The compound was extracted from a five-day culture of B. amyloliquefaciens Cas02 and purified by preparative high-performance liquid chromatography. High-performance liquid chromatography indicated a purity of approximately 90%, with three peaks corresponding to the Iturin A2, A3 and A4 isomers in a peak-area ratio of 4.9:1.2:1. In disc-diffusion assays, a loading of 20 milligrams per millilitre produced a 26-millimetre inhibition zone around R. solanacearum. Only weak inhibition, with zones no larger than 10 millimetres, was observed against Pseudomonas syringae, Pectobacterium aroidearum and Pectobacterium carotovorum. Within the four plant-pathogenic bacteria tested, the results therefore showed strongest activity against the tomato wilt pathogen rather than equally broad inhibition across the panel.

Scanning and transmission electron microscopy showed that damage increased with the Iturin A concentration. Untreated R. solanacearum cells displayed the plump, short-rod morphology associated with the species. Exposure to 16 micrograms per millilitre Iturin A caused cell shrinkage, while higher concentrations produced more severe deformation and adhesion between neighbouring cells. Transmission electron microscopy showed membrane invagination at 16 micrograms per millilitre. At 25 micrograms per millilitre, membranes were ruptured and cytoplasmic material leaked from treated cells. A separate experiment used a green-fluorescent-protein-producing strain because intracellular fluorescence decreases when the protein escapes through a damaged membrane. Fluorescence fell by 19.90%, 36.10% and 74.21% after 24 hours at 16, 25 and 50 micrograms per millilitre, respectively, providing complementary evidence of a dose-dependent loss of membrane integrity.

Iturin A also reduced the capacity of R. solanacearum to form an attached microbial community. Crystal-violet measurements showed that biofilm formation declined by 31.89% and 56.65% at concentrations of 10 and 16 micrograms per millilitre, while 25 micrograms per millilitre produced near-complete inhibition in the laboratory assay. Biofilm formation supports bacterial persistence in soil, xylem colonisation and tolerance of environmental stress and antimicrobial agents, making its suppression biologically relevant to tomato wilt. The experiment, however, measured attached biomass in laboratory wells and did not directly quantify bacterial survival or xylem colonization in tomato plants. In parallel, the fluorescent probe DCFH-DA detected a concentration-dependent increase in intracellular reactive oxygen species. Fluorescence was 1.6-, 2.2- and 3.0-fold higher than in untreated cultures after exposure to 16, 25 and 50 micrograms per millilitre Iturin A, respectively, consistent with the induction of oxidative stress.

Physiological measurements indicated that Iturin A disturbed the machinery responsible for cellular energy production. Succinate dehydrogenase activity declined as the concentration increased: treatments of 16 and 25 micrograms per millilitre reduced activity by 38.99% and 78.84%, respectively, while activity was undetectable at 50 micrograms per millilitre. Succinate dehydrogenase links the tricarboxylic acid cycle to the electron transport chain, so the measurement indicated impairment in both metabolic systems. Total NAD+ and NADH contents also decreased, including reductions of 43.65% and 55.35% at 25 and 50 micrograms per millilitre. Cytochrome c oxidase staining moved in the opposite direction, with a larger positively stained area in treated cells. The investigators interpreted this pattern as accumulation or abnormal localisation of terminal oxidase components when upstream electron transport is impaired, rather than as evidence of increased normal respiration.

The researchers also profiled the pathogen’s transcriptional response to a subinhibitory Iturin A concentration. RNA was extracted from R. solanacearum exposed to 16 micrograms per millilitre for 24 hours and from untreated controls, with three biological replicates per group. Sequencing identified 168 upregulated and 336 downregulated genes using thresholds of more than a twofold absolute change and an adjusted p-value below 0.05. Enriched pathways included ribosome function, secondary-metabolite biosynthesis, amino-acid biosynthesis, flagellar assembly and oxidative phosphorylation. Quantitative reverse-transcription PCR of seven selected genes agreed with the sequencing results. Among the validated downregulated genes were galU, reduced by 9.5-fold, which participates in the production of precursors used in surface and extracellular polysaccharides; YscC, reduced by 3.41-fold, which encodes an outer-membrane ring channel protein of the type III secretion system; sbnA, reduced by 36.3-fold, which belongs to a siderophore-biosynthesis pathway associated with iron acquisition; and exaC, reduced by 3.4-fold, an aldehyde dehydrogenase involved in acetaldehyde detoxification during ethanol metabolism.

