High-Throughput Screening of Plant Extracts for Targeted Control of Burkholderia glumae, Causing Rice Sheath and Panicle Blight

Article information

Plant Pathol J. 2025;41(1):112-119

Publication date (electronic) : 2025 February 1

doi : https://doi.org/10.5423/PPJ.NT.10.2024.0167

Seungchul Lee¹, Yong Tae Jeong², Seokhun Jang¹, Taeho Jung¹, Buyng Su Hwang², Ji Su Choi², Young Taek Oh², Mohamed Mannaa^1,3,4,, Young-Su Seo^1,3,

¹Department of Integrated Biological Science, Pusan National University, Busan 46241, Korea

²Nakdonggang National Institute of Biological Resources, Sangju 37242, Korea

³Institude of System Biology, Pusan National University, Busan 46241, Korea

⁴Department of Plant Pathology, Faculty of Agriculture, Cairo University, Giza 12613, Egypt

^*Co-corresponding authors. M. Mannaa, Phone) +82-51-510-2195, FAX) +82-51-514-1778, E-mail) mannaa_mohamed@yahoo.com, Y.-S. Seo, Phone) +82-51-510-2267, FAX) +82-51-514-1778, E-mail) yseo2011@pusan.ac.kr

Handling Editor : Hun Kim

Received 2024 October 28; Revised 2024 December 17; Accepted 2024 December 17.

Rice (Oryza sativa), one of the most important staple crops globally, is threatened by various diseases that severely reduce yields and affect food security. Among the bacterial pathogens, Burkholderia glumae is recognized as a key agent responsible mainly for bacterial panicle blight, causing grain losses of up to 75% under favorable conditions, especially in warm and humid environments (Fory et al., 2014). Burkholderia species, including B. glumae, exhibit a remarkable adaptability to diverse ecological niches, which is largely attributed to their genomic plasticity, characterized by multi-replicon genomes rich in insertion sequences and genomic islands (Mannaa et al., 2018). This phytopathogenic B. glumae is prevalent in nearly all rice-growing regions globally and has also been detected in association with other crops, including eggplant, sesame, paprika, and tomato (Jeong et al., 2003; Zhu et al., 2008). The disease control remains challenging primarily due to the lack of highly effective treatment options (Pedraza-Herrera et al., 2020).

Currently, no reliable control method has been established for B. glumae. Some chemicals, like oxolinic acid, have shown temporary effectiveness in reducing disease severity, but their use is limited due to the development of resistance and potential environmental harm (Hikichi, 1993; Maeda et al., 2004). Moreover, there is no commercially available rice cultivar resistant to B. glumae, adding to the complexity of managing this disease (Mizobuchi et al., 2016; Nandakumar et al., 2009). These challenges highlight the urgent need for alternative approaches, particularly those based on natural and eco-friendly solutions.

Plants produce a wide array of secondary metabolites with known antimicrobial properties, making them valuable resources for developing biological control strategies (González-Lamothe et al., 2009; Palombo and Semple, 2001). Plant extracts have demonstrated effectiveness against various bacterial and fungal pathogens in crops (Abbassy et al., 2014; Hernández-Ceja et al., 2021; Lee et al., 2022). However, the systematic screening of plant-derived compounds specifically targeting B. glumae and other rice pathogens remains underexplored. This research seeks to address this gap by investigating a broad range of plant extracts for their antibacterial activity against B. glumae and related rice pathogens.

Therefore, the objectives of this study were to systematically screen a large number of plant extracts to identify those with significant antibacterial activity against B. glumae and to further evaluate their effects on bacterial swimming motility and disease severity in rice plants. This work aims to provide new insights into the potential of plant-based solutions for managing B. glumae, offering promising alternatives to chemical treatments.

The plant extracts used in this study were obtained from diverse sources over a collection period spanning 2016 to 2023. A total of 1,134 extracts were prepared from 292 plant species originating from various geographic regions across Korea. The extracts were deposited and catalogued in the plant extract repository at the Nakdonggang National Institute of Biological Resources (NNIBR), Sangju, Korea, under their unique accession codes (Supplementary Table 1). Plant extracts were obtained using two methods: solvent extraction with 70% aqueous ethanol and heat extraction using distilled water. Each extract was prepared from specific plant parts, including whole plants, roots, and aerial tissues, following standardized protocols. Detailed information regarding plant species, parts used, extraction methods, and collection dates is provided in Supplementary Table 1. Bacterial strains used in this study are shown in Supplementary Table 2.

