The presence of plant secondary metabolites (PSMs) such as flavonoids, coumarins, phenolic compounds, and terpenes, is known to modify the chemical and physical properties of soils. PSMs also serve as allelochemicals, protect plants against pathogens, act as substrates, and can induce pollutant catabolic pathways of soil microorganisms (Pilon-Smits, 2005; Singer, 2006; Zhou and Wu, 2012; Pino et al., 2016). They also shape the structure and function of plant-associated bacterial communities, i.e., rhizobacteria and endophytes, favoring the growth of suitable pollutant consumers (Uhlik et al., 2013; Qin et al., 2014; Eevers et al., 2016), which directly use their degradative capabilities to metabolize a given pollutant (Glick, 2010; Pawlik et al., 2015; Sauvêtre and Schroder, 2015; Khare et al., 2018; Wu et al., 2018). Thus, the role of PSMs in the degradation of pollutants can be substantial.

Due to their structural similarity, PSMs may have a considerable influence on the removal of structurally related pollutants (Uhlik et al., 2013; Hu et al., 2014). PSMs typically provide the energy for microorganisms to perform cometabolism, while the structurally similar pollutant is degraded as a secondary substrate (Musilova et al., 2016). Common examples of cometabolites are biphenyl and PCBs (Singer et al., 2003). However, PSMs can also be used as a primary source of carbon and energy by bacterial communities to support their growth and stimulate the expression of desirable genes involved in the catabolism of structurally similar pollutant. The degree of similarity between a PSM and a pollutant has been found to influence the rate of pollutant removal (Mierzejewska et al., 2019; Urbaniak et al., 2019a).

Earlier studies described syringic acid (SA) as a characteristic PSM for cucurbits (Blum et al., 2000; Campos et al., 2009; Kruczek et al., 2015; Shi et al., 2016), which are themselves known as effective phytoremediators of organic pollutants (Eevers et al., 2018; Urbaniak et al., 2019b,2020). The addition of SA to bacterial cultures not only enhanced MCPA removal but more importantly increased the number of detected functional genes responsible for the initiation of phenoxy herbicide biodegradation (Mierzejewska et al., 2019; Urbaniak et al., 2019a). Additionally, SA application and zucchini cultivation was found to decrease the toxicity of MCPA-contaminated soil (Mierzejewska et al., 2022).

4-chloro-2-methylphenoxyacetic acid (MCPA) is one of the most commonly used herbicides in Europe, and it is particularly persistent under the low winter temperatures, low soil organic carbon content, and acidic pH that are typical for Europe (Paszko et al., 2016). In areas with high contamination levels, heavy rainstorms facilitate the transport of MCPA residues from soil to water, resulting in the contamination of aquatic ecosystems (Matamoros et al., 2012; Rheinheimer dos Santos et al., 2020) to levels exceeding permissible threshold concentrations (Ignatowicz and Struk-Sokołowska, 2004; Rippy et al., 2017). These are believed to account for the adverse effects on living organisms reported in several worldwide studies (Pereira and Cerejeira, 2000; Nielsen and Dahllof, 2007; Palma et al., 2018; Morton et al., 2020).

Despite the large body of research performed on the effects of MCPA on soil- and water-inhabiting organisms, little is known about its influence on soil and plant-associated bacterial communities. Also, little attention has been devoted to the role of PSMs in the remediation of MCPA-contaminated soil. While our prior research has demonstrated that SA treatment enhanced MCPA transformation (Mierzejewska et al., 2019; Urbaniak et al., 2019a), no studies have examined the effects of such treatment on the structure of plant-associated bacterial communities (rhizobacteria and endophytes).

Consequently, this study aimed to determine whether adding SA, which is structurally like the herbicide, to unplanted and zucchini-planted soil contaminated with MCPA shapes the bacterial communities within the unplanted soil, rhizosphere, and plant endosphere (i.e., endophytic communities in roots and leaves). The obtained results could be applied to develop nature-based solutions for the removal of MCPA residues and to prevent their dispersal in the environment.

