Skip to main content
NCBI home page
Brazilian Journal of Microbiology logoLink to Brazilian Journal of Microbiology
. 2022 Jan 10;53(1):19–32. doi: 10.1007/s42770-021-00657-2

Antibacterial, anti-biofilm, and anti-virulence potential of tea tree oil against leaf blight pathogen Xanthomonas oryzae pv. oryzae instigates disease suppression

Kumari Vishakha 1, Shatabdi Das 1, Sudip Kumar Das 1, Satarupa Banerjee 1, Arnab Ganguli 1,✉
PMCID: PMC8882498  PMID: 35001350

Abstract

Bacterial leaf blight (BLB) disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), causes major annual economic losses around the world. Inorganic copper compounds and antibiotics are conventionally used to control BLB disease. They often cause environmental pollution, contributing to adverse effects on human health. Therefore, research is now leading to the search for alternative control methods. Tea tree oil (TTO) is obtained from a traditional medicinal plant, Melaleuca alternifolia, with antibacterial properties. In this study, we found that TTO showed antibacterial activity against Xoo with a minimum inhibitory concentration (MIC) of 18 mg/ml. These antagonistic activities were not limited only to planktonic cells, as further studies have shown that TTO effectively eradicated sessile cells of Xoo in both initial and mature biofilms. Furthermore, it was also observed that TTO reduced various key virulence properties of Xoo, such as swimming, swarming motility, and the production of extracellular polymeric substances, xanthomonadin, and exoenzymes. TTO triggered ROS generation with cell membrane damage as an antibacterial mode of action against Xoo. The in silico study revealed that 1,8-cineole of TTO was effectively bound to two essential proteins, phosphoglucomutase and peptide deformylase, responsible for the synthesis of EPS and bacterial survival, respectively. These antibacterial and anti-virulence activities of TTO against Xoo were further confirmed by an ex vivo virulence assay where TTO significantly reduced the lesion length caused by Xoo on rice leaves. All these data concluded that TTO could be a safe, environment-friendly alternative approach for the comprehensive management of BLB disease.

Graphical abstract

graphic file with name 42770_2021_657_Figa_HTML.jpg

Keyword: Xanthomonas oryzae pv. oryzae (Xoo); Bacterial leaf blight (BLB); Anti-biofilm; Anti-virulence; Tea tree oil

Highlights

  • Tea tree oil (TTO) is effective against planktonic and sessile forms of Xanthomonas oryzae pv. oryzae (Xoo).

  • TTO shows antimotility activity and reduces EPS, xanthomonadin, and virulent exoenzyme production.

  • TTO damages the cell membrane and induces oxidative stresses as its mode of antibacterial action.

  • 1,8-Cineole was found to be a potent compound, as it binds to peptide deformylase (PDF) and phosphohexose mutase (PHM).

  • In the ex vivo study, TTO also reduces the virulence properties of Xoo.

Introduction

Rice is the most commonly used crop in the world. More than half of the population is dependent on rice [26]. Worldwide, every year at least 40% of the rice crop is lost because of spontaneous attacks by various pathogens, such as fungi and bacteria. These phytopathogens cause severe diseases, thereby disrupting global agriculture and food safety [26]. Rice was found to be more susceptible to bacterial pathogens [18]. One of the most harmful diseases, bacterial leaf blight, is caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) which destroys up to 80% of the crop. As a consequence, both the quality and yield of the crop can be reduced [6]. Xoo is a rod-shaped Gram-negative bacterium that enters and multiplies plant tissue through wounds or hydathodes, resulting in leaf wilting, which is a typical symptom of blight disease [24].

Xoo produces several virulence factors, such as motility, production of extracellular polymeric substance (EPS), exoenzymes, and xanthomonadin. Among the different virulence factors of Xoo, the most important one is the ability to make biofilms [22, 40]. As a pathogenic mechanism, Xoo produces biofilms within the xylem vessel of rice plants. Xylem vessels are crucial for the transport of nutrients and water in plants. During the blight infection, Xoo blocked the xylem vessel by forming biofilms and consequently reduced the overall yield of the rice [3]. In addition, biofilms also protect Xoo from different environmental stresses and antibacterial agents. Therefore, inhibition of biofilm formation can also be an essential criterion for an agent used for the treatment of BLB disease. Motility and EPS production are both important virulence factors, as they are both responsible for the adhesion of the surface of the bacterial leaf and the formation of biofilms in the vessels of the xylem [3, 41].

Nowadays, chemical pesticides are used as a convenient treatment to protect crops from the attack of insects, weeds, and other pests. Pesticides are mixtures of chemical compounds that are potentially harmful to humans and can cause both acute and chronic health effects [37]. In addition, pesticides have a significant effect on biodiversity as a whole and, most importantly, hinder the nitrogen fixation process, which is very important for crop development. [25]. Pesticides such as bactericides have been used as foliar treatment in agriculture to protect crops from the bacterial disease [34]. Antibiotics and chemicals are used primarily to treat the BLB disease in rice, but recent studies have shown that Xoo has developed a high resistance to these bactericides [42]. Therefore, very few alternatives are available to deal with the control and management of BLB disease.

It is very important to use natural antibacterials to manage the damage caused by BLB disease because natural compounds are less harmful and environmentally friendly. Tea tree oil (TTO) is an essential oil derived from the leaves of Melaleuca alternifolia [8]. TTO is a complex mixture of volatile compounds, the main components of which are terpinen-4-ol, γ-terpinene, α-terpinen, terpinolene, and α-terpineol [4, 27]. TTO has a diverse range of pharmacological activities, including antibacterial, antifungal, antiviral, antiprotozoal, and immune effects [5]. As a result, TTO has been widely used as an antimicrobial agent in Australia and, more recently, worldwide [4]. However, to the best of our knowledge, no systematic research has been undertaken to date on the mode of antimicrobial and anti-biofilm activity of TTO against Xoo and the consequent control of BLB disease. This study aimed to determine whether TTO could reduce the pathogenicity of Xoo and reduce the symptoms of BLB disease. Therefore, this study investigates how TTO (I) inhibits growth, (II) affects the production of biofilms, and (III) affects the pathogenicity of Xoo.

