Maize Root Exudates Promote Bacillus sp. Za Detoxification of Diphenyl Ether Herbicides by Enhancing Colonization and Biofilm Formation

Diphenyl ether herbicides are extensively employed in agriculture to control various broadleaf weed species in crops such as soybeans, rice, and peanuts (Liang et al. 2021). Furthermore, agricultural production involves crop rotation systems such as the soybean-maize rotation system to improve crop yield (Benitez et al. 2021). However, the increasing use of these herbicides has increased their residues in the soil. Diphenyl ether herbicide residues are phytotoxic in sensitive crops, such as maize and cucumbers, adversely affecting agricultural economies (Li et al. 2022). Additionally, diphenyl ether herbicides can disrupt the structure of the soil microbial community, significantly threatening plant health and environmental microorganisms (Zhao et al. 2022). Consequently, various remediation strategies are being developed to address the contamination of sites by diphenyl ether herbicides.

One avenue of addressing the contamination is using microbes (Zhang et al. 2022a). Microbes are essential for organic pollutant degradation due to their high diversity, activity, and abundance in soil (Jiang et al. 2021). However, various factors in natural agricultural environments constrain some isolated degradation strains, decreasing their degradation ability. Therefore, rhizoremediation is often applied to maintain microbial activity.

Rhizoremediation involves using microorganism-associated plants within the rhizosphere to treat environmental pollutants in situ (Li et al. 2019). Microbes directly metabolize organic pollutants during this process, while plants enhance their overall degradation efficiency (Jiang et al. 2022). The soil contaminated with diphenyl ether herbicides harbors numerous bacterial species, including Bacillus, Pseudomonas, Lysinibacillus, Chryseobacterium, and Edaphocola that possess the capability to degrade these compounds. The genus Bacillus, such as Bacillus sp. YS-1 (Shang et al. 2022), Bacillus sp. FE-1 (Cui et al. 2018), and strain Za, predominates other diphenyl ether herbicide-degrading bacteria (Zhang et al. 2018).

Bacillus contributes to biocontrol, biofertilization, and the induction of systemic resistance in plants (Jiang et al. 2018; Sun et al. 2022). Bacillus is prevalent in the rhizosphere and root endosphere, significantly promoting plant development (Naik et al. 2019). The efficacy of Bacillus relies on efficient colonization and biofilm formation, which protects its cells under adverse conditions (Yahav et al. 2018). Biofilm formation on roots enhances resource absorption and increases the production of exopolysaccharides and plant hormones, ultimately inducing systemic resistance (Pagnani et al. 2020).

The rhizosphere, a complex ecosystem surrounding the roots, contains root exudates and is rich in nutrients (Xie et al. 2012). Root exudates contain organic acids, carbohydrates, amino acids, and fatty acids that are in transit from the roots to the rhizosphere (Sun et al. 2010). Thus, they are essential carbon and energy sources for rhizospheric microorganisms, promoting microbial growth and activity (Liao et al. 2021; Preece and Peñuelas 2020). Root exudates provide nutritional resources and signaling molecules for soil microorganisms, influencing various rhizosphere processes (Chaparro et al. 2012). The organic acids within root exudates mediate the intricate relationship between plants and microbes (Zhang et al. 2014). These processes encompass the recruitment of plant-beneficial microorganisms and the regulation of biofilm formation on roots and also enhance the degradation efficiency of microorganisms (Zhou et al. 2023). However, further investigation is required to determine whether root exudates can reduce or eliminate the harmful effects of diphenyl ether herbicides on crops and to elucidate the interactions between degrading strains and plants in the rhizosphere.

This study focused on Bacillus sp. Za, a strain capable of degrading diphenyl ether herbicides (Zhang et al. 2018). The study investigated the interactions among diphenyl ether herbicide degradation, maize root exudates, and Bacillus sp. Za. Maize root exudates enhanced strain Za's capability to degrade diphenyl ether herbicides. This in turn mitigated the inhibition of organic acid in root exudates secretion in maize caused by diphenyl ether herbicide. During this process, root exudates effectively promoted the colonization and biofilm formation of strain Za, which was beneficial for degrading strains to function in the rhizosphere. A beneficial interaction was observed between strain Za and maize. These results provide valuable insights into the plant-microbe relationships and hold promise for advancing phytoremediation efforts.

