Hydrogen sulphide alleviates Fusarium Head Blight in wheat seedlings

Regents and media

Sodium hydrosulphide (NaHS) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) served as the donor for H₂S. Carboxymethyl cellulose (CMC) medium (15 g/L carboxymethyl cellulose, 1 g/L yeast extract, and 1 g/L NH₄NO₃) was used for liquid cultures of fungi.

Plant materials and treatments

The local virulent F. graminearum Schw., was kindly provided by Professor Xiue Wang, Nanjing Agricultural University, was used for FHB evaluation. F. graminearum inoculum was prepared in accordance with the method described by Buhrow et al. (2016). F. graminearum conidia collected from PDA agar plates were transferred into liquid medium (CMC) and cultivated at 28 °C for 5 d with shaking at 200 rpm. Then conidial suspension was filtrated through six layers of gauze and centrifuged at 6500 rpm for 6 min, and the spore concentration was adjusted to 3.2 ×10⁷ conidia/mL (0.1% Tween 80) with a haemocytometer and light microscopy.

Two-day-old seedlings of the FHB-susceptible wheat variety ‘Bainong 207’ were tested for coleoptile infection analysis by using the method described by Wu et al. (2005) with minor alterations. Two days post-sowing (dps), the top 1–2 mm of the coleoptiles was cut off, and 2 µL of F. graminearum inoculum was added to the top of the remaining seedlings. Coleoptiles inoculated with 0.1% Tween-80 served as controls. Then, the inoculated seedlings were grown in controlled environmental conditions (temperature: 25 °C; photoperiod: 16 h light/8 h dark; relative humidity: 95%).

NaHS was used as the H₂S donor according to the method described by Hosoki, Matsuki & Kimura (1997). In order not to affect the growth of plants, we first determined the application concentration range of NaHS. Two-day-old wheat seedlings were culture in 0.0 (control), 0.1, 0.3, 0.5, and 1.0 mM NaHS respectively for 3 days, then the growth index was investigated on 9 dps (days post sowing). To explore the potential functions of H₂S in FHB resistance of wheat, the seedlings were pre-treated with 0, 0.1, 0.3, and 0.5 mmol/L (mM) NaHS for 6 h; exposed to F. graminearum inoculation; and then continuously cultured in NaHS at the pre-treatment concentrations. After continuous treatment for 3 days, the treatment was stopped, and sterile distilled water was used to irrigate the wheat. The optimal NaHS concentration, that effectively relived the FHB symptoms, was determined according to the disease index combined with growth indexes in wheat seedlings. To confirm the hypothesis that NaHS-mediated disease suppression may be attributable to the production of H₂S or HS⁻, various sodium salts were used as controls for H₂S donors, in accordance with the method described by Zhang et al. (2008) with some modifications. Wheat was treated in the same way as in the aforementioned experiment with water (CK), 0.3 mM NaHS (the optimal concentration), Na₂SO₄, Na₂SO₃, NaHSO₄, NaHSO₃, or NaAC. All measurements were collected with three replicates.

Disease assessment

On 7 days post-inoculation (dpi), visible necrosis symptoms induced by F. graminearum on the wheat seedling stems were scored using the disease scoring system described by Nicholson et al. (1998). Each disease index assessement was performed by taking 15 coleoptiles per replicate.

Lesion colour grade:

Grade 0: no disease.

Grade 1: very slight brown necrosis.

Grade 2: slight/moderate brown necrosis.

Grade 3: extensive brown necrosis.

Grade 4: extensive black necrosis.

Disease index (DI) = $\frac{\sum \left(lesioncolourgrade\times lesionlength\right)}{totalnumblesofplant}$

Note: lesion length (mm).

Determination of the wheat growth index

Wheat growth indexes were determined to evaluate the effects of different concentrations of NaHS on wheat growth. Each growth index was assessed by taking 15 seedlings per replicate. Subsequently, the dry weights of the samples were measured after 72 h in an oven at 65 °C.

Relative quantification of F. graminearum DNA on the inoculated stem base of wheat

A real-time quantitative PCR (RT-qPCR) assay was performed for relative quantification of F. graminearum based on specific primers such as Fg16F/Fg16R according to Nicholson et al. (1998). The inoculated plant sheath and frozen mycelium (PDA: potato dextrose agar, control) were finely ground with a grinder for 2 min to a fine powder. Then, fungal DNA was extracted from 100 mg of samples with the HP Fungal DNA Mini Kit (Omega, Shanghai, China).

Before qPCR, the quality of the qPCR primers was evaluated by PCR to determine whether they could yield target fragment and if the product was unique. qPCR was performed on a Roche LighterCycler 96 qPCR machine based on the protocol previously described by Tang et al. (2020).

Measurement of endogenous H₂S in wheat

H₂S content data were collected as previously described by Sekiya et al. (1982) with some modifications. Wheat leaves (0.3 g) were sampled and snap-frozen in liquid nitrogen. The samples were ground and treated with 1.5 mL of Tris hydrochloride (Tris–HCl) buffer (pH 8.0), after which the supernatant was collected and mixed with a zinc acetate trap in a test tube after centrifugation. After adding 0.1 mL of 20 mM dimethyl-p-phenylenediamine dissolved in 7.2 M HCl, the test tube was sealed quickly. Then, 0.1 mL of 30 mM ferric chloride in 1.2 M HCl was injected into the test tube, and the mixture was incubated for 30 min at 25 °C. Measurement of H₂S was performed using a UV–VIS spectrophotometer (ScanDrop, Analytikjena, Germany) at a wavelength of 667 nm. The blank was prepared with unused zinc acetate solution by using the same procedures.

Determination of the H₂O₂ and MDA contents

The hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents were measured in accordance with the instructions of the H₂O₂ kit and MDA assay kit (Jiancheng Bioengineering Institute, Nanjing, China), respectively.

Measurement of SOD, CAT, APX, and POD activities

The activities of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol-dependent peroxidase (POD) were assayed by the spectrophotometric method according to the kits provided by Jiancheng Bioengineering Institute.

RNA isolation and RT-qPCR

Total RNA was isolated from samples according to the instructions provided with the Plant RNA Rapid Extraction Kit (Mei5 Biotechnology, Beijing, China). The quantity and quality of total RNA were tested by gel electrophoresis and spectrophotometry. Genes selected for further analysis included PR1.1, PR2, PR3, and PR4, with the internal control being the glyceraldehyde-3-phosphate dehydrogenase gene from T. aestivum (TaGAPDH). The primer sequences are listed in Table S1. All procedures for RT-qPCR were conducted as mentioned above.

Evaluation of H₂S- induced FHB resistance in wheat heads

To further evaluate the FHB resistance regulation by H₂S in wheat, wheat plants at the flowering stage were selected and transplanted under controlled environmental conditions. The wheat was subjected to the treatment described above with an optimum concentration of NaHS (0.3 mM). During this period, four spikes per plant were inoculated with 10 µL of spore suspension (3.2 ×10⁷ conidia/mL) of F. graminearum by using a syringe. Each treatment was repeated with four spikes, represent disease phenotype of wheat spikes were observed at 4 dpi.

Effect of H₂S on fungal growth

The spore suspension of F. graminearum was prepared in accordance with the methods described above. Four aliquots of the spore suspension (4 µL) were placed on 9-cm diameter Petri dishes maintained in sealed 1-L containers (Hu et al., 2014). NaHS solutions, at concentrations of 0, 0.01, 0.075, 0.1, and 0.3 mM were used to fumigate F. graminearum at relative humidity of 90%–95% at the bottom of the sealed containers. Spore germination (24 h) and hyphal diameter (3 d) were counted according to the method described by Fu et al. (2014).

Statistical analysis

All data were analysed by IBM SPSS 19.0 and graphed with Origin 8.5. The experimental data values, which were expressed as the mean ± standard error of the mean, were the average of the measurements obtained with triplicate independent assays. The data were evaluated by one-way ANOVA at a 95% confidence level followed by Tukey’s test, where differences were considered significant at P < 0.05. The data collected from RT-qPCR were analysed using the 2^−ΔΔCT method.

Induction of endogenous H₂S content after infection with F. graminearum

To explore the role of endogenous H₂S in the response to F. graminearum, the endogenous H₂S content in wheat was examined. After infection with F. graminearum, the endogenous H₂S content increased quickly in comparison with that in the control, and the H₂S content in infected wheat at 12 h was approximately twice that in the control (p < 0.05), and it peaked at 24 h (Fig. 1). As the disease symptoms increased, the H₂S content in wheat decreased. This result showed that endogenous H₂S was strongly induced by F. graminearum, and might play an important role in resistance to F. graminearum in wheat.

Effects of NaHS treatments on the resistance of wheat to F. graminearum

To determine the toxic effects of the H₂S donor NaHS on wheat growth under normal conditions, wheat was watered with solutions containing 0, 0.1, 0.3, 0.5, and 1.0 mM NaHS for 3 d. As shown in Table 1, low concentrations of NaHS (0–0.5 mM) had no significant effect on the growth parameters of wheat, whereas the 1.0 mM NaHS concentration was observed to inhibit wheat growth to a certain extent, especially root length.

To explore the effect of external application of H₂S on FHB pathogenicity, a standard method involving inoculation of seedlings (wheat coleoptiles), which has been proven to be a reliable prescreening method for FHB resistance (Li et al., 2019; Shin et al., 2014; Soresi et al., 2015; Wu et al., 2005), was used to assess FHB severity. Photographs of representative wheat seedlings on 7 dpi were taken (Fig. 2A). After coleoptile inoculation, the leaves of plants without NaHS application turned yellow with observable fungal hyphae. In addition, a large number of hyphae and brown lesions could be observed in the diseased stems. Figure 2A showed that the application of NaHS at different concentrations (0.0, 0.1, 0.3, and 0.5 mM) reduced the disease symptoms to varying degrees. In comparison with the untreated plants, those treated with 0.5 mM NaHS subgroup showed the maximum decrease (85.85%) in the disease index (p < 0.001), followed by an 85.60% decline in the 0.3 mM NaHS subgroup (p < 0.001) (Fig. 2B). Furthermore, RT-qPCR analysis showed a significantly lower quantity of F. graminearum in the sheath of the NaHS/infected groups in comparison with the non-NaHS/infected group (Fig. 2C), especially at concentrations of either 0.3 or 0.5 mM (p < 0.01). These findings suggest that NaHS could ameliorate the negative effects of F. graminearum stress.

As shown in Table 2, the non-NaHS/infected group showed a notable decrease in plant height, fresh weight and dry weight, whereas exogenous NaHS inhibited the decline in wheat seedling growth indexes under F. graminearum stress. Moreover, an increase in the concentration of NaHS from 0.3 mM to 0.5 mM led to reductions in plant height, root length, and fresh weight. On the basis of these results, we chose a working concentration of 0.3 mM NaHS for the subsequent analyses.

To further verify the resistance regulation of H₂S to FHB in wheat, spike inoculated wheat was treated with 0.3 mM NaHS (Fig. 2D). The result showed that NaHS significantly reduced FHB severity in spike, which was consistent with the phenomenon in coleoptile. On the basis of these results, we could hypothesise that the application of NaHS may confer resistance to FHB in wheat and result in suppression of fungal propagation.

Protective roles of H₂S in amelioration of F. graminearum stress in wheat

To investigate the effect of H₂S in alleviating disease symptoms induced by NaHS in wheat inoculated with F. graminearum, 0.3 mM Na₂SO₄, Na₂SO₃, NaHSO₄, NaHSO₃, and NaAC were applied in the same experimental system. As shown in the data in Fig. 3A, the control groups with Na⁺ or sulphur-containing components did not show reduced the disease severity as the NaHS group did, which indicated that H₂S or HS⁻, rather than other derivatives, promoted the role of NaHS in heightening FHB resistance in wheat.

The endogenous H₂S level in wheat seedlings was measured under various treatment conditions (Fig. 3B). Marked modulation of endogenous H₂S content in the results showed that NaHS pre-treatment caused a change in the H₂S content of wheat seedlings. Briefly, the H₂S content in pre-treated plants was higher than that the control plants within 36 h, especially at 6′/0 h and 9 h, and almost tripled in comparison with the values in the control group (p < 0.001). Meanwhile, the time-course of changes in the endogenous H₂S content in the control group in response to F. graminearum was consistent with the results in Fig. 1. These results showed that the increase in endogenous H₂S levels through exogenous application of NaHS could contribute to resistance against F. graminearum in wheat seedlings.

Exogenous H₂S modulated oxidative stress in wheat seedlings under biotic stress conditions

To further understand the mechanism underlying H₂S-induced resistance against F. graminearum, the H₂O₂ and MDA contents were determined. An H₂O₂ burst occurred in the control group after infection, while the H₂O₂ content was notably reduced in the NaHS group (Fig. 4A). The H₂O₂ levels increased by approximately 59.6% in the control group (p < 0.001), whereas NaHS treatment decreased the H₂O₂ content by 10.4% after 12 h of inoculation (p < 0.05).

MDA, a product of membrane lipid peroxidation, is one of the most commonly used indexes of lipid peroxidation. The MDA content in the control wheat samples increased significantly up to 36 h of inoculation, which was twice that of the treatment group (p < 0.001), and subsequently decreased (Fig. 4B). In addition, it was lower in the NaHS group than in the control group during the entire treatment period. These results indicated that F. graminearum could cause H₂O₂ accumulation and membrane damage in wheat seedlings, while exogenous application of H₂S could relieve the oxidative stress caused by H₂O₂ and MDA.

Effects of H₂S on the activities of the antioxidant enzymes in wheat grown under biotic stress

The major antioxidant enzymes include SOD, POD, APX, and CAT, which detoxify reactive oxygen species (ROS) in various plant tissues. In our experiments, the activities of antioxidant enzymes (SOD, POD, APX, and CAT) in the NaHS group were substantially higher than those in the control group during most of the treatment period (Fig. 5). SOD activity in the NaHS group and control group increased sharply and reached the maximum level at 48 h, which was 22.09% higher than that in the control (p < 0.01), and then rapidly declined from 48 h to 72 h (Fig. 5A). Additionally, NaHS treatment activated POD activity, which was approximately 1.34 times higher than that in the control group at 36 h (p < 0.01). APX activity varied similarly in the control and NaHS groups, while APX activity in the NaHS group was higher than that in the control group during the entire treatment period (10′/4 and 36 h, p < 0.05), except for 72 h (Fig. 5C). Furthermore, there was no obvious difference in CAT activity between the NaHS and control groups at the beginning. This gap narrowed from 0 to 12 h, after which CAT activity in the NaHS group increased faster than that in the control group, and was 1.71 times the activity in the control group at 72 h (p < 0.01) (Fig. 5D). Unambiguously, these results demonstrated that alleviation of oxidative stress by H₂S in wheat under FHB stress was closely related to improvement of antioxidant system activities in wheat seedlings.

Activation effects of H₂S on defence response genes

Defence response genes, including genes encoding pathogenesis-related proteins (PRs), are involved in the resistance response to many plant-pathogen interactions (Hammond-Kosack & Jones, 1996; Pritsch et al., 2000; Pritsch et al., 2001; Stevens et al., 1996). To examine the association between H₂S-induced resistance and PR gene expression in wheat, we conducted a quantitative analysis for the PR1.1, PR2, PR3 and PR4 genes after NaHS application (Fig. 6). The results showed that H₂S upregulated the transcription levels of PR1.1, PR2, PR3 and PR4, which peaked at 12 h and 48 h, with the degree of between 2 and 4 times (PR1.1, PR2, and PR3, peaked at 12 h, p < 0.001; PR4, peaked at 48 h, p < 0.05). Moreover, the relative expression levels of PRs in the NaHS group were higher than those in the control group within 48 h. Thus, our results demonstrated that exogenous H₂S improved disease resistance against F. graminearum by advancing the accumulation of defence-related genes.

