans [26,27].
Ethylenediaminetetraacetic acid (EDTA) is a chelating agent targeting divalent cations and has been previously used in oil fields to increase oil production and inhibit scale formation [28,29]. EDTA has also been shown to possess antimicrobial activities against bacteria and C. albicans because it can limit the availability of essential cations. The chelation of cations causes a separation of lipopolysaccharides from the outer membrane of microbial cells and thus increases the membrane permeability and subsequently leads to cell death in bacteria [30][31][32][33][34][35]. Generally considered safe, EDTA has been used intensively in the food and therapeutic industry [33,36,37]. Here, we demonstrated that EDTANa 2 had antifungal activity against Fusarium graminearum and DON biosynthesis. Our study contributes to the understanding of the mechanisms underlying EDTANa 2 control of FHB and provides a fungicide candidate molecule against Fusarium graminearum and its mycotoxin biosynthesis.

Ethylenediaminetetraacetic Acid Disodium Salt Exhibits Various Antifungal Activities against Fusarium Species
Mycelial growth was inhibited with 0.15 mM ethylenediaminetetraacetic acid or ethylenediaminetetraacetic acid sodium salt ( Figure 1A). The effectiveness of ethylenediaminetetraacetic acid sodium salt was affected by its number of sodium ions. For all tested reagents, EDTANa 2 had the best antifungal activity in terms of growth inhibition as well as cell swelling effects, closely followed by EDTANa 3 . The antifungal activity of EDTANa 4 was similar to that of EDTA. Moreover, the mycelial growth and morphology with the 0.3, 0.45, 0.6 mM sodium ion treatments showed no difference from the control group (only the treatment with 0.6 mM sodium ion is shown in Figure 1A), indicating that the sodium ions had no effect on Fusarium growth at a concentration less than 0.6 mM.
To For F. graminearum PH-1 strain, a linear regression of the percentage inhibition related to the control of mycelial growth versus the log10 transformation for each of EDTANa2 concentration was obtained. The median effective concentration (EC50) was calculated for each strain using a linear equation. The EC50 value of EDTANa2 for F. graminearum PH-1 strain was 0.29 mM (107.88 mg L −1 ) ( Figure 1B). For F. graminearum PH-1 strain, a linear regression of the percentage inhibition related to the control of mycelial growth versus the log 10 transformation for each of EDTANa 2 concentration was obtained. The median effective concentration (EC 50 ) was calculated for each strain using a linear equation. The EC 50 value of EDTANa 2 for F. graminearum PH-1 strain was 0.29 mM (107.88 mg L −1 ) ( Figure 1B).

The Control Effect and Phytotoxicity Test of Ethylenediaminetetraacetic Acid Disodium Salt
The EDTANa 2 for control of seedling blight in wheat was effective, reducing disease severity by 59%, 79%, 92% at 1 mM, 2 mM and both 4 mM and 8 mM, relative to the inoculated control, respectively ( Figure 2A). No significant different effect was observed among 1 mM to 8 mM EDTANa 2 , indicating that the control effect was stable within that range under controlled conditions. To ascertain whether the EDTANa 2 molecule could inhibit Fusarium infection under natural conditions, a crop phytopathogen, F. graminearum, was chosen for pathogenicity assays by spray inoculation experiments. The disease incidences were recorded 21 days post inoculation (dpi). The field experiment was conducted for two years (2018 and 2019) and produced similar results to the previously described experiments. As shown in Figure 2B, EDTANa 2 significantly reduced Fusarium head blight in the field. After spray treatment with 7 g ha −1 EDTANa 2 , F. graminearum caused 52% and 45% spikelets infection at 21 dpi, 45% and 49% reduction compared to the sterile water control in 2018 and 2019, respectively. When the dosage of EDTANa 2 increased to 70 g ha −1 , Toxins 2021, 13, 17 4 of 15 the incidence of disease decreased to 12% and 8%, 87% and 91% reduction compared to the sterile water control in 2018 and 2019, respectively. In 2019, the incidence of disease treated with 70 g ha −1 EDTANa 2 had a 70% reduction compared to the 140 g ha −1 carbendazim treatment ( Figure 2B). To further analyze the influence of EDTANa 2 treatments on spikelet morphology, we sprayed a series of EDTANa 2 concentrations (7 g ha −1 to 4000 g ha −1 ) onto wheat spikelets, and the results showed that there was little change in spikelets morphology following spray application of 7 g ha −1 to 1600 g ha −1 EDTANa 2. However, when the dosage of EDTANa 2 increased to 2000 g ha −1 , phytotoxicity was observed ( Figure 2C). These results suggested that EDTANa 2 could be used as a safe antifungal agent at low concentration. Wheat spikelets (cultivar Huaimai33) were sprayed with water, 40% carbendazim (140 g ha −1 ), or EDTANa2. Twenty-four hours later, the spikelets were inoculated via a spray inoculation experiment with a conidial suspension. Each combination of fungicide treatment and fungus was represented by 30 heads. After 21 days, the percentages of infected spikelets were determined, and representative heads were photographed. Values are means ± SD. Different letters represent a significant difference at P < 0.05. (C) Phytotoxicity test of EDTANa2 in the field. Wheat spikelets (cultivar Huaimai33) were sprayed with EDTANa2 and photographed at 21 days post inoculation. The data are an average ± standard error from 30 randomly selected heads. The experiment was replicated three times.