Materials and methods

Materials

TTO was purchased from Sigma-Aldrich Chemicals (St. Louis, USA). The purity of TTO is up to 98%, and the main components of the TTO sample are terpinen-4-ol (38.1%), γ-terpinene (22.2%), α-terpinene (9.1%), terpinolene (3.5%), 1,8-cineole (6.3%), and α-terpineol (4.6%) [13]. The peptone sucrose broth (PSB) and the peptone sucrose agar (PSA) were purchased from Sigma-Aldrich Chemicals Company (St. Louis, USA). Eppendorf (1.5, 2 ml) 96-well microtiter plate, 24-well microtiter plate, and 12-well microtiter plate were purchased from Tarsons, India.

Microorganisms and growth condition

The Xanthomonas oryzae pv. oryzae (Xoo) strain SboBLB3 was isolated and characterized from a susceptible rice crop infected with bacterial leaf blight disease at Bihar Agricultural University, Sabour, Bihar, India. The NCBI accession number for SboBLB3 was MH986180. The bacterial strain was routinely grown in PSA plates (0.5% peptone, 3% sucrose, 1.5% agar) at 28 ± 2° C.

Antibacterial activity of tea tree oil (TTO) against Xoo

Determination of MIC

The antibacterial effect of tea tree oil (TTO) on Xoo was determined by the broth microtiter dilution method, a standard procedure given by the Clinical and Laboratory Standards Institute (CLSI), USA. To determine the minimal inhibitory concentration (MIC) of TTO for the Xoo cells, the Xoo cells were cultured overnight at 30 °C under shaking conditions. The culture was then diluted with PSB at a final concentration of 105 CFU ml−1. TTO was prepared in PSB to obtain sub-inhibitory concentrations by serial dilution in 96-well microtiter plates. A total of 100 µl of the bacterial suspension and 100 µl of TTO dilutions of different sub-inhibitory concentrations were added to individual wells of 96-well microtiter plates. Wells without TTO treatment served as negative growth controls. The plates were then incubated at 28 °C for 24 h. The MIC was defined as the lowest TTO concentration that resulted in complete inhibition of visible growth [39]. As a positive control, we used streptomycin sulphate, and we calculated the MIC of streptomycin against Xoo in the same manner as stated here. All tests were performed three times, independently, in triplicate.

Disc diffusion assay

Xoo was taken from an overnight culture (inoculated from a single colony) and was freshly grown for 4 h with 105 CFU ml−1. This culture was used to prepare a bacterial lawn on nutrient agar. Six-mm filter paper discs were used to examine antibiotic susceptibility patterns to TTO at MIC and half MIC. The filter paper discs were prepared by absorbing 10 μl of each solution. The diameter of the bacterial growth inhibition zone around the disc (including the disc) was measured. All tests were performed three times, independently, in triplicate [9].

Time-kill assay

Xoo was taken from an overnight culture (inoculated from a single colony) and was freshly grown for 4 h with 105 CFU ml−1. The cells were then treated with different concentrations of TTO (MIC and half MIC) in a final volume of 10 ml. In control, no TTO was added. The 1 ml sample was taken at various time intervals and an optical density (OD) of 600 nm was obtained using a spectrophotometer. All tests were performed three times, independently, in triplicate [39].

Anti-biofilm activity of TTO against Xoo

Inhibition of the initial cell attachment assay

The inhibitory effect of TTO on the initial attachment of the cell to the substratum or surface was investigated using 24-well (flat bottom) cell culture plates. Xoo overnight-grown cultures were inoculated into each well with PSB containing TTO MIC and half MIC and then incubated at 30 °C for 72 h without agitation. To remove planktonic and nonadherent cells, the wells were thoroughly washed three times with 500 μl of sterile phosphate-buffered saline (PBS).

Sessile cells were stained with 0.05% crystal violet to assess biofilm biomass, and the excess was subsequently rinsed off with distilled water. The crystal violet associated with biofilm biomass was then solubilized with 95% ethanol and finally quantified by taking OD at 595 nm. The percentage of biomass formation was determined using the following equation.

Biofilm formation (%) = [{Test sample OD595 nm / Control sample OD595 nm} × 100].

To investigate cell viability, adherent bacteria in each well were resuspended by vigorous pipetting and vortexing followed by 30 s sonication and then serially diluted 106- through 108-fold and then plated onto PSA plates. Agar plates had been incubated at 30 °C for 24 h before counting bacterial colonies [1].

Disruptive potential of TTO in preformed Xoo biofilms

The biofilms were grown in peptone sucrose broth at 30 °C in 24-well polystyrene microtiter plates without shaking. After 72 h of incubation, the cultures were removed, and the biofilms were rinsed with PBS and then supplemented with fresh PSB broth and TTO with different concentrations. The developed biofilms were washed with PBS after another 24 h of incubation and subsequently fixed with methanol, stained with crystal violet, solubilized with ethanol, and eventually quantified using a microplate reader at 595 nm.

For cell viability, sessile cells were washed with PBS, and then resuspended with vigorous pipetting and vortexing, and then sonicated for 30 s. The number of CFU/biofilm was quantified using the PSA agar plate method [1].

Visualization of biofilm

Light microscopy

In order to investigate the anti-biofilm activity of TTO against Xoo by light microscopy, the biofilm assay was performed with small glass slides (1 × 1 cm) placed in the wells of the 12-well polystyrene microtiter plate. Half-MIC TTO was added to the biofilms and incubated at 37 °C for 24 h. After incubation, the planktonic cells were removed, and the biofilm formed on the glass slides was stained with crystal violet dye for 5 min. It was gently washed with deionized water and allowed to dry for 5 min. The slides were then viewed under a 40× magnification light microscope, and images were taken using a digital camera [1].

Confocal laser scanning microscopy (CLSM) analysis

The surface topology of the Xoo biofilm architecture was visualized under CLSM by forming a biofilm on a glass slide placed on a 12-well polystyrene microtiter plate. To determine the effect of TTO on disrupting the preformed biofilms, TTO was added to the preformed biofilms and incubated overnight at 30 °C. The glass slides were taken and washed with phosphate saline buffer (PBS), accompanied by staining with acridine orange, and analyzed under a confocal laser scanning microscope (CLSM; Carl Zeiss LSM700, Jena, Germany) [1].

Effect of TTO on virulence morphological traits of Xoo

Swimming and swarming

Xoo was taken from an overnight culture (inoculated from a single colony) and was freshly grown for 4 h having 106 CFU/ml. For motility assays, Xoo cells mixed with half-MIC TTO were gently inoculated in the center of solidified PSA agar plates. A total of 1 μl suspension droplets were carefully placed in the middle of soft swimming plates (3% sucrose, 0.5% peptone, 0.3% agar) and swarming plates (3% sucrose, 0.5% peptone, 0.5% agar) and kept for incubation at 28 ± 2 °C. Swimming and swarming characteristics were evaluated after 24 h by measuring the diameter of the bacterial zone. The equivalent quantity of only Xoo inoculum without TTO in the respective media was used as control [32].