In the preliminary screening, plant extracts were tested for antibacterial activity against B. glumae BGR1 using an agar disc diffusion assay (Fig. 1). Luria-Bertani (LB) agar plates (1.5%, w/v) were prepared, and 1% from overnight culture of B. glumae (OD₆₀₀ = 2.0) was spread over the plates. Sterilized 8 mm paper discs were placed on the plates, and 20 μl of each plant extract (20 mg/ml) was applied to the discs. Kanamycin (5 mg/ml) was used as a positive control, and 1× phosphate buffered saline (PBS) as a negative control. Plates were incubated at 37°C for 12 h, and presence of clear inhibition zone was considered positive for selection.

Fig. 1

Overview of the screening and selection process of plant extracts for controlling Burkholderia glumae. (A) Study workflow: A total of 1,134 plant extracts were screened against B. glumae using an agar diffusion assay. From these, 33 extracts were selected for liquid broth testing, leading to the identification of 2 extracts for further investigation. These extracts demonstrated significant inhibition of bacterial growth, motility, and reduced disease severity in rice plants. (B) Inhibition criteria: Only extracts showing clear, strong inhibition zones (red boxes) were selected for further analysis, indicating effective bacterial growth suppression. Kanamycin was used as a positive control, applied to the central discs.

The screening process and experimental design are summarized in Fig. 1A. Based on preliminary testing, a concentration of 20 mg/ml was selected for this screening. Among the tested plant extracts, 33 showed significant inhibitory effects, indicated by clear inhibition zones from the agar disc diffusion assay (Fig. 1B). The size and clarity of the inhibition zones varied, with certain extracts demonstrating stronger antibacterial activity, as reflected by larger and more distinct zones. These extracts were subsequently selected for further analysis. The agar disc diffusion method provided a rapid and effective means of identifying potential antibacterial candidates, consistent with other studies that have highlighted its utility in screening for antimicrobial properties (Mougou and Boughalleb-M’hamdi, 2018; Zaidan et al., 2005).

Our screening yielded effective plant extracts, suggesting that the diverse range of species we tested may have contributed to the observed antibacterial activity. This could be due to the diverse geographic origins and species characteristics of the plants we utilized, spanning multiple ecosystems, which likely increased the chemical diversity of the extracts.

The extracts selected from the first screening were subjected to further testing in liquid LB broth. Single colonies of B. glumae BGR1 were cultured in LB broth at 37°C with shaking at 200 rpm until OD₆₀₀ reached 2.0. Bacterial cultures were inoculated at 1% into fresh LB broth containing 0.2 mg/ml plant extract diluted in 1× PBS. The negative control contained 0.01% PBS, and the positive control used 50 μg/ml kanamycin. The OD₆₀₀ values were measured at various time points over 24 h to monitor bacterial growth.

The liquid broth assay provided a more controlled and quantitative assessment of bacterial inhibition. Of the 33 extracts selected from the agar disc diffusion assay, five extracts were found to significantly inhibit bacterial growth compared to the PBS control, with optical density (OD₆₀₀) values remaining low throughout the incubation period (Fig. 2). This confirmed the potent antibacterial effects observed during the agar diffusion assays. Details on the five plant extracts with potential antimicrobial activity against B. glumae is shown in Supplementary Table 3.

Fig. 2

Second screening of selected plant extracts in liquid Luria-Bertani broth on the growth of Burkholderia glumae BGR1. (A) Growth curves of B. glumae treated with 33 plant extracts selected from the initial screening. Optical density (OD₆₀₀) was measured over time to evaluate bacterial growth inhibition. Plant extracts were tested at 0.2 mg/ml in phosphate buffered saline (PBS). Controls included PBS as the negative control and 50 μg/ml kanamycin as the positive control. (B) Growth curves of B. glumae treated with 5 plant extracts selected for significant inhibition in the second screening.