EDTANa2 Decreases DON Biosynthesis and TRI Gene Expression of Fusarium Graminearum In Vitro
Because the mycotoxin DON is a virulence factor, we investigated the mycotoxin biosynthesis potential of strains under EDTANa2 treatment. To verify the ability of the EDTANa2 to limit toxin production, DON amounts were measured using a competitive The test was designed with two random replications for each race of plant. Ten seedlings per treatment were inoculated with macroconidia suspension 24 h after EDTANa 2 spray. The lesions of diseased leaves were measured and photographed on sixth day post inoculation. Different letters represent a significant difference at p < 0.05. A linear regression equation of the percentage control effect for each EDTANa 2 concentration was developed using the SPSS 20.0 (IBM, Chicago, IL, USA) statistical package (y = 4.67 + 3.329x, R = 0.9476). (B) Control effect of EDTANa 2 on Fusarium head blight in the field. Wheat spikelets (cultivar Huaimai33) were sprayed with water, 40% carbendazim (140 g ha −1 ), or EDTANa 2 . Twenty-four hours later, the spikelets were inoculated via a spray inoculation experiment with a conidial suspension. Each combination of fungicide treatment and fungus was represented by 30 heads. After 21 days, the percentages of infected spikelets were determined, and representative heads were photographed. Values are means ± SD. Different letters represent a significant difference at P < 0.05. (C) Phytotoxicity test of EDTANa 2 in the field. Wheat spikelets (cultivar Huaimai33) were sprayed with EDTANa 2 and photographed at 21 days post inoculation. The data are an average ± standard error from 30 randomly selected heads. The experiment was replicated three times.

EDTANa 2 Decreases DON Biosynthesis and TRI Gene Expression of Fusarium Graminearum In Vitro
Because the mycotoxin DON is a virulence factor, we investigated the mycotoxin biosynthesis potential of strains under EDTANa 2 treatment. To verify the ability of the EDTANa 2 to limit toxin production, DON amounts were measured using a competitive ELISA-approach. As shown in Figure 3A, the DON production in TBI media was significantly inhibited by 0.4 mM and 0.8 mM EDTANa 2 , about 47% and 57.3% lower than the control group, respectively. and TRI genes expression (C). DON content was determined using a competitive ELISA approach 7 d after start of the experiments. The experiment was repeated three times. H2O2 was measured at 4 h and 12 h and calculated based on a standard curve included in each experiment. TRI gene expression was assayed by qRT-PCR. Hyphae were harvested from 2-day-old TBI cultures (1 day after EDTANa2 adding). Data are represented as the means ± SD of three biological replicates (significant differences at * P < 0.05).