Results

Root exudates enhanced the diphenyl ether herbicide degradation efficiency of strain Za

Strain Za effectively degrades various stable diphenyl ether herbicides in soil, including nitrofen, fluoroglycofen, lactofen, and bifenox and reduces the toxicity of these herbicides to maize (Zhang et al. 2018; Zhao et al. 2022). Maize has a rich root system and produces a significant amount of root exudates, with organic acids constituting an important component. The shake flask degradation experiment was conducted to evaluate the influence of additional root exudates and organic acids on the degradation ability of strain Za towards diphenyl ether herbicides. When root exudates were present, strain Za exhibited significantly enhanced degradation rates, degrading 85.2% of fluoroglycofen after 18 h (37.7% increase compared to the control) and 76.2% of lactofen after 12 h (67.5% increase compared to the control) (Fig. 1A and D). Fluoroglycofen and lactofen were completely degraded within 48 and 36 h, respectively. The residual levels of nitrofen and bifenox were reduced to less than 2% within 48 h, indicating a significant decrease in their concentrations. These results conclusively showed that root exudates enhanced the diphenyl ether herbicide degradation efficiency of strain Za.

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Maize root exudates had a spectrum of organic acids, mainly including oxalic, malic, succinic, fumaric, and trans-aconitic acids (Supplementary Fig. S1). We examined the impact of five organic acids on the degrading ability of strain Za (Supplementary Fig. S2). Organic acids enhanced the degradation ability of strain Za displayed towards all four diphenyl ether herbicides. For example, within 48 h, malic acid improved the degradation ability of strain Za towards lactofen, nitrofen, and bifenox. Fumaric acid exhibited superior enhancement in the degradation of strain Za compared to the other four organic acids. Specifically, strain Za degraded 89.5% nitrofen after 48 h and completely degraded nitrofen. These results demonstrated that all five organic acids improved the degradation efficiency of strain Za towards diphenyl ether herbicides.

Strain Za induced organic acid in root exudate re-secretion in herbicide-polluted maize

Considering the positive impact of organic acids in root exudates on the degradation efficiency of diphenyl ether herbicides by strain Za, we wondered how the organic acids of the root exudation were influenced by the diphenyl ether herbicides. After adding the herbicides, changes in organic acids secreted by maize roots were analyzed under hydroponic conditions. The reduction in oxalic, malic, succinic, fumaric, and trans-aconitic acid levels under 0.02 mg/liter of lactofen indicated that even low concentrations of diphenyl ether herbicides inhibited the secretion of root exudates in maize. At 0.10 mg/liter, only oxalic and fumaric acid were detectable. However, no organic acid was detectable at 0.5 mg/liter of lactofen. These results confirmed that the increased herbicide concentration reduced the variety and quantity of organic acids secreted by maize roots, including lactofen, nitrofen, bifenox, and fluoroglycofen (Fig. 2). Strain Za did not significantly change the types and quantities of organic acids in the absence of diphenyl ether herbicides. The strain exhibited restored secretion of organic acids across various levels (Fig. 2). At herbicides concentration of 0.02 mg/liter, strain Za increased the concentration of all five organic acids. Secretion of organic acids could be partially restored with the addition of strain Za when the herbicide concentration was increased to 0.5 mg/liter. For example, oxalic and fumaric acids, which were inhibited by lactofen, were detected again after inoculation of strain Za. Bifenox and fluoroglycofen inhibited trans-aconitic acid secretion, which was restored by Za. The recovery effect of strain Za on organic acid secretion was influenced by herbicide types and concentrations. These findings confirmed that strain Za has a positive effect on the secretion of organic acids inhibited by diphenyl ether herbicides, suggesting the establishment of a beneficial interaction between strain Za and maize roots.

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Organic acids in root exudates promoted colonization and biofilm formation of strain Za

The colonization strength of functional bacterial strains in maize rhizospheres is a crucial factor influencing their expected functions (Kakembo and Lee 2019). Root colonization analysis showed that Bacillus sp. Za-gfp effectively colonized and formed biofilms in maize roots in hydroponic and soil cultures (Supplementary Fig. S3A). The strain Za-gfp migrated from the rhizosphere soil to the roots, colonizing the surface of maize roots for over 21 days (Supplementary Fig. S3B and C). Root exudates and the five kinds of organic acids were used to test whether they affect colonization of strain Za on roots under hydroponic conditions. The results showed that root exudates and fumaric and succinic acids significantly increased strain Za colonization on the maize root surface to 6.80, 6.02, and 5.99 log colony forming units (CFU)/g of roots, respectively, compared to the control (Fig. 3A and B). These results confirmed that organic acids in root exudates significantly promoted the colonization of strain Za in maize roots.