The sequencing results also included changes in other genes associated with secretion and virulence. The type III secretion-system genes YscJ, popA and popB were identified as differentially expressed, but they were not among the seven genes validated by quantitative reverse-transcription PCR. Separately, the validated transcriptome analysis showed increases in pilQ, secY and cspA, by 9.3-, 36.44- and 28.93-fold, respectively. The authors proposed that the increases in pilQ and secY might represent compensatory responses intended to preserve secretion and surface structures under stress, while higher cspA expression was compatible with activation of a general bacterial stress response. These were measurements of gene expression; the study did not directly measure corresponding changes in protein abundance, secretion activity or the resulting magnitude of pathogen virulence.

The pot experiment assessed preventive rather than curative performance. Groups of 20 tomato seedlings of the Guifei cultivar at the four-true-leaf stage received Iturin A, kasugamycin or sterile water by soil drench, followed two days later by inoculation with R. solanacearum Cas800. Iturin A and kasugamycin were each applied at 20 millilitres of a 100-micrograms-per-millilitre solution per plant. Disease severity was scored from seven days after inoculation, and outcomes were calculated on day 15. Disease incidence was 24.07 ± 0.93% in the Iturin A group, compared with 84.12 ± 0.88% in the untreated control and 39.63 ± 0.37% in the kasugamycin group. Relative to the control, disease index decreased by 74.27% with Iturin A and by 50.96% with kasugamycin. Iturin A therefore produced 1.45-fold the reported control efficacy of kasugamycin under the experimental conditions, although the finding came from one controlled pot setup rather than a multi-season field trial.

Together, the observations identify several complementary processes through which Iturin A may explain its activity in the pot assay: direct damage to the bacterial envelope, reduced biofilm formation, accumulation of reactive oxygen species, interference with the electron transport chain and energy metabolism, and altered expression of genes connected to secretion, surface structures and iron acquisition. The investigators propose that this multitargeted mode of action could make the development of bacterial resistance less likely, but resistance evolution was not directly tested. Important limitations remain. Most mechanistic experiments used cultured bacteria, the efficacy result came from controlled pots, and treatment was applied before pathogen inoculation; the ability of Iturin A to cure already symptomatic plants was not evaluated. Multi-season field trials are needed to establish performance under natural conditions, while the authors identified fermentation optimisation and production-cost reduction as prerequisites for practical development of Iturin A-based microbial pesticides.

Subject of Research: Multitargeted antibacterial effects of Iturin A against the tomato bacterial wilt pathogen Ralstonia solanacearum

Article Title: Multitargeted antibacterial mechanisms of Iturin A derived from Bacillus amyloliquefaciens against tomato bacterial wilt

Article References: Yang, J., Liu, T., Li, Z., Zhu, T., Chi, L., Yan, X., Pan, B., Liang, J., & Wang, X. (2026). Multitargeted antibacterial mechanisms of Iturin A derived from Bacillus amyloliquefaciens against tomato bacterial wilt. Frontiers in Microbiology, 17, Article 1944894. https://doi.org/10.3389/fmicb.2026.1944894

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1944894

Keywords: Iturin A, tomato bacterial wilt, Ralstonia solanacearum, Bacillus amyloliquefaciens, cyclic lipopeptide, membrane disruption, biofilm inhibition, electron transport chain, plant pathogen, biocontrol, Multitargeted, antibacterial