Fusaric acid mediates the assembly of disease-suppressive rhizosphere microbiota via induced shifts in plant root exudates

Plant health is significantly impacted by the complex interplay between the plant itself, its associated microbiota, and soil-borne pathogens. *Fusarium oxysporum*, a widespread soilborne fungus, causes Fusarium wilt, a devastating disease affecting various crops and resulting in substantial economic losses. While plants possess inherent defense mechanisms, the rhizosphere, the narrow region of soil directly surrounding plant roots, harbors a diverse microbial community, some members of which can promote plant health through various mechanisms, including antibiosis, competition, and induced systemic resistance. The balance between these beneficial microbes and the pathogen determines the ultimate health of the plant. Recent research emphasizes the plant's active role in shaping its rhizosphere microbiome, recruiting beneficial microbes in response to pathogen attack, a concept aligned with the 'cry-for-help' hypothesis. Plant genotype plays a crucial role in this interaction, with resistant and susceptible cultivars exhibiting distinct rhizosphere microbial communities. However, the mechanisms underlying these interactions, particularly the influence of pathogen virulence factors, remain largely unexplored. This study focuses on the role of fusaric acid (FA), a well-known phytotoxin and virulence factor produced by *Fusarium* species, in shaping the rhizosphere microbiota of resistant and susceptible tomato cultivars upon infection by FOL. The study hypothesizes that resistant and susceptible tomato cultivars differ in their ability to recruit beneficial microbes upon pathogen infection, and that this microbial recruitment is modulated by pathogen virulence factors like FA. Using a tomato-FOL model system, the researchers aimed to investigate how FOL infection differentially alters the rhizosphere microbiota, determine FA's effect on microbiota alteration, and evaluate how root exudate changes affect beneficial bacterial colonization.

The literature extensively documents the importance of the rhizosphere microbiome in plant health and disease resistance. Studies highlight the dynamic interactions between plants and their associated microorganisms, ranging from mutualistic to pathogenic relationships. Soilborne pathogens, such as *Fusarium oxysporum*, pose a significant threat to plant health and cause substantial economic losses globally. The infection process typically involves asymptomatic root penetration, xylem vessel colonization, and subsequent spread to aboveground tissues. Plants counter these pathogens through diverse mechanisms, including the recruitment of beneficial rhizosphere microbes that can inhibit pathogens via competition, antibiosis, or by inducing systemic resistance. Research shows that plants actively influence their rhizosphere microbiome composition, manipulating it to enhance nutrient acquisition and disease suppression. Pathogen attack often triggers a selective recruitment of beneficial microbes as a defense mechanism. The plant genotype and the specific pathogen involved are crucial factors influencing this microbial assemblage. Furthermore, pathogen virulence factors, such as effector proteins and mycotoxins, can directly or indirectly alter the composition and function of the rhizosphere microbiota, influencing the outcome of plant-pathogen interactions. Fusaric acid (FA), a well-studied virulence factor of *Fusarium* species, is known for its phytotoxic effects and ability to induce wilt symptoms in plants. However, its impact on the rhizosphere microbiota remains an area of active investigation.

The study employed a combination of experimental approaches using two tomato cultivars (Z19, resistant; D72, susceptible) and the *Fusarium oxysporum* f. sp. *lycopersici* (FOL) pathogen. Initially, the disease severity and pathogen density were compared in both cultivars grown in natural and sterile soil to assess the contribution of the rhizosphere microbiota to disease resistance. A split-root system was utilized to separate the direct and indirect effects of FOL on the rhizosphere microbiota. In this system, one part of the root system was inoculated with FOL, and the other part remained non-inoculated. Rhizosphere samples were then collected from both parts and used in a rhizosphere transplant experiment to evaluate the disease-suppressive potential of the microbiota. The effect of FA, produced by FOL, was tested by amending the soil with FA in a similar split-root system and rhizosphere transplant experiment. 16S rRNA gene amplicon sequencing was employed to profile the bacterial communities in the rhizosphere samples, allowing for the identification of OTUs altered by FOL and FA in both cultivars. A collection of bacterial isolates was obtained from the tomato rhizosphere, and their disease-suppressive abilities were evaluated using pot experiments. A synthetic community (SynCom) was created using selected isolates to test their combined effect on disease suppression. The influence of individual isolates was assessed through drop-out experiments. Quantitative PCR was used to quantify FOL abundance and bacterial counts, and HPLC-MS analysis was performed to analyze FA content in the rhizosphere and metabolite profiles in root exudates. The effect of root exudates on bacterial growth and biofilm formation was determined through in vitro assays. A FOL mutant lacking the FA biosynthetic gene (*Δfub1*) was generated and used to investigate the role of FA in shaping the rhizosphere microbiota. Statistical analyses, including PERMANOVA, OPLS-DA, and DESeq2, were used to analyze the data.