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Mature biofilm formation typically indicates successful colonization of the rhizosphere (Li et al. 2013). To investigate the impact of root exudates on strain Za's biofilm formation, a 48-well plate was utilized to simulate biofilm formation, and subsequent dynamic changes in biofilm formation were observed. The biofilm formed within 24 h and began to disperse after 60 h. Consequently, the time points of 24 and 60 h were critical for the establishment of biofilm formation in strain Za (Fig. 3C). The effects of root exudates and organic acids during the initial stages of biofilm formation were investigated. At 24 h, under the treatment of root exudates and fumaric acid, the biofilm biomass of strain Za increased by 38.5 and 27.7%, respectively, compared to the control. After 24 h, the biofilm biomass of strain Za exhibited a significant increase of 38.5 and 27.7% when treated with root exudates and fumaric acid, respectively (Fig. 3D). These results indicated the facilitative role of root exudates and fumaric acid in the process of biofilm formation.

Root exudates enhanced the growth and chemotaxis of strain Za

The mechanism of root exudates promoting biofilm formation of strain Za was investigated through RNA sequencing (RNA-seq). At 24 and 60 h, 532 and 2,206 differentially expressed genes (DEGs) were identified (log₂ fold change > 1, P value < 0.05), respectively (Supplementary Fig. S4A and B). Hierarchical clustering groups the DEGs by their expression patterns (Supplementary Fig. S4C). At 24 h, the significant DEGs were enriched in biosynthesis and metabolism of amino acid, organic acid, and carboxylic acid, indicating that root exudates might facilitate biofilm formation by promoting the increase biomass of Za (Fig. 4A; Supplementary Fig. S4D and E). Induction of root exudates for 60 h significantly induced the expression of genes linked to ribonucleoprotein complex synthesis, carbohydrate binding, nucleotide binding, and catalytic activity in the cells, promoting biofilm formation. Chemotaxis (mcpC, fliG, and cheA) and flagellum synthesis (fliZ, fliD, fliP, fliH, and fliF) genes were repressed, indicating the completion of bacterial cell aggregation and limiting movement aids in preserving the mature aggregation phase. These results indicated that maize root exudates stimulated the expression of metabolism-related genes important for growth and reproduction and regulated motility-related genes, including motility- and chemotaxis-related genes.

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Bacterial growth is necessary for biofilm formation (Liu et al. 2024). During the adhesion stage of biofilm formation, chemotaxis controls the direction of flagellum rotation, and bacterial movement cessation is often coupled with biofilm maturation (Guo et al. 2020). As many DEGs were associated with growth metabolism, chemotaxis, and motility in response to root exudates, the impact of root exudates on strain growth and chemotaxis was investigated. Root exudates were added to mineral salt medium (MSM) to investigate their potential in promoting the growth of strain Za. When cultured for 48 h, the biomass of strain Za with root exudates was three times higher than that of the control (Fig. 4B). Chemotaxis experiments showed that fumaric acid, succinic acid, and oxalic acid elicited stronger chemotaxis in strain Za than other organic acids, especially at 25 to 50 μM (Fig. 4C), displaying a concentration-dependent chemotactic behavior. Additionally, strain Za demonstrated a higher chemotactic response towards maize root exudates (Fig. 4D). These results suggested that root exudates promoted the growth of strain Za, which demonstrated chemotaxis to root exudates.