Quantification of exopolysaccharides (EPS) and xanthomonadin production

To determine EPS production, Xoo culture with and without half-MIC TTO was grown on PSB in a 24-well plate for 24 h at 28 ± 2 °C. Nonadherent cells were aspired and removed to detect the production of EPS from bacterial cells. A total of 500 µl of 0.5% NaCl was added to each well. These suspended cells were transferred to fresh sterile test tubes containing 5% phenol as an equal volume of 0.5% NaCl. To this solution, 5 volumes of concentrated sulfuric acid containing 0.2% hydrazine sulphate were added and incubated in the dark for 1 h and the absorbance was measured at 490 nm (Sahu, Zheng & Yao, 2018).

For the measurement of xanthomonadin pigment, Xoo cells were collected by centrifuging a 4 ml broth suspension with and without TTO treatment and mixed with 1 ml of 100% methanol. The concoctions were further incubated in the dark for 10 min, and kept on a rotating shaker followed by centrifugation at 12,000 × g for 8 min to collect the supernatant. The xanthomonadin pigment was estimated by measuring the absorbance at OD445 and the result was denoted relative to the cell density measured before the assay [30].

Various extracellular enzymatic assays

The fresh colony of Xoo was grown in 10 ml of liquid PSB medium in the presence and absence of TTO at a half-MIC TTO starting with an OD600 of 0.05. After incubation for 24 h, the Xoo culture at OD600 of 1.8 was centrifuged at 12,000 rpm for 12 min and the obtained supernatant was used for enzymatic plate assays.

Extracellular cellulase activity was calculated by a radial diffusion assay using 2% CMC (Sigma-Aldrich) as a substrate. The media were poured into the plates and wells with 6 mm diameter were cut out with the help of a cork borer. The cell suspension (with or without TTO) was applied to the wells of the plate and incubated for 24 h at 28 ± 2 °C. Positive cellulase activity was indicated by the production of a white halo around the well in the plate.

For endoglucanase activity, Xoo culture with and without TTO has been pipetted into 6-mm-diameter wells cut into CMC agar plates (1% agar, 0.125% CMC in 0.05 M potassium phosphate buffer, pH 6.0). The plates were incubated for 48 h at 28 °C and then developed with Congo red dye.

Extracellular lipase/esterase activity was tested in a PSA medium containing 0.01% CaCl2 and 1% Tween 80. The white crystals surrounding the colonies were measured in a medium containing CaCl2 and Tween 80 indicating positive lipase activity.

To assess the zone of activity of various enzymes, 104 cells (both control and treatment) were used in all experiments. All assays were repeated three times, independently, in triplicate [32].

Mechanism of action of TTO against Xoo

Determination of ROS generation

DCFH2-DA passively enters the cell and reacts with ROS to form the highly fluorescent compound 2,7-dichlorofluorescein. At the end of the treatment, the bacterial cells were harvested and washed three times with PBS. The cell pellet was collected, and a homogeneous suspension was made by PBS up to 1 ml. The cells were then incubated with 1.5 ml 100 µM DCFH2-DA at 37 °C for 30 min. After that, ROS generation was analyzed by flow cytometry (Model: FACSVerse flow cytometer, Becton Dickinson). Data were analyzed by FCS Express Software [39].

Determination of membrane potential

Rh123 is a dye that stains the depolarized membrane. Bacteria with membrane potential prohibit the dye to enter, but bacteria with depolarized membranes allow it to enter the cell. At the end of the treatment, the bacterial cells were harvested and washed three times with PBS. The cell pellet was collected, and a homogeneous suspension was made by PBS up to 1 ml. Rh 123 reagent was added and incubated in the dark for 10 min, and then analyzed by flow cytometry (Model: FACSVerse flow cytometer, Becton Dickinson). Data were analyzed by FCS Express Software [39].

Study of membrane permeabilization by propidium iodide (PI) uptake assay

PI can enter the bacterial cell membrane only when it has been permeabilized through an agent, binds to DNA, and gives fluorescence. Fluorescence emissions can be detected with a flow cytometer. After treatment, cells were washed in PBS buffer and incubated with PI (1.3 μg/ml) at 37 °C for 20 min in the dark. PI fluorescence was measured in the flow cytometer (Becton Dickinson (BD) FACSVerse). Data were analyzed by FCS Express Software [39].

Docking of different components of TTO with peptide deformylase (PDF) and phosphohexose mutase (PHM) proteins of Xoo

The protein sequence of XanA (accession number: ACD61040) was used as input to the Phyre2 server [20]. An intensive mode was used for modeling, where the server attempts to create a complete full-length sequencing model through a combination of multiple template modeling and simplified ab initio folding simulation. The pdb file for peptide deformylase (PDF) (PDB ID: 6IL0) was downloaded from the protein data bank. The prepared protein module of Discovery Studio 2.5 was used to prepare the structure of both proteins by removing heteroatoms by adding hydrogen, deleting water molecules, and assigning partial charges by using the CHARMM force field, and then assigning protonation states [11]. The binding sites of each protein were obtained by discovery studio [11] and CASTp server [35].

The 2D structure of the ligand was downloaded from the PubChem compound database (https://pubchem.ncbi.nlm.nih.gov) in SDF format. Then, the Prepare Ligand Module of Discovery Studio 2.5 was used to generate the different conformations of each ligand [11]. The stable conformer of each ligand with the minimum potential energy was further processed.

Molecular docking was performed using the AutoDock Vina program [36]. Based on the binding sites obtained using discovery studio 2.5 and CASTp server for each protein, the ligands were individually docked to each binding site with grid coordinates (grid center) and grid boxes. After docking, the binding affinities of the ligand were predicted as negative Gibbs free energy scores (kcal/mol), which were calculated based on the AutoDock Vina scoring function [10]. Post-docking analyses were visualized using PyMOL and LigPlot [21], which showed the locations of binding sites, hydrogen-bond interactions, hydrophobic interactions, and bonding distances as interaction radii of < 5 Ᾰ from the position of the docked ligand.

