Crop losses to plant pathogens are a growing threat to global food security and more effective control strategies are urgently required. Biofumigation, an agricultural technique where Brassica plant tissues are mulched into soils to release antimicrobial plant allelochemicals called isothiocyanates (ITCs), has been proposed as an environmentally friendly alternative to agrochemicals. Whilst biofumigation has been shown to suppress a range of plant pathogens, its effects on plant pathogenic bacteria remain largely unexplored. Here, we used a laboratory model system to compare the efficacy of different types of ITCs against Ralstonia solanacearum plant bacterial pathogen. Additionally, we evaluated the potential for ITC‐tolerance evolution under high, intermediate, and low transfer frequency ITC exposure treatments. We found that allyl‐ITC was the most efficient compound at suppressing R. solanacearum growth, and its efficacy was not improved when combined with other types of ITCs. Despite consistent pathogen growth suppression, ITC tolerance evolution was observed in the low transfer frequency exposure treatment, leading to cross‐tolerance to ampicillin beta‐lactam antibiotic. Mechanistically, tolerance was linked to insertion sequence movement at four positions in genes that were potentially associated with stress responses (H‐NS histone like protein), cell growth and competitiveness (acyltransferase), iron storage ([2‐Fe‐2S]‐binding protein) and calcium ion sequestration (calcium‐binding protein). Interestingly, pathogen adaptation to the growth media also indirectly selected for increased ITC tolerance through potential adaptations linked with metabolism and antibiotic resistance (dehydrogenase‐like protein) and transmembrane protein movement (Tat pathway signal protein). Together, our results suggest that R. solanacearum can rapidly evolve tolerance to allyl‐ITC plant allelochemical which could constrain the long‐term efficiency of biofumigation biocontrol and potentially shape pathogen evolution with plants.

Crop losses to plant pathogens are a growing threat to global food security, and hence, more effective control strategies are urgently required. Biofumigation, an agricultural technique, where Brassica plant tissues are mulched into soils to release antimicrobial plant allelochemicals called isothiocyanates (ITCs), has been proposed as an environmentally friendly alternative to synthetic agrochemicals. While biofumigation has been shown to suppress a range of plant pathogens, its effects on plant pathogenic bacteria remain largely unexplored. Here we used a laboratory model system to compare the efficacy of different types of ITCs against Ralstonia solanacearum plant bacterial pathogen. Additionally, we evaluated the potential for ITC-tolerance evolution under high, intermediate and low transfer frequency ITC exposure treatments. We found that allyl-ITC was the most efficient compound at suppressing R. solanacearum growth, and its efficacy was not improved when combined with other types of ITCs. Despite consistent pathogen growth suppression, ITC tolerance evolution was observed in the low transfer frequency exposure treatment. Mechanistically, tolerance was associated with parallel mutations in a gene linked to glucose/sorbonose dehydrogenase, resulting in cross-tolerance to ampicillin beta-lactam antibiotic. Interestingly, pathogen adaptation to the growth media also indirectly selected for increased ITC tolerance through potential metabolic adaptations linked with cell wall structure (serine/threonine kinase) and DNA replication, recombination and repair (deoxyribonucleases). Together, our results suggest that R. solanacearum can rapidly evolve tolerance to allyl-ITC plant allelochemical, which could constrain the long-term efficiency of biofumigation biocontrol and potentially shape pathogen evolution with plants.