The strain Za biofilm reduced the herbicide phytotoxicity

Given the observed stimulation of biofilm formation in strain Za by root exudates, we sought to investigate whether this phenomenon contributes to the mitigation of herbicide phytotoxicity. The gene fliZ plays a pivotal role in the biofilm formation process of strain Za. To this end, a biofilm deficient mutant, ZaΔfliZ, was deployed. Biofilm formation and colonization of maize roots were reduced in the ZaΔfliZ mutant, while its growth remained normal (Fig. 5A and B; Supplementary Fig. S5). The ability of wild-type (WT) Za and its mutant counterpart to mitigate herbicidal damage on maize was investigated in the presence of diphenyl ether herbicides. Diphenyl ether herbicides (0.5 mg/liter) significantly hampered maize growth, highlighting its susceptibility to the adverse effects of these compounds (Fig. 5). In contrast, strain Za and the mutant ZaΔfliZ relieved the growth inhibition of diphenyl ether herbicides in maize. The mutant exhibited reduced efficacy in remediating the damage caused by herbicides compared to strain Za. Specifically, lactofen-treated mutant ZaΔfliZ significantly differed from WT Za in maize seedling length (Fig. 5A). When maize was treated with herbicides nitrofen, bifenox, and fluoroglycofen, ZaΔfliZ also displayed notable variations in maize seedling length (Fig. 5). These findings indicated that ZaΔfliZ exhibited reduced efficacy in mitigating the damage caused by diphenyl ether herbicides compared to WT Za, which formed biofilms and degraded these herbicides.

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Based on the above results, we proposed a pattern to explain the degradation process of diphenyl ether herbicides in maize roots by strain Za (Fig. 6). The degradation ability of strain Za towards diphenyl ether herbicides was enhanced by root exudates, which induced the re-secretion of organic acids in herbicide-polluted maize plants. During this process, strain Za exhibited positive chemotaxis towards root exudates and successfully formed biofilm, colonizing maize roots. Consequently, the presence of root exudates significantly augmented the degradation capacity of strain Za while also attracting additional strains for colonization, thereby establishing a mutually beneficial partnership with maize. This information improved the theoretical foundation for comprehending the interactions between functional microorganisms and plants.

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Discussion

Diphenyl ether herbicide residues pose a threat to subsequent crop growth and cause economic losses in agriculture. Current research on the degradation of diphenyl ether herbicides primarily focuses on isolating degrading strains. However, the relationship between these strains and crops during the degradation process remains unclear. Strain Za degraded diphenyl ether herbicides in the rhizosphere, reducing their phytotoxicity on maize and restoring the secretion of root exudates. This recovery enhanced the degradation function of strain Za by promoting its colonization and biofilm formation, forming a beneficial positive feedback effect. The mutual interaction between strain Za and plants created a beneficial root environment, enhancing plant growth.

The reciprocal interaction between plants and their microbiota could constitute a mechanism that facilitates their adaptation to pesticide-induced stress. Root exudates serve as a mechanism for plants to “cry for help” under pesticide stress (Feng et al. 2021). The release of root exudates enhances the bacterial-mediated degradation of chemical pollutants, thereby augmenting their remediation potential (Chen and Liu 2024). For instance, maize root exudates were found to enhance the degradation of hexachlorocyclohexane isomers by Sphingobium sp. D4 and the degradation of bensulfuron-methyl by strain S113 (Alvarez et al. 2022; Zhang et al. 2022b). The secretion of root exudates by plants facilitated the microbial degradation process, thereby indirectly facilitating the elimination of sulfonamides (Zhou et al. 2024). In this study, the degradation efficiency of strain Za of diphenyl ether herbicides was improved with the participation of root exudates and organic acids. As a critical intermediate within the tricarboxylic acid cycle, fumaric acid promotes pollutant degradation and enhances the degradation efficiency of the rhizosphere community to phenanthrene (Jiang et al. 2021). Fumaric acid enhanced the degradation efficiency of strain Za against resistant diphenyl ether herbicides. Furthermore, the responses of plants to various stressors significantly modified root exudation patterns, thereby exerting discernible impacts on the interactions between root exudates and rhizobacteria (Chai and Schachtman 2022). Chlorpyrifos induced the release of linolenic acid in root exudates, thereby recruiting Sphingomonas to degrade residual pesticides and mitigating pesticide-induced stress (Li et al. 2023). Diphenyl ether herbicides inhibited the secretion of organic acids in root exudates, but the strain Za degraded diphenyl ether herbicides in the rhizosphere, thereby alleviating this inhibitory effect. Differences in the secretion of organic acids indicated that the characteristics of root exudates might be influenced by the specific pesticide variety employed. These results suggest that the mutual interaction between strain Za and plants creates a beneficial root environment, mitigating the damage caused by herbicides to plants.

Typically, the first step in rhizosphere bacterial colonization involves chemotaxis to the rhizosphere and subsequent adhesion to roots, followed by biofilm formation (Liu et al. 2024). Organic acids in root exudates act as distinctive signal molecules capable of initiating microbial colonization in the maize rhizosphere (Bukhat et al. 2020). Strain Za exhibited chemotaxis towards fumaric acid, which significantly enhanced its colonization levels and biofilm formation. Presumably, fumaric acid functions as a nutrient and signaling molecule for soil microorganisms (Korenblum et al. 2022). Meanwhile, succinic acid only promoted strain Za colonization of the roots. The citric acid in cucumber exudates attracted strain SQR9 isolated from cucumber and induced biofilm formation, but it only induced chemotaxis in strain N11 from banana, without biofilm formation (Zhang et al. 2014). These findings suggest a potential mutual selection between the strain and the host. Root exudates and organic acids chemotactically attracted and induced bacterial responses, promoting strain Za colonization and biofilm formation, which played a crucial role in establishing a mutualistic relationship between bacteria and host plants (Huang et al. 2022; Liu et al. 2024). Biofilms improved the degradation efficiency of strain Za, which was consistent with the biofilm formed by strain RS1-gfp, which enhanced the degradation rate of phenanthrene (Chen et al. 2019). Additionally, the biofilm formation of strain Za was related to its growth and chemotaxis under the action of root exudates (Fig. 4). Root exudates represent the primary nutrient source in the rhizosphere, providing a niche for the growth of rhizospheric bacterial (Zhalnina et al. 2018). Therefore, the improved efficiency of diphenyl ether herbicide degradation by root exudates may be related to its promotion of biofilm formation. These results demonstrated the significance of root exudates in the colonization process of strain Za, implying that the enrichment of beneficial bacteria in the rhizosphere can be modulated by regulating root exudation.

In this study, strain Za and crops established beneficial interactions based on their efficient degradation ability and secretion of root exudates, respectively. The colonization and biofilm formation of strain Za in the root system was key to this effect.

Materials and Methods

Experimental materials

Lactofen, bifenox, fluoroglycofen, and organic acids were purchased from J&K Scientific Co., Ltd. (Beijing, China). Nitrofen was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All other organic reagents are chromatographic grade.

The strain Za, maintained in our laboratory, was cultured in Luria-Bertani (LB) medium (5.0 g/liter of yeast extract, 10.0 g/liter of tryptone, and 10.0 g/liter of NaCl) at 37°C, with 180 rpm. The strain Za-gfp constructed in our laboratory was cultured in an LB medium containing zeocin (Zhao et al. 2022). Next, the cultured strains Za and Za-gfp were washed with sterile water and resuspended in sterile water. The chemotaxis buffer consisted of 0.20 g/liter of KCl, 1.44 g/liter of Na₂HPO₄, 8 g/liter of NaCl, 0.24 g/liter of KH₂PO₄, 0.029 g/liter of EDTA, 1.00 ml/liter of glycerol, and 0.441 ml/liter of sodium lactate. Hoagland medium used for maize cultivation contained 0.945 g/liter of Ca(NO₃)₂·4H₂O, 0.506 g/liter of KNO₃, 0.08 g/liter of NH₄NO₃, 0.136 g/liter of KH₂PO₄, 0.493 g/liter of MgSO₄, and 2.5 ml of mixed solution (5.56 g/liter of FeSO₄·7H₂O and 7.46 g/liter of EDTA-Na₂, pH 5.5). The mineral salt medium, MSgg medium, and phosphate-buffered saline (PBS) formulations followed the Zhang et al. (2022b) protocol.

Maize seeds were placed on gauze and soaked overnight. The seeds were sterilized by shaking with 3% H₂O₂ and spread on sterile gauze with double-distilled water (ddH₂O) in Petri dishes at 25°C for 2 days. The germinating seeds were selected and planted in Hoagland medium at 25°C with 16 h of light and 22°C with 8 h of darkness in a plant growth chamber. Maize seedlings exhibiting consistent growth characteristics were chosen for subsequent experimental analyses.