Virulence assay on rice leaves

Leaf samples were taken from 40–60-day-old susceptible rice cultivar plants grown in greenhouse conditions. The leaves were washed twice with sterile distilled water followed by clipping tips of leaves with sterile scissors dipped in four groups: Group 1: leaves were treated with TTO, Group 2: leaves were treated with only Xoo, Group 3: leaves were co-treated with both Xoo and TTO, Group 4: leaves were pretreated with TTO and then treated with Xoo. After 3 days, the length of the lesion was measured on 0.5% (w/v) water agar plates. The experiment was repeated three times in triplicates [33].

Statistical analysis

Each experiment was performed in three technical replicates and three biological replicates. Mean significant values were determined using Student’s t test. Analysis of variance (ANOVA) followed by the post-test (Duncan’s multiple comparison test) was also used to analyze significance between more than two treatments. ∗ P < 0.05 and ∗  ∗ P < 0.01 were considered statistically significant.

Results

Antibacterial activity of TTO against Xoo

Determination of MIC of TTO against Xoo

The antibacterial efficacy of TTO against Xoo was calculated by estimating the minimum inhibitory concentration (MIC) value using the broth microdilution assay. To determine the MIC value, different concentrations of TTO were used for the treatment of Xoo. We have observed that the antibacterial efficacy of TTO against Xoo has increased in a concentration-dependent manner. The MIC value was obtained as 18 mg/ml after 24 h of incubation by assessing the turbidity of bacterial growth as shown in Fig. 1A. We also used streptomycin as a positive control. The MIC value of streptomycin against Xoo was 0.175 mg/ml.

Fig. 1.

Fig. 1

Open in a new tab

Antibacterial activity of TTO. A Graphical representation of the minimal inhibitory concentration (MIC) of TTO against Xoo. Data are represented as the mean ± SEM where n = 3. B Graphical representation of the minimal inhibitory concentration (MIC) of streptomycin (positive control) against Xoo. Data are represented as the mean ± SEM where n = 3. C Pictorial representation of disc diffusion assay using untreated (control) and MIC of TTO against Xoo. D Graphical representation of formation of zone of inhibition after treatment with different concentrations of TTO. Data are represented as the mean ± SEM where n = 3. E Graphical representation of antibacterial activity of different concentrations of TTO against Xoo by measuring OD at 600 nm. Data are represented as the mean ± SEM where n = 3

Disc diffusion assay

The antibacterial activity of TTO was further re-evaluated by disc diffusion assay using a previously determined half of the MIC and MIC concentrations. The diameters of the inhibition zone were estimated to be 13 ± 1.7 mm and 21 ± 1.3 mm at half of the MIC and MIC of TTO, respectively, as shown in Fig. 1C and D. Thus, both the microbroth dilution assay and the disc diffusion assay indicated that TTO has substantial antibacterial activity against Xoo.

Time-kill assay

After evaluating the concentration-dependent antibacterial action of TTO against Xoo, we tested the time-dependent effect of TTO on Xoo growth inhibition. For this, we used the time-kill assay. Visible growth inhibition was observed in both half of the MIC and MIC concentrations of TTO within 3–4 h compared to control as shown in Fig. 1E. Approximately 60% growth inhibition of Xoo was observed in half of the MIC and an almost complete reduction of Xoo growth was observed in the MIC after 24 h.

Anti-biofilm activity of TTO against Xoo

Inhibition of initial biofilm formation

Initial adhesion and subsequent biofilm formation of planktonic cells to the surface are an initial step toward the pathogenicity of Xoo [7]. Targeting the initial biofilm formation can provide an excellent way to minimize pathogenicity. The effect of TTO treatment on the inhibition of the initial biofilm formation by Xoo was evaluated by counting sessile cells and measuring biofilm biomass using a crystal violet assay. As shown in Fig. 2A, the biomass of the biofilm was reduced by half of the MIC and MIC of the TTO compared to the control. The initial biofilm biomass of Xoo was reduced to 18 ± 1.9% when treated with the MIC of TTO. Similarly, there is a major decrease in the viability of sessile cells found in TTO treatment as seen in Fig. 2B.

Fig. 2.

Fig. 2

Open in a new tab

Anti-biofilm activity of TTO against Xoo. A Graphical representation of effect of TTO on the biofilm biomass of initial and mature biofilms. Data are represented as the mean ± SEM where n = 3. B Graphical representation of effect of TTO on the viability of sessile cells of initial and mature biofilms. Data are represented as the mean ± SEM where n = 3. C Light microscopic observation of TTO-induced biofilm inhibition. (C1) represents untreated cells and (C2) represents treated cells. D Confocal microscopic observation of TTO-induced biofilm inhibition. (D1) represents untreated cells and (D2) represents treated cells

Disruptive potential of TTO in preformed biofilms of Xoo

Next, we have also extended our research to explore the disruptive capabilities of TTO against preformed Xoo biofilms. A substantial reduction in preformed biofilm biomass was observed in both half of the MIC and MIC treatment of TTO as shown in Fig. 2A. Similarly, decreases in viable sessile cells were observed in TTO treatment in a concentration-dependent manner as shown in Fig. 2B.

Microscopic observation of TTO-treated biofilm

In order to further verify the anti-biofilm activity of TTO, we performed a microscopic visualization of the preformed biofilm status in the presence and absence of MIC of TTO. The light microscopic study showed that the biofilm-forming ability of Xoo was substantially reduced when cells were treated with the MIC of TTO, as fewer crystal violet stains were retained in the TTO-treated cells compared to the control (Fig. 2(C1), (C2)). CLSM analysis confirmed that biofilm formation decreased as green fluorescence decreased in TTO-treated cells compared to the control (Fig. 2(D1), (D2)). From both light microscopy and CLSM observations, it can be said that TTO successfully eradicated the Xoo biofilm formation.

Effect of TTO on the virulence traits of Xoo

Swimming and swarming

The efficacy of swimming and swarming motility of Xoo was tested in the presence of MIC of TTO. After incubation at 28 ± 2 °C for 24 h, the result showed that the MIC of TTO significantly decreased the swimming and swarming ability of Xoo. Little swimming and swarming motility were seen on the agar plates in the TTO treatment, which contrasted sharply with the control, where a large diffused colony was observed on the PSA plates as shown in Fig. 3A and B. It can be hypothesized that the antimotility activity of TTO may have reduced the amount of Xoo bacterial cells that access the adhesion site and consequently reduced the development of biofilm.

Fig. 3.

Fig. 3

Open in a new tab

Anti-virulence activity by TTO against Xoo. A Pictorial representation of inhibition of swimming motility of Xoo by TTO. (A1) represents untreated cells; (A2) represents treated cells. B Pictorial representation of inhibition of swarming motility of Xoo by TTO. (B1) represents untreated cells; (B2) represents treated cells. C Graphical representation of effect of TTO on EPS production of Xoo. Data are represented as the mean ± SEM where n = 3. D Graphical representation of effect of TTO on xanthomonadin production of Xoo. Data are represented as the mean ± SEM where n = 3. E Graphical representation of effect of TTO on different exoenzymes’ production of Xoo. Data are represented as the mean ± SEM where n = 3

Quantification of extracellular polymeric substances (EPS) and xanthomonadin production

EPS is considered a key component of bacterial biofilm and is also responsible for the motility of Xoo. Therefore, this study has further explained the effect of TTO on EPS and xanthomonadin production by Xoo. Next, we determined the effect of TTO treatment on EPS production of Xoo. For this, the Xoo culture was grown in the presence of TTO. We observed that when cells were treated with TTO, there was a significant reduction in EPS after 24 h of incubation compared to the control (Fig. 3C). In our study, when Xoo was treated with TTO, xanthomonadin production was also significantly reduced compared to the control (Fig. 3D).

Various extracellular enzymatic assays

Exoenzymes are another major virulence factor responsible for pathogenicity. The activities of these extracellular enzymes were evaluated in the presence and absence of different concentrations of TTO. As shown in Fig. 3E, the activities of lipase, endoglucanase, and cellulase were significantly reduced in TTO treatment compared to the control.

Mechanism of action of TTO against Xoo

TTO induced oxidative stress in Xoo

Next, we determined the mechanism for the antibacterial and anti-biofilm activity of TTO against Xoo. For this reason, we have determined the generation of reactive oxygen species in Xoo treated with TTO using the DCFDA method. As shown in Fig. 4A, we found that DCFDA green fluorescence intensity in TTO-treated Xoo cells increased six times compared to the green fluorescence intensity in untreated cells. This suggested a significant generation of ROS in TTO-treated Xoo cells.

Fig. 4.

Fig. 4

Open in a new tab

Mechanism of antibacterial action of TTO against Xoo. A ROS generation was flow cytometrically evaluated for untreated and TTO-treated Xoo cells by using DCFDA. Data are represented as the mean ± SEM where n = 3. B Membrane potential was flow cytometrically evaluated in untreated and TTO-treated Xoo cells by using Rh123. Data are represented as the mean ± SEM where n = 3. C Cell membrane damage was flow cytometrically for untreated and TTO-treated Xoo cells by PI uptake. Data are represented as the mean ± SEM where n = 3

Treatment with TTO reduced the membrane potential of Xoo

ROS generation is often associated with cell membrane damage. Membrane integrity is an essential element in the survival of bacteria. Loss of membrane integrity is the predominant onset of bacterial death. The integrity of the membrane of bacteria cells can be determined by the membrane potential. Change in membrane potential indicates a loss of integrity of the cell membrane. In this study, the influence of TTO on the cell membrane potential of the Xoo cell membrane was evaluated by the flow cytometry method using rhodamine 123. In untreated cells, the mean fluorescence intensity of rhodamine 123 was 25.6 and that was decreased to 6.7 in TTO-treated Xoo cells (Fig. 4B). This finding suggested that TTO could target Xoo cell membranes.

TTO induced membrane damage and increased membrane permeability of Xoo

Change in membrane potential is the primary sign of damage to the membrane. In this study, we used the propidium iodide uptake method to validate TTO-induced cell membrane damage. Using flow cytometry analysis, we found that only 11.4% of PI-positive cells are present in control cells. However, as our experiment has shown, TTO could damage the cell membrane and increase the permeability of the PI to the cell membrane. As a result, 67.8% of PI-positive cells were identified by the MIC of TTO treatment (Fig. 4C).

TTO binds to two important Xoo proteins involved in survival and biofilm production

We used in silico analysis to understand the antibacterial and anti-biofilm effects of TTO against Xoo. Here, peptide deformylase (PDF) and phosphohexose mutase (PHM) were two essential proteins of Xoo used as targets, and different chemicals of TTO were used as ligands; these are terpinen-4-ol, γ-terpinene, α-terpinen, terpinolene, 1,8-cineole, and α-terpineol [31].

We found all chemicals bound to both PDF and PHM with binding energy greater than − 4.8 kcal/mol (Table 1). In comparison, among the different TTO chemicals, 1,8-cineole has the highest interactions with PDF and PHM. The binding energies of 1,8-cineole with PDF and PHM were − 7.2 kcal/mol and − 7.0 kcal/mol respectively (Table 1). The binding of 1,8-cineole to the PDF and PHM binding sites was regulated by multiple hydrogen bonding and hydrophobic interactions. Using the LigPlot software, we observed that amino acids of PDF were hydrogen-bonded to 1,8-cineole (Fig. 5A). Similarly, in PHM 1,8-cineole hydrogen-bonded to amino acids (Fig. 5B). These docking experiments have shown that 1,8-cineole and other compounds present in TTO bind to PDF and PHM and may inhibit its function. This may be one of the explanations for TTO’s antibacterial and anti-biofilm activity against Xoo.

Table 1.

Tabular representation of binding energy of interaction between peptide deformylase and phosphohexose mutase with different constituent sites of TTO

graphic file with name 42770_2021_657_Tab1_HTML.jpg

Open in a new tab
Fig. 5.

Fig. 5

Open in a new tab

Antibacterial and anti-biofilm mechanism action of TTO against Xoo. A In silico study of interaction of 1,8-cineole with peptide deformylase. (A1) picture represents binding sites of 1,8-cineole in PDF. (A2) picture represents interactions of 1,8-cineole with amino acids of PDF. B In silico study of interaction of 1,8-cineole with phosphohexose mutase. (B1) picture represents binding sites of 1,8-cineole in PHM. (B2) picture represents interactions of 1,8-cineole with amino acids of PHM

Virulence assay on rice leaves

Finally, the study of the virulence potential of Xoo in the presence and absence of MIC concentration of TTO was carried out using the leaf clip method. The findings showed that after 3 days of inoculation on agar plates, the lesion length of Xoo + TTO co-treated leaves was significantly decreased relative to the only Xoo-treated leaves (Fig. 6(A3), (A2)). We also pretreated leaves with TTO before exposing them to Xoo (Fig. 6(A4)) to further validate our findings. In comparison to only Xoo-treated leaves, we found that pre-treatment of TTO decreased the lesion length significantly. These findings have shown that TTO also decreased the virulence properties of Xoo under ex vivo conditions. Only TTO treatment (Fig. 6(A1)) did not cause any harmful impacts in rice leaves, indicating that TTO is non-toxic and could serve as a strong weapon for controlling BLB disease.