Ethylenediaminetetraacetic Acid Disodium Salt Affected Cell Wall Formation and Cell Permeability
The inhibition of Fusarium growth by EDTANa2 was observed, so we analyzed the  As several lines of evidence in the literature corroborate an important role for H 2 O 2 in induction of toxin production, the accumulation of H 2 O 2 upon EDTANa 2 application was monitored using an in vitro assay. It showed that adding 0.4 mM and 0.8 mM EDTANa 2 resulted in a decreased H 2 O 2 content in the medium compared to the control as fast as 4 h after the start of the assay ( Figure 3B). This indicated that EDTANa 2 decreased the intracellular oxygen content within a short time after adding to the medium, which may reduce the activation of oxygen to toxin synthesis.
To further reveal the expression profiles of individual genes and their coordination in the trichothecene biosynthesis pathway, we measured the expression of several TRI genes after treating with EDTANa 2 . We found four genes in the trichothecene biosynthesis pathway were significantly down-regulated compared with the control group ( Figure 3C). After treating with 0.4 mM EDTANa 2 at 12 h, the expression of the TRI6 and TRI10 genes, which have been identified as positive transcription factor genes for trichothecene biosynthesis in F. graminearum, decreased by 2-and 2.13-times; The TRI12 gene, which is associated with trichothecene accumulation and resistance in F. graminearum, decreased by 3.45-times compared with the control group; The expression of the TRI11 gene also decreased by 1.56-times. The inhibitory effect of 0.8 mM EDTANa 2 on the TRI genes expression was consistent with that of 0.4 mM EDTANa 2 . It should be mentioned that the Tri101 gene encoding a trichothecene 3-O-acetyltransferase showed a remarkable upregulation, with 2.24-and 3.02-fold more transcripts after treating with 0.4 mM and 0.8 mM EDTANa 2 , respectively ( Figure 3C). These in vitro results suggested that EDTANa 2 can indeed inhibit DON biosynthesis and may be useful for reducing DON contamination in grains caused by F. graminearum.

Ethylenediaminetetraacetic Acid Disodium Salt Affected Cell Wall Formation and Cell Permeability
The inhibition of Fusarium growth by EDTANa 2 was observed, so we analyzed the cell wall formation and cell permeability of cells grown in the presence of EDTANa 2 . The mycelia treated by EDTANa 2 were sensitive to the preparation condition of scanning electron microscopy (SEM), and were destroyed and lysed with SEM observation ( Figure 4A). As shown in Figure 4B, the ultra-structures of the untreated cells exhibited normal electrondense layers and patterns under transmission electron microscopy. In contrast, the cells cultured in 0.075 mM EDTANa 2 had a clearly altered cell wall electron density, thickness and ultrastructure. The most notable visual alteration was a thicker cell wall in the treated cells, which topped 573 nm compared with that of the control cell, which had an average of 142 nm (the biggest thickness was 153 nm) (n = 20). Furthermore, the number of layers in the treated cell wall was reduced, and the electron density declined, whereas three layers were easily recognized in the normal cell wall. In filamentous fungi, chitin, a β-1,4-linked polysaccharide of N-acetylglucosamine, is a key structural component of the cell wall [38]. To further analyze the effect of EDTA and EDTANa 2 on cell wall formation, we measured the chitin content of F. graminearum cell walls after EDTA or EDTANa 2 treatment. The results illustrated that the EDTA and EDTANa 2 treatment groups produced only 33.43% and 25.23% chitin relative to the hyphal dry weight, values 35.35% and 51.2% lower than those of the control group, respectively ( Figure 4C). It is noteworthy that EDTANa 2 was more effective than EDTA, which concurred with our above results.  In most cases, the mode of action of antimicrobials against pathogens depends on the destruction of the fungal cell membrane and the resulting increase in cell permeability. The change in electrical conductivity reflects the change in the cell membrane permeability of Fusarium. Additionally, our data showed that the relative conductivity of hypha was change in electrical conductivity reflects the change in the cell membrane permeability of Fusarium. Additionally, our data showed that the relative conductivity of hypha was significantly increased by 11% and 15% after 0.15 mM EDTA and 0.15 mM EDTANa 2 treatment compared with that of the control group, respectively ( Figure 4D). There was no difference between 0.6 mM NaCl treatment and the control group. Therefore, EDTA and EDTANa 2 , especially EDTANa 2 , indeed affected both cell wall formation and cell permeability and resulted in a decreasing resistance to the external environment in F. graminearum.
2.5. The Inhibitory Effects of Ethylenediaminetetraacetic Acid Disodium Salt against Fusarium are Ameliorated by Mn 2+ but not Mg 2+ and Ca 2+ In order to find out the mechanism of inhibiting effect of EDTANa 2 , we examined the effect of the saturation of EDTANa 2 (0.15 mM) by the addition of excess cations (0.15 mM to 1.2 mM). As shown in Figure 5A, the adding of Mn 2+ increased the biomass of mycelia, but the adding of Mg 2+ and Ca 2+ had no significant effect on mycelia biomass. In addition, when an additional 0.3 mM MgCl 2 , 0.3 mM CaCl 2 or 0.15 mM FeCl 3 was added into the media as sources of divalent ions to bind 0.15 mM EDTANa 2 , mycelial growth was not resumed. 0.15 mM EDTANa 2 plus 0.3 mM Mn 2+ , however, rescued mycelial growth and morphology, which was similar to that in control wells not given EDTANa 2 ( Figure 5B). That indicated that the inhibiting effect of EDTANa 2 was related to its function of chelation.
significantly increased by 11% and 15% after 0.15 mM EDTA and 0.15 mM EDTANa2 treatment compared with that of the control group, respectively ( Figure 4D). There was no difference between 0.6 mM NaCl treatment and the control group. Therefore, EDTA and EDTANa2, especially EDTANa2, indeed affected both cell wall formation and cell permeability and resulted in a decreasing resistance to the external environment in F. graminearum.