The resistant cultivar (Z19) exhibited significantly greater resistance to FOL than the susceptible cultivar (D72), irrespective of the presence of soil microbiota, suggesting the contribution of plant innate immunity. The rhizosphere microbiota from FOL-infected resistant plants demonstrated disease-suppressive properties, whereas that from susceptible plants exhibited disease-conducive effects. Fusaric acid (FA), produced by FOL, was detected in the culture filtrate and in the rhizosphere of infected plants. FA treatment resulted in similar patterns of disease severity and pathogen density as observed with FOL infection, with opposite effects in the resistant and susceptible cultivars. 16S rRNA gene amplicon sequencing revealed significant alterations in bacterial community composition in the systemic pot, influenced by cultivar, FOL infection, and their interaction. Specific OTUs belonging to *Streptomyces*, *Arthrobacter*, *Lysobacter*, *Sphingomonas*, and *Flavobacterium* showed contrasting relative abundance changes in response to FOL infection between the cultivars. The analysis using FA yielded corroborating results. Bacterial isolates from the genera *Flavobacterium*, *Arthrobacter*, *Streptomyces*, *Lysobacter*, *Sphingobium*, and *Sphingomonas* were isolated, and most showed disease-suppressive effects. *Sphingomonas* isolates exhibited the strongest disease suppression. The synthetic community (SynCom) significantly reduced disease severity in both cultivars, with *Sphingomonas* sp. Sm12 contributing the most. *Sphingomonas* colonization was cultivar-specific in response to FOL infection and FA amendment, being suppressed in the susceptible and enhanced in the resistant cultivar. HPLC-MS analysis of root exudates revealed significant alterations induced by FOL and FA, with distinct changes in metabolite profiles between cultivars. Specific metabolites, including α-tomatine, tryptophan, rutin, 2-hydroxyglutaric acid, tyramine, leucine, fumaric acid, and xylose, were shown to influence Sm12 growth and biofilm formation. The FOL *Δfub1* mutant, lacking FA production, caused less severe disease symptoms and had a reduced impact on *Sphingomonas* colonization compared to the wild type.

This study provides compelling evidence for the crucial role of fusaric acid (FA), a key virulence factor of *Fusarium oxysporum* f. sp. *lycopersici* (FOL), in shaping the rhizosphere microbiome and influencing disease outcome in tomato. The findings demonstrate that FA, produced by FOL, differentially alters root exudate profiles in resistant and susceptible cultivars, which in turn affects the recruitment of beneficial bacterial taxa. The resistant cultivar effectively recruits disease-suppressive bacteria, such as *Sphingomonas*, which enhance plant defense through induced systemic resistance. Conversely, in the susceptible cultivar, FA hinders the recruitment of these beneficial bacteria, promoting pathogen infection. This work highlights the intricate interplay between the host plant, the pathogen, and the rhizosphere microbiota in determining disease susceptibility and resistance. The ability of the resistant cultivar to manipulate its root exudates to recruit beneficial bacteria provides a critical first line of defense against pathogen invasion. The direct and indirect effects of FA on the rhizosphere microbiota, including phytotoxicity and antimicrobial activity, are significant factors in the pathogenesis of FOL. These findings underscore the importance of understanding plant-pathogen-microbiota interactions in developing sustainable disease management strategies.

This research demonstrates a novel mechanism by which a pathogen, through its virulence factors, manipulates the plant rhizosphere microbiota to promote disease development. The study highlights the critical role of fusaric acid (FA) in mediating the differential recruitment of disease-suppressive bacteria in resistant and susceptible tomato cultivars. The findings emphasize the importance of considering the plant-pathogen-microbiota interaction in developing effective disease management strategies. Future studies could focus on identifying additional virulence factors and their impact on the rhizosphere, further investigation of the specific mechanisms by which identified metabolites influence bacterial growth and biofilm formation, and testing the effectiveness of manipulating the rhizosphere microbiota as a disease control approach.

The study primarily focused on bacterial communities in the rhizosphere; therefore, other components of the rhizosphere microbiome, such as fungi and mycorrhizal associations, were not explicitly investigated. The experiments were conducted under controlled greenhouse conditions, potentially limiting the generalizability of the findings to natural field settings. The mechanisms by which specific metabolites identified in root exudates influence bacterial growth and biofilm formation require further investigation.