Climate change poses a range of challenges to essential agricultural crops, including extreme weather events, temperature fluctuations, and, most notably, an increase in drought frequency (Zhou et al., 2024). Tomato (S. lycopersicum) is the most widely cultivated horticultural crop, produced at 186.1 million tons in 2022 (FAO, 2024; Hammond et al., 2022; Kopecká et al., 2023). However, drought stress severely impacts tomato production by disrupting key physiological processes, such as reducing photosynthesis, impairing water and nutrient uptake, and inducing oxidative stress through reactive oxygen species (ROS) accumulation (Li et al., 2024). These disruptions alter gene expression and metabolic pathways crucial to maintain osmotic balance, leading to stunted plant growth (Francesca et al., 2022; Landi et al., 2023). Consequently, tomato, which is particularly sensitive to water scarcity, experiences significant yield reductions, with losses reaching up to 40% (Hammond et al., 2022; Kopecká et al., 2023; Cruz-López et al., 2024).

To address this constraint, the use of biostimulants—substances or microbial inoculants that improve nutrient efficacy, stress tolerance, and crop quality—has gained attention (du Jardin, 2015). These sustainable products are increasingly important in mitigating the adverse effects of climate change, including the growing severity of droughts (du Jardin, 2015; Rouphael and Colla, 2018). Humic substances (HS), major components of soil organic matter resulting from the decomposition of plant, animal, and microbial residues, are one of the main categories of plant biostimulants (du Jardin, 2015). HS are a mixture of supramolecular conformations composed of relatively small and heterogeneous molecules associated by weak bonds (Piccolo, 2002; Mahler et al., 2021; Tiwari et al., 2023). They benefit plants indirectly by enhancing soil quality and its microbial community, providing carbon, improving soil aeration and stability, and facilitating nutrient uptake (Elkins and Nelson, 2002; Magdoff and Weil, 2004; Nardi et al., 2002; Piccolo, 2002). They also directly influence plant growth by activating hormonal pathways, particularly those of auxin, abscisic acid, and ethylene (Canellas et al., 2015). Some studies highlighted that HS contain auxins or have structural similarities with auxin and interact with hormone-cell receptors to initiate hormonal pathways (Canellas et al., 2002; Piccolo, 2002; Trevisan et al., 2010). Such activation leads to the stimulation of proton pumps, improving nutrient import within plants, increasing cell wall loosening and division, and enhancing root system development (Mora et al., 2010, 2012; Morsomme and Boutry, 2000; Muscolo et al., 2013; Nardi et al., 2002; Olaetxea et al., 2019; Quaggiotti, 2004; Trevisan et al., 2010; Zandonadi et al., 2007). The impact of HS on plants also comes from the activation of ROS pathways and the overexpression of antioxidant enzymes, which prime the plant without causing irreversible damage (Berbara and García, 2014; García et al., 2016; Schiavon et al., 2010). The beneficial effects of HS under water stress have been observed in various plants, including rice—where it helped protect cell membrane permeability—and Brassica napus, with reported increases in chlorophyll content, as well as in maize and tomato among others (Eyheraguibel et al., 2008; Galambos et al., 2020; Garcia et al., 2014; Lotfi et al., 2015). Despite in-depth studies on these products, our understanding of their biostimulation mechanisms remains incomplete. Their impacts are probably not only due to a direct influence on the plant but might also involve interactions with intermediary microorganisms (da Silva et al., 2021b).

Plants are known to harbor microbial communities within their tissues, known as endophytes, whose composition is shaped by various biotic and abiotic factors (Lengrand et al., 2024). These plant–microbe interactions are crucial for the adaptation and survival of both plants and microbes under stressful conditions (Meena et al., 2017; Meenakshi et al., 2019; Ullah et al., 2019)—for instance, bacterial endophytes can enhance plant growth through several mechanisms, such as improving nutrient uptake, producing hormones, and generating beneficial metabolites (Malinowski and Belesky, 2000; Ullah et al., 2019). Endophytes can be acquired from the environment or transmitted through seeds, with each group playing a vital role in plant health. Seed microbiota serve as a reservoir of microbial taxa that have co-evolved with plant hosts, providing functions that can support plant survival (Geisen et al., 2017; Hardoim et al., 2012; Johnston-Monje and Raizada, 2011). Moreover, this vertical transmission ensures the continuity of certain microorganisms from parent plants to their offspring, potentially promoting their beneficial growth. This process contributes to overall plant health and fosters beneficial endosymbiotic relationships (Barret et al., 2016; Cope-Selby et al., 2017; Hardoim et al., 2012; Rudgers et al., 2009; Shade et al., 2017).