Collection of root exudates and identification of organic acids

Root exudates were collected following the previous method (Zhang et al. 2015). To collect root exudates, maize plants were removed from the hydroponic Hoagland medium after 3 days of cultivation and washed with water. The maize seedling was transferred into a centrifuge tube filled with 50 ml of sterile water and was incubated for 24 h in a plant growth chamber. Root exudates from 40 maize seedlings were collected and centrifuged at 12,000 rpm for 20 min to obtain 20 ml of concentrated 100× root exudates.

The compositions of organic acids in root exudates were identified using CNWBOND SCX and CNWBOND WAX SPE Cartridge (Anpel, China). The purified samples were filtered through a 0.22-μm membrane and analyzed by high-performance liquid chromatography (HPLC) using a Phecda C18 reversed-phase column (4.6 × 250 mm, 5 μm) (Thermo Fisher Scientific, MA, U.S.A.). The mobile phase was 10 mM K₂HPO4 (pH 2.9) at a flow rate of 1 ml/min, and the column temperature was kept at 35°C. The compounds were detected at a wavelength of 210 nm, with an injection volume of 10 μl. The type and content of the organic acids were determined by comparing the sample with organic acid standards. There were three replicates for each identification.

Degradation experiment

Strain Za was cultured in MSM medium with 1× root exudates and 50 mg/liter of diphenyl ether herbicides (lactofen, nitrofen, bifenox, and fluoroglycofen) at 37°C and 180 rpm to determine the impacts of maize on the degradation capability of strain Za for diphenyl ether herbicides. The cultures were incubated for 60 h, with herbicide concentration measurements taken every 6 h. The effect of organic acids, such as oxalic (50 μM), malic (25 μM), fumaric (25 μM), succinic (50 μM), and trans-aconitic (100 μM) acids on Za was investigated. After dichloromethane extraction, the samples were centrifuged at 12,000 rpm to separate the organic phase. Subsequently, the organic phase was evaporated, and the resulting residues were reconstituted in methanol before being filtered through a 0.22-μm membrane for subsequent HPLC analysis. The HPLC analytical column was the Acclaim C18 reversed-phase column (4.6 × 250 mm, 5 μm). The mobile phase consisted of methanol, water, and acetic acid (80:19:1) at a flow rate of 1 ml/min, and the column temperature was kept at 40°C. The compounds were detected at two different wavelengths (230 and 280 nm), with an injection volume of 20 μl. Each degradation treatment was replicated three times.

Restoration of organic acids

The types and concentrations of organic acids were investigated to evaluate the restorative effect of strain Za on the secretion of organic acids under diphenyl ether herbicide damage. Maize seedlings were planted in Hoagland solution containing lactofen, nitrofen, bifenox, and fluoroglycofen (0, 0.02, 0.10, and 0.50 mg/liter, respectively). Each pot contained six maize plants. Simultaneously, strain Za (10⁷ CFU/ml) was added to the herbicide treatments. After 7 days, the root exudates were collected, and the types and concentrations of organic acids were measured accordingly. Each treatment contained three replicates.

Strain Za-gfp visualization and enumeration on maize roots

The selected, physically uniform, and normally growing seedlings were inoculated with strain Za-gfp and observed in hydroponic and soil cultures after 3 days. The yellow brown soil (Luvisols) was collected from the Garden of Nanjing Agricultural University and passed through a 2-mm sieve. The root surface was washed off with ddH₂O to remove soil, and root segments were taken at 0.5-cm intervals from the root tip and fixed with 2.5% glutaraldehyde. The fixed roots were examined using a confocal laser scanning microscope (Leica TCS SP3). Bacterial DNA was extracted from soils and roots using the FastDNA SPIN Kit for Soil (MP Biomedicals, CA, U.S.A.) and Plant DNA Isolation Kit (FORE GENE, China), respectively. The number of Za-gfp was detected by the gfp-F/R qPCR primers (Supplementary Table S1). Each treatment was determined with three replicates.

Colonization measurements

To investigate the influence of root exudates and organic acids on the root surface colonization of strain Za, the maize roots were fully immersed in 1× root exudates, 50 µM oxalic acid, 25 µM malic acid, 50 µM succinic acid, 25 µM fumaric acid, and 100 µM trans-aconitic acid, respectively. After 30 min, the treated roots were placed in Hoagland solution with 10⁸ CFU/ml strain Za-gfp. After cultivation for 3 days, the quantity of strain Za-gfp colonizing maize roots was quantified by qPCR. Each set of data had three replicates.

