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. 2015 Apr 19;16(8):882–892. doi: 10.1111/mpp.12248

Two thiadiazole compounds promote rice defence against X anthomonas oryzae pv. oryzae by suppressing the bacterium's production of extracellular polysaccharides

Xiaoyu Liang 1, Xiaoyue Yu 1, Wenxia Dong 1, Shijian Guo 2, Shu Xu 1, Jianxin Wang 1, Mingguo Zhou 1,✉
PMCID: PMC6638481  PMID: 25727092

Summary

Thiazole, isothiazole, thiadiazole, and their derivatives are used to control various human, animal and plant diseases. In addition to having direct anti‐microbial and anti‐fungal properties, these compounds are thought to induce host defences, but the mechanism of defence induction remains poorly understood. This article reports that the thiadiazoles of zinc thiazole and bismerthiazol induce H2O2 accumulation, up‐regulation of defence‐related genes, callose deposition and hypersensitive response‐like cell death in rice leaves infected with Xanthomonas oryaze pv. oryzae (Xoo) strain ZJ173, but not in non‐infected leaves. These defence responses in Xoo‐infected leaves were suppressed by the exogenous application of catalase, which reduces H2O2 accumulation. The application of extracellular polysaccharides (EPSs) extracted from strain ZJ173 significantly compromised rice defence against ZJ173 with or without thiadiazole treatment. The EPS‐deficient Xoo mutant ∆ gumH triggered a stronger defence than its parent strain ZJ173. The thiadiazole treatments reduced EPS production by strain ZJ173, but not by the thiadiazole‐resistant strain 2‐1‐1, which is thiadiazole resistant in vivo, but not in vitro; moreover, enhanced defence was not detected in thiadiazole‐treated rice inoculated with 2‐1‐1. Based on these data, we infer that zinc thiazole and bismerthiazol promote rice defence against Xoo by inhibiting the production of bacterial EPS.

Keywords: bismerthiazol, defence responses, extracellular polysaccharides, rice, thiadiazole, Xanthomonas oryzae pv. oryzae, zinc thiazole

Introduction

Thiazole, isothiazole, thiadiazole and their derivatives not only have anti‐microbial and anti‐fungal properties, but also induce systemic acquired resistance (SAR) in host plants, and so are widely used to reduce disease in plants and other organisms (Leoni et al., 2014; Oostendorp et al., 2001; Turan‐Zitouni et al., 2004). Although two novel thiadiazoles—zinc thiazole and bismerthiazol—have been used to control bacterial leaf blight (BLB) of rice in China for several years (Chen et al., 2014; Zhang et al., 2005, 2013; Fig. S1, see Supporting Information), their modes of action are poorly understood.

When attacked by certain pathogens, plants may recognize the pathogens and activate complex defence responses by the initiation of appropriate signalling processes (Durrant and Dong, 2004). The defence responses mainly include reactive oxygen species (ROS) bursts, callose deposition, hypersensitive response‐like cell death and the expression of defence genes (Hammond‐Kosack and Jones, 1996; Lamb and Dixon, 1997). The defence genes, which mainly include those encoding pathogenesis‐related (PR) proteins, play an important role in defence responses. For example, the genes OsPR1a and OsPR1b are commonly used as markers for defence responses in many plant species (Mitsuhara et al., 2008; Van Loon and Van Strien, 1999). POX22.3, a peroxidase gene, is expressed during resistant responses of rice to the BLB pathogen Xanthomonas oryaze pv. oryzae (Xoo) (Chittoor et al., 1997; Liu et al., 2005). The induction of defence responses in rice occurs through a complex signal transduction network, which mainly involves salicylic acid (SA) and jasmonic acid (JA) (De Vleesschauwer et al., 2013; Fitzgerald et al., 2004; Qiu et al., 2007). OsPAL (encoding phenylalanine ammonia‐lyase, PAL) and OsLOX (encoding lipoxygenase, LOX) are key genes in the synthesis of SA and JA, respectively (Coquoz et al., 1998; Lee et al., 2004; Mase et al., 2005).

As an important virulence factor of pathogenic bacteria, extracellular polysaccharide (EPS) helps pathogenic bacteria avoid and delay the activation of plant defence responses (Jahr et al., 1999; Parniske et al., 1994). For example, EPS cloaks Ralstonia solanacearum from recognition by its plant host (Milling et al., 2011); EPS induces plant susceptibility to Xanthomonas campestris pv. campestris by chelating calcium and suppressing callose deposition (Aslam et al., 2008; Yun et al., 2006). However, the role of EPS produced by Xoo in its interaction with rice plants is poorly understood.

Previous research has shown that Xoo isolates induced on nutrient agar (NA) medium are resistant based on growth in bismerthiazol‐amended medium, but are not resistant on bismerthiazol‐treated rice and, remarkably, show a decrease or even loss of virulence; moreover, field isolates resistant to bismerthiazol based on pathogenicity are sensitive in vitro (Zhu et al., 2013). These results indicate that resistance based on direct biocidal effects can be separated from resistance based on the induction of host defences. The mutant Xoo 2‐1‐1, which was selected on bismerthiazol‐treated rice inoculated with wild‐type Xoo ZJ173, shows cross‐resistance to zinc thiazole in vivo, but not in vitro (Zhu et al., 2014). The mechanism by which Xoo 2‐1‐1 resists these thiadiazoles in vivo is unknown.

The objectives of this study were: (i) to determine whether zinc thiazole and bismerthiazol affect the defence responses of rice against Xoo; (ii) to determine whether Xoo EPS is involved in Xoo resistance against rice defence; (iii) to investigate the relation between thiadiazole compounds, EPS and rice defence; and (iv) to study the mechanism by which Xoo isolates resist thiadiazole compounds.

Results

Effects of zinc thiazole on H2O2 levels and BLB lesions in Xoo‐inoculated rice leaves

An increase in H2O2 is one of the earliest defence responses in plants to pathogen infection or elicitor treatment (Orozco‐Cárdenas et al., 2001). In the current study, we determined whether zinc thiazole (at 200 mg/L) and Xoo inoculation, alone or in combination, induced the generation of extracellular H2O2 in rice plants. The results showed that zinc thiazole alone did not trigger the release of H2O2, but that zinc thiazole applied 1 day after Xoo inoculation triggered a sustained release of H2O2 (Fig. 1a). The application of zinc thiazole 1 day before Xoo inoculation also tended to induce H2O2 production, but the increase in H2O2 was significant only on the third day after treatment (Fig. 1a).

Figure 1.

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Effects of zinc thiazole on H2O2 levels, bacterial leaf blight (BLB) lesions, Xanthomonas oryaze pv. oryzae (Xoo) density, expression of defence genes and signalling pathways in rice leaves. Leaves were not inoculated or were inoculated with Xoo on day 0 or day 2. Leaves were not treated with zinc thiazole or were treated with 200 mg/L zinc thiazole on day 1. The five treatments were as follows: CK, not inoculated with Xoo and not treated with zinc thiazole; T1, not treated with zinc thiazole, but inoculated with Xoo; T2, treated with zinc thiazole, but not inoculated with Xoo; T3, treated with zinc thiazole 1 day after Xoo inoculation; T4, treated with zinc thiazole 1 day before Xoo inoculation. (a) H2O2 levels at 2‐day intervals after zinc thiazole treatment. (b) Zinc thiazole inhibition of lesion expansion on leaves; inhibition was calculated relative to the controls that were not treated with zinc thiazole, and was determined 7 days after zinc thiazole application. H2O2 levels (c) and Xoo density (d) in Xoo‐inoculated rice leaves, 3 days after application of different concentrations of zinc thiazole. Expression of defence genes (e), phenylalanine ammonia‐lyase (PAL) activity (f) and salicylic acid (SA) levels (g) were determined 3 days after Xoo‐inoculated rice leaves had been treated with 200 mg/L zinc thiazole. Values are the means of three experiments, with three replicate plants per experiment. For (a) and (c–g), means with different letters are significantly different according to Fisher's least‐significant difference test (P < 0.05). For (b), an asterisk indicates a significant difference (P < 0.05) between application time according to Student's t‐test.

We also investigated the effect of zinc thiazole application time on BLB lesion expansion on rice leaves. Zinc thiazole inhibited BLB lesion expansion when applied 1 day before or 1 day after Xoo inoculation, but the inhibition was greater when the compound was applied before rather than after Xoo inoculation (Fig. 1b).

H2O2 levels and Xoo density in rice leaves are affected by zinc thiazole concentration

To determine the effect of zinc thiazole concentration on defence responses of rice against Xoo, we assessed the effect of zinc thiazole concentration on H2O2 generation and on the density of Xoo in rice leaves. H2O2 levels increased gradually as the zinc thiazole concentration increased from 0 to 200 mg/L, but then decreased when the concentration was raised to 300 mg/L (Fig. 1c). In contrast, the density of Xoo in the leaves decreased steadily as the zinc thiazole concentration increased from 0 to 300 mg/L (Fig. 1d).

Effects of zinc thiazole on the expression of defence‐related genes in Xoo‐inoculated leaves

The induction of H2O2 by elicitors is often followed by the expression of defence genes (Apel and Hirt, 2004). Therefore, we measured the effect of zinc thiazole treatment on the expression of five defence‐related genes (OsPR1a, OsPR1b, POX22.3, OsPAL and OsLOX) in rice. The transcription levels of all five genes were increased slightly by Xoo alone, but not by zinc thiazole alone (Fig. 1e). Transcription levels of OsPR1a, OsPR1b, POX22.3 and OsPAL, but not OsLOX, were greatly increased when Xoo inoculation was followed by zinc thiazole treatment (Fig. 1e). PAL activity and SA levels were increased when Xoo‐inoculated plants were treated with zinc thiazole, but not when inoculated rice plants were not treated with zinc thiazole or when non‐inoculated rice plants were treated with zinc thiazole (Fig. 1f,g).

Effects of bismerthiazol on defence‐related gene expression and H2O2 levels in Xoo‐inoculated rice leaves

We also assessed whether the expression of the five defence‐related genes and the generation of H2O2 in plants was affected by bismerthiazol application alone, Xoo inoculation alone or bismerthiazol application 1 day after Xoo inoculation. The expression of OsPR1a, OsPR1b, POX22.3 and OsPAL, but not OsLOX, was strongly increased when plants were inoculated with Xoo and then treated with bismerthiazol; however, expression was not increased by bismerthiazol alone (Fig. 2a). In addition, bismerthiazol treatment triggered a sustained release of H2O2 in Xoo‐inoculated rice leaves, but not in non‐inoculated leaves (Fig. 2b).

Figure 2.

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Defence‐related gene expression and levels of H2O2 in rice leaves on application of bismerthiazol following Xanthomonas oryaze pv. oryzae (Xoo) (strain ZJ173) inoculation. Leaves were not inoculated (CK), were inoculated with Xoo alone (ZJ173), were treated with bismerthiazol alone (Bismerthiazol) or were treated with bismerthiazol 1 day after Xoo inoculation (ZJ173 + Bismerthiazol). (a) Expression of defence‐related genes. (b) Changes in H2O2 levels over time. Values are means and standard errors (SEs) of three independent experiments (results were similar for each experiment). Means with different letters are significantly different according to Fisher's least‐significant difference test (P < 0.05).

Cellular defence responses of rice leaves against Xoo when treated with the two thiadiazoles

To determine whether zinc thiazole and bismerthiazol are involved in the cellular defence responses of rice, we examined the H2O2 burst, callose deposition and cell death in plants that were treated with Xoo alone, zinc thiazole alone, bismerthiazol alone, or zinc thiazole or bismerthiazol 1 day before Xoo inoculation. The H2O2 burst, callose deposition and cell death were substantially greater in leaves that were treated with zinc thiazole or bismerthiazol following Xoo inoculation than in leaves treated with Xoo alone or with the compounds alone (Fig. 3).

Figure 3.

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Effects of the two thiadiazoles on the cellular defence responses against Xanthomonas oryaze pv. oryzae (Xoo) in rice. Rice plants were treated with zinc thiazole, bismerthiazol or water. One day later, the plants were inoculated or not inoculated with Xoo, and the inoculated plants were immediately treated with a catalase solution at 5000 units/mL or water. ‘+’ indicates that the plants were inoculated or treated with the indicated compounds, and ‘–’ indicates that the plants were not inoculated or were treated with water instead of the indicated compounds. Leaves were stained and photographed 24 h after inoculation to observe the H2O2 burst (indicated by brown staining), callose deposition (indicated by fluorescence) and cell death (indicated by blue staining). Bar, 100 μm.

To determine whether H2O2 is necessary in the defence induced by zinc thiazole and bismerthiazol, we used catalase to decompose H2O2 without inhibiting pathogen proliferation (Ahn et al., 2007; Van Wees and Glazebrook, 2003). Catalase applied alone to plants did not induce any evident responses, whereas catalase suppressed the H2O2 burst, callose deposition and cell death in leaves treated with the compounds and Xoo (Fig. 3).

Inhibition of EPS production by the two thiadiazoles

Both thiadiazoles reduced the production of EPS by Xoo (Fig. 4a). The reduction in EPS production was not increased by using a higher concentration of zinc thiazole, probably because the solubility of zinc thiazole is low. In contrast, the reduction in EPS production was increased by using a higher concentration of bismerthiazol. The expression of EPS synthesis genes was usually suppressed by one or both of the thiadiazoles (Fig. 4b).

Figure 4.

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Inhibition of extracellular polysaccharide (EPS) production by the two thiadiazoles. (a) Production of EPS in thiadiazole‐treated Xanthomonas oryaze pv. oryzae (Xoo) cultures relative to production in non‐treated cultures. (b) Effects of the two thiadiazoles on the transcription level of six EPS synthesis genes in Xoo. The treatments were as follows: CK, not treated with zinc thiazole and bismerthiazol; a1, 45 mg/L zinc thiazole; a2, 90 mg/L zinc thiazole; b1, 6 mg/L bismerthiazol; b2, 12 mg/L bismerthiazol. Values are means and standard errors (SEs) of three independent experiments. Datasets marked with an asterisk are significantly different from the control as assessed by Student's t‐test: *P < 0.05.