Although HS and beneficial bacteria are effective as stand-alone biostimulants, numerous studies have shown that their positive impact on plant growth and stress tolerance is enhanced when used in co-inoculation (Canellas et al., 2013, 2015; da Silva et al., 2021c, da Silva et al., 2021a; Olivares et al., 2015)—for instance, Galambos et al. (2020) reported the combined use of HS with Paraburkholderia phytofirmans and Pantoea agglomerans that significantly promoted tomato growth, suggesting a complementary effect. This finding aligns with research by de Melo et al. (2018) and Olivares et al. (2015), which demonstrated improved plant vitality and stress tolerance when HS were used alongside microbial inoculants. In maize, Canellas et al. (2013) observed that HS application with Herbaspirillum seropedicae activated key metabolic pathways, enhancing the plant response to environmental challenges. Similarly, Baldotto et al. (2010) observed that such co-inoculation strategies led to significant improvements in nutrient uptake and overall plant health. The beneficial effects of co-inoculation of HS and plant-growth-promoting bacteria (PGPB) are likely due to multiple factors: (1) increased bacterial colonization and penetration (Nardi et al., 2009; Olivares et al., 2015, 2017; Piccolo, 2002; Canellas et al., 2008; Puglisi et al., 2008; Canellas et al., 2013), (2) activation of plant metabolic pathways that complement those induced by bacteria and HS individually (Galambos et al., 2020; Aguiar et al., 2016), and (3) direct effect of HS on bacterial metabolism (Wang et al., 2024). Since HS are known to influence co-inoculated bacteria, it stands to reason that they would similarly impact the communities of endophytic bacteria within plants.

In the present work, we hypothesize that HS positively influence plant growth and stress resilience concurrently with modifications in bacterial endophytic communities and that investigating these effects could help in understanding the mechanism of action of these molecules. To distinguish the direct effect of HS on the plant and their indirect effect via interactions with the soil microbiome, we developed a hydroponic system in sterile conditions. This setup allowed us to focus exclusively on seed-transmitted endophytic bacteria, avoiding any influence from the soil microbiome. This study analyzed the effects of HS on tomato growth under both non-stress and osmotic stress conditions, comparing the endophytic communities in roots across these different scenarios. The objectives were to determine (i) the impact of osmotic stress, (ii) the influence of HS, and (iii) the combined effects of HS and osmotic stress on both plant growth and the composition of the bacterial endophytic community. The results highlight the distinct effects of osmotic stress, HS, and their combined interaction on plant growth and the composition of the bacterial endophytic community.

This study employed a sterile hydroponic system to isolate the direct effects of HS on plant physiology from interactions with soil microbes. In this particular system, the results indicate that, under osmotic stress, HS application enhanced tomato resilience, evidenced by increased shoot and root weights, leaf area, shoot length, total chlorophyll content, and stomatal conductance. This growth enhancement is likely due to HS’ protective effects, activating antioxidant enzymes and thereby reducing oxidative stress, as indicated by the lower MDA content in leaves. The observed decrease in MDA levels post-HS application points to reduced lipid membrane peroxidation, a marker of oxidative damage. This aligns with other studies showing that HS application under stress conditions leads to a healthier plant state through enhanced antioxidant enzyme activities such as those of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POX) (Aguiar et al., 2016; García et al., 2012; Lotfi et al., 2015; Matuszak-Slamani et al., 2022)—for example, in the experiment of Matuszak-Slamani et al. (2022) on soybeans under osmotic stress in hydroponics, humic acid (HA) application led to an increase in plant length and chlorophyll content parallel with an upregulation of the antioxidant defense system. Similar results were observed in sugarcane treated with HA under water stress, which displayed better growth than control plants with increased stomatal conductance, transpiration, and net photosynthesis. This induced tolerance was also correlated with the activation of antioxidant enzymes’ activities (Aguiar et al., 2016). On the contrary, HS application did not significantly affect tomato growth under non-stress conditions. This result aligns with existing literature in hydroponics showing that HS-containing biostimulants have limited effects in unstressed environments (Aguiar et al., 2016; García et al., 2012; Lüdtke et al., 2021; Matuszak-Slamani et al., 2022). The effects of HS as biostimulants are highly dependent on factors such as dosage, plant species, and the experimental system used (Jindo et al., 2020). In this case, the concentration selected was optimized to demonstrate the beneficial effects of HS under osmotic stress.