Fig. 6.

Fig. 6

Open in a new tab

Effect of TTO on rice disease responses to the Xoo. A Pictorial representation of virulence assay on rice plants. (A1) TTO: figure represents leaves were only treated with TTO. (A2) Xoo: figure represents leaves were only treated with Xoo. (A3) Xoo + TTO (co-treated): figure represents leaves were co-treated with both Xoo and TTO. (A4) TTO + Xoo (pretreated): figure represents leaves were pretreated with TTO and then treated with Xoo. Photographs were taken at 3 days after pathogen inoculation. B Graphical representation of lesion development, examined by measuring lesion length. Data are represented as the mean ± SEM where n = 3

Discussion

At present, these prevalent BLB disease pathogens are difficult to control for a variety of significant reasons, such as ever-changing climate, insufficient cultivation practices, high tolerance to common treatments, and, in particular, limited quantities of agrochemicals targeted at them [38]. Due to this serious situation, novel and high-efficiency alternatives targeting phytopathogens should be actively developed. Some essential oils have made an enormous contribution to plant disease management strategies due to their high efficiency as antimicrobial agents and their considerable potential to reduce the unnecessary use of environmentally toxic agrochemicals and antibiotics [19, 29]. The antimicrobial activity of TTO has previously been reported against various human as well as phytopathogens [13, 23, 31], but the management of BLB disease by inhibition of survival, biofilm, and virulence of Xoo by TTO has never been reported. Thus, for the first time, in this study, the antibacterial, anti-biofilm, and anti-virulence activities of TTO were evaluated against Xoo.

The broth dilution and time-kill assay verified the antibacterial efficacy of TTO against the planktonic form of Xoo. However, the disc diffusion assay gives us a hint that TTO might inhibit Xoo on a solid surface. For the control of BLB disease, this is a very interesting finding. Since, Xoo planktonic cells are initially attached to the xylem vessel of rice leaves, and the latter form biofilms within the vessel [3, 41]. The formation of biofilm is a crucial virulence factor for Xoo since the formation of biofilm gives the microbes an extra benefit over their planktonic counterparts by blocking the entry of antibacterial compounds [2, 7]. Therefore, an antibacterial agent should effectively remove Xoo biofilms for the complete management of BLB disease. In this research, TTO was found to effectively remove the development of initial and mature Xoo biofilms. Flagella are considered the most important player for the recognition of appropriate adhesion sites, early attachment, biofilm formation, and, most importantly, swimming and swarming motility [17]. Hence, it is also very important for the antibacterial agent to reduce the motility of Xoo to prevent BLB disease effectively. We observed in our study that TTO significantly reduced both swimming and swarming motility of Xoo. It can be hypothesized that the antimotility activity of TTO may have reduced the amount of Xoo bacterial cells that access the adhesion site and consequently reduced the development of biofilm. This study was extended by assessing the impact of TTO on EPS and xanthomonadin production by Xoo. EPS is a key component of bacterial biofilm and is also connected with the motility of Xoo. Since EPS acts as a binding matrix for sessile cells [3], the decrease in EPS in the current investigation may be a possible cause for the decrease in Xoo biofilm formation ability. On the other hand, xanthomonadin pigment protects Xoo from phototoxicity and is therefore considered an important virulence factor for the survival of Xoo in host plants [12, 17]. In this study, TTO significantly reduced the pigment of xanthomonadin and may increase the susceptibility of Xoo to light-induced cytotoxicity. Besides the production of EPS and xanthomonadin, other important virulence factors are the production of exoenzymes. TTO reduced the production of several exoenzymes like lipase, endoglucanase, and cellulase. These extracellular enzymes are mainly responsible for the pathogenicity that helps Xoo to invade, colonize, and subsequently form biofilms within plant tissue [14]. All of these data suggest that TTO not only eradicates planktonic and sessile cells of Xoo but also inhibits several virulence factors that are important for survival and pathogenicity.

We also assessed the mode of action behind TTO’s antibacterial activity. Using the flow cytometry experiment, we observed in the TTO-treated cells a significant increase in the green fluorescence intensity of DCFDA relative to the control. This suggests that the cells treated with TTO generate significant ROS and this can contribute to severe oxidative damage. The disruption of the bacterial cell membrane is one of the consequences of this oxidative damage [28]. As an indication of membrane disruption in TTO-treated cells, we have seen changes in the membrane potential of bacterial cells. We subsequently verified that TTO caused substantial membrane damage using a propidium iodide uptake study, as cells with red fluorescence intensity in TTO-treated cells were considerably higher than in control. We should also hypothesize that membrane damage can result in the cellular material being released outside, leading to bacterial cell death.

ROS generation and membrane damage may not be the only antibacterial and anti-biofilm mechanism of action of TTO. We used in silico analysis to explore other pathways for the antibacterial and anti-biofilm effects of TTO against Xoo. Two essential proteins of Xoo, namely peptide deformylase (PDF) and phosphohexose mutase (PHM), have been used as targets. Different chemicals of TTO have been used as ligands; these are terpinen-4-ol, γ-terpinene, α-terpinen, terpinolene, 1,8-cineole, and α-terpineol [31]. Peptide deformylase (PDF) is known to be an excellent target for the production of antibacterial agents, as this enzyme is important for bacterial growth as it catalyzes the elimination of N-formyl from N-terminal methionine following translation [43]. On the other hand, phosphohexose mutase (PHM) is a key controller of the production of main exopolysaccharides xanthan and xanthomonadin, as reported earlier [15]. Xanthan is needed for initial cell adhesion with subsequent biofilm production within the plant tissue [16]. All the chemicals of TTO were bound to both of these proteins and 1,8-cineole was identified as the most active chemical among the different components of TTO. Therefore, we should hypothesize chemical components of TTO that are targeting some important proteins of Xoo for their antibacterial and anti-biofilm activities.

Briefly, this study highlights TTO appears to have significant antibacterial, anti-biofilm, and anti-virulence efficacy against Xoo, a major agricultural phytopathogen of BLB disease. Innovative methods to counter the phytopathogens are urgently needed in agriculture. Since to our knowledge there is no report of TTO as a biofilm inhibitor of phytopathogenic bacteria, the study will open the path for the development of products to treat the devastating Xoo. In addition, due to the potential multifarious activity possessed by TTO, future research could be oriented towards lowering the dosage of prevalent bactericides by seeking the additive activity of the molecule with them.

Acknowledgements

The authors are thankful to Chancellor, Techno India University, West Bengal for providing the necessary infrastructure and laboratory facilities. The authors are grateful to Dr. Bishun Deo Prasad (Bihar Agricultural University, Sabour, Bihar, India) for providing the Xanthomonas oryzae pv. oryzae (Xoo) strain. The authors are also thankful to Mrs. Sheolee Ghosh Chakraborty for supporting CLSM and Dr. Ritesh Tiwari for supporting flow cytometry facilities at Centre for Research in Nanoscience and Nanotechnology (CRNN), University of Calcutta, Kolkata, West Bengal.

Author contribution

AG and KV designed the experiments; KV, SD, and SKD performed the experiments; AG, KV, and SD analyzed the data; AG and KV wrote the drafts of the article; AG supervised the manuscript. All authors revised and approved the final version of the manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.

Footnotes

Responsible Editor: Fernando R. Pavan

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Banerjee S, Vishakha K, Das S, et al. Antibacterial, anti-biofilm activity and mechanism of action of pancreatin doped zinc oxide nanoparticles against methicillin resistant Staphylococcus aureus. Colloids Surf, B. 2020;190:110921. doi: 10.1016/j.colsurfb.2020.110921. [DOI] [PubMed] [Google Scholar]
  • 2.Bjarnsholt T, Ciofu O, Molin S, et al. Applying insights from biofilm biology to drug development — can a new approach be developed? Nat Rev Drug Discov. 2013;12:791–808. doi: 10.1038/nrd4000. [DOI] [PubMed] [Google Scholar]
  • 3.Bogino P, Oliva M, Sorroche F, Giordano W. The role of bacterial biofilms and surface components in plant-bacterial associations. IJMS. 2013;14:15838–15859. doi: 10.3390/ijms140815838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Brun P, Bernabè G, Filippini R, Piovan A. In vitro antimicrobial activities of commercially available tea tree (Melaleuca alternifolia) essential oils. Curr Microbiol. 2019;76:108–116. doi: 10.1007/s00284-018-1594-x. [DOI] [PubMed] [Google Scholar]
  • 5.Carson CF, Hammer KA, Riley TV. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. CMR. 2006;19:50–62. doi: 10.1128/CMR.19.1.50-62.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chukwu SC, Rafii MY, Ramlee SI, et al. Bacterial leaf blight resistance in rice: a review of conventional breeding to molecular approach. Mol Biol Rep. 2019;46:1519–1532. doi: 10.1007/s11033-019-04584-2. [DOI] [PubMed] [Google Scholar]
  • 7.Costerton JW. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284:1318–1322. doi: 10.1126/science.284.5418.1318. [DOI] [PubMed] [Google Scholar]
  • 8.Cox SD, Mann CM, Markham JL, et al. The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil): S.D. COX ET AL. J Appl Microbiol. 2001;88:170–175. doi: 10.1046/j.1365-2672.2000.00943.x. [DOI] [PubMed] [Google Scholar]
  • 9.Das S, Vishakha K, Banerjee S, et al. Sodium alginate-based edible coating containing nanoemulsion of Citrus sinensis essential oil eradicates planktonic and sessile cells of food-borne pathogens and increased quality attributes of tomatoes. Int J Biol Macromol. 2020;162:1770–1779. doi: 10.1016/j.ijbiomac.2020.08.086. [DOI] [PubMed] [Google Scholar]
  • 10.AD ElmezayenA Al-Obaidi AT Şahin K Yelekçi 2020 Drug repurposing for coronavirus (COVID-19): in silico screening of known drugs against coronavirus 3CL hydrolase and protease enzymes J Biomol Struct Dyn 1–13 10.1080/07391102.2020.1758791 [DOI] [PMC free article] [PubMed]
  • 11.Ganguli A, Choudhury D, Chakrabarti G. 2,4-Dichlorophenoxyacetic acid induced toxicity in lung cells by disruption of the tubulin-microtubule network. Toxicol Res. 2014;3:118. doi: 10.1039/c3tx50082a. [DOI] [Google Scholar]
  • 12.Goel AK Rajagopal L Nagesh N Sonti RV 2002 Genetic locus encoding functions involved in biosynthesis and outer membrane localization of xanthomonadin in Xanthomonas oryzae pv. oryzae JB 184 3539 3548 10.1128/JB.184.13.3539-3548.2002 [DOI] [PMC free article] [PubMed]
  • 13.Groppo FC, Ramacciato JC, Simões RP, et al. Antimicrobial activity of garlic, tea tree oil, and chlorhexidine against oral microorganisms. Int Dent J. 2002;52:433–437. doi: 10.1111/j.1875-595X.2002.tb00638.x. [DOI] [PubMed] [Google Scholar]
  • 14.Hu J, Qian W, He C. The Xanthomonas oryzae pv. oryzae eglXoB endoglucanase gene is required for virulence to rice: Xoo eglXoB gene required for virulence to rice. FEMS Microbiol Lett. 2007;269:273–279. doi: 10.1111/j.1574-6968.2007.00638.x. [DOI] [PubMed] [Google Scholar]
  • 15.Hung C-H, Wu H-C, Tseng Y-H. Mutation in the Xanthomonas campestris xanA gene required for synthesis of xanthan and lipopolysaccharide drastically reduces the efficiency of bacteriophage φL7 adsorption. Biochem Biophys Res Commun. 2002;291:338–343. doi: 10.1006/bbrc.2002.6440. [DOI] [PubMed] [Google Scholar]
  • 16.Jansson P, Kenne L, Lindberg B. Structure of the extracellular polysaccharide from Xanthomonas campestris. Carbohyd Res. 1975;45:275–282. doi: 10.1016/S0008-6215(00)85885-1. [DOI] [PubMed] [Google Scholar]
  • 17.Jarrell KF, McBride MJ. The surprisingly diverse ways that prokaryotes move. Nat Rev Microbiol. 2008;6:466–476. doi: 10.1038/nrmicro1900. [DOI] [PubMed] [Google Scholar]
  • 18.Jiang N, Yan J, Liang Y, et al. (2020) Resistance genes and their interactions with bacterial blight/leaf streak pathogens (Xanthomonas oryzae) in rice (Oryza sativa L.)—an Updated Review. Rice 13:3. 10.1186/s12284-019-0358-y [DOI] [PMC free article] [PubMed]
  • 19.Khaledi N, Taheri P, Tarighi S. Antifungal activity of various essential oils against Rhizoctonia solani and Macrophomina phaseolina as major bean pathogens. J Appl Microbiol. 2015;118:704–717. doi: 10.1111/jam.12730. [DOI] [PubMed] [Google Scholar]
  • 20.Kelley LA, Mezulis S, Yates CM, et al. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015;10:845–858. doi: 10.1038/nprot.2015.053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Laskowski RA, Swindells MB. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. J Chem Inf Model. 2011;51:2778–2786. doi: 10.1021/ci200227u. [DOI] [PubMed] [Google Scholar]
  • 22.Lee B-M. The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice. Nucleic Acids Res. 2005;33:577–586. doi: 10.1093/nar/gki206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Li W-R, Li H-L, Shi Q-S, et al. Erratum to: the dynamics and mechanism of the antimicrobial activity of tea tree oil against bacteria and fungi. Appl Microbiol Biotechnol. 2017;101:2201–2201. doi: 10.1007/s00253-017-8119-6. [DOI] [PubMed] [Google Scholar]
  • 24.Mansfield J, Genin S, Magori S, et al. Top 10 plant pathogenic bacteria in molecular plant pathology: top 10 plant pathogenic bacteria. Mol Plant Pathol. 2012;13:614–629. doi: 10.1111/j.1364-3703.2012.00804.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Meena R, Kumar S, Datta R, et al. Impact of agrochemicals on soil microbiota and management: a review. Land. 2020;9:34. doi: 10.3390/land9020034. [DOI] [Google Scholar]
  • 26.Niño-Liu DO, Ronald PC, Bogdanove AJ. Xanthomonas oryzae pathovars: model pathogens of a model crop. Mol Plant Pathol. 2006;7:303–324. doi: 10.1111/j.1364-3703.2006.00344.x. [DOI] [PubMed] [Google Scholar]
  • 27.Puvača N, Čabarkapa I, Petrović A, et al. Tea tree (Melaleuca alternifolia ) and its essential oil: antimicrobial, antioxidant and acaricidal effects in poultry production. Worlds Poult Sci J. 2019;75:235–246. doi: 10.1017/S0043933919000229. [DOI] [Google Scholar]
  • 28.Rajagopal L, Sundari CS, Balasubramanian D, Sonti RV. The bacterial pigment xanthomonadin offers protection against photodamage. FEBS Lett. 1997;415:125–128. doi: 10.1016/S0014-5793(97)01109-5. [DOI] [PubMed] [Google Scholar]
  • 29.Raveau R, Fontaine J, Lounès-Hadj Sahraoui A. Essential oils as potential alternative biocontrol products against plant pathogens and weeds: a review. Foods. 2020;9:365. doi: 10.3390/foods9030365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Sahu SK, Zheng P, Yao N. Niclosamide blocks rice leaf blight by inhibiting biofilm formation of Xanthomonas oryzae. Front Plant Sci. 2018;9:408. doi: 10.3389/fpls.2018.00408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Shi C, Zhang X, Guo N. The antimicrobial activities and action-mechanism of tea tree oil against food-borne bacteria in fresh cucumber juice. Microb Pathog. 2018;125:262–271. doi: 10.1016/j.micpath.2018.09.036. [DOI] [PubMed] [Google Scholar]
  • 32.Singh A, Gupta R, Tandon S, Pandey R. Thyme oil reduces biofilm formation and impairs virulence of Xanthomonas oryzae. Front Microbiol. 2017;8:1074. doi: 10.3389/fmicb.2017.01074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Singh A, Gupta R, Tandon S, et al. Anti-biofilm and anti-virulence potential of 3,7-dimethyloct-6-enal derived from Citrus hystrix against bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae. Microb Pathog. 2018;115:264–271. doi: 10.1016/j.micpath.2017.12.051. [DOI] [PubMed] [Google Scholar]
  • 34.Sundin GW, Castiblanco LF, Yuan X, et al. Bacterial disease management: challenges, experience, innovation and future prospects: challenges in Bacterial Molecular Plant Pathology. Mol Plant Pathol. 2016;17:1506–1518. doi: 10.1111/mpp.12436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Tian W, Chen C, Lei X, et al. CASTp 3.0: computed atlas of surface topography of proteins. Nucleic Acids Res. 2018;46:W363–W367. doi: 10.1093/nar/gky473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.O Trott AJ Olson 2009 AutoDockVina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading J Comput Chem NA-NA 10.1002/jcc.21334 [DOI] [PMC free article] [PubMed]
  • 37.Tago D, Andersson H, Treich N (2014) Pesticides and health: a review of evidence on health effects, valuation of risks, and benefit-cost analysis. In: Blomquist GC, Bolin K (eds) Advances in health economics and health services research. Emerald Group Publishing Limited, pp 203–295 [PubMed]
  • 38.Velásquez AC, Castroverde CDM, He SY. Plant–pathogen warfare under changing climate conditions. Curr Biol. 2018;28:R619–R634. doi: 10.1016/j.cub.2018.03.054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Vishakha K, Das S, Banerjee S, et al. (2020) Allelochemical catechol comprehensively impedes bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae. Microbial Pathogenesis 149:104559. 10.1016/j.micpath.2020.104559 [DOI] [PubMed]
  • 40.White FF, Yang B. Host and pathogen factors controlling the rice-Xanthomonas oryzae Interaction. Plant Physiol. 2009;150:1677–1686. doi: 10.1104/pp.109.139360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Xu J, Zhou L, Venturi V, et al. (2015) Phytohormone-mediated interkingdom signaling shapes the outcome of rice-Xanthomonas oryzae pv. oryzae interactions. BMC Plant Biol 15:10. 10.1186/s12870-014-0411-3 [DOI] [PMC free article] [PubMed]
  • 42.Xu Y, Zhu X-F, Zhou M-G, et al. (2010) Status of streptomycin resistance development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their resistance characters. Journal of Phytopathology. 10.1111/j.1439-0434.2009.01657.x
  • 43.Yuan Z, Trias J, White RJ. Deformylase as a novel antibacterial target. Drug Discov Today. 2001;6:954–961. doi: 10.1016/S1359-6446(01)01925-0. [DOI] [PubMed] [Google Scholar]

Associated Data

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

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Brazilian Journal of Microbiology are provided here courtesy of Brazilian Society of Microbiology

RESOURCES