Effects of EPS on defence responses in rice

To determine whether EPS produced by Xoo suppresses rice defence responses, we treated Xoo‐inoculated plants with EPS extracted from Xoo and then measured the expression of defence‐related genes. The transcription levels of three PR genes and the OsPAL gene were increasingly suppressed as the EPS concentration increased, but OsLOX expression was not affected by EPS (Fig. 5a). In addition, EPS treatment enhanced the formation and expansion of BLB lesions on inoculated rice leaves (Fig. 5b).

Figure 5.

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Effects of exogenous extracellular polysaccharide (EPS) on the defence responses in rice. For CK, rice plants were not inoculated with Xanthomonas oryaze pv. oryzae (Xoo) or treated with EPS. For the EPS treatment, non‐inoculated plants were treated with 50 mL of 200 mg/L EPS. For the ZJ173 treatment, plants were inoculated with Xoo (strain ZJ173). For the three other treatments, the Xoo‐inoculated plants were treated with 50 mL of EPS (50, 100 or 200 mg/L) or water 1 day after inoculation. (a) Defence‐related gene expression was measured 3 days after the treatments had been applied. (b) Lesion lengths of the leaves were measured 7 days after EPS application. (c) OsPR1b gene expression of EPS‐treated and untreated rice leaves that were inoculated with ∆ gumH, which is a Xoo mutant that is deficient in EPS production; OsPR1b gene expression is relative to that in ZJ173‐inoculated rice leaves. Values are means and standard errors (SEs) of three independent experiments.

We also examined the expression level of the OsPR1b gene in rice plants inoculated with ∆gumH, which is an EPS‐deficient mutant of Xoo. ∆gumH induced stronger expression of defence genes than ZJ173 when EPS was not added (Fig. 5c). When 200 mg/L EPS was added to the ∆gumH‐inoculated rice plants, ∆gumH induced equal or even lower defence gene expression than ZJ173 (Fig. 5c).

Effect of exogenous EPS on defence responses induced by the two thiadiazoles

To determine whether exogenous EPS could reverse the defence induced by the thiadiazoles, we assessed the defence responses of rice against Xoo on plants treated with zinc thiazole or bismerthiazol and with or without 200 mg/L EPS. The H2O2 production and transcription levels of three PR genes and the OsPAL gene were suppressed by EPS in both zinc thiazole‐ and bismerthiazol‐treated plants, but OsLOX gene expression was not affected (Fig. 6a,b). In addition, the inhibitory effects of the two compounds on BLB lesion formation were reduced by the addition of EPS (Fig. 6c).

Figure 6.

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Effect of exogenous extracellular polysaccharide (EPS) on defence responses induced by the two thiadiazole compounds. Rice plants were inoculated with Xanthomonas oryaze pv. oryzae (Xoo) and, 1 day later, were treated with 50 mL of zinc thiazole or bismerthiazol and with or without 200 mg/L EPS. H2O2 levels (a) and defence‐related gene expression (b) were measured 3 days after treatments. The lengths of bacterial leaf blight (BLB) lesions were measured 7 and 15 days after treatments, and the inhibition rate (c) was calculated. Values are the means and standard errors (SEs) of three replicates. Bars with asterisks are significantly different from the control according to Student's t‐test at P < 0.05.

Effects of the two thiadiazoles on defence responses of rice leaves inoculated with the mutant 2‐1‐1

The Xoo mutant 2‐1‐1 is resistant to zinc thiazole and bismerthiazol in vivo, but not in vitro (Zhu et al., 2014), suggesting that its in vivo resistance involves the failure to induce host defences rather than a reduced sensitivity to the compounds. We found that EPS production by 2‐1‐1 was not affected by zinc thiazole or bismerthiazol (Fig. 7a). Similarly, the expression of EPS synthesis genes was seldom increased by the two thiadiazoles (Fig. 7b), the expression of defence‐related genes in rice inoculated with 2‐1‐1 was not increased by the two thiadiazoles (Fig. 7c), and lesion formation by 2‐1‐1 was not reduced by the two thiadiazoles (Fig. 7d).

Figure 7.

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Effect of the two thiadiazoles on extracellular polysaccharide (EPS) production of the Xanthomonas oryaze pv. oryzae (Xoo) mutant 2‐1‐1, and defence‐related gene expression in rice leaves inoculated with 2‐1‐1. (a) Effect of the two thiadiazoles on EPS production of the thiadiazole‐resistant 2‐1‐1. (b) Effect of the two thiadiazoles on the transcription level of EPS synthesis genes in the thiadiazole‐resistant 2‐1‐1. The treatments were divided into five groups: CK, control (not treated with thiadiazole compounds); a1, treated with 45 mg/L of zinc thiazole; a2, treated with 90 mg/L of zinc thiazole; b1, treated with 6 mg/L of bismerthiazol; b2, treated with 12 mg/L of bismerthiazol. Datasets marked with an asterisk are significantly different from the control as assessed by Student's t‐test: *P < 0.05. (c) Effect of the two thiadiazoles on defence genes in thiadiazole‐resistant 2‐1‐1‐inoculated rice leaves. (d) Effect of the two thiadiazoles on the formation of bacterial leaf blight (BLB) lesions in thiadiazole‐resistant 2‐1‐1‐inoculated rice leaves. The four treatments were as follows: CK, control (not inoculated with Xoo and not treated with thiadiazoles); T1, not treated with thiadiazoles, but inoculated with Xoo 2‐1‐1; T2, treated with zinc thiazole 1 day after Xoo 2‐1‐1 inoculation; T3, treated with bismerthiazol 1 day after Xoo 2‐1‐1 inoculation; T4, not treated with thiadiazoles, but inoculated with Xoo ZJ173; T5, treated with zinc thiazole 1 day after Xoo ZJ173 inoculation; T6, treated with bismerthiazol 1 day after Xoo ZJ173 inoculation. Values are means and standard errors (SEs) of three independent experiments.

Discussion

Previous research has indicated that thiadiazole compounds protect rice plants against Xoo by inhibiting the pathogen's growth (Chen et al., 2014; Yakushiji and Wakae, 1971; Zhang et al., 2005; Zhu et al., 2014). In this study, we discovered another mechanism by which thiadiazole compounds inhibit Xoo. Our results showed that zinc thiazole and bismerthiazol enhance H2O2 production, cellular defence responses and defence‐related gene expression in Xoo‐inoculated rice leaves. These results indicate that zinc thiazole and bismerthiazol can enhance rice defence against Xoo.

Because the rapid production and accumulation of H2O2 is a recognized part of various defence processes (Low and Merida, 1996; Tanaka et al., 2003), we infer that the increased H2O2 production by thiadiazole‐treated and Xoo‐inoculated rice represents a defence response. In support of this inference, we found that the removal of H2O2 by catalase nullified the defence responses of rice induced by the two thiadiazole compounds. The results indicate that H2O2 acts as a trigger and mediator of thiadiazole‐induced defence responses.

H2O2 production and inhibition were greater when zinc thiazole was applied 1 day after rather than 1 day before Xoo inoculation; moreover, when the concentration of zinc thiazole applied was increased from 200 to 300 mg/L, H2O2 production decreased. These data suggest that thiadiazole‐induced defence depends on the application time and concentration of thiadiazole. However, Xoo density in leaves decreased as the thiadiazole concentration increased. As an aminoglycoside used for BLB control in China, streptomycin did not enhance the defence of Xoo‐inoculated rice (Xu et al., 2010; Fig. S2, see Supporting Information). Overall, these findings indicate that zinc thiazole and bismerthiazol enhance rice defence against Xoo and inhibit Xoo reproduction.

Some thiazole and thiadiazole compounds, such as probenazole, vitamin B1 and benzothiadiazole, can directly induce SAR‐mediated protection against BLB (Ahn et al., 2007; Lin et al., 2008; Mohan Babu et al., 2003). Moreover, probenazole and benzothiadiazole can induce plant defence responses in rice by acting either upstream or downstream of SA (De Vleesschauwer et al., 2012, 2013). Although the two thiadiazoles might work through the SA signalling pathway in Xoo‐inoculated rice, treatment with zinc thiazole or bismerthiazol only did not induce rice defence, indicating that zinc thiazole and bismerthiazol failed to induce SAR in rice plants. Therefore, the mechanism of plant defence induction may differ between zinc thiazole and bismerthiazol vs. probenazole, vitamin B1 and benzothiadiazole.

Because it is needed for the production of bacterial biofilms, EPS is an important virulence factor, especially for vascular pathogens (Li and Wang, 2011; Milling et al., 2011; Torres et al., 2006). Previous reports have shown that EPS protects R. solanacearum and X. campestris pv. campestris from host defences (Aslam et al., 2008; Milling et al., 2011; Yun et al., 2006). Until now, no reports have evaluated the role of EPS in the susceptibility of rice to Xoo. Here, we found that treatment of rice leaves with EPS extracted from Xoo inhibited the expression of defence genes in Xoo‐inoculated rice plants and enhanced the formation and expansion of BLB lesions. The EPS‐deficient mutant ∆gumH induced much stronger defence than ZJ173, and the addition of EPS to rice plants decreased the defence induced by ∆gumH. These results suggest that EPS produced by Xoo suppresses rice defence and enhances the susceptibility of rice to Xoo.

Several recent studies have shown that some thiazole compounds have substantial anti‐biofilm activity against Gram‐positive and Gram‐negative bacteria (Mohammad et al., 2014; More et al., 2014; Rane et al., 2012). Another report has indicated that bismerthiazol significantly decreases EPS production (Ma et al., 1997). The current study confirmed these results in that zinc thiazole and bismerthiazol inhibited EPS production by Xoo. Interestingly, exogenous EPS did not affect the inhibitory effects of either compound in vitro (Fig. S3, see Supporting Information), whereas EPS addition to the plants strongly decreased the induction of defence responses. Therefore, we infer that zinc thiazole and bismerthiazol promote rice defence against Xoo by reducing EPS production.

A previous report has described field isolates of Xoo that are bismerthiazol resistant in the field, but bismerthiazol sensitive in vitro (Zhu et al., 2013). The mutant 2‐1‐1, which was screened from the wild‐type Xoo (strain ZJ173) in bismerthiazol‐treated rice, is resistant to zinc thiazole and bismerthiazol in vivo, but not in vitro (Zhu et al., 2014). These results suggest that the resistance mechanism of Xoo against thiadiazoles can differ in vivo vs. in vitro. In our research with 2‐1‐1, we found that zinc thiazole and bismerthiazol did not reduce EPS production of 2‐1‐1 and did not enhance the defence responses of rice leaves inoculated with 2‐1‐1. These findings may explain why 2‐1‐1 is resistant to thiadiazoles in vivo.

In summary, the data indicate that EPS helps Xoo to resist rice defences, and the two thiadiazoles promote rice defence against Xoo by inhibiting the production of bacterial EPS. In addition, the mechanism by which Xoo isolates resist thiadiazole compounds in vivo is related to the induction of rice defence and requires further work.

Experimental Procedures

Bacterial strain, rice cultivar and culture conditions

The wild‐type strain Xoo ZJ173, which is sensitive to zinc thiazole, was kindly provided by Professor Zhi‐Gang Xu of Nanjing Agricultural University, Nanjing, Jiangsu Province, China. The EPS‐deficient mutant ∆gumH was previously constructed on the basis of ZJ173 in our laboratory, and the mutant 2‐1‐1 was screened from ZJ173 on bismerthiazol‐treated rice leaves. Xoo was grown on NA containing 5 g of polypeptone, 1 g of yeast powder, 3 g of beef extract, 10 g of sucrose and 16 g of agar powder per litre. Nutrient broth (NB) contained the same ingredients, but lacked agar. Rice cultivar IR24, which is susceptible to Xoo, was used in this study when it had grown to the tillering stage. IR24 was grown in 500‐cm3 plastic pots (three plants per pot) containing autoclaved soil in a glasshouse at 20–30 °C with ambient light and relative humidity.

Compounds

Zinc thiazole (purity, 98.7%) and bismerthiazol (purity, 92.5%) were provided by Xinnong Chemical Co. Ltd., Hangzhou, Zhejiang Province, China and Longwan Agrichemical Co. Ltd., Wenzhou, Zhejiang Province, China, respectively, and were dissolved in N,N‐dimethylformamide as stock solutions. N,N‐Dimethylformamide was purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. Tween 80 at 250 mg/L was added to the solutions when the compounds were applied to plants.

Plant treatment

Rice plants were treated with 50 mL of water or 50 mL of compounds at 200 mg/L on day 1. Rice plants were inoculated with a suspension of 109 colony‐forming units (CFU)/mL of Xoo (or with NB alone as a control) on day 0 or day 2 using the leaf clipping method (Kauffman et al., 1973). Each treatment was represented by three replicate pots, and the experiment was repeated three times.

Measurement of H2O2 levels in rice leaves

H2O2 levels in Xoo‐inoculated rice leaves were determined 1, 3, 5 and 7 days after the compounds had been applied. A 50‐mg quantity of the washed leaves was ground in 750 μL of sodium phosphate buffer (50 mm, pH 7.4) with a mortar and pestle. The homogenate was centrifuged at 10 000 g for 20 min at 4 °C, and the supernatants were collected for assays. H2O2 levels were measured with reagent kits (Nanjing Jiancheng Biology Institution, Nanjing, Jiangsu Province, China) according to the instruction manual. H2O2 levels were determined by monitoring the absorbance of the titanium peroxide complex at 405 nm.

Gene expression in rice leaves

Total RNA was isolated from leaves using the TIANGEN PLANT RNA Kit (Tiangen Biotech, Beijing, China) and reverse transcribed to cDNA using the cDNA Synthesis Kit (TakaRa, Dalian, China). PCR primers were designed using Primer 5.0 (Table S1, see Supporting Information). Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) was performed with the ABI 7500 Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA). 18s RNA was used as a reference gene (Kim et al., 2003). qRT‐PCRs were performed with SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol. Each experiment was repeated at least three times with similar results.