The result of this experiment revealed that the increase of tomato resilience to osmotic stress is associated with its direct interaction with plants or its phytobiome and not with an indirect effect through the soil. Since seed-transmitted endophytic communities of plants are now well recognized to play a role in plant health (Pandey et al., 2022; War et al., 2023), we deepened the taxonomic characterization of these communities by PCR amplification and sequencing from total DNA extracted from disinfected roots. The use of PNA-chr11, tailored specifically to reduce S. lycopersicum var. Moneymaker amplification (Lengrand et al., manuscript submitted for publication), and primers targeting the 16S-ITS-23S rrn operon enabled us to achieve species-level identification of endophytes. The primary microbiota present in the roots and transmitted through seeds were identified as Bradyrhizobium, Methylobacterium, Ralstonia, Frigoribacterium, Hymenobacter, and Sphingomonas, with specific reads assigned to B. sp. SK17, M. oryzae, M. sp. FF17, M. sp. NMS14P, R. pickettii, F. sp. NBH87, H. sp. BRD128, and S. aerolata. This finding aligns with the results of Lengrand et al. (manuscript submitted for publication) which identified the core microbiome of S. lycopersicum var. Moneymaker transmitted from seed to shoots and roots as R. pickettii, S. aerolata, B. sp. SK17, and P. aquatile. Additionally, the genera Frigoribacterium and Hymenobacter were also found in the leaves and roots of plantlets grown in sterile conditions.

The composition of the endophytic microbiome in tomato plants is highly sensitive to environmental conditions. In this study, the tomatoes were grown under sterile conditions, and the hydroponic solution containing PEG, HS, or both was autoclaved before use. Therefore, the observed shifts in the microbiome are not attributable to external microorganisms. The sterile conditions likely reduced the diversity of the endophytic communities by preventing the introduction of soil-derived microbes.

Notably, osmotic stress alters bacterial community composition by reducing the alpha diversity in both HS-treated and untreated plants. A similar reduction of bacterial richness in the roots of Atractylodes lancea grown under PEG-induced osmotic stress was observed in the experiment of Wang et al. (2022). Additionally, the application of PEG alone induced a notable shift in the root endophytic community composition, specifically reducing the prevalence of the Bradyrhizobium genus. This reduction may be due to the greater resilience of genera like Sphingomonas and Ralstonia under stress conditions, which could outcompete Bradyrhizobium for resources in stressful environments.

Under both stress and non-stress conditions, the application of HS led to an increase of Bacteroidetes, especially of Hymenobacter, with a reduction in Proteobacteria—with an almost complete suppression of Sphingomonas. Interestingly, the same conclusion was drawn in the experiment of da Silva et al. (2021b) in rice root endophytic communities, suggesting a consistent effect across different plant species. This influence of HS on bacterial communities seems to extend beyond plant-associated microbiota in the literature, as a similar shift after HS application was also observed in wastewaters and lakes (Hutalle-Schmelzer and Grossart, 2009; Luo et al., 2019). Indeed in ammonium-rich wastewater from landfill leachate, HS application diminished Proteobacteria while enhancing Bacteroidetes with a significant change for aerobic ammonium-oxidizing bacteria (AOB), crucial for nitritation-anammox processes (Luo et al., 2019). Similar adjustments were also observed in the microbiota of oligotrophic Lake Stechlin, where HS addition specifically enriched groups within Alphaproteobacteria, Bacteroidetes, and Deltaproteobacteria, indicating a direct HS effect on microbiota rather than an indirect plant-mediated one (Hutalle-Schmelzer and Grossart, 2009). The interaction between HS and microbial communities may be explained in part by the degradation of HS by bacteria. Under anaerobic conditions, bacteria involved in the anaerobic ammonium oxidation (Anammox) process contribute to HS degradation (Liang et al., 2009). Indeed reduced HS can act as electron donors for anaerobic organisms utilizing alternative electron acceptors, allowing these microorganisms to derive energy from HS by using carbon sources as acetate (Liang et al., 2009; Lipczynska-Kochany, 2018). This hypothesis might account for the observed shift, particularly for Hymenobacter, the predominantly favored genus, which includes numerous species known to function as ammonia-oxidizing bacteria (Garcia-Lopez et al., 2019; Zhang et al., 2020). In addition, HS contain a high concentration of dissolved organic carbon (DOC) ( Supplementary Table S2 ), which can contribute to microbial nutrition (Tahiri et al., 2016a, b). Apart from DOC, the other nutrients in HS should be negligible in plant or microbial nutrition compared with the nutrients present in the hydroponic solution since they represent, for those present in larger quantities (N and Fe), concentrations a thousand times lower.

