## 2 Results and Discussion
### Ailanstigols A and B Inhibit the Formation of Bacterial Biofilm  
Bacterial biofilms are primarily composed of bacterial cells and extracellular polymeric substances (EPS), and the formation of bacterial biofilms significantly enhanced bacterial viability and resistance to antibiotics. To investigate whether compounds **1** and **2** could affect bacterial biofilm formation, these compounds were added to cultures of the ES4326, PXO 71A, or PXO 86A strain. The cultures were then stained with crystal violet to visualize the biofilm. The results demonstrated that compounds **1** and **2** at a concentration of 4 times IC\({}_{50}\) could inhibit the formation of ES4326 biofilms, with inhibition rates of 37.68 \(\pm\) 5.23% and 39.88 \(\pm\) 11.96%, respectively, and the higher the compound concentration, the more pronounced the inhibitory effects (Figure 7A,D). At a concentration of 4 times IC\({}_{50}\), compound **1** significantly inhibited the formation of PXO 71A biofilm, with an inhibition rate of 51.22 \(\pm\) 3.47% (Figure 7B). However, compound **2** had only a slight influence on the formation of the PXO 71A biofilm (Figure 7B). Both compounds **1** and **2** could inhibit the formation of PXO 86A biofilms. Following treatment with compound **1** at a concentration of IC\({}_{50}\), PXO 86A biofilms demonstrated the strongest inhibition, with a rate of 53.72 \(\pm\) 4.72% (Figure 7C). Specialized metabolites play a crucial role in plant defenses against pathogen infection and can inhibit the growth and pathogenicity of pathogens through various mechanisms. Steroidal hydrazones interact with the cell membranes and cell walls of _Bacillus subtilis_, _Escherichia coli_, and _Staphylococcus aureus_, disrupting bacterial structure and function. Additionally, they can inhibit bacterial metabolic pathways, suppressing bacterial growth [41]. Cationic steroids interact with bacterial cell membranes, damaging the integrity of the membrane and causing leakage of cellular contents and killing the bacteria [42]. Juglone at a concentration of 20 \(\upmu\)g/mL is known to significantly damage the permeability and integrity of the _P. syringae_ cell membrane. In addition, juglone effectively inhibits the pro  
Figure 7: Determination of biofilm formation of _Xanthomonas oryzae_ pv. _oryzae_ PXO 71A, PXO 86A, and _Pseudomonas syringae_ pv. _maculicola_ ES4326 after treatment with compounds **1** and **2**. **(A–C)** The results of the biofilm formation assay in ES4326, PXO 71A, or PXO 86A treated with compound **1** for 48 h. (**D–F**) The results of the biofilm formation assay of ES4326, PXO 71A, or PXO 86A treated with compound **2** for 48 h. IC\({}_{50}\): Half maximal inhibitory concentration. The IC\({}_{50}\) values of compound **1** against the PXO 71A, PXO 86A, and ES4326 strains were determined to be 485.85 \(\upmu\)g/mL, 314.16 \(\upmu\)g/mL, and 289.23 \(\upmu\)g/mL, respectively. The IC\({}_{50}\) values of compound **2** against the PXO 71A, PXO 86A, and ES4326 strains were determined to be 354.09 \(\upmu\)g/mL, 274.12 \(\upmu\)g/mL, and 476.18 \(\upmu\)g/mL, respectively. One-way ANOVA and Tukey’s tests were used to compare mean differences, and different lowercase letters (a, b, and c) represent significant differences (\(p<0.05\)). duction of extracellular polymeric substances, affecting the formation of the cell membrane and inhibiting bacterial growth [43]. In this study, steroid compounds were found that inhibited the growth of _X. oryzae_ pv. _oryzae_ and _P. syringae_ pv. _maculicola_ and reduced the cell viability of these two bacteria. After being treated with these compounds, the bacterial cell membranes underwent significant changes in both fluidity and integrity, which in turn disrupted their normal functioning and resulted in the leakage of intracellular proteins. Furthermore, as the concentration of the compounds increased, the amount of protein leakage also gradually grew, demonstrating a dose-dependent relationship.