Antibacterial Activity and Mechanism of Action of Sulfone Derivatives Containing 1,3,4-Oxadiazole Moieties on Rice Bacterial Leaf Blight

In this study, sulfone derivatives containing 1,3,4-oxadiazole moieties indicated good antibacterial activities against rice bacterial leaf blight caused by the pathogen Xanthomonas oryzaepv. pv. oryzae (Xoo). In particular, 2-(methylsulfonyl)-5-(4-fluorobenzyl)-1,3,4-oxadiazole revealed the best antibacterial activity against Xoo, with a half-maximal effective concentration (EC50) of 9.89 μg/mL, which was better than those of the commercial agents of bismerthiazole (92.61 μg/mL) and thiodiazole copper (121.82 μg/mL). In vivo antibacterial activity tests under greenhouse conditions and field trials demonstrated that 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole was effective in reducing rice bacterial leaf blight. Meanwhile, 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole stimulate the increase in superoxide dismutase (SOD) and peroxidase (POD) activities in rice, causing marked enhancement of plant resistance against rice bacterial leaf blight. It could also improve the chlorophyll content and restrain the increase in the malondialdehyde (MDA) content in rice to considerably reduce the amount of damage caused by Xoo. Moreover, 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole, at a concentration of 20 μg/mL, could inhibit the production of extracellular polysaccharide (EPS) with an inhibition ratio of 94.52%, and reduce the gene expression levels of gumB, gumG, gumM, and xanA, with inhibition ratios of 94.88%, 68.14%, 86.76%, and 79.21%, respectively.


Introduction
Rice is one of the most important staple crops around the world. Unfortunately, grain yield has decreased significantly because of rice bacterial leaf blight, which is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), the most important and well-known bacterial disease of rice in rice-growing regions. Bacterial leaf blight can cause leaf wilting, affect photosynthesis, reduce 1000-grain weight, and generally result in yield losses by 20%-30% and even 100% under severe conditions [1][2][3][4][5]. Although bismerthiazole and streptomycin are the main tools for controlling rice bacterial leaf blight in China, Xoo has developed high resistance to both these bactericides [6,7]. Therefore, the search for new antibacterial agents remains a difficult task, and such agents are greatly needed in the field of agricultural bactericides.

Determination of SOD and POD Activities
SOD, a key enzyme that resists biological oxidation in plant, catalyzes the reduction of superoxide anions (O2 − ) to hydrogen peroxide (H2O2). The diminished capacity for O2 − removal causes a decreased ability of progeria cells to minimize oxidative damage may be a key factor in the disease. It plays a critical role in the defense of cells against the toxic effects of oxygen radicals [17].
POD constitutes a class of enzymes extensively distributed in plants and it has been shown that POD plays an active role in metabolism. An important function attributed to POD in plants concerns lignin synthesis. In many cases, particularly for plant-microbe interactions, this has been suggested as defense responses of plants to the stress [18].
As shown in Figure 2, SOD and POD activities in rice were enhanced by 2-(methylsulfonyl)-5-(4fluorophenyl)-1,3,4-oxadiazole at all sampling times, and the contents changed approximately in a Λ-shape manner and peaked on the 5th day with a rate of increase of 62.67% and 50.65%, respectively.
Nevertheless, SOD and POD activities showed a declining tendency as time progressed from 5th day to 7th day. The value during this period was observed to be higher than the one inoculated by Xoo and treated with water. These results showed that 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazolecould improve the disease resistance of plants, which rely on inducible defense responses in the form of enzymes that are activated for controlling rice bacterial leaf blight.

Determination of Chlorophyll Content in Rice
Photosynthesis is a special, and the most basic, life process of green plants providing themselves necessary growth and energy [19]. Chlorophyll is the photosynthetic organelle of green plants whose content is closely related to photosynthesis, extent of bacterial infection in plants leading to proliferation and destruction of the plant chloroplasts and factors retarding the synthesis of chlorophyll causing leaf chlorosis [20].

Determination of MDA Content in Rice
Increasing appreciation of the causative role of oxidative injury in many disease states places great importance on the reliable assessment of lipid peroxidation. MDA is one of several low-molecular-weight end products formed via the decomposition of certain primary and secondary lipid peroxidation products [21].
As shown in Figure 4, the MDA content in rice was increased in different treatment groups at all sampling times. It reached the highest value at 7th day, and it increased to 6.39 μM/L after treatment with 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole. This value was higher than that in rice treated with water (6.39 μM/L) but lower than the one inoculated with Xoo (7.30 μM/L) and the one treated with bismerthiazole (7.17 μM/L) during 1-7 days. The MDA content demonstrated that 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole could restrain the increase in the MDA content in rice, thereby enhancing the host's resistance to the disease.

Quantitative Determination of EPS Production
EPS, high-molecular weight compounds secreted by microorganisms into their environment, establish the functional and structural integrity of biofilms. EPS are considered the fundamental component that determines the physiochemical properties of a biofilm. EPS can protect pathogenic bacteria and contribute to their pathogenicity. A previous study reported that EPS, one pathogenic factor of Xoo, can lead to wilting of rice leaves and reduce EPS production to decrease the pathogenicity.

EPS Gene Expression Level in Xoo
The biosynthetic pathway by which EPS is produced involves three steps: the first step is the conversion of simple sugars to nucleotidyl derivative precursors; the second step is the assembly of pentasaccharide subunits attached to an inner-membrane polyprenol phosphate carrier, with addition of acetyl and pyruvate groups; and the third step is the polymerization of the pentasaccharide repeating units and secretion of EPS [22]. XanA coding for phosphoglucomutase/phosphomannomutase, which can be glucose-6-phosphate into glucose-1-phosphate in the first step of EPS biosynthesis [23]. The genes encoding the proteins related to the last two steps of EPS biosynthesis are encoded by the gum gene cluster [24]. GumM (the protein of gumM) plays an important role in the process of the biosynthesis of the pentasaccharide, GumG (the protein of gumG) is partly responsible for modification of the pentasaccharide, and GumB (the protein of gumB) is a key role in the process of EPS polymerization and transport [25].

Bacterial Strains and Culture Conditions
PXO99A strain of Xoo was grown at 28 ± 1 °C in nutrient broth (NB) medium in conical flasks or on nutrient agar (NA) medium in Petri dishes [6]. NA medium was prepared with 1 g of yeast extract, 3 g of beef extract, 5 g of polypeptone, 10 g of sucrose, and 15 g of agar powder per 1000 mL of distilled water, pH 7.0-7.2. NB medium contained the same components but lacked agar powder.

In Vitro Antibacterial Activity
In this study, eight sulfone derivatives containing 1,3,4-oxadiazole moieties were evaluated for their antibacterial activities against Xoo via the turbidimeter test [26] in vitro. Dimethylsulfoxide (DMSO) in sterile distilled water served as an untreated blank control. Bismerthiazole and thiodiazole copper which are the principal tools used for controlling rice bacterial leaf blight in China at present served as positive controls,. Approximately 40 μL of solvent NB containing Xoo, incubated on the phase of logarithmic growth, was added to 5 mL of solvent NB containing the test compounds. The inoculated test tubes were incubated at 28 °C and continuously shaken at 180 rpm for 24-48 h until the bacteria were incubated on the logarithmic growth phase. The growth of the cultures was monitored on a Model 680 microplate reader (BIO-RAD, Hercules, CA, USA) by measuring the optical density at 595 nm (OD595). The inhibition rate I was calculated by the following formula: Inhibition rate I (%) = (C − T)/C × 100 (1) where C is the corrected turbidity values of bacterial growth on untreated NB (blank control), T is the corrected turbidity values of bacterial growth on treated NB, and I represents the inhibition rate. Based on previous bioassays, the results of antibacterial activities (expressed by EC50) of the title compounds against Xoo were also evaluated and calculated with SPSS 17.0 software. The experiment was repeated three times.

In Vivo Antibacterial Activity
To determine the control efficiency of antibacterial potency in vivo at the greenhouse condition, the curative activity of 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole against rice bacterial leaf blight was analyzed in potted plants using a complete randomized block design [27]. Seeds of Nipponbare were sowed in plastic pots that contained field soil and thinned to six to ten rice seedlings. Xoo, the pathogen of the rice disease of bacterial leaf blight, was cultured in solvent NB at 28 ± 1 °C overnight at 180 rpm, and the concentrations were then adjusted to 10 8 CFU/mL. Five weeks after sowing, Xoo of rice bacterial leaf blight was inoculated on the rice plant. We used scissors, which were sterilized using 70% ethanol prior to use and then dipped in bacterial solution, to inoculate the rice plant with Xoo. After 7 d of Xoo inoculation, 200 μg/mL 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4oxadiazole solution was sprayed until run-off onto the leaves, whereas the control plants were sprayed with the same volume of distilled water. All inoculated plants were incubated in a growth chamber at 28 °C and 90% relative humidity. At 15th day after inoculation, the average lesion length was observed and scored, and the control efficiency was calculated. The experiment was repeated three times.
Meanwhile, the protective activity of 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole against rice bacterial leaf blight was also analyzed under greenhouse conditions. Five weeks after sowing the rice plant, 200 μg/mL 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole solution was sprayed onto the leaves until run-off, whereas the control plants were sprayed with the same volume of distilled water. Seven d after 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole spraying, Xoo rice bacterial leaf blight was inoculated on the rice plant. All inoculated plants were incubated in a growth chamber at 28 ° C and 90% relative humidity. At 28th day after inoculation, the average lesion length was observed and scored, and the experiment was repeated three times. The control efficiency was calculated as follows: Control efficiency I (%) = (C − T)/C × 100 (2) where C represents the disease incidence of the untreated blank control, T represents the disease incidence of the treatment, and I represents the control efficiency.

Field Trial against Rice Bacterial Leaf Blight
To further determine the activities of 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole, field trials against rice bacterial leaf blight were conducted. The effect of the natural infection of Xoo was studied in a field with rice having suffered rice bacterial leaf blight for several years. Sterile distilled water served as an untreated blank control, whereas the commercial bactericides bismerthiazole and zhongshengmycin were the positive controls. 2-(Methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole (150 g ai/ha) and the commercial bactericides bismerthiazole (375 g ai/ha) and zhongshengmycin solutions (45 g ai/ha) were sprayed on the foliage of the rice once every 7 days three times. For each treatment, three replicates were conducted. The disease incidence of the rice plants was investigated 15th day after the third spraying. The control efficiency was calculated using the following formula: where C represents the disease incidence of the untreated blank control, T represents the disease incidence of the treatment, and I represents the control efficiency.

Determination of SOD Activity
Rice samples (0.5 g) were homogenized in 0.05 M phosphate buffer (5 mL, pH 7.8) and centrifuged at 4000 g for 10 min. The supernatant was used as an enzyme source. The reaction mixture (3 mL) contained phosphate buffer (1. Phosphate buffer instead of enzyme liquid was set as blank control. The mixture was illuminated under a fluorescent lamp (4000 LUX, Ningbo Jiangnan Instrument Plant, Ningbo, China) for 20 min, and the absorbance was read at 560 nm. For the blank, identical solutions were kept under the dark. SOD activity was expressed as the change in absorbance g −1 fresh tissue [28].

Determination of POD Activity
Rice samples (1 g) were homogenized in 20 mM KH2PO4 (5 mL) and then centrifuged at 4000 g for 15 min at 4 °C. The supernatant was used as an enzyme source. The mixed reaction solution of POD consisted of 0.1 M phosphate buffer (500 mL, pH 6.0), guaiacol (280 μL), 30% H2O2 (190 μL) and 20 mM KH2PO4. To initiate the reaction, mixed reaction solution of POD (3 mL) and enzyme solution (0.1 mL) were added to the sample cuvette. Mixed reaction solution of POD and KH2PO4 were set as blank control. The absorbance was read at 470 nm about once a minute. POD activity was expressed as U·g −1 FW min −1 [29].

Determination of Chlorophyll Content
According to the work conducted by Peng [30], rice samples (10 mg) were homogenized in a 5 mL mixture of acetone and ethanol with a volume ratio of 4:1. Dark extraction was carried out for 1 h, and the extract was centrifuged at 4000 g for 5 min. The chlorophyll extract was then placed in a 1 cm-thick cuvette, a mixture of acetone and ethanol with a volume ratio of 4:1 was used as a reference. The absorbance was read at 645 and 663 nm: Cb (mg·L −1 ) = 0.0229A645 − 0.00468 A663 Total chlorophyll content = Ca + Cb (6) In the above equation, A645 and A663 are the absorbance values at the wavelengths 645 and 663 nm, respectively.

Biofilm Assays
Biofilm formation in glass test tube was quantified as described previously [32,33]. Bacteria were grown in NB with shaking to the mid-exponential growth phase and then diluted to 1:100 in fresh NB. About 2 mL of a diluted bacterial suspension was placed in each glass tube, incubated with 10 μg/mL 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole,and shaken at 28 °C for 72 h. The culture medium was poured out, and attached bacterial cells were gently washed three times with distilled water. The cells were then stained with 0.1% crystal violet (2 mL) for 15 min. Unbound crystal violet was poured out, and the glass tubes were washed three times with water. The crystal violet-stained cells were solubilized in DMSO (2 mL). Biofilm formation was quantified by measuring the absorbance at 570 nm using a Synergy H1 detector (BioTek, Winooski, VT, USA). Three replicates were used for quantitative measurement.

Quantitative Determination of EPS Production
To measure the influence of EPS production of Xoo in culture supernatants, bacterial cells were grown in NB supplemented with different concentrations (20, 10, 5, and 2.5 μg/mL) of 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole at 28 °C for 72 h. Subsequently, 10 mL portions of the cultures were collected, and the cells were removed by centrifugation at 8000 g for 20 min [34]. Finally, three volumes of ethyl alcohol were added to the supernatants [35]. The precipitated EPS were pelleted via centrifugation, dried, and weighed. The test was performed three times independently.

RNA Extraction, cDNA Synthesis, and RT-qPCR Analysis
Bacteria were grown in NB medium at 28 °C with shaking at 180 rpm, and 1 mL samples of Xoo strain cultures were collected at 12 h after bacterial cells were incubated with different concentrations (20, 10, 5, and 2.5 μg/mL) of 2-(methylsulfonyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole. Bacterial cells were centrifuged at 12,000 g for 10 min, and the cell pellets were treated with a Trizol reagent kit (TaKaRa, Dalian, China) [36]. Total RNA purity was estimated by calculating OD260/OD280 using an ultraviolet spectrophotometer (ACTGene, Piscataway, NJ, USA). All OD260/OD280 values of RNA were between 1.8 and 2.2. The concentration of total RNA was calculated according to the dilution ratio and OD260.
cDNAs were synthesized using a cDNA synthesis kit (TaKaRa). H2O was added up to 6 μL to the solution containing 1000 ng of RNA and 1 μL of random primers, and heated to 70 °C for 10 min. The solution was rapidly placed on ice for 2 min. Up to 2 μL of MLV buffer, 0.5 μL of 10 mM each dNTP, 0.25 μL of RRI, and 0.25 μL of M-MLV were added. The reaction mixture was heated to 30 °C for 10 min, 42 °C for 1 h, and to 70 °C for 15 min.
RT-qPCR was carried out using SYBR Premix Ex TaqII (TaKaRa). The reaction solution contained 10 μL of SYBR, 0.8 μL of primer pair, 1 μL of cDNA, and 8.2 μL of H2O. Four target genes were chosen for expression analysis with 16S rRNA as the endogenous control. The primers, shown in Table 5, were designed using the sequences of the Xoo genome. The PCR cycle consisted of the following steps: 30 s at 95 °C and 40 cycles of 20 s at 95 °C and 30 s at 59 °C. After each run, a dissociation curve was designed to confirm specificity of the product and avoid production of primer dimers. Relative amounts of amplification products were calculated with the comparative 2 −∆∆Ct method. A total of three independent biological replicates were used for each treatment.