In parallel, Actinobacteria have also been found to be active in degrading HS in freshwater and estuarine environments (Haukka et al., 2005; Hutalle-Schmelzer and Grossart, 2009; Rocker et al., 2012; Lipczynska-Kochany, 2018). However, an increase in the Actinobacteria phylum, particularly the Frigoribacterium genus, was only observed in plants under osmotic stress that were treated with HS. This increase was notably absent when either the biostimulant or the osmotic stress was applied alone. Interestingly, an increase in Actinobacteria within the root microbiome has already been correlated with enhanced drought resilience in plants (Fitzpatrick et al., 2018a; Martins et al., 2023; Naylor et al., 2017; Santos-Medellín et al., 2017; Schimel et al., 2007; Tocheva et al., 2016). Xu et al. (2021) demonstrated that Actinobacteria enrichment in drought stress conditions may be partially due to a reduction of iron homeostasis (increase in insoluble Fe³⁺ in dried soils and reduction of plant Fe transporters) within the root under drought stress. Indeed the root’s reduced iron uptake and increased iron storage lead to low available iron concentrations in the root, benefiting bacteria proficient in scavenging the limited iron and to the detriment of less capable ones. This hypothesis gained support from the observation of Xu et al. (2021), where external application of iron interrupts the enrichment of Actinobacteria and, in parallel, reduces the drought stress tolerance of sorghum. In our experiment, osmotic stress induced in hydroponic systems might not affect Fe³⁺ availability, but HS could create soluble nutrient complexes (with Fe and phosphate among others) absorbable by plants (Spark et al., 1997). However, this absorption mainly involves fulvic acids and not larger humic acid molecules that only interact with cell walls and cannot be absorbed (Nardi et al., 2002). Consequently, the formation of such complexes with Fe, coupled with diminished water uptake due to osmotic stress, could mimic a drought stress effect on reduced Fe availability and increase Actinobacteria’s relative abundance. The increased abundance of Actinobacteria in line with higher plant drought tolerance is reported in numerous studies (Ebrahimi-Zarandi et al., 2023). A variety of mechanisms are used by these bacteria, including increasing nutrient availability via phosphate and potassium solubilization, siderophore production, and nitrogen fixation as well as the production of ACC deaminase, phytohormones, volatile organic compounds, extracellular polysaccharides, and osmolytes (Boukhatem et al., 2022). They also activate the plant’s antioxidant system and induce the expression of stress-responsive genes. A meta-transcriptomic analysis conducted by Xu et al. (2018) on sorghum roots under drought stress revealed significant alterations in actinobacterial gene expression across different functional categories, highlighting the critical roles of carbohydrates, amino acid transport, and ATP-binding cassette (ABC) transporters. The Frigoribacterium genera, representing the major increase of Actinobacteria in this study, were already known to solubilize phosphate and to produce siderophores with ACC deaminase activity (Boukhatem et al., 2022; Ebrahimi-Zarandi et al., 2023) as exemplified by the strain F. faeni 801, isolated from halophytes and with high tolerance to salt stress (Zhou et al., 2017).

Finally, the genus Roseateles, whose abundance was increased under combined osmotic and HS treatment, encompasses species known for their remarkable degradation capabilities. Among these, the strain R. depolymerans KCTC42856 stands out for its ability to break down aliphatic polymers in biodegradable plastics (Lee et al., 2016). Similarly, R. chitinivorans HWN-4^T has been identified as a promising candidate for bioremediation, capable of mitigating environmental contaminants (Sisinthy and Gundlapally, 2020). In the experiment of Le et al. (2023), further insights from antiSMASH analysis highlighted the significant variability in secondary metabolite profiles across Roseateles strains, suggesting a rich repertoire of defense mechanisms against competitive microbial species (Le et al., 2023). Considering the characteristics of this genus, it was probably more resilient to osmotic stress induced by PEG and had the ability to surpass competing bacteria by producing certain metabolites to gain access to limited resources.