Recent research has provided a comprehensive insight into the interactions between bacteria and plants in contaminated environments (Tardif et al., 2016; Thijs et al., 2018). It appears that the structure of bacterial communities in soils and plants is determined by the presence of pollutants. However, our knowledge about the effects of plant root exudates (including PSMs, such as SA) on the communities of bacteria in contaminated environments remains limited. Some studies have shown that certain phenolic compounds, such as benzoic acid, can enhance the biodegradative activity of bacteria in soil (Mandal et al., 2010; Zwetsloot et al., 2020). However, phenolic compounds can exert contrasting effects on microbial communities depending on the prevailing conditions (Zwetsloot et al., 2020).

While most studies have examined the biodegradation of phenoxy herbicides, especially 2,4-D (2,4-dichlorophenoxy acid) in soil, the most widely-used pesticide in European agriculture is MCPA. MCPA is usually sprayed as a commercial formulation in the form of amines, sodium salts, or esters to control perennial and broadleaf annual weeds (Paszko et al., 2016). Higher phenoxy herbicide (i.e., MCPA) concentrations are mostly detected in regions with ongoing intensive agriculture (Bianchi et al., 2017), although residuals of MCPA can be transported with intensive surface run-off and become a threat to non-target organisms. Monitoring studies showed that MCPA was present in 33–60% of the studied sites along the river tributaries in Poland (Zagibajło et al., 2017; Jarosiewicz et al., 2018). Thus, the development of methods for the prevention of MCPA residuals leaching is of the highest importance. Still, little attention has been paid to the potential of selected plants as phytoremediators of soils contaminated with phenoxy herbicides. The members of the Cucurbitaceae family are particularly promising candidates due to their ability to extract, translocate, and accumulate highly toxic persistent organic pollutants from soils (Hülster et al., 1994; White et al., 2003; Inui et al., 2008; Zhang et al., 2009; Wyrwicka et al., 2014; Urbaniak et al., 2016) and several cucurbit species, such as zucchini (C. pepo cv “Atena Polka”), have demonstrated resistance to MCPA (Mierzejewska et al., 2022).

Therefore, this study investigates the effects of a specific PSM (SA) and zucchini cultivation on the removal of MCPA from the soil. In unplanted MCPA-contaminated soil, amendment with SA led to a higher decrease of MCPA (30%) than in untreated soil (18%) (Figure 1). However, higher MCPA removal (50%) was observed in the planted condition amended with SA. Another study showed that the removal of 2,4-D from water, using the plant species Plectranthus neochilus, was 49% after 30 days (Ramborger et al., 2017). Furthermore, Germaine et al. (2006) demonstrated that endophyte-enhanced phytoremediation significantly improves the capacity for removal of 2,4-D. Our study showed that SA improves the removal of MCPA from unplanted soil. However, when SA application and zucchini cultivation are combined, the decrease of the herbicide concentration in the soil is significantly greater.

Although SA enhanced the removal of MCPA from soil (Figure 1), MCPA had a detrimental effect on fresh biomass (Table 2). Indeed, MCPA application was associated with 90% lower aboveground biomass in the planted variants. Phenoxy herbicides are transported to meristems, causing uncontrolled growth and consequently damaging the development of plant tissues (Grossmann, 2003). SA has been found to alleviate the toxic effects of MCPA (Mierzejewska et al., 2022), however, these studies investigated higher initial concentrations of both compounds. Kováčik et al. (2010) showed that certain phenolic acids, e.g., salicylic acid, can alleviate stress symptoms in plants. On the other hand, Zhou et al. (2014) suggested that SA can exhibit phytotoxic effects. Blum (1996) reported the concentration, composition, and synergism of individual phenolic compounds to be crucial for plant growth inhibition in the environment. Hence, we claim that despite improving MCPA-removal efficiency, the application of SA throughout the incubation time did not enhance the plant growth-promoting properties of the system.

The above-mentioned observations are as per earlier studies indicating that SA significantly enhanced the removal of MCPA from liquid cultures enriched with microorganisms derived from agricultural soil (Mierzejewska et al., 2019; Urbaniak et al., 2019a). Hence, this study also examines the effects of SA and MCPA, used individually or in combination, on the composition of bacterial communities within the soil, rhizosphere, and the zucchini plant itself.