Two extracts, FBCC-EP312 and FBCC-EP487, were selected for further analysis: one showed the lowest OD₆₀₀ values across all time points, while the other displayed a strong initial reduction. To assess their specificity, the extracts were tested against other rice pathogens (Burkholderia gladioli, Burkholderia plantarii) and the non-rice pathogen Escherichia coli. Single colonies of each species were grown on LB agar at 28°C or 37°C and inoculated into LB broth containing 0.2 mg/ml of the plant extracts. Negative control was 0.01% PBS, and positive controls was 50 μg/ml kanamycin. The OD₆₀₀ values were measured at 12-h intervals up to 36 h to evaluate bacterial growth inhibition. Both extracts, FBCC-EP312 and FBCC-EP487, significantly inhibited the rice pathogens but had no effect on E. coli (Fig. 3A), suggesting a specific mode of action targeting Burkholderia species.

Fig. 3

Effects of selected plant extracts on the growth of rice pathogens and bacterial motility. (A) Growth curves of the rice pathogens Burkholderia glumae, Burkholderia gladioli, Burkholderia plantarii, and the non-rice pathogen Escherichia coli in response to two plant selected extracts: FBCC-EP312 (Trapa japonica) and FBCC-EP487 (Rumex crispus). Optical density (OD₆₀₀) was measured at 12-h intervals up to 36 h, with phosphate buffered saline as the negative control. Different letters on error bars indicate significant differences based on the least significant difference (LSD) test (P < 0.05). (B) Swimming motility assay of B. glumae BGR1 in response to FBCC-EP312 and FBCC-EP487. Images show swimming zones of B. glumae after incubation for 24 h. (C) Quantification of the swimming motility zones (mm²). Different letters on error bars indicate significant differences (P < 0.05) according to the LSD test.

This specificity is critical in developing biocontrol agents, as broad-spectrum antimicrobials can disrupt beneficial microbiota and lead to ecological imbalances. Our results align with studies showing plant extracts can selectively inhibit certain bacterial strains based on biochemical interactions (Álvarez-Martínez et al., 2021). The lack of effect on E. coli in our study indicates that the active compounds may interact with specific metabolic or structural features of Burkholderia species. While many plant extracts exhibit broad-spectrum activity, affecting a wide range of bacteria (Atef et al., 2019; Upadhyay et al., 2014), our findings highlight the targeted potential of these particular extracts.

Bacterial motility is a critical virulence factor for many plant pathogens, including B. glumae. By reducing swimming motility, the pathogen’s ability to colonize and infect plant tissues is significantly diminished. The effect of FBCC-EP312 and FBCC-EP487 on the swimming motility of B. glumae BGR1 was evaluated using LB agar plates (0.25%, w/v). Single colonies of B. glumae were grown overnight, centrifuged, and resuspended in 1 mM MgSO₄. Bacterial suspensions were normalized to an OD₆₀₀ of 2.0 prior to inoculation onto soft agar plates to minimize the impact of cell density on motility measurements. Three μL of the suspension were spotted onto the center of LB agar plates and incubated at 37°C for 24 h. After incubation, plates were imaged, and swimming zones were measured using ImageJ software. Data were statistically analyzed to assess significant differences in motility.

The conducted motility assays showed that both FBCC-EP312 and FBCC-EP487 drastically reduced the motility of B. glumae compared to the PBS control (Fig. 3B). This reduction in motility was further quantified, demonstrating a significant difference (P < 0.05) between the treated and untreated bacteria (Fig. 3C). Our results align with previous studies showing that plant extracts can inhibit bacterial motility, thus reducing the pathogen’s virulence (Magnini et al., 2021).

The ultimate test of the efficacy of the selected plant extracts was their ability to reduce disease severity in rice plants inoculated with B. glumae. A rice stem inoculation assay was conducted to evaluate the effect of the selected two plant extracts on the pathogenicity of B. glumae BGR1. Rice plants (Oryza sativa L. cv. Dongjin) were grown in a greenhouse under controlled conditions (37°C during the day, 25°C at night). A puncture was made in the rice stem, and single colony from B. glumae was inoculated using sterilized toothpick. The plant extracts (FBCC-EP312 and FBCC-EP487) were applied at 0.2 mg/ml in 1× PBS, while PBS was used as the control. After 7 days, lesion areas were measured. Bacterial populations were assessed by excising 1 cm stem segments above and below the puncture site, homogenizing them in 1 mM MgSO₄, and performing serial dilutions for colony forming unites (cfu) analysis.

