Study of the biochemical activity and plant growth promoting bacteria in soils polluted with oxyfluorfen

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=oafa20 Cogent Food & Agriculture ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/oafa20 Study of the biochemical activity and plant growth promoting bacteria in soils polluted with oxyfluorfen Salvadora Navarro-Torre, María Aguilera-Salas, Paloma Ávila-Pozo, Juan Parrado & Manuel Tejada To cite this article: Salvadora Navarro-Torre, María Aguilera-Salas, Paloma Ávila-Pozo, Juan Parrado & Manuel Tejada (2023) Study of the biochemical activity and plant growth promoting bacteria in soils polluted with oxyfluorfen, Cogent Food & Agriculture, 9:1, 2247171, DOI: 10.1080/23311932.2023.2247171 To link to this article: https://doi.org/10.1080/23311932.2023.2247171 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 13 Aug 2023. Submit your article to this journal Article views: 362 View related articles View Crossmark data Citing articles: 1 View citing articles SOIL & CROP SCIENCES | RESEARCH ARTICLE Study of the biochemical activity and plant growth promoting bacteria in soils polluted with oxyfluorfen Salvadora Navarro-Torre 1 , María Aguilera-Salas 1,2 , Paloma Ávila-Pozo 2 , Juan Parrado 3 and Manuel Tejada 2 * Abstract: There is currently a great deal of information available about the toxic effects of oxyfluorfen on soil biochemical activity and microbial biodiversity. However, there is no information about how this herbicide affects plant growth promoting bacteria (PGPB) or their properties, such as biofilm formation, nitrogen fixation, siderophore and auxin production, and phosphate solubilisation. As such in this study an agricultural soil was polluted with the oxyfluorfen herbicide every 30 days at a dose of 4 L ha −1 for a total period of 90 days. During this experimental period, the dehydrogenase activity was determined, a count and isolation of cultivable bacteria was performed, and the PGPB and their properties were characterized. The results indicated that oxyfluorfen inhibits the dehydrogenase activity, with this inhibition increasing with herbicide concentration in the soil (69.9% compared to non-polluted soil). It also causes changes in the population diversity of cultivable bacteria in soils. Regard to the evolution of isolated PGPB, it was found that oxyfluorfen induces the growth of nitrogen-fixing, biofilm-forming, and siderophoreproducing bacteria, while negatively affecting the growth of auxin-producing and phosphate-solubilising bacteria. These results suggest that oxyfluorfen modulates the properties of PGPB in a concentration-dependent manner. Subjects: Agriculture & Environmental Sciences; Soil Sciences; Environmental Studies Keywords: oxyfluorfen; dehydrogenase activity; plant growth-promoting (PGP) properties 1. Introduction Pesticides currently play an important role in agriculture as they enhance crop development and productivity by minimizing the economic losses caused by weeds, insects and pathogens (Tejada et al., 2016, 2017). However, excessive pesticide use is affecting the environment negatively since it is deteriorating soil quality (biochemical activity and microbial biodiversity) and increasing the contamination of surface and groundwater as a result of diffuse pollution (e.g., run-off and leaching) (Khalid et al., 2020; Ogura et al., 2021; Yu et al., 2019). Oxyfluorfen is a herbicide used worldwide (Sheng et al., 2020) that inhibits protoporphyrinogen oxidase (PPO), thereby increasing the levels of protoporphyrin IX in the plant cells (Wang et al., 2016). High amounts of this compound cause peroxidation of the fatty acids in the cell membrane, Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Received: 09 June 2023 Accepted: 08 August 2023 *Corresponding author: Manuel Tejada, Grupo de Investigación Edafología Ambiental, Departamento de Cristalografía, Mineralogía y Química Agrícola, E.T.S.I.A, Universidad de Sevilla, Crta. De Utrera Km. 1, Sevilla 41013, Spain E-mail: [email protected] Reviewing editor: Pedro González-Redondo, University of Seville, Spain Additional information is available at the end of the article Page 1 of 17 thereby weakening it and resulting in a loss of pigmentation in the leaves, necrosis, and death of the plant (Liu et al., 2016). This herbicide has a degradation rate of 35–138 days (depending on the environmental conditions), thus meaning that it persists in soils (Lewis et al., 2016). Indeed, several authors have described the pollution of surface and ground water with this herbicide as a consequence of run-off, and even its accumulation in crops (Sondhia, 2010; Tudi et al., 2021; Zhang et al., 2018). In addition, the Environmental Protection Agency (EPA) has classified oxyfluorfen as carcinogenic for humans (EPA, 2002). In Spain, oxyfluorfen is currently applied in quantities of up to 150 g·ha −1 ·year −1 and its approval has been renewed until 2024, although it is a candidate for substitution by another less toxic herbicide (MAPA, 2022). In addition to crop and human damage, oxyfluorfen also affects soil microorganisms, altering their activities and their biodiversity (Gómez et al., 2014; Rodríguez-Morgado et al., 2014; Tejada et al., 2022). These microorganisms are essential for good crop development given their multiple interactions with plants, especially those that promote plant growth, which are known as PGPB (Plant Growth Promoting Bacteria) (Chamkhi et al., 2022). Such bacteria exhibit numerous properties involved in the enhancement of plant growth, such as their ability to facilitate the absorption of nutrients, improve plant development, and protect plants against different biotic and abiotic stressors, while also acting as natural fertilizers (Goswami et al., 2016; Navarro-Torre et al., 2020; Olanrewaju et al., 2017). These plant growth-promoting (PGP) properties include phosphate solubilisation, a property. Through this property, bacteria can solubilise phosphates by producing organic acids, hydroxyl ions, protons, and carbon dioxide, thus making phosphorous more readily available for plants (Alori et al., 2017; Goswami et al., 2015). Nitrogen fixation is another key PGP property involved in made more available the nitrogen uptake by plants. Nitrogen-fixing bacteria can fix nitrogen from the atmosphere (not assimilated by plants) and turn it into ammonia, which is assimilated by plants, thanks to the enzyme nitrogenase (Backer et al., 2018; Goswami et al., 2015). To make iron available for plants, some PGPB produce siderophores, small molecular weight peptides that are able to chelate Fe 3+ ions with high affinity and form a siderophore-Fe +3 complex that is easily assimilated by plants via specific receptors (Alori & Babalola, 2018). In addition, this property also acts as a biocontrol by preventing phytopathogen growth (Shen et al., 2013). The PGPB can also synthesize and produce phytohormones such as auxins, gibberellins, cytokinins, ethylene, and abscisic acid, with the former, in particular indole-3-acetic acid (IAA), being the most abundant phytohormone produced by bacteria (Goswami et al., 2015; Navarro-Torre et al., 2020; Olanrewaju et al., 2017). IAA is involved in root elongation, thus meaning that IAA producing bacteria can improve the root architecture in the way that plants have more accessibility to water and nutrients (Alemneh et al., 2020; Turan et al., 2016). Finally, biofilm formation is another important trait present in some PGPB. During this process, the bacteria form a biofilm around that may help to protect them against biotic and abiotic stress and favour nutrient uptake (Rossi, 2020). In light of the above, it can be concluded that a decrease or disappearance of this bacterial function from soils due to oxyfluorfen contamination is likely to result in a marked loss of soil quality, thus preventing it from being used for agricultural purposes (Filimon et al., 2021). We hypothesized that oxyfluorfen herbicide negatively affects the biochemical activity of the soil as well as PGