Introduction

Black shank, caused by Phytophthora nicotianae, is among the most widespread and damaging diseases of cultivated tobacco (Nicotiana tabacum) worldwide (Csinos et al. 1999). The pathogens infect plants at any stage of their growth, with the field stage being the most damaging, causing root rot, stem lesions, leaf necrosis and plant death. Disease damage can spread rapidly under conditions of high temperature (23 °C–28 °C) and high soil moisture, causing serious yield losses (Gallup et al. 2018; Vontimitta et al. 2012). Traditional control strategies, including crop rotation, fungicide applications, and the use of resistant cultivars, are not sufficient to control this soil-borne disease (Haas et al. 2005). Fungicides have become increasingly unwelcome, although they are currently used in most cases because of their long time in the field, and can lead to resistant pathogen strains (Aguiar et al. 2017). Some recent studies have shown that several years of monoculture plants can recruit growth-promoting rhizobacteria (PGPR) to prevent losses caused by soil-borne pathogens. However, this discovery is not practical for preventing soil-borne pathogens for certain economic reasons (Haas et al. 2005). Thus, it is urgent to explore more efficient and sustainable control methods for these soil-borne diseases.

The plant defence system usually consists of preexisting physical and chemical barriers as well as inducible defence responses (Jackson et al. 1996). A plant’s successful defence against invading pathogens depends on early recognition of the pathogens and initiation of the appropriate signalling processes to activate a multicascade defence response (Ryals et al. 1996; Meng et al. 2013). Local or systemic two-layer defence mechanisms produce systemic acquired immune resistance (SAR) to produce resistance to protect themselves against pathogen invasion (Dempsey et al. 1997; Bigeard et al. 2015). Similarly, upon perception of certain stimuli, such as the colonization of beneficial microorganisms and the application of chemical substances, plants can activate their own defence system and enhance their defence ability against future pathogens and pests, namely induced resistance (IR) (De Kesel et al. 2021). A number of chemical inducers are used for IR stimulation, such as SA, benzothiadiazole (BTH), and β-aminobutyric acid (BABA), have been proven to induce plant disease resistance (Barilli et al. 2010; Frąckowiak et al. 2019). Inducers restrict pathogen development by activating host celldefence responses, including oxidative burst, lignin and callose deposition, and a complex signal transduction network (SA JA and ET) (Ryals et al. 1996; Thomma et al. 1998; Kunkel et al. 2002). The IR phenotype is associated with both direct induction of defence responses, and primed defence responses (Wilkinson et al. 2019; Balmer et al. 2015). Direct defensive responses are defensive responses that are locally or systematically induced in plants when exposed to IR stimuli, and these responses can be detected prior to any challenge (Mauch-Mani et al. 2017; Conrath et al. 2002; Van Hulten et al. 2006). In the priming defence response, the plant's defence system is not directly affected by IR stimulation but can be activated in late challenge (Mauch-Mani et al. 2017). Studies have shown that a low concentration of MeJA (10 μmol/L) triggers a priming defence mechanism, while a higher concentration of MeJA (50 or 100 μmol/L) directly activates defence responses, thus enhancing disease resistance in grape berries (Wang et al. 2015). Protecting plants by inducible resistance mechanisms may be a more environmentally friendly control method. The metabolic investment of the plant is reduced compared to constructional defence activation (Conrath et al. 2006).

Thiamine is a water-soluble B-complex vitamin that is produced in plants and microbes (Burrows et al. 2000). Many studies have documented the beneficial roles of thiamine in enhancing plant disease resistance (including to bacterial wilt) (Ahn et al. 2005, 2007; Jung et al. 2003). It has been shown that thiamine can directly inhibit the activity of Plasmopara viticola in vitro (Boubakri et al. 2012). Some early experiments demonstrated that thiamine can activate PR-1 gene expression in tobacco and stimulate resistance to TMV in a SA-dependent manner (Malamy et al. 1996). Subsequent investigations revealed that thiamine primes the pathogen-induced expression of PR-1 and PAL as well as callose deposition and an oxidative burst associated with the HR in Arabidopsis (Ahn et al. 2007). When infected by the blast fungus Magnaporthe grisea, the basic resistance level of rice plants supplied with thiamine was increased, which was attributed to the formation of a physical barrier that prevented or slowed fungal penetration and stronger and more rapid pathogenesis-related (PR) gene expression and activity of protein kinase C (Ahn et al. 2005). Recent studies have shown that thiamine induces rice to inhibit nematode infection by enhancing lignin production mediated by H2O2 and phenylpropanol (Huang et al. 2016). Although thiamine has been found to induce resistance to different pathogens, the molecular mechanisms underlying this have not been completely elucidated (Asensi-Fabado et al. 2010), especially Phytophthora infection, which has not been tested previously.

Because of the protective activity of thiamine in inducing plant priming against disease, we hypothesized that thiamine might have the potential to activate plant disease resistance against P. nicotianae infections in the thiamine nonaccumulator plant tobacco. Therefore, related experiments were conducted using N. tabacum (tobacco) to investigate the related underlying mechanisms of thiamine in inducing plant disease resistance. We monitored the content of H2O2 and SA and the activity of defence enzymes in tobacco leaves. In addition, we also examined the expression of a set of pathogenesis-related genes in SA defence signalling pathways, the ET pathway and the HR pathway, or encoding the proteins participating in antifungal defence by quantitative real-time PCR (RT–qPCR).

Materials and methods

Plant material and inoculum preparation

The tobacco cultivar Honghua Dajinyuan (HD), which is highly sensitive to black shank, was used as the material. The seeds (gifted by tobacco companies) were sown in seedling trays, and the seedlings were transferred to larger pots (diameter = 30 cm) at the 5–6 true-leaved stage, keeping one seedling per pot, and grown in a greenhouse (25 ± 3℃), Yunnan Agricultural University, Kunming, Yunnan Province, China. The plantlets were used within two weeks of transplanting.

The P. nicotianae strain maintained in our laboratory was routinely recultured on potato dextrose agar (PDA: 200 g of potato, 20 g of glucose and 15 g of agar in 1000 ml water) at 27 °C in the dark for 15 days (Huang et al. 2015; Gallup et al. 2018).

Effect of thiamine at different concentrations on mycelial growth and sporangium production of P. nicotianae in vitro

Thiamine stock solution was prepared in sterile distilled water, and the solution was filtered through a microfiltration membrane(0.45 μm). The effect of thiamine on the mycelial growth of P. nicotianae was evaluated on PDA plates according to the method of Zhang et al. (2018; 2020). Mycelial plugs (3 mm diameter) from actively growing the pathogen for 15 days were transferred into a new PDA plate with different concentrations of thiamine (0, 1, 2, 5, 10, 20 and 50 mM). The mycelial disk was placed in the centre of the plate (diameter 90 mm). After 15 days of incubation at 28 °C, the colony diameters were measured by the perpendicular diameters, took the mean of diameters measured at right angles to each other, and the inhibition rate was calculated. Each of the thiamine concentrations was replicated on four plates, and the experiment was repeated three times.

According to the reported method of Dalio et al. (2014), with slight modifications, the effect of thiamine on the sporangia of P. nicotianae was studied. Briefly, 0.1% KNO3 was used to prepare induction solutions containing different concentrations of thiamine (0, 1, 2, 5, 10, 20, 50 mM), and a 7-mm P. nicotianae agar disk from actively growing 15-day mycelium was transferred to a petri dish supplemented with induction solution (10 mL), followed by culturing at 28 °C for 48 h. In an aseptic operating environment, the agar medium at the lower part of the fungal mycelia disk was cut off in parallel to make the thickness approximately 1 mm and was placed on the slide to observe the number of sporangia under a 10 × 20 optical microscope, and pictures were taken and recorded. All treatments consisted of four replicates, and the experiment was repeated three times.

Induction of ‘HD’ tobacco resistance against P. nicotianae by thiamine

Thiamine Treatment: Four- to five-leaf tobacco seedlings were uniformly sprayed with either distilled water (DW) or thiamine at concentrations of 20 mM. After that, the treated seedlings in each treatment group were separately covered with plastic bags to maintain high humidity and incubated in a climate-controlled room. A second spray was given seven days later. Each treatment was conducted in triplicate, and each replicate contained 10 plants. Leaves were detached from the same layer of plants for assays at 0, 6, 24, 72 and 120 h (Deenamo et al. 2018).

P. nicotianae inoculation: Three days after the second spray of distilled water (DW) or thiamine (20 mM) on the leaves, P. nicotianae was inoculated according to the method of Keller (Sullivan et al. 2005). Mycelial plugs (7 mm diameter) from actively growing the pathogen for 15 days were placed on the base surface of the petiole that was scratched with the blade first (the 4th extended leaf from top to bottom) and then moisturized with sterile cotton. After inoculation, the seedlings were kept in a plastic bag with high humidity and placed in a greenhouse. Tobacco stalk symptoms were evaluated at 0, 2, 6, 8, 10 and 15 dpi. At 15 dpi, the morphological indexes were measured, and the disease condition was recorded and classified by plant. The disease severity was based on the degree of spread of disease spots at the stem base and leaf wilting. The disease classification standard and investigation method were carried out according to the provisions of the tobacco industry standard YC/T 39–1996 of the People's Republic of China. Ten plants were used for each treatment. The experiment was repeated three times. Leaves were collected from plants at different intervals (1, 5, 10 dpi), immediately frozen in liquid nitrogen, and stored at -80 °C until use for enzyme assays and gene expression analysis.

Protein extraction, H2O2 content and enzyme activity assays

Protein content, H2O2 content, and the activities of main antioxidant enzymes, including CAT, POD and PAL, were determined using a protein assay kit, an H2O2 content assay kit, a CAT activity assay kit, a POD activity detection kit, and a PAL activity assay kit (Suzhou Greys Biological Technology Co., Ltd, Suzhou, China), respectively.

Total phenolic content and lignin detection

Leaf samples (0.1 g fresh weight) were ground to a fine powder in liquid nitrogen with a frozen mortar and pestle and then homogenized with 1.5 mL of 60% ethanol. The homogenate was centrifuged at 12,000 rpm for 10 min at room temperature. The total phenol content of the extract was determined by the Folin phenolic method (Blainski et al. 2013). Under alkaline conditions, the phenolic substances reduced tungstomolybdic acid to produce blue compounds. The absorbance value was read at 760 nm to determine the total phenol content. The absorbance values were calibrated against a standard curve and expressed as µg per millilitre (µg ML−1). For the determination of lignin content, the leaf samples (1.5 mg dry weight) were ground into a fine powder with a mortar and pestle, homogenized with 1.5 mL of 80% ethanol and centrifuged at 12,000 rpm for 10 min at room temperature. The total phenolic content of the extract was determined by the acetylation method (Moreira-Vilar et al. 2014), and the phenol hydroxyl group in the lignin was acetylated. The absorbance value was read at 280 nm to determine the lignin content. The absorbance values were calibrated against a standard curve and expressed as milligrams per gram of dry weight (mg g−1 DW).

SA and scopoletin measurements

The contents of SA and scopoletin in the leaves of tobacco seedlings were measured by high-performance liquid chromatography (HPLC). The SA content was determined as described in previous studies (Wen et al. 2005; Drzewiecka et al. 2012) with some slight modifications. Leaf samples (0.2 g fresh weight) were ground to a fine powder in liquid nitrogen with a frozen mortar and pestle and then homogenized with 1 mL of 70% methanol and extracted overnight at 4 °C. After the supernatant was centrifuged at 8000 × g for 10 min, it was subsequently filtered through a 0.45-µm membrane.

Scopoletin content was determined according to the method of Lerat et al. (2009). Leaf samples of 0.1 g fresh weight were ground into a fine powder with a mortar and pestle and then dissolved in a flask with 20 mL of 50% methanol. The mixture was ultrasonically extracted at room temperature for 20 min, centrifuged at 3000 rpm for 5 min and filtered through a 0.45-µm aqueous phase membrane.

Chromatographic separation was performed on a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm) using a Jingdao LC-20AT high-performance liquid chromatograph. The compound in the sample (10 µL) was separated in a mobile phase containing methanol and 0.1% acetic acid water. The flow rate was 0.8 mL min−1, the column temperature was controlled at 35 °C, and the retention time was 40 min. The SA UV detection wavelength was 306 nm, and the scopoletin detection wavelength was 340 nm. Each sample was subjected to HPLC with three independent replicates.

Analysis of gene expression by quantitative real-time PCR

Transcription of defence-related genes was determined, and the expression levels of SA pathway, ET pathway and HR pathway genes listed in Table 1 were detected (Guo et al. 2020). Total RNA was extracted from tobacco leaf tissue with a MagenHiPure HP Plant RNA Mini Kit (R4165-02, Magen, China). The RNA samples were measured for quality and quantity by measuring the ratio of 260/280 nm absorption, and their integrity was evaluated by visualizing the bands following electrophoresis on a 1% agarose gel. cDNA for RT–qPCR was synthesized from 2 µg of total RNA using the ABM 5 × All-in-One RT MasterMix (with AccuRT Genomic DNA Removal Kit) kit according to the manufacturer's instructions.

The PCR conditions were as follows: an initial incubation at 95℃ for 3 min, followed by 45 cycles of 95℃ for 10 s and 60℃ for 30 s and then by a melting curve cycle. The threshold period (CT) and melting curve of each gene were analysed. The relative mRNA amount was calculated by the 2−△△Ct method. Three biological replicates were performed for each experiment.

Statistical analysis

All of the data were analysed using SPSS 20.0. Significant differences between each experimental value between treatments were analysed at p < 0.05 by Student’s t-test. Data are presented as means ± SE. The graphs were generated using Origin 2018.

Results

In vitro inhibition of mycelial growth and sporangium production of P. nicotianae by thiamine

The results of in vitro inhibition test showed that the mycelial growth and sporangium production of P. nicotianae were inhibited by thiamine in a dose-dependent manner. Inhibition of mycelial growth started at 1 mm/L and reached 93% inhibition with 50 mm/L thiamine (Fig. 1a, b). The number of sporangia released was significantly lower for all thiamine treatments (Fig. 1c). These data indicated that thiamine has a significant inhibitory effect on P. nicotianae in vitro.

Fig. 1
figure 1

Effect of thiamine on in vitro growth and sporangium production of P. nicotianae a P. nicotianae cultures in Petri dishes illustrating the inhibition of mycelial radial growth with increasing thiamine concentrations. The mycelial colonies were 15 days old. b Inhibition of P. nicotianae mycelial growth using different thiamine concentrations. c Inhibition of P. nicotianae sporangium production at different thiamine concentrations. These assays were repeated three times showing similar results. Bars show means ± SE (n = 12). Values with the same letter are not significantly different at P < 0.05

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Induced resistance of thiamine-pretreated tobacco against P. nicotianae

The tobacco leaves were sprayed with 20 mM thiamine, and there was no negative effect on tobacco growth during the whole experimental period (Table 2). However, the tobacco leaves were slightly damaged in the early stage of thiamine treatment and gradually returned to normal in the later stage.

At 2 dpi, small black and necrotic lesions were observed on the stems inoculated with the pathogen. In contrast, 20 mM thiamine pretreatment significantly inhibited the growth of P. nicotianae on infected stems, reduced the area of disease patches to varying degrees (Fig. 2a), and significantly reduced the severity of the disease by 37% (Fig. 2b). The results indicated that thiamine pretreatment could effectively inhibit P. nicotianae growth on tobacco stalk.

Fig. 2
figure 2

Effect of exogenous thiamine treatment on tobacco against P. nicotianae. a Disease symptom b disease severity (%) of tobacco leaves pretreated with either DW or 20 mM thiamine for 3 day prior to subsequent inoculation with P. nicotianae at 15 dpi. Bars show means ± SE (n = 30). Different letters indicate significant diferences among treatments (P < 0.05; Duncan’s multiple-range test)

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Effects of thiamine priming on H2O2 content, lignin content, CAT and POD activities, total protein content, and total phenolic content in tobacco leaves after P. nicotianae challenge.

The leaves were collected at 5 dpi, and the test results are shown in Fig. 3. H2O2 did not change with thiamine or inoculation wih P. nicotianae alone, but increased significantly with both thiamine and P. nicotianae together (Fig. 3a). There were similar results with lignin content (Fig. 3b). By contrast, POD activity did not change with P. nicotianae alone but increased significantly with thiamine, to the same extent, alone and with P. nicotianae (Fig. 3c). CAT activity did not change with thiamine alone but showed small but significant increases with P. nicotianae, alone and with thiamine (Fig. 3d). Protein content did not change either when inoculated with P. nicotianae alone or with thiamine, but significantly reduced protein content with thiamine alone(Fig. 3e). Total phenol content did not change with thiamine or inoculation with P. nicotianae (Fig. 3f).

Fig. 3
figure 3

Effect of exogenous thiamine pretreatment on H2O2 content, lignin content, POD and CAT activities, total phenolic content and total protein content in tobacco leaves after inoculation with P. nicotianae (P. nic). The leaves of tobacco were sprayed with either distilled water (DW) or Thiamine. After treatment, leaves were subsequently treated with either DW or P. nicotianae. After 5 day, the leaf samples were collected for determining a H2O2 content; b lignin content; c POD activity; d CAT activity staining; e protein content; f total phenolic content; Bars show means ± SE (n = 4). Different letters indicate significant diferences among treatments (P < 0.05; Duncan’s multiple-range test)

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Effects of thiamine priming on salicylic acid and scopoletin contents in tobacco leaves after P. nicotianae challenge

The treated leaves were collected at 5dpi, and the contents of endogenous SA and Scp were determined. The results showed that the content of SA did not change with thiamine or inoculation with P. nicotianae alone, but but increased significantly with both thiamine and P. nicotianae together (Fig. 4a). In addition,

Fig. 4
figure 4

Effect of exogenous thiamine pretreatment on salicylic acid content and scopoletin contents in tobacco leaves after inoculation with P. nicotianae (P. nic). The leaves of tobacco were sprayed with either distilled water (DW) or Thiamine. After treatment, leaves were subsequently treated with either DW or P. nicotianae. After 5 day, the leaf samples were collected for determining a SA content; b scopoletin content; Bars show means ± SE (n = 4). Different letters indicate significant diferences among treatments (P < 0.05; Duncan’s multiple-range test)

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thiamine alone decreased SCP content, but inoculation with P. nicotianae increased it, with and without thiamine(Fig. 4b).

Effects of thiamine on the kinetics of H2O2 content, defence enzyme activities and lignin contents in tobacco leaves

The results revealed that thiamine treatment caused an accumulation of H2O2 in leaves at 6 h until 72 h, which was significantly higher than the control (DW), and then gradually decreased (Fig. 5a). CAT activity was slightly increased from 6–24 h, peaked at 24 h (1.54-fold) and subsequently remained at the same level until 120 h, and was significantly higher than the control within 24–120 h (Fig. 5b). POD activity continuously increased at 6 h until 120 h and reached its highest level at 120 h (4.11-fold), and was significantly higher than that of the control (DW) within 6 to 120 h (Fig. 5c). PAL activity increased continuously from 6 to 24 h, peaked at 24 h (5.03-fold), and then decreased gradually, and was significantly higher than that of the control (DW) in 6–72 h (Fig. 5d). At 24 h and 72 h, the lignin contents remained at the same level as the control (Fig. 5e), indicating that thiamine did not induce lignin deposition in tobacco leaves.

Fig. 5
figure 5

The effect of Thiamine on a H2O2 content; b CAT activities; c POD activities; d PAL activities and e lignin contents in tobacco leaves. The leaves were sprayed with either distilled water (DW) as control or 20 mM Thiamine and harvested at different points of time (6, 24, 72 and 120 h) for enzyme activity measurements, H2O2 content and lignin content. Bars show means ± SE (n = 4). Different letters indicate significant diferences among treatments (P < 0.05; Duncan’s multiple-range test)

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Effects of thiamine on PR1, PR5, NPR1, PAL, CM1, H1N1, and EFE26 expression in tobacco leaves

To investigate the effects of thiamine on the expression of tobacco defence genes, the expression levels of SA pathway (PR1, PR5, NPR1, PAL, CM1), ET pathway (EFE26) and HR pathway (H1N1) genes were determined by qRT–PCR. The expression of PR1 was slightly induced at 6 h until 24 h and greatly induced by 90.49-fold and 30.92-fold at 72 h and 120 h, respectively (Fig. 6a). The expression of PR5 increased gradually from 6 to 72 h, reached a maximum at 72 h (39.4-fold), and then decreased but was still higher than that of the control (Fig. 6b). For NPR1, the expression was downregulated at 6 h and increased by 1.55-fold at 72 h. After 72 h, the expression was inhibited to maintain the same level as the control (Fig. 6c). For PAL, the expression was significantly upregulated by 2.44-fold at 6 h and remained the same at 24 h when compared to the control plants. Subsequently, the expression was increased by 1.53-fold and 1.30-fold at 72 h and 120 h, respectively (Fig. 6d). The expression of CM1 was significantly upregulated at 6–72 h, then the expression decreased, but still higher than that of the control (Fig. 6e). In thiamine-treated plants, the expression of H1N1 increased at 6 h and decreased at 24 h but was still higher than that in the control. The expression increased again by 8.92- and 14.62-fold at 72 h and 120 h, respectively (Fig. 6f). The expression of EFE26 reached the highest level at 6 h (9.13-fold), after which the expression decreased. However, compared to the level of the control, the expression of EFE26 was still higher at 24 h until 120 h (Fig. 6g). These data indicated that 20 mM thiamine treatment resulted in significant upregulation the expression levels of genes related to the SA, ET and HR pathways.

Fig. 6
figure 6

Effect of Thiamine on transcript abundant of a PR1; b PR5; c NPR1; d PAL; e CM1; f H1N1 and g EFE26 genes in tobacco leaves. The leaves were sprayed with either distilled water or 20 mM Thiamine. qRT-PCR were taken at various time points (6, 24, 72 and 120 h). The expression levels of genes were expressed as a relative transcript fold change to their controls. Bars show means ± SE (n = 4). Different letters indicate significant diferences among treatments (P < 0.05; Duncan’s multiple-range test)

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Effects of thiamine priming on defence genes in tobacco leaves after P. nicotianae challenge

The expression profiles of PR1 and PR5 exhibited similar patterns in the pathogen-inoculated leaves. Compared with the control, with thiamine alone application induced 23.69- and 32.01-fold increases in PR1 and PR5 transcripts at 1 dpi, respectively. However, the different treatments triggered enhanced gene expression at various time points. The transcript levels of PR genes in leaves treated with thiamine and inoculated with P. nicotianae increased earlier and higher than other treatments. At 5 dpi, an additive effect was observed with both thiamine and P. nicotianae together, and the expression levels of PR1 and PR5 were increased by 14.91- and 5.70-fold compared with thiamine alone, respectively (Fig. 7a, b).

Fig. 7
figure 7

Effect of Thiamine on transcript abundance of a PR1; b PR5; c NPR1; d PAL; e CM1; f H1N1 and g EFE26 genes in tobacco leaves after inoculation with P. nicotianae. Total RNA was extracted from leaf tissues taken at different time points, converted to cDNA, and subjected to quantitative real-time PCR. Bars show means ± SE (n = 4). Different letters indicate significant diferences among treatments (P < 0.05; Duncan’s multiple-range test)

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The expression levels of PAL, NPR1 and CM1 were up-regulated at 1 dpi 10 dpi and 1 dpi with thiamine alone, respectively, and inoculation with P. nicotianae did not significantly affect their transcript levels. At 5 dpi, the expression of NPR1 was significantly upregulated (2.37-fold) after inoculation with P. nicotianae compared with thiamine alone(Fig. 7c-e). At 1 and 10 dpi, the expression of the H1N1 gene were up-regulated with thiamine alone. At 10 dpi, all treatments had significantly higher expression than the control (Fig. 7f). For EFE26, an additive effect was observed with both thiamine and P. nicotianae together at 1 dpi, with a 2.57-fold increase in EFE26 expression compared to thiamine alone (Fig. 7g).

Discussion

In this study, we found that pretreatment with 20 mM thiamine could effectively improve tobacco resistance to P. nicotianae without toxic effects, indicating that 20 mM thiamine was a safe concentration range for tobacco cultivation. Previous studies have shown that 30 mM thiamine can protect grapes from Plasmopara viticola (Boubakri et al. 2012), and 50 mM thiamine can replace carbendazim as a systematic fungicide to effectively control sheath blight disease in rice (Bahuguna et al. 2012), indicating that thiamine meets the requirements of being an activator of plant-induced resistance and is a systematic broad-spectrum persistent drug resistance inducer, which can be further developed and used.

It has been reported that thiamine can inhibit the growth of Plasmopara viticola and grey mould in vitro (Boubakri et al. 2012; Hong et al. 2016). Our results demonstrated that the mycelial growth and sporangium production of P. nicotianae were inhibited by thiamine in a dose-dependent manner (Fig. 1). Surprisingly, the growth of P. nicotianae was not completely inhibited, even at the highest thiamine concentrations, possibly indicating that the P. nicotianae isolate was able to detoxify thiamine to a certain amount. These results were first reported about the effect of thiamine on P. nicotianae growth, at least showing that P. nicotianae isolates isolated by us were sensitive to thiamine.

The rapid accumulation of ROS is one of the earliest responses when plants are attacked by pathogens at attack sites. Increasing ROS can participate in the orchestration of the hypersensitive response and can be used to destroy invading pathogens (Bastas et al. 2014). H2O2 is a stable intermediate of ROS and has been shown to inhibit the viability of diverse microbial pathogens, and its oxidative potential contributes to plant wall strengthening during plant-pathogen interactions (Wu et al. 1997). H2O2 could also induce the expression of genes encoding proteins involved in defensive and antioxidant processes and has been reported by some studies as a diffusible selective signal (Bhattacharjee 2005). Compared to the inoculated with P. nicotianae without thiamine pretreatment tobacco plants, H2O2 accumulation was significantly increased in the thiamine-treated tobacco plants with P. nicotianae inoculation, indicating a priming effect mediated by thiamine on the P. nicotianae-induced oxidative burst (Fig. 3a). Ahn et al. also reported thiamine-induced H2O2 generation in Arabidopsis plants but only following pathogen invasion. These results suggest that H2O2 plays a key role in thiamine signal transduction, leading to the induction of defence responses that ultimately inhibit the pathogen.

Lignin is a high-molecular-weight polymer that consists of β-(1,4)-glucan (Luna et al. 2011). Lignin deposition could be seen as a way to strengthen the plant cell wall. It is also an evaluation method widely used to judge plant-triggered immunity (PTI) (Bittel and Robatzek 2007). It is well known that monolignols are efficiently polymerized by peroxidase with H2O2 consumption, leading to lignin formation (Pauwels et al. 2008). In noninoculated plants, no significant differences in lignin levels were measured in thiamine-treated versus nontreated plants (Fig. 5e). However, significantly higher lignin levels were measured at 5 dpi in thiamine-treated plants that were inoculated than in those that were inoculated but not treated, indicating that thiamine can induce cell wall lignification after pathogen challenge (Fig. 3b). This result is consistent with the result reported by Huang et al. (2016).

It is known that plant cells can be injured by excess H2O2. Thus, plants must employ some mechanism to detoxify excess H2O2, such as the antioxidant enzyme system, of which CAT is a well-known member (Wojtaszek 1997). Our data showed that the activity change of CAT was according to H2O2 concentration (Fig. 3), which may indicate CAT’s degrading function on H2O2 at relatively high H2O2 concentrations. The fact that massive accumulation of H2O2 caused less damage during the pathogen-induced oxidative burst might be due to the induction of CAT activities. CAT and POD are considered the main antioxidant systems to protect cells against oxidative damage (De Gara et al. 2003). Thiamine application could induce increases in CAT and POD activity levels (Figs. 3 and 5). Studies have shown that thiamine can increase H2O2 and lignin contents and the activity of defence compounds (Boubakri et al. 2012; Huang et al. 2016; Bahuguna et al. 2012). Our results also showed that thiamine preconditioning induced a series of defence responses, including H2O2 accumulation, and significantly increased CAT, POD and PAL activity and SA content (Figs. 3 and 4), which in turn induced lignin production and increased the physical barrier of the cell wall to restrict the penetration of pathogens.

The response of defence-related genes to exogenously applied thiamine was examined in tobacco leaves. Our results showed that the expression of the PR1, PR5, NPR1, PAL, CM1, H1N1, and EFE26 genes was significantly induced by thiamine (Figs. 6 and 7). Chemical elicitors have been reported to induce resistance against fungal pathogen infection in crops by stimulating PR gene expression (Yu et al. 2014). The increased expression of PR1, PR5 and others is widely accepted as a hallmark of plant defence induction (Aćimović et al. 2015). In addition, the PR1, PR5, NPR1, PAL, and CM1 genes mediated the defence response through the SA pathway. The results showed that the thiamine-induced expression of the PR gene might be related to the tobacco resistance conferred on it, and the PR gene was rapidly activated at 24 h (Fig. 6). The NPR1 regulator mediates SAR to a broad spectrum of plant pathogens by activating defence genes (PRs) in SAR, including PR-1 and PR-5, by virtue of their particular structures and functions (Mach et al. 2015; Yang et al. 2018). Here, the results showed that thiamine treatment enhanced the expression of NPR1, PR1, and PR5 in tobacco leaves to confer resistance against P. nicotianae, although the reaction was dependent on the days after P. nicotianae infection (Fig. 7). EFE26, H1N1 and CM1 are ET biosynthesis and signalling marker genes (Chen et al. 2003), and our results also showed that tobacco resistance against P. nicotianae was related to their expression. We also observed a significant upregulation of the PAL gene (Fig. 6), which was correlated with the increase in PAL activity (Fig. 5) by thiamine treatment. As mentioned above, PAL is considered to be a key enzyme in catalysing various phenylpropanoid defence metabolites, such as lignin. Furthermore, it has been proven that PAL functions as a rate-limiting enzyme in the phenylpropanoid pathway (Kolahi et al. 2013). Therefore, we can conclude that cell wall reinforcement by the accumulation of lignin supports mechanical resistance to pathogen penetration, which may be attributed to the increased expression of the PAL gene.

The IR phenotype is associated with both direct induction of defence responses, which can be transient or long-lasting, and primed defence responses (Wilkinson et al. 2019; Balmer et al. 2015). Ahn et al. (2007). reported for the first time that thiamine induced resistance in Arabidopsis against P. syringae through priming defence responses. Boubakri et al. (2012). showed that thiamine effectively induces resistance against P. viticola in grapevine through a dual mode of action involving direct antifungal activity and induction of host defence mechanisms. Studies have also shown that inducers can trigger priming defence mechanisms at low concentrations and directly activate defence responses at high concentrations (Wang et al. 2015; van Hulten et al. 2006). Our data showed that 20 mM thiamine could directly inhibit the activity of P. nicotianae, and regardless of pathogen infection, thiamine could induce the accumulation of a large amount of H202 in tobacco that would be expected to increase the activity of defence-related enzymes and the expression of related defence genes. These defence genes were upregulated or downregulated to different degrees during subsequent P. nicotianae infection. It was suggested that thiamine acted as an inducer in tobacco, inducing a direct defence response in tobacco, and may initiate a defence response to the subsequent attack of P. nicotianae through the SA, ET and HR pathways.

In conclusion, the findings of this study clearly demonstrate that thiamine can effectively induce resistance against P. nicotianae in tobacco under glasshouse-controlled conditions through a dual mode of action involving direct antifungal activity and induction of host defence mechanisms. The thiamine-induced defence response included the generation of H2O2, lignin deposition, defence enzyme activity enhancement and the expression of a number of defence-related genes (SA pathway, ET pathway and HR pathway). These results suggest that thiamine can be used in tobacco cultivation to test its effectiveness under field conditions.

Table 1 Primer pairs used for qRT-PCR
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Table 2 Effects of different treatments on tobacco morphology indexes
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