The Inhibitory Effects of Ethylenediaminetetraacetic Acid Disodium Salt against Fusarium are Ameliorated by Mn 2+ but not Mg 2+ and Ca 2+
In order to find out the mechanism of inhibiting effect of EDTANa2, we examined the effect of the saturation of EDTANa2 (0.15 mM) by the addition of excess cations (0.15 mM to 1.2 mM). As shown in Figure 5A, the adding of Mn 2+ increased the biomass of mycelia, but the adding of Mg 2+ and Ca 2+ had no significant effect on mycelia biomass. In addition, when an additional 0.3 mM MgCl2, 0.3 mM CaCl2 or 0.15 mM FeCl3 was added into the media as sources of divalent ions to bind 0.15 mM EDTANa2, mycelial growth was not resumed. 0.15 mM EDTANa2 plus 0.3 mM Mn 2+ , however, rescued mycelial growth and morphology, which was similar to that in control wells not given EDTANa2 ( Figure 5B). That indicated that the inhibiting effect of EDTANa2 was related to its function of chelation.
We further measured the trace element content of Fusarium hyphae in different treatments. As shown in Figure 5C, when EDTA and EDTANa2 were added to the medium, the Mg and Mn elements decreased as expected. However, the contents of Ca and Fe elements were not significantly changed after the addition of EDTA and EDTANa2. When an additional Mg 2+ or Ca 2+ was added into the medium containing EDTANa2, all the trace elements, except Mn, increased or showed no difference compared with the untreated group. The Mn was always in a deficient state in the mycelia treated by EDTA and EDTANa2 unless Mn 2+ was added into the medium. These results suggested that intracellular Mn 2+ was chelated by EDTANa2, resulting in cell wall and cell membrane defects.  We further measured the trace element content of Fusarium hyphae in different treatments. As shown in Figure 5C, when EDTA and EDTANa 2 were added to the medium, the Mg and Mn elements decreased as expected. However, the contents of Ca and Fe elements were not significantly changed after the addition of EDTA and EDTANa 2 . When an additional Mg 2+ or Ca 2+ was added into the medium containing EDTANa 2 , all the trace el-ements, except Mn, increased or showed no difference compared with the untreated group. The Mn was always in a deficient state in the mycelia treated by EDTA and EDTANa 2 unless Mn 2+ was added into the medium. These results suggested that intracellular Mn 2+ was chelated by EDTANa 2 , resulting in cell wall and cell membrane defects.