Measurement of BLB lesions on rice leaves

The lengths of lesions on the clipped leaves were measured on day 7 or day 15. The inhibition rate (Zhu et al., 2013) was calculated as follows: 1 − (length of lesions treated with compounds/length of lesions not treated with compounds) × 100.

Measurement of Xoo density in rice leaves

Rice plants were inoculated with Xoo or NB medium and, 1 day later, were treated with 50 mL of zinc thiazole at five concentrations (0, 25, 100, 200 and 300 mg/L). Three days after zinc thiazole treatment, the density of Xoo in the leaves was determined. A 50‐mg quantity of washed leaves was soaked in a 5% sodium hypochlorite solution for 10 min, washed in sterile distilled water for 30 s and then homogenized in 2 mL of distilled water using a sterile mortar and pestle. The homogenate was serially diluted and 50 μL of each dilution was spread onto NA plates. After incubation for 4 days at 28 °C, the bacterial colonies on the plates were counted and the bacterial density per gram of leaf was calculated. Each treatment was represented by three replicate pots, and the experiment was repeated three times.

Observation of cellular defence responses of rice leaves

Rice plants were treated with 50 mL of water, or zinc thiazole or bismerthiazol at 200 mg/L, 1 day before the upper leaves (length, 3 cm) were inoculated with Xoo or NB medium using a needle‐less syringe (Mukoo and Yoshida, 1951). At 12 h after inoculation, the leaves of rice plants were sprayed with 50 mL of water or catalase at 5000 units/mL. Cellular defence responses (H2O2 burst, callose deposition and cell death) were assessed 24 h after inoculation. H2O2 accumulation was assessed after staining leaves with 3,3‐diaminobenzidine tetrachloride and examining them under visible light with a 10× objective (Rea et al., 2004). Callose deposition was assessed by staining leaves with aniline blue and examining them under ultraviolet light with a 10× objective (Reuber et al., 1998). Leaves with symptoms of hypersensitive response‐like cell death were photographed after staining with trypan blue (Peng et al., 2003).

Measurement of PAL activity and SA

Apical segments (length, 3 cm) of rice leaves were removed and washed thoroughly with deionized water. A 50‐mg quantity of washed leaf tissue was then homogenized with a mortar and pestle under chilled conditions in 0.65 mL of 50 mm Tris‐HCl buffer (pH 8.8) containing 15 mm of β‐mercaptoethanol. The homogenate was centrifuged at 10 000 g for 20 min at 4 °C, and the supernatant was retained as the enzyme extract. A 0.1‐mL volume of the enzyme extract, plus 1 mL of the extraction buffer, 0.5 mL of 10 mm l‐phenylalanine and 0.4 mL of deionized water, was incubated at 37 °C for 30 min. The reaction was terminated by the addition of 0.5 mL of 6 m HCl, and the product was extracted with 15 mL of ethyl acetate using a rotary evaporator. The solid residue was suspended in 3 mL of 0.05 m NaOH, and the cinnamic acid concentration was measured spectrophotometrically by the absorbance at 290 nm. One unit of PAL activity was defined as 1 μmoL of cinnamic acid produced per minute.

Total SA was extracted and measured as described by Verberne et al. (2002). The sample was dissolved in 2.4 mL of the eluent. Samples were passed through a 0.22‐μm filter, and 20 μL was used for high‐performance liquid chromatography. The eluent was 0.2 m sodium acetate buffer (pH 5.5) (75%) with methanol (25%) at a flow rate of 0.80 mL/min. The absorbance of the eluted samples was recorded at 295 nm, and SA concentrations were determined by comparison with SA standards.

Measurement of EPS production and expression of genes responsible for EPS synthesis in Xoo

EPS was extracted as described previously (Vojnov et al., 1998). Xoo was grown in NB at 28 °C to the late logarithmic growth phase, and the suspension was diluted to approximately 107 CFU/mL. A 100‐μL volume of Xoo suspension was added to 30 mL of NB in 50‐mL Erlenmeyer flasks containing different concentrations of zinc thiazole (0, 45 or 90 mg/L) or bismerthiazol (0, 6 or 12 mg/L). EPS production and the expression of EPS synthesis genes were determined when thiadiazole‐treated and untreated bacteria grew to population densities of 109 CFU/mL. For the EPS production assay, the cells were removed from 20 mL of culture by centrifugation (10 000 g for 5 min). EPS in the supernatant was centrifuged at 10 000 g for 5 min at 4 °C after the addition of three volumes of ethyl alcohol. EPS production was determined with the phenol–sulfuric acid method (Dubois et al., 1956).