In summary, endophytic communities carried by tomato seeds and transmitted to the roots were distinctly influenced by osmotic stress and the application of HS, either individually or in combination. The variations following the application of HS can be attributed to several hypotheses: (1) the composition of the HS may favor certain genera capable of using these compounds, indicating a direct nutritional impact, specifically for the Hymenobacter genus (Liang et al., 2009; Lipczynska-Kochany, 2018; Luo et al., 2019). Additionally, the effect of HS might be mediated by the release of metabolites, which could influence microbial community dynamics by promoting specific microbial taxa or altering interactions within the community; (2) Indirectly, HS could modify plant metabolism such as root iron homeostasis and favor specific phyla such as Actinobacteria with the Frigoribacterium genus (Fitzpatrick et al., 2018a; Nardi et al., 2002; Xu et al., 2021); (3) The recruitment of certain microbes could respond to HS-induced mild stress, activating plant defense mechanisms (Berbara and García, 2014; da Silva et al., 2021b; García et al., 2016); (4) The effects of HS could also be independent of endophytic composition, leading to a parallel benefit for plants and a change in their microbiota communities (Berbara and García, 2014; da Silva et al., 2021b; García et al., 2016).

In conclusion, this study investigated the combined and individual impacts of HS and osmotic stress on plant physiology and endophytic communities, revealing a possible correlation between endophytic communities and plant physiology, even in sterile environments. This underscores the importance of seed-borne bacteria as a primary inoculum. Moreover, HS application under osmotic stress increased tomato growth parameters in concert with the abundance of Frigoribacterium sp., Roseateles sp., and Hymenobacter sp. and reduction of Sphingomonas sp., highlighting the potential of HS to enhance certain plant–endophyte relationships. This opens new perspectives on the mode of action of HS, possibly by interacting with endophytes. Based on the findings of this study, the application of HS in agriculture and hydroponic systems should be carefully tailored to maximize both plant health and the beneficial effects on endophytic communities. Optimal concentrations of HS should be determined based on specific crop and environmental conditions to ensure that the right balance is achieved. Additionally, combining HS applications with bacterial inoculants like Frigoribacterium and Hymenobacter could further enhance plant resilience under stress.

Future research should explore the role of plants in the shifts observed in endophytic bacterial communities following the application of HS and PEG. This could involve creating synthetic microbial communities based on metabarcoding results to directly test the effects of HS and PEG on bacterial strains and communities as well as using RNAseq to identify the most expressed bacterial genes under different conditions. Additionally, the current study is limited by its 3-week duration, which may not have allowed the full capture of the long-term effects of HS and osmotic stress on plant growth and endophytic communities, particularly beyond the vegetative stage. Future studies should consider extending the experimental period to observe potential impacts throughout the entire cultivation cycle.

Moreover, a promising avenue for future research involves exploring how HS induce shifts in endophytic bacterial communities, guided by several key hypotheses. One hypothesis suggests that HS may favor bacterial species capable of using HS as electron donors in the anammox process. This could be tested by creating knockout mutants of specific genes involved in this pathway, such as hydrazine synthase (hzsA, hzsB, and hzsC) and hydrazine dehydrogenase (hdh) (Wang et al., 2016; Kuenen, 2008). Another hypothesis posits that HS form complexes with iron, reducing its availability and potentially increasing the presence of Actinobacteria (Xu et al., 2021). Testing this could involve reducing iron levels in hydroponic solutions instead of applying HS to see if the same effects are observed. Additionally, it is also hypothesized that HS might induce mild stress in plants, leading to shifts in bacterial communities. This could be investigated using tomato mutants lacking the rop9 gene, which regulates ROS formation (Puli et al., 2024). These lines of inquiry could provide valuable insights into the mechanisms behind HS-induced microbial shifts and their impact on plant health and stress tolerance. Finally, studying different tomato cultivars, particularly those with varying stress tolerances and responses to HS, could reveal whether the microbial shifts and beneficial effects of HS are consistent across genetic backgrounds.