Chitin Synthases are More Active in F. graminearum When Mn 2+ Is Used as a Cofactor
To further study the mechanism of the EDTANa 2 control effect, chitin synthases were extracted for enzymatic analysis. Unlike most chitin synthases in other fungi that used Mg 2+ as the cofactor, chitin synthases in F. graminearum were more active when Mn 2+ was used at the active site. The chitin synthases with 5 mM Mn 2+ had a 26% higher activity than that with 5 mM Mg 2+ , indicating that chitin synthases in F. graminearum may use Mn 2+ as the cofactor. The addition of 0.25 mM EDTANa 2 into the reaction systems could decrease enzymatic activity by 40% and 44% after chelation of Mn 2+ and Mg 2+ , respectively. However, chitin synthase in the Mn 2+ reaction system still had a 33% higher activity than that in the Mg 2+ reaction system. When the EDTANa 2 concentration was increased to 1 mM, the activity of chitin synthase in the Mn 2+ and Mg 2+ reaction systems decreased to 0.09 and 0.11 nmol GlcNAc h −1 mg −1 , respectively ( Figure 6A). Consistent with this, the metal ion addition experiment showed that the chitin content returned to normal levels after the addition of 0.15 mM Mn 2+ to SNA culture medium (containing 0.15 mM EDTANa 2 ). While wells adding Mg 2+ or Ca 2+ produced equivalent chitin to wells adding EDTANa 2 only, a 35-50% reduction was observed compared to the control group ( Figure 6B). To further study the mechanism of the EDTANa2 control effect, chitin synthases were extracted for enzymatic analysis. Unlike most chitin synthases in other fungi that used Mg 2+ as the cofactor, chitin synthases in F. graminearum were more active when Mn 2+ was used at the active site. The chitin synthases with 5 mM Mn 2+ had a 26% higher activity than that with 5 mM Mg 2+ , indicating that chitin synthases in F. graminearum may use Mn 2+ as the cofactor. The addition of 0.25 mM EDTANa2 into the reaction systems could decrease enzymatic activity by 40% and 44% after chelation of Mn 2+ and Mg 2+ , respectively. However, chitin synthase in the Mn 2+ reaction system still had a 33% higher activity than that in the Mg 2+ reaction system. When the EDTANa2 concentration was increased to 1 mM, the activity of chitin synthase in the Mn 2+ and Mg 2+ reaction systems decreased to 0.09 and 0.11 nmol GlcNAc h −1 mg −1 , respectively ( Figure 6A). Consistent with this, the metal ion addition experiment showed that the chitin content returned to normal levels after the addition of 0.15 mM Mn 2+ to SNA culture medium (containing 0.15 mM EDTANa2). While wells adding Mg 2+ or Ca 2+ produced equivalent chitin to wells adding EDTANa2 only, a 35%-50% reduction was observed compared to the control group ( Figure 6B).

Discussion
The genus Fusarium, which is pathogenic and toxic to plants and humans, is one of the most economically damaging fungal genera. Fusarium colonizes a wide range of environments, and control of Fusarium floral infections remains problematic. Some fungicides, including triazoles, benzimidazoles and strobilurins, are moderately effective, but reports of fungicide failure resulting from resistance have increased since the late 1960s [39,40]. To make matters worse, the development of new fungicides is much slower than the appearance of fungicide resistance in Fusarium populations [41,42]. Here, a nonantibiotic drug, EDTANa2, exhibited novel antifungal activity against F. graminearum and DON production. Through combined analyses of morphology, DON content and TRI gene expression detection, chitin detection, cell permeability, transmission electron microscopy and field experiments, we demonstrated that EDTANa2 destroyed cell wall and cytomembrane integrity and inhibited TRI gene expression in F. graminearum, and the antifungal