Plants treated with FBCC-EP312 and FBCC-EP487 showed a marked reduction in lesion area compared to untreated controls (Fig. 4A and B), indicating that these extracts can significantly mitigate the effects of bacterial panicle blight. Furthermore, the bacterial populations in the treated plants were significantly reduced, as evidenced by CFU counts from rice tissues (Fig. 4C and D). These findings demonstrate the practical application of these plant extracts in reducing disease severity in rice, offering a sustainable alternative to synthetic chemicals.

Fig. 4

Effects of the selected plant extracts on rice disease severity caused by Burkholderia glumae BGR1. (A) Photographs of rice stems showing the progression of disease after stem inoculation with B. glumae BGR1 and treatment with the selected plant extracts (FBCC-EP312 and FBCC-EP487). Control plants were treated with phosphate buffered saline. (B) Quantitative analysis of disease severity, represented by lesion area (mm²) on rice stems. Different letters on error bars indicate statistically significant differences (P < 0.05) according to the least significant difference (LSD) test. (C) Spotting assay on agar plates showing bacterial populations from the rice stem extracts after treatment. (D) Bacterial population quantified from rice stem suspensions, presented as colony forming unites (cfu)/ml. Error bars represent standard deviations, and different letters indicate statistically significant differences (P < 0.05) based on the LSD test.

Interestingly, while FBCC-EP312 showed a stronger reduction in bacterial motility compared to FBCC-EP487, the latter had a greater effect on controlling disease severity in rice plants. This suggests that motility is not the only virulence factor at play. FBCC-EP487 may target other virulence mechanisms, such as toxin production or biofilm formation, which could explain its superior disease control. These results highlight the complexity of B. glumae pathogenicity and suggest that the extracts may act on different pathways, warranting further investigation into their specific modes of action.

Statistical analysis was conducted using the Statistical Analysis Systems (SAS Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was performed utilizing the GLM procedure, and differences between means were determined using the least significant difference test at P < 0.05.

Both Trapa japonica (FBCC-EP312) and Rumex crispus (FBCC-EP487) have been studied for their medicinal properties. T. japonica, commonly known as water chestnut, has demonstrated various biological activities, with recent studies highlighting its antibacterial potential. For instance, extracts from T. japonica have been found to alter microbial communities in aquatic environments, particularly by inhibiting harmful cyanobacterial species, which suggests possible biocontrol applications (Liu et al., 2021). Furthermore, other research underscores the significant antimicrobial effects of a related species, Trapa natans, which has shown activity against several bacterial species, including Staphylococcus aureus (Kiruba et al., 2023).

R. crispus, another widely recognized medicinal herb, also possesses remarkable antimicrobial properties. Several studies have identified bioactive compounds in R. crispus, such as anthraquinones like emodin, which exhibit strong antimicrobial effects against resistant bacterial strains, including methicillin-resistant S. aureus (Pelzer et al., 2021). Additionally, research has demonstrated the broad-spectrum antimicrobial activity of R. crispus extracts, which have proven effective against various microbial species, including S. aureus and Bacillus subtilis, further highlighting the plant’s versatility in combating bacterial infections (Wegiera et al., 2011; Yıldırım et al., 2001). These findings are consistent with our results, where both plant extracts effectively inhibited rice-pathogenic Burkholderia species.

While our study emphasizes the selective antibacterial action of these plant extracts, it is important to recognize that both T. japonica and R. crispus have been documented to possess broad-spectrum antimicrobial activity in other contexts. This suggests that their selectivity may vary depending on conditions or extraction methods. Further research is needed to isolate and characterize the active compounds to better understand their mode of action.

Taken together, this study identified two potent plant extracts, T. japonica (FBCC-EP312) and R. crispus (FBCC-EP487), that demonstrated strong antibacterial activity against B. glumae and other related rice pathogens. These extracts not only inhibited bacterial growth but also reduced motility, a key virulence factor, and significantly decreased disease severity in rice plants. Future research should focus on isolating and characterizing the active compounds within these extracts to better understand their mechanisms of action. Additionally, field trials are needed to confirm the efficacy of these extracts under natural agricultural conditions. If successful, these plant-derived compounds could provide an eco-friendly alternative to synthetic bactericides, reducing the environmental and health risks associated with chemical treatments.