The effects on the diversity of bacteria were found to be compartment-specific, with an interaction between SA and MCPA (Table 5). The highest diversity indices values were found for unplanted bulk and rhizospheric soil, and the lowest in leaves. Approximately, 10% lower values of diversity indices were observed after SA application (Table 3). Indeed, it has previously been reported that SA has antimicrobial activity against some bacterial strains (Shi et al., 2016). Our findings also indicate that the amendment of soil with MCPA did not affect diversity indices significantly (Table 3). Also, Ławniczak et al. (2016) found that the application of oligomeric herbicidal ionic liquids with MCPA did not cause any changes in the diversity indices. In variants amended with SA and MCPA diversity indices (i.e., Obs.ASV) were higher than in untreated variants. Also, Lipthay et al. (2004) reported higher species richness and Shannon index in herbicide-acclimated sediments. This effect can be explained by the direct and indirect effects of the applied compounds (MCPA and SA).

Formed metabolites and root exudates produced in response to stress conditions can also influence the structural diversity (Uhlik et al., 2013; Musilova et al., 2016). It is important to emphasize that interactions between the studied compartments and the application of both compounds, MCPA and SA, determined the diversity of bacteria.

After the addition of phenolic compounds (SA and MCPA), the bacterial communities in the unplanted soil, rhizospheric soil, and root endosphere were found to be dominated by Proteobacteria and differed only slightly from those of the untreated variant (Figures 2A, 3A, 4A). Also, the addition of DDE (0.0001 mg/L) to vermiculite led to the dominance of Proteobacteria in the roots endosphere of zucchini (Eevers et al., 2016). Similarly, Tejada et al. (2010) report that the use of MCPA (1.5 L/ha) did not appear to affect microbial structural diversity in soil. In contrast, it has been found that microbial communities isolated from the rhizosphere of soil contaminated with mecocrop (MCPP; 0.53 or 1.06 g/L) differed substantially from those isolated from unplanted soil (Lappin et al., 1985). The above-mentioned results show that in unplanted soil, rhizospheric soil, and roots endosphere, the amendment of soil with SA and MCPA not significantly affect the predominant taxa.

However, the application of MCPA and SA, either separately or together, considerably affected the composition of the endophytic bacterial community in leaves, although they demonstrated the lowest diversity indices (Figures 2–4). MCPA is absorbed through both leaves and roots and is translocated throughout the plant (via xylem and phloem) to the meristematic regions (Polit et al., 2014). The induced changes result in a series of biochemical and physiological processes which influence and disturb the morphology of plant roots, stems, and leaves (Grossmann, 2009). The observed changes in the composition of the plant endosphere microbiome can be due to the application of herbicides. Endophytes in plants can directly contribute to the detoxification of pollutants in plants or promote plant growth in stress conditions (Tétard-Jones and Edwards, 2016). However, the latter was not observed, since fresh weight was not higher after the simultaneous amendment of SA and MCPA. To the best of our knowledge, this is the first study showing that after the application of phenolic compounds (i.e., SA and MCPA) to soil, the most profound changes in structural diversity across the plant microbiome are observed in the leaves.

Further investigation of structural diversity showed that amendment of the soil with SA or MCPA led to the prevalence of the Actinobacteria (genus Pseudarthrobacter) in the leaves endosphere (Figures 2D, 3D). Similarly, Zhou et al. (2018) found that amendment of soil with the phenolic compound p-coumaric acid enhanced the relative abundances of Actinobacteria (i.e., Pseudarthrobacter) in the rhizosphere. Actinobacteria are described as common endophytic microorganisms, which can have multiple beneficial functions in plants, such as the production of antimicrobial agents (Musa et al., 2020) and biodegradation of petroleum and plastic compounds (Singh and Dubey, 2018). Combined MCPA and SA treatment, however, resulted in Firmicutes (genus Psychrobacillus) becoming the predominant taxon in leaves endosphere (Figure 4D). Regar et al. (2019) suggest that an increase in the abundance of Firmicutes can be a response to environmental stress such as herbicide contamination. Rangjaroen et al. (2019) mentioned that endophytic strains of Bacillus isolated from an herbicide-treated environment can enhance the resistance of plants to fungal pests. Our observations confirmed that the amendment of the soil with SA and MCPA favored the presence of Actinobacteria (genus Pseudarthrobacter) and Firmicutes (genus Psychrobacillus) in leaves.