Discussion
The genus Fusarium, which is pathogenic and toxic to plants and humans, is one of the most economically damaging fungal genera. Fusarium colonizes a wide range of environments, and control of Fusarium floral infections remains problematic. Some fungicides, including triazoles, benzimidazoles and strobilurins, are moderately effective, but reports of fungicide failure resulting from resistance have increased since the late 1960s [39,40]. To make matters worse, the development of new fungicides is much slower than the appearance of fungicide resistance in Fusarium populations [41,42]. Here, a nonantibiotic drug, EDTANa 2 , exhibited novel antifungal activity against F. graminearum and DON production. Through combined analyses of morphology, DON content and TRI gene expression detection, chitin detection, cell permeability, transmission electron microscopy and field experiments, we demonstrated that EDTANa 2 destroyed cell wall and cytomembrane integrity and inhibited TRI gene expression in F. graminearum, and the antifungal effect of EDTANa 2 relied on Mn 2+ -chelating abilities.
EDTA is considered as an ion chelator, and it has been recommended as an antimicrobial agent against bacteria and C. albicans [43][44][45]. The EC 50 value of EDTANa 2 for F. graminearum is 107.88 mg L −1 , which is higher than that of carbendazim (about 0.5 mg L −1 ). The field test, however, shows that 70 g ha −1 EDTANa 2 decreased the incidence of disease by 70% compared to 140 g ha −1 carbendazim treatment. As DON is an important virulence factor in wheat, previous studies suggest that significant decreases in FHB incidence in field situations are possible with proper DON inhibited fungicide applications [46]. In our study, the DON production was significantly inhibited by EDTANa 2 , which may increase the antifungal effects on FHB.
The fungal cell wall protects the cell against osmotic pressure and other environmental stresses and is considered the carbohydrate armor of the fungal cell [47]. After EDTANa 2 treatment, all layers across the cell wall were affected, and the chitin content was decreased in Fusarium. In addition, the membrane permeability increased significantly when Fusarium was cultured in medium containing EDTANa 2 . This suggests that the fungal cells became more sensitive to changes in environmental stresses with EDTANa 2 treatment.
Our next question was which divalent cation was chelated by EDTANa 2 and then caused chitin content reduction and cell wall defects in Fusarium. There have been reports that calcium-binding agents inhibit Cryptococcus neoformans and C. albicans by disrupting the assembly of the polysaccharide capsule through Mg 2+ and Ca 2+ chelation [31,48,49]. However, the results in our paper showed that EDTANa 2 chelated Mn 2+ and resulted in a reduction of chitin synthesis. In fungi, chitin is synthesized by chitin synthase, whose activities are known to depend upon the presence of a divalent cation [50,51]. Chitin synthases (CHSs) in Fusarium were previously classified into seven categories [52]. Different chitin synthases are distinct in their responses to the divalent cation, for example, Chs2 and Chs3 are stimulated, while Chs1 is inhibited by Co 2+ in Saccharomyces cerevisiae [53]. On the basis of the data obtained in our study, it may be logical to assume that Mn 2+ is essential for the main chitin synthase of Fusarium. EDTA had a fungistatic effect on F. fujikuroi growth, a pathogen causing bakanae disease, and its action was largely suppressed by Mn 2+ and slightly by Ca 2+ [54]. Combining with our findings, we can speculate that this kind of chelating agent, such as EDTA and EDTANa 2 inhibits Fusarium spp. mainly because of Mn 2+ deficiency.
In summary, EDTANa 2 inhibits DON production and disrupts the cell wall and cell membrane functionality of Fusarium, an effect that appears to mainly result from Mn 2+ chelation. The results of our study provided new material and candidate compound against Fusarium in crop protection.

Control Effect Measurement on Wheat Seedling Blight
The test was evaluated under controlled conditions using a completely randomized design with two replications for each treatment. Ten seedlings per treatment were inoculated on the fully expanded primary leaves 8 d after Huaimai33 planting. The EDTANa 2 was sprayed on leaves at different concentration from 0.5 to 8 mM. After 24 h, leaves were punctured and inoculated with three microliter of macroconidia suspension (1 × 10 6 spores mL −1 ). The lesions of diseased leaves were measured and photographed at 6th day post inoculation. Duncan's multiple comparison test (SPSS20.0, IBM, Chicago, IL, USA) with a significant difference set as P < 0.05 was used to compare sample means. Mean values and standard deviations were reported. The differences between means with P less than 0.05 were considered statistically significant. The control effect was characterized by linear regression analysis (R = 0.94) using the SPSS statistical package.

Control Effect Measurement on Fusarium Head Blight and Phytotoxicity Field Test
The field study was conducted for two years (2018 and 2019) at the same location with different randomizations for each year (Table 1). Wheat (Triticum aestivum L. cv. Huaimai33) was grown on the experimental farm of Nanjing Agricultural University. At Zadok's growth stage (ZGS) 65, while more than half of the wheat spikes were in bloom, field plots were arranged in a randomized block, which was designed with three 3 plots (each plot was 4 × 5 m). The treatments were as follows: (1) a control consisting of water; (2) 140 g ha −1 carbendazim; (3) 7 g ha −1 EDTANa 2 ; (4) 70 g ha −1 EDTANa 2 . 7-4000 g ha −1 EDTANa 2 were used for phytotoxicity assays. For floral spray inoculations, each plant was sprayed with 0.5 mL of 1 × 10 4 spores mL −1 F. graminearum strain PH-1 conidia 24 h after EDTANa 2 spray treatment. Pathogenicity assays were performed 14 or 21 days after EDTANa 2 spray treatment as described previously [57]. The influence of EDTANa 2 on wheat was tested by assessing browning spikelets. Thirty wheat heads were randomly selected to calculate the browning spikelets ratio for each concentration of EDTANa 2 . The browning spikelets ratio was defined as follows: browning spikelet (%) = browning spikelets/total spikelets. The experiment was replicated three times.

Mycelial Growth Inhibition by EDTANa 2
For a fungicide-sensitivity assay in the laboratory, a three-day-old mycelial plug (5 mM in diameter) was placed in the center of a minimal medium (MM) ( [59]. Ten microliter of conidia (1.5 × 10 7 /mL) were inoculated in 30 mL TBI and cultured at 28 • C for 24 h in dark, and then 0.4 mM or 0.8 mM EDTANa 2 was added and cultured for additional 6 days. The experiment was repeated three times. To assay TRI gene expression, hyphae were harvested from 2-day-old TBI cultures (1 day after EDTANa 2 adding) and used for RNA isolation. qPCR was performed as previously describe (Table S3) [57]. The tubulin gene of F. graminearum was used as the internal control. The results were calculated with the data from three biological replicates.

Chitin Content, Chitin Synthase Activity and Cell Membrane Permeability Measurement
Macroconidia (10 4 mL −1 ) were cultured in SNA, SNA amended with 0.15 mM EDTA or SNA amended with 0.15 mM EDTANa 2 for 7 days and were used for chitin determination as previously described [57]. Fresh mycelium cultured in Czapek's medium (3 g L −1 of NaNO 3 , 1.31 g L −1 of K 2 HPO 4 , 0.5 g L −1 of KCl, 0.5 g L −1 of MgSO 4 ·7H 2 O, 0.01 g L −1 of FeSO4·7H 2 O, 30 g L −1 of sucrose, pH 7.2) for 5 d were collected and finely ground with liquid nitrogen for chitin synthase extraction. Chitin synthase activity was extracted and measured according to Song et al. [57] with some modifications. Chitin synthase activity was measured by following ( 14 C) GlcNAc incorporation into the filter-retainable polymer in the presence of 0-1 mM EDTANa 2 plus 5 mM Mg 2+ or Mn 2+ . To measure the cell membrane permeability, macroconidia (final concentration was 10 3 mL −1 ) were inoculated into SNA, SNA with 0.3 mM NaCl, SNA with 0.15 mM EDTA or SNA with 0.15 mM EDTANa 2 at 25 • C for 7 days. The conductivity was measured with a conductometer (CON510 Eutech/Oakton, Singapore) as described previously [60]. The conductivity of mycelia boiled for 5 min represented the final conductivity. The relative conductivity was calculated as follows: relative conductivity (%) = conductivity/final conductivity × 100. Three biological replicates were tested for each treatment.

Fungicidal Activity of EDTANa (n) against Fusarium spp.
Ten microliters of spores (10 4 mL −1 ) of F. graminearum PH-1 were plated in a 96-well flat bottom culture plate containing 0.15 mM EDTA, EDTANa 2 , EDTANa 3 or EDTANa 4 in 100 µL of SNA medium (pH 4.2). The plates were cultured at 25 • C for 24 h and observed using an inverted microscope. The growth and morphology of mycelia were photographed and compared among treatments using NIS-Elements AR software (version 3.2, Nikon, Tokyo, Japan). The experiment was replicated three times.
The determination of the minimum inhibitory concentration (MIC) was performed using 96-well microtiter plates. The Fusarium spp. (listed in 'Fungi, plants, and culture conditions') fungal inoculated in 96-well microtiter plates were treated with EDTANa 2 at different concentrations and incubated for 24 or 36 h. The lowest concentration that demonstrated no visible growth was determined as the MIC. Measurements were repeated three times.

Effect of Metal Ions on EDTANa 2 Activity
To assess whether cations would ameliorate the inhibitory effects of EDTANa 2 on mycelial growth and chitin synthesis, an additional 0.3 mM MgCl 2 , 0.3 mM CaCl 2 , 0.3 mM MnCl 2 or 0.15 mM FeCl 3 was added into SNA medium separately to bind the preadded 0.15 mM EDTANa 2 , followed by inoculation at 25 • C for 24 h and subsequent microexamination. Corresponding amounts of MgCl 2 , CaCl 2 , MnCl 2 or FeCl 3 were used as controls. Subsequently, a series of MgCl 2 , CaCl 2 or MnCl 2 concentrations (0, 0.15, 0.3, 0.45, 0.6, 1.2 mM) was added to the media to saturate the 0.15 mM EDTANa 2 to different degrees, and the fungi were cultured for 52 h to measure the mycelial biomass at OD 290 . The mycelia that were treated with 0.3 mM cations were collected at 7 d, frozen and dried for chitin content and trace element measurements [61]. Three independent experiments were performed, and the average was calculated.

Statistical Analysis
Statistical analysis was performed using Duncan's multiple comparison test (for multiple comparisons) and Student's t-test, all at a significance level of 0.05.