The standard strain of F. graminearum was PH-1 (NRRL31084), which is used to investigate the detailed physiological and biochemical responses under guaiacol treatment (Gu et al., 2015). The strains were grown on Potato Dextrose Agar (PDA) medium and Carboxymethyl Cellulose (CMC) medium for mycelial growth assay and sporulation assay, respectively. Trichothecene biosynthesis inducing (TBI) media was used for the DON production assay (Gardiner et al., 2009). Yeast extract peptone dextrose (YEPD) medium was used for conidial germination assays and fungal culture prior to quantitative real-time RT-PCR (qRT-PCR) analysis for gene expression (Gao et al., 2016a). Guaiacol (a.i. 99%;100 ml; Shanghai Macklin Biochemical Co., Ltd.) was stored at 4°C in darkness.

The mycelial growth of PH-1 in PDA with or without guaiacol was measured after incubation for 3 days at 25°C. Growth inhibition as a percent of the control was calculated. The median effective concentration (EC₅₀) of guaiacol was calculated based on linear regression of colony diameter vs. log-transformed fungicide concentration (Thomulka et al., 1996). The bioassay data were obtained as the mean of three replicates. The morphology of mycelia treated with guaiacol was observed by scanning electron microscopy (SEM, EVO-LS10, ZEISS, Jena, Germany), and sample drying was carried out according to a previously described method (Gao et al., 2016b).

For the conidiation assay, five 5-mM mycelial plugs were taken from the edge of a 3-day-old colony into 150-ml flasks containing 50 ml of CMC culture. The flasks were incubated at 25°C for 5 days with shaking (175 rpm). The number and morphology of the conidia were determined with a hemocytometer and microscope, respectively (Duran et al., 2010). Conidia (10⁵) of F. graminearum strain PH-1 were inoculated into 30 ml of YEPD medium with or without guaiacol. Microscopic examination was carried out using germ tubes germinated from spores for 2, 4, 6, 8, 10, 12, and 24 h.

The relative conductivity and glycerol content of guaiacol-treated F. graminearum were measured according to a method described by Gao et al. (2016b). Conidia (10⁵) of PH-1 were placed in 250-ml flasks containing 100 ml of YFPD medium at 25°C with shaking (175 rpm). After 24 h, some of the flasks were treated with guaiacol and shaken for an additional 24 h. Then, the mycelia were washed and suspended at 0.5 g of mycelia per sample in 20 ml of double-distilled water. A conductivity meter (CON510 Eutech/Oakton, Singapore) was used to measure the electrical conductivity after 0, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, and 180 min. After 180 min, the final conductivity of the mycelia was measured after boiling for 5 min. The relative conductivity was calculated with the following formula:

Glycerol in the mycelia was measured with a glycerol detection kit (Beijing Applygen Technologies Inc., Beijing, China), and mycelia were treated as described above.

To observe the impact of guaiacol on the antioxidant system in F. graminearum, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels were compared. Conidia (10⁵) of PH-1 were cultured on YEPD medium for 24 h and then treated with guaiacol at different concentrations for another 24 h. A peroxidase assay kit (A084-3-1), catalase assay kit (A007-1-1) and superoxide dismutase assay kit (A001-1-1) from Nanjing Jiancheng Bioengineering Institute were used to measure enzyme activities. An MDA detection kit (D799761; Shanghai Sangon Biotech Institute, Shanghai, China) was used to determine the MDA level based on the spectrophotometric determination of the reaction between MDA and 1,3-diethyl-2-thiobarbituric acid (TBA) assisted by trichloroacetic acid (TCA) (Yang et al., 2012). Enzymatic activities and the MDA content were normalized according to their mycelial dry weight.

The mycelia were treated with guaiacol as described above, and the calcium indicator Fluo-3-acetoxymethyl (Fluo-3-AM; S1056; Shanghai Beyotime Biotechnology Institute, Shanghai, China) was used for detecting the changes of Ca²⁺ concentration in the mycelia with guaiacol-treated. All mycelia were loaded with 5 μM Fluo-3-AM ester, a membrane-permeable calcium indicator, for 40 min at 37°C. After loading with the Fluo-3 dye, the mycelia were washed with ddH₂O. Fluorescence was observed using a PerkinElmer UltraVIEW VoX system equipped with a 60 × objective. Fluo-3 was excited by argon laser light at 488 nm (Li et al., 2009).