The shifts in bacterial communities that occur after SA treatment could influence the degradative capacity against MCPA. Various genes belonging to the tfd cluster (responsible for initial steps of phenoxy herbicides biodegradation) have been identified in several taxa, e.g., Burkolderia, Pseudomonas, Achromobacter, Delftia, Bradyrhizobium, and Cupriavidus (Suwa et al., 1996; Kamagata et al., 1997; Baelum et al., 2010). Previous studies also demonstrated that SA doubled the presence of tfdA genes (Mierzejewska et al., 2019; Urbaniak et al., 2019a). In our study, the enrichment of soil with SA induced the occurrence of certain Proteobacteria genera such as Pseudomonas in leaves endosphere (Table 4), while the addition of MCPA + SA positively influenced the presence of Pseudomonas and Burkholderia in the rhizosphere (Table 4). Germaine et al. (2006) demonstrated that the endophytic strain Pseudomonas putida VM1450 enhanced the removal of 2,4-D from soil and demonstrated high biodegradative potential. In addition, the presence of Burkholderia in soils has been confirmed in our previous studies (Mierzejewska et al., 2019; Urbaniak et al., 2019a). Our findings indicate that combined MCPA + SA treatment induced the presence of Sphingomonas and Pandoraea in the roots endosphere (Table 4). Several Sphingomonas strains have been reported as being MCPA-degraders (Gözdereliler et al., 2013; Liu et al., 2013; Nielsen et al., 2013), and Pandoraea was found to substantially contribute to the degradation of lindane (HCH) in water and soil slurries (Okeke et al., 2002); however, to our knowledge, no studies have examined whether this genus was able to degrade phenoxy herbicides or related phenolic compounds. The above findings suggest that the amendment of the soil promoted the growth of specialized taxa; however, they were not the predominant taxa in the investigated compartments. Still, the functions of the identified strains need to be further confirmed by culturing techniques and molecular as well as metabolic analysis.

The above findings show that the addition of SA can enhance the removal of MCPA from unplanted soil. This removal is further enhanced by combining SA amendment with the cultivation of zucchini; although SA itself did not have a positive effect on the zucchini growth. The application of both phenolic compounds (i.e., MCPA and SA) led to changes in bacterial diversity: the highest bacterial diversity indices were observed for unplanted soil and rhizospheric soil, and the lowest in leaves. Despite the lowest diversity, the leaf endophytes were strongly affected by the addition of the studied phenolic compounds favoring the growth of the phyla Actinobacteria (especially Pseudarthrobacter spp.) and Firmicutes (especially Psychrobacillus). SA also promoted the growth of specific genera in the rhizosphere (Burkholderia, Pseudomonas, Sphingomonas) and in leaves endosphere (Pseudomonas), which were previously reported to harbor functional genes for MCPA biodegradation. Hence, it appears that SA not only influenced the structure of the bacterial communities in either MCPA-contaminated soil and zucchini, but also enhanced the bacterial degradation of the herbicide in the soil–plant system.

This is the first study to collectively examine different aspects of MCPA removal from soil: biostimulation (use of the PSM as an enhancer of MCPA removal) and phytoremediation (use of zucchini as a potential tool for removal of the herbicide) and to study the composition of soil and endophytic bacterial communities as an effect of addition of structurally related phenolic compounds (SA and MCPA). However, to disentangle the mechanisms behind the shifts in bacterial communities and their functions, additional research is needed.

The datasets presented in this study can be found in online repositories. The latest version of link to repository of our sequencing data is https://www.ncbi.nlm.nih.gov/bioproject/PRJNA809520.

EM, ST, MU, and JV designed the experimental setup and analysis and wrote the manuscript. EM and MU performed the experiments and prepared the samples. EM and ST prepared the samples for DNA extraction and sequencing and analyzed the results using bioinformatics tools. EM determined the fresh biomass of plants and analyzed the fresh biomass and MCPA removal data. KZ prepared the samples and measured the MCPA concentration in the soil. All authors agreed on the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.