For the determination of the expression of the genes responsible for the synthesis of EPS, total RNA was isolated from leaves using the TIANGEN BACTERIA RNA Kit (Tiangen Biotech) and was reverse transcribed to cDNA using the TaKaRa cDNA Synthesis Kit. The qRT‐PCR assay was performed as described previously. The PCR primers are listed in Table S2 (see Supporting Information). The experiments were repeated three times independently with three replicates each time.

The method of purification and application of EPS

EPS was extracted from Xoo cultures as described earlier. To increase the purity of EPS, ethanol precipitation was performed one to three times to precipitate EPS. After centrifugation (10 000 g for 5 min), the resulting EPS pellet was solubilized in sterile distilled water and purified by dialysis, using a 10 kDa molecular weight cutoff (MWCO) cellulose membrane, against cooled and stirred distilled water for 2 days. After determination of EPS concentrations, EPS was diluted to three concentrations (50, 100 and 200 mg/L) and sprayed onto the leaves of rice plants.

Supporting information

Fig. S1 Chemical structure of zinc thiazole (A) and bismerthiazol (B).

Click here for additional data file. (76.6KB, tif)

Fig. S2 Defence‐related gene expression and levels of H2O2 in rice leaves on application of streptomycin following Xanthomonas oryaze pv. oryzae (Xoo) (strain ZJ173) inoculation. Rice plants were inoculated with Xoo and then treated with 50 mL of 200 mg/L streptomycin or water. As control, rice leaves were not inoculated with Xoo and not treated with streptomycin. Gene expression was measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), 3 days after treatment. Values are means and standard errors (SEs) of three independent experiments.

Click here for additional data file. (77.9KB, tif)

Fig. S3  In vivo inhibitory effects of the two thiadiazoles and exogenous extracellular polysaccharide (EPS) on Xanthomonas oryaze pv. oryzae (Xoo) growth. A 100‐μL Xoo suspension [107 colony‐forming units (CFU)/mL] was added to 30 mL of nutrient broth (NB) medium in 50‐mL Erlenmeyer flasks containing serial concentrations of zinc thiazole (0, 45 mg/L) or bismerthiazol (0, 6 mg/L) in the presence of EPS. When the cell density of the suspension in the control flask increased to approximately 108 CFU/mL, optical density values were regressed on thiadiazole concentration to obtain the inhibition rate. Letters Z and B represent zinc thiazole and bismerthiazol, respectively. Values are means and standard errors (SEs) of three independent experiments.

Click here for additional data file. (62.9KB, tif)

Table S1 Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) primers for plant defence‐related genes in rice.

Table S2 Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) primers for extracellular polysaccharide (EPS) synthesis genes in Xanthomonas oryaze pv. oryzae (Xoo).

Click here for additional data file. (41KB, doc)

Acknowledgements

This study was supported by the Special Fund for Agro‐scientific Research in the Public Interest (No. 201303023) and the National Science and Technology Support Program (2012BAD19B01).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 Chemical structure of zinc thiazole (A) and bismerthiazol (B).

Click here for additional data file. (76.6KB, tif)

Fig. S2 Defence‐related gene expression and levels of H2O2 in rice leaves on application of streptomycin following Xanthomonas oryaze pv. oryzae (Xoo) (strain ZJ173) inoculation. Rice plants were inoculated with Xoo and then treated with 50 mL of 200 mg/L streptomycin or water. As control, rice leaves were not inoculated with Xoo and not treated with streptomycin. Gene expression was measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), 3 days after treatment. Values are means and standard errors (SEs) of three independent experiments.

Click here for additional data file. (77.9KB, tif)

Fig. S3  In vivo inhibitory effects of the two thiadiazoles and exogenous extracellular polysaccharide (EPS) on Xanthomonas oryaze pv. oryzae (Xoo) growth. A 100‐μL Xoo suspension [107 colony‐forming units (CFU)/mL] was added to 30 mL of nutrient broth (NB) medium in 50‐mL Erlenmeyer flasks containing serial concentrations of zinc thiazole (0, 45 mg/L) or bismerthiazol (0, 6 mg/L) in the presence of EPS. When the cell density of the suspension in the control flask increased to approximately 108 CFU/mL, optical density values were regressed on thiadiazole concentration to obtain the inhibition rate. Letters Z and B represent zinc thiazole and bismerthiazol, respectively. Values are means and standard errors (SEs) of three independent experiments.

Click here for additional data file. (62.9KB, tif)

Table S1 Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) primers for plant defence‐related genes in rice.

Table S2 Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) primers for extracellular polysaccharide (EPS) synthesis genes in Xanthomonas oryaze pv. oryzae (Xoo).

Click here for additional data file. (41KB, doc)

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