Effects of H₂S on mycelial growth and spore germination

To further understand the mechanism underlying the regulatory effects of H₂S in resistance to F. graminearum in wheat, the effects of H₂S on mycelial growth and spore germination were investigated (Fig. 7). As shown in Fig. 7, with increasing NaHS concentration, the mycelial diameter of F. graminearum on PDA decreased, and mycelial growth was inhibited by 37.66% even at 0.075 mM NaHS treatment (p < 0.001) (Fig. 7B). At 0.3 mM NaHS, no signs of mycelial growth were observed. Moreover, in our assessment of spore germination after 24-h incubation, and most of the fungal spores germinated at 0.01 mM. However, treatment with 0.1 mM NaHS completely restrained spore germination of F. graminearum (Figs. 7A, 7C). These data showed that exposure to H₂S notably inhibited mycelial growth and spore germination of F. graminearum in a dose-dependent manner, suggesting that H₂S had an effectively fungicidal role.

Application of exogenous H₂S reduced FHB symptoms in wheat

Fusarium diseases resulted in severe economic losses for the production of cereal crops. Global climate change has increased the contribution of weather conditions to epidemics of wheat FHB, and control of this disease has proven to be exceedingly challenging (Zhang et al., 2014). H₂S is an essential signalling molecule that exhibits positive effects on numerous physiological functions, including those serving against environmental stress and pathogens. Treatments of H₂S combined with silicon have been reported to alleviate the negative effects of drought stress and rust infection in wheat (Naz et al., 2021). Our study demonstrated that exogenous H₂S alleviated FHB symptom in wheat seedlings, enhancing the plant defense mechanism and inhibiting fungal growth.

In this study, H₂S content was conspicuously induced after F. graminearum infection, suggesting that H₂S may be partly responsible for the defence response to F. graminearum in wheat (Fig. 1). This result was supported by previous studies in Arabidopsis showing that the endogenous H₂S content increased significantly after pathogen infection (Shi et al., 2015). Nevertheless, previous to this study, the role of H₂S in wheat resistance to FHB was not clear.

Coleoptile inoculation method can be used as a rapid and reliable method for FHB pathogenicity research, and it has been described in many studies in the literature (Wu et al., 2005; Yang et al., 2018). Comparative pathogenic analyses indicated that the application of NaHS effectively reduced the disease symptoms of wheat seedlings in a dose-dependent manner, and at an optimal concentration of 0.3 mM, the FHB severity of wheat spikes was also alleviated (Fig. 2). NaHS is a commonly used H₂S donor in biological systems that decomposes into Na⁺ and HS⁻ in solution, of which HS⁻ combines with H⁺ to generate H₂S (Zhang et al., 2010a; Zhang et al., 2010b). The role of NaHS as an H₂S donor, in reducing the negative effects of F. graminearum in wheat was supported by two sets of evidence. First, only NaHS could effectively reduce the FHB severity of wheat in the solutions we tested. Moreover, as shown in Table 1, NaHS itself did not negatively affect seedling growth at low concentrations, implying that NaHS counteracts the pathogen-induced stresses in plants in a way that was independent of any “cross-tolerance”. On the other hand, wheat seedlings treated with NaHS showed markedly higher the levels of extractable H₂S compared with the control (Fig. 3). These data provided new insights, hinting at a possible role of H₂S in the plant defence signalling of wheat against F. graminearum.

H₂S enhanced the resistance of wheat to F. graminearum by enhancing the defense mechanism

ROS, especially H₂O₂, are known to be important in plant-pathogen interactions. Low levels of H₂O₂ are essential for peroxidase-dependent lignification, which hinders penetration by a pathogen. However, necrotrophic pathogens induced H₂O₂ at toxic levels, which could lead to cell death, and the pathogens could use these dead cells as an entry point into plants or obtain nutrients from dead cells for further invasion and growth (Hückelhoven, 2007). Correspondingly, during the process of infection and colonisation of grain hosts, Fusarium causes oxidative stress in the host by secreting toxic levels of ROS including H₂O₂, which acts as weapons to attack plants (Walter, Nicholson & Doohan, 2010). In this study, F. graminearum-infected wheat showed a marked increase in H₂O₂ levels, and this increase was mitigated by treatment with NaHS (Fig. 4). Furthermore, considering the MDA content, control plants showed elevated MDA levels after infection, which was likely connected with the remarkable accumulation of H₂O₂, implied that rapid oxidative explosion destroyed membranes. Plants receiving NaHS treatment showed significantly lower post-infection MDA levels in comparison with the control group, indicating decreased ROS levels, and a reduced degree of membrane peroxidation. Taken together, these data support the idea that H₂S appears to be an antioxidant signalling molecule participating in the resistance against F. graminearum in wheat.

To further examine the role of NaHS in relieving ROS toxicity, the activities of various antioxidant enzymes, including SOD, POD, APX, and CAT, were evaluated. As shown in Fig. 5, the activities of SOD, POD, APX, and CAT were upgraded by NaHS treatment, indicating that H₂S could enhance the activities of antioxidant enzymes to scavenge ROS, which was consistent with findings showing H₂S attenuated the rot of freshly cut pears by enhancing the antioxidant system to exterminate excessive ROS (Hu et al., 2014). In vitro assays showed that APX and CAT were targets of persulfidation, which were positively modulated by persulfidation in the presence of H₂S (Corpas, 2019). Moreover, exogenous H₂S treatment has also been shown to relieve the ROS burst and cell damage induced by drought, salinity, temperature, and heavy metals in plants (Corpas, 2019). The present data also speculated that the resistance of the FHB-resistant variety ‘Vulkan’ was associated with antioxidative enzyme activity (Spanic et al., 2017). These results may partly account for the effects of H₂S treatment in inducing resistance to F. graminearum in wheat.

PRs are related to the development of systemic acquired resistance (SAR) against further infection enforced by pathogeny. Additionally, published papers have described that the genes encoding PRs in wheat were activated after F. graminearum attack (Pritsch et al., 2000; Pritsch et al., 2001). However, physiological concentrations of H₂S have been shown to upregulate the expression levels of disease-related genes (Shi et al., 2015). For example, the auxin signalling pathway mediated by MIR393 includes MIR393a/b and its target genes (TIR1, AFB1, AFB2, and AFB3), which were all transcriptionally regulated by H₂S (Shi et al., 2015). In this study, we noted that, the PR gene transcript accumulation in NaHS-treated wheat was greater than that in control plants (Figs. 6A–6D). Taken together, these results could indicate that H₂S displayed the ability to enhance the expression of defence response genes, thereby possibly contributing to greater disease resistance in wheat.

The effects of H₂S on the growth of F. graminearum in vitro

We also studied the toxicity of H₂S to F. graminearum, and found that H₂S released by 0.3 mM NaHS hindered the development of F. graminearum during the testing period (Figs. 7A–7C). Our results suggested that H₂S exerted its antifungal effect by influencing various aspects of fungal development, including inhibition of spore germination and retardation mycelial growth. Similar effects of H₂S have been confirmed by Fu et al. (2014) and Hu et al. (2014).

The potential application of H₂S in wheat FHB in the future

Our experiments showed that H₂S could be a potential superior candidate for controlling FHB. NaHS has been widely used as the rapid H₂S donor that generates a large amount of H₂S over a short time period (Sun et al., 2017). The rapid generation and loss of H₂S limit its application in agriculture. Controlled release of H₂S could make it safer and more cost-effective to use. With this in mind, Lee et al. (2011) previously reported that the slow-releasing H₂S donor, GYY4137, exhibited novel anti-cancer effects in vitro and in vivo. Likewise, in the work of Liu et al. (2021), they provided an infrared-light-responsive pesticide delivery system to intentionally regulate imidacloprid release and enhance utilization efficiency. Hence, it would be valuable to develop a delivery system that could be manipulated to achieve sustained release of H₂S from its donor NaHS. In the future, reformulated NaHS could be applied to plants or sprayed directly into the soil to inhibit the fungal, which might have an application value.