Biofilm formation measurements

The biofilm formation of strain Za was measured in a 48-well plate. Each well of the plate was inoculated with 200 μl of strain Za and 800 μl of MSgg film-forming medium. The final concentration of strain Za was 10⁸ CFU/ml. Biofilms were determined as described in previous studies (Mosharaf et al. 2018). The liquid below the biofilm was removed, and the biofilm was rinsed twice with ddH₂O. Subsequently, the biofilm was resuspended in 1 ml of PBS buffer, the suspension was transferred to a 2-ml centrifuge tube containing stainless steel beads, and the biofilm was homogenized using a Bioprep-24 Homogenizer. The absorbance at 600 nm was measured to determine the biofilm biomass. An MSgg film-forming medium containing Za-gfp and 10 μl of 1× root exudates or various organic acid solutions (10, 25, 50, and 100 μM) was analyzed to study the influence of root exudates and organic acids on biofilm formation by strain Za. Samples were taken every 12 h to measure the biofilm quantitatively, and each treatment included three replicates.

Strain Za RNA-seq

The RNA-seq was employed to analyze strain Za under the 1× root exudate treatment and unraveled how root exudates regulate strain Za behavior in the rhizosphere, especially on biofilm promotion mechanisms. After 24 and 60 h, the biofilm of strain Za with or without root exudates was collected and stored at −80°C until RNA extraction. The cDNA synthesis, cDNA library construction, and DNA sequencing were executed by Novogene Co., Ltd. (Beijing, China). Some significant DEGs for qPCR validation were selected to validate the RNA-seq results. The results showed that qPCR exhibited similar expression trends as the original RNA-seq results (Supplementary Table S2). Primers are listed in Supplementary Table S1.

Growth and chemotaxis measurements

A concentrated suspension of strain Za and 2 ml of maize root exudates were added to the MSM medium to make 0.1 OD₆₀₀, the final concentration of strain Za, to evaluate the impact of strain Za on growth at 37°C and 180 rpm for 48 h. The OD₆₀₀ was measured every 6 h with three replicates.

Next, 200 μl Za-gfp was absorbed with a pipette tip, and 100 μl of 1× root exudates or organic acid solutions with concentrations of 10, 25, 50, and 100 µM were absorbed with a needle to investigate the chemotaxis of root exudates and organic acids to strain Za. The pipette and the needle tips were connected and incubated overnight. Afterwards, the samples were diluted and plated onto LB plates. The quantification of CFU was performed after 12 h of incubation, and each treatment included three replicates.

Construction of the mutant

The ZaΔfliZ mutant strain was constructed to examine the influence of biofilm formation on diphenyl ether herbicide detoxification and how biofilm form is inhibited. The gene fliZ is associated with biofilm formation. Primers fliZ-1F/R and fliZ-3F/R were used to amplify the homology arms of fliZ-1 and fliZ-3 (approximately 1,000 bp upstream and downstream of the fliZ gene, respectively), using Za genomic DNA as the template. Furthermore, gene Zeo was amplified with fliZ-2F/R primers using Za-gfp genomic DNA as the template. An overlap PCR was then performed to fuse these three fragments, and the resultant product was sequenced and introduced directly into strain Za. Transformants were cultured selectively on LB plates containing Zeocin, successfully creating the ZaΔfliZ mutant.

Detoxification assay

Evaluations were performed to determine the effects of strain Za and the ZaΔfliZ mutant biofilm formation on the degradation of diphenyl ether herbicides and their potential to mitigate the damage to maize growth in a Hoagland solution. Strains Za and the ZaΔfliZ mutant were added to 230 ml of Hoagland solution with 0.5 mg/liter of diphenyl ether herbicides (lactofen, nitrofen, bifenox, and fluoroglycofen, respectively). After 7 days, the length of the seedlings was measured as indicators. Each treatment included three replicates.

Statistical analysis

All experiments were conducted at least three times independently. The data were analyzed for one-way analysis of variance (ANOVA) using IBM SPSS Statistics 20. Significant differences were determined with Duncan's test (P < 0.05). Data are presented as mean ± standard deviation (SD).