The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was selected to quantify the expression levels of Ca²⁺-ATPase (PMCA) genes. The sequences of four tested genes (accession number: FGSG_00508, FGSG_04245, FGSG_04919, and FGSG_08985) were retrieved from NCBI Database¹ for the design of primers. Total RNA was extracted from the mycelia of each sample by using TRIzol (Invitrogen, Waltham, MA, United States) according to the manufacturer’s instructions (Gao et al., 2016a). The first-strand cDNA synthesized with the PrimeScript^® RT reagent kit (TaKaRa, Kusatsu, Japan) was used as template for qRT-PCR analysis (Roche Molecular Systems, LightCycler^® 96, Life Technologies, United States). The primers used for amplifying the target genes are listed in Supplementary Table 1. To standardize the results, the relative abundance of elongation factor 1-α (EF1-α, FGSG_08811) was also determined and used as the internal standard.

To quantify DON production in vitro, conidia (10⁵) of PH-1 were inoculated in 30 ml of liquid trichothecene biosynthesis-inducing (TBI) medium with or without guaiacol at different concentrations and cultured at 28°C for 7 days in the dark. Then, the solution was filtered, and the mycelia were dried and weighed. The filtrate was redissolved in methanol for DON analysis, and the mycelia were used for RNA extraction. The expression levels of two trichothecene biosynthesis genes (Tri5 and Tri6) were determined with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The primers used to amplify the Tri5 and Tri6 genes are listed in Supplementary Table 1. The amount of DON in each sample was determined using a high-performance liquid chromatography-mass spectrometer/mass spectrometer (HPLC-MS/MS) system (a Shimadzu 30A LC system coupled to a Triple Quad 6500+, Sciex, United States). The mass spectrometric parameters were set according to a previously described method (Dong et al., 2016). The experiment was repeated two times.

To quantify DON production in wheat, 20 g of finely ground samples were extracted with 80 ml of acetonitrile:water (84:16, v/v) for 60 min at 200 rpm on an orbital shaker. The extracts were filtered and passed through a MycoSep 226 AflaZon + multifunctional column (Romer Labs, Beijing, China), and 4 ml of the purified extract was evaporated to dryness under a nitrogen stream. The residue was dissolved in 1 ml of methanol:water (40:60, v/v), followed by centrifugation at 10,000 rpm and subsequent analysis by LC-MS/MS analysis of DON (Dong et al., 2016). The recovery of DON in wheat ranged from 82.1 to 95.7%. The limit of detectionm (LOD) quantification (LOQ) of DON were estimated based on signal-to-noise ratios of 3/1 (10 μg/kg) and 10/1 (30 μg/kg), respectively.

Field trials were conducted in the village of Jinci in Zhuzhen (Luhe District, Nanjing, Jiangsu Province, China). The local common wheat cultivar, Zhengmai 10, which is moderately resistant to F. graminearum, was chosen to test the efficacy of guaiacol. The three following treatments were applied: for treatments 1 and 2, guaiacol diluted 1:200 (40 mM Gua) and 1:100 (80 mM Gua), respectively, was sprayed during the flowering period, and for treatment 3, water was sprayed as a control. Each treatment was performed in four replications, and the plot area was 5 m × 5 m. The experiments were conducted as a randomized complete block design with a 1-m protection row surrounding each plot.

After 7 days, all treatments were applied for a second time. It should be noted that all the treatments were conducted on a sunny day in the late afternoon to avoid the spray being washed off of the plants by rainfall. The experimental site was naturally infected. Two weeks after the final treatments, wheat heads were collected from five sampling locations in each plot, and 100 heads of wheat were collected at each sampling location. Finally, the disease incidence (percentage of diseased heads), disease index (DI), and control efficacy were calculated. The DI in the wheat heads was assessed using five evaluation classes according to the percentage of the wheat head surface that showed the symptoms of FHB (0: 0%; 1: 1–25%; 2: 26–50%; 3: 51–75%; and 4: > 75%). The DI of each plot was calculated using the following formula: [(number of wheat heads in each class × class number)/(total number of wheat heads × 4)] × 100.

All data are expressed as the mean ± standard deviation (SD). The statistical significance of differences was determined by the unpaired Student’s t-test with GraphPad Prism software (La Jolla, CA, United States). A p-value less than 0.05 was used to indicate a statistically significant difference.

Treatment of F. graminearum PH-1 with guaiacol at different concentrations showed that guaiacol inhibited hyphal growth in a dose-dependent manner and that 6.4 mM guaiacol completely inhibited mycelial growth (Figure 1A). The EC₅₀ value of guaiacol for PH-1 was 1.838 mM. Furthermore, assessment of the mycelial growth of F. graminearum by SEM clearly revealed morphological alterations in the hyphae upon guaiacol treatment. Unlike hyphae in the control group, which were regular and smooth, treatment with 16 mM guaiacol led to significant collapse of the hyphae (Figure 1B).

