Suppression of Xanthomonas oryzae pv. oryzae biofilm formation by Acacia mangium methanol leaf extract

Abstract Xanthomonas oryzae pv. oryzae (Xoo), a pathogen responsible for rice bacterial leaf blight, produces biofilm to protect viable Xoo cells from antimicrobial agents. A study was conducted to determine the potency of Acacia mangium methanol (AMMH) leaf extract as a Xoo biofilm inhibitor. Four concentrations (3.13, 6.25, 9.38, and 12.5 mg/mL) of AMMH leaf extract were tested for their ability to inhibit Xoo biofilm formation on a 96-well microtiter plate. The results showed that the negative controls had the highest O.D. values from other treatments, indicating the intense formation of biofilm. This was followed by the positive control (Streptomycin sulfate, 0.2 mg/mL) and AMMH leaf extract at concentration 3.13 mg/mL, which showed no significant differences in their O.D. values (1.96 and 1.57, respectively). All other treatments at concentrations of 6.25, 9.38, and 12.5 mg/mL showed no significant differences in their O.D. values (0.91, 0.79, and 0.53, respectively). For inhibition percentages, treatment with concentration 12.5 mg/mL gave the highest result (81.25%) followed by treatment at concentrations 6.25 and 9.38 mg/mL that showed no significant differences in their inhibition percentage (67.75% and 72.23%, respectively). Concentration 3.13 mg/mL resulted in 44.49% of biofilm inhibition and the positive control resulted in 30.75% of biofilm inhibition. Confocal laser scanning microscopy (CLSM) analysis of Xoo biofilm inhibition and breakdown showed the presence of non-viable Xoo cells and changes in aggregation size due to increase in AMMH leaf extract concentration. Control slides showed the absence of Xoo dead cells.

摘要 水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae，Xoo）是引发水稻细菌性叶枯病的病原菌，可通过产生生物被膜（biofilm）保护存活的Xoo细胞免受抗菌剂侵害。本研究旨在探究马占相思（Acacia mangium）叶片甲醇提取物（AMMH）对Xoo生物被膜的抑制活性。本实验设置3.13、6.25、9.38及12.5 mg/mL共4种浓度的AMMH叶片提取物，检测其对96孔微孔板上Xoo生物被膜形成的抑制能力。结果显示，阴性对照组的光密度（Optical Density，O.D.）值在所有处理组中最高，表明生物被膜形成旺盛；紧随其后的为阳性对照组（硫酸链霉素，0.2 mg/mL）与3.13 mg/mL浓度的AMMH叶片提取物组，三者的O.D.值无显著差异（分别为1.96与1.57）。其余浓度为6.25、9.38及12.5 mg/mL的处理组，其O.D.值无显著差异（分别为0.91、0.79与0.53）。在生物被膜抑制率方面，12.5 mg/mL浓度处理组的抑制效果最优，抑制率达81.25%；其次为6.25 mg/mL与9.38 mg/mL处理组，二者抑制率无显著差异（分别为67.75%与72.23%）。3.13 mg/mL浓度处理组的生物被膜抑制率为44.49%，阳性对照组的抑制率为30.75%。通过共聚焦激光扫描显微镜（Confocal Laser Scanning Microscopy，CLSM）对Xoo生物被膜的抑制与解离效果进行分析，结果显示：随着AMMH叶片提取物浓度升高，样本中出现失活的Xoo细胞，且菌体聚集规模发生改变；对照组玻片未检测到Xoo死细胞。