The conidial production and germination of F. graminearum were also depressed by guaiacol. Compared to that in the control, the number of conidia was significantly decreased by 97.8% upon treatment with 4.8 mM guaiacol (Figure 2A), and guaiacol at 1.6–6.4 mM delayed the process of conidial germination. In the control group, all the conidia germinated completely after 8 h, and the germination rate decreased gradually with increasing guaiacol concentration (Figure 2B). Light microscopy was used to observe the morphology of conidia under guaiacol treatment. After treatment for 24 h, the conidia treated with guaiacol became wider and shorter than those of the control (Figure 2C). All these results suggest that guaiacol had a remarkable effect on hyphae growth and conidial development in F. graminearum.

To test the effect of guaiacol on the F. graminearum cell membrane, we measured relative conductivity, the MDA concentration, and the glycerol and hydrogen peroxide (H₂O₂) content of F. graminearum upon guaiacol treatment. Intracellular glycerol has been shown to play an important role in the response of fungi to osmotic stress (Duran et al., 2010). In the present study, we found that the glycerol content was significantly higher in guaiacol-treated F. graminearum compared to the control group (Figure 3A), suggesting that glycerol accumulation was induced by guaiacol to maintain osmotic balance. Treatment with guaiacol resulted in a remarkable increase in the relative conductivity of F. graminearum compared to that of the control group (Figure 3B), suggesting that guaiacol can induce electrolyte leakage by enhancing cell membrane permeability in F. graminearum. Electrolyte leakage resulting from damage to the cell membrane can trigger osmotic stress responses in fungi. The MDA concentration has been frequently used as an index of lipid peroxidation, which indicates oxidative injury of the cell plasma membrane (Tsikas, 2017). In this study, we found that the MDA and H₂O₂ content was significantly lower in guaiacol-treated F. graminearum compared to the control group (Figures 3C,D), suggesting that the guaiacol-induced cell membrane damage was not related to oxidative damage. Indeed, as a strong oxidant, guaiacol can also effectively remove reactive oxygen species from F. graminearum.

To gain more information on the possible link between guaiacol and oxidation, POD, CAT and SOD activities were measured in untreated F. graminearum samples and F. graminearum samples treated with 1.6–6.4 mM guaiacol, followed by 24 h of cultivation. The results shown in Figure 4 demonstrate that the POD, CAT, and SOD activities were also inhibited in the guaiacol-treated samples compared with the untreated samples. These results indicate that guaiacol may restore homeostasis during oxidative stress in F. graminearum.

Consistent with the results of morphological analysis, treatment of F. graminearum PH-1 spores with guaiacol at different concentrations (0, 1.6, 3.2, 4.8, and 6.4 mM) showed that guaiacol inhibited DON synthesis in a dose-dependent manner (Figure 5A). Furthermore, the expression levels of two trichothecene biosynthesis genes, Tri5 and Tri6, were investigated by qRT-PCR. Compared to that in the untreated control, the relative expression of these two genes was significantly downregulated (p < 0.001) in the guaiacol-treated mycelia (Figure 5B).

In F. graminearum, Tri1 encodes the oxygenase cytochrome P-450, which is involved in the late steps of biosynthesis of the trichothecene DON (Zhang et al., 2015). Upon culture under conditions that induce DON biosynthesis (liquid TBI medium), colocalized fluorescent signals were detected in a mutant tagged with Tri1-GFP (ΔTri1:FgTri1-GFP), indicating DON toxisomes; however, 3.2 or 4.8 mM guaiacol treatment strongly inhibited DON toxisome formation, as only faint fluorescent signals were observed in the guaiacol-treated mycelia (Figure 5C).

Zhi et al. (2020). found that guaiacol can suppress osteoclastogenesis by inducing Ca²⁺ oscillation. Therefore, in this study, we analyzed changes in the Ca²⁺ concentration in the mycelia of F. graminearum treated with guaiacol at different concentrations (0, 1.6, 3.2, 4.8, and 6.4 mM). The Fluo-3-AM-stained hyphae of F. graminearum showed more extensive green fluorescence in the presence of guaiacol than in its absence (Figure 6A), indicating that guaiacol treatment led to intracellular Ca²⁺ oscillation. Furthermore, the expression levels of four Ca²⁺-ATPase (PMCA) genes involved in Ca²⁺ transport were determined by qRT-PCR (Bruce, 2018). Compared to that in the untreated control, the relative expression of FGSG_00508, FGSG_04919, FGSG_08985, and FGSG_04245 was significantly downregulated (p < 0.001) in mycelia treated with 6.4 mM guaiacol, although the expression of FGSG_04245 was upregulated upon treatment with guaiacol at low concentrations (Figure 6B).

A field experiment was conducted to test the efficacy of guaiacol in controlling FHB and wheat DON contamination. This study was conducted in the dry wheat season; therefore, the FHB and DON concentrations in the grain were relatively low. The results in Figure 7 indicate that guaiacol treatment significantly inhibited FHB infection, and significant decreases in DON content of 17.2 and 23.4% were observed following the Gua-200 and Gua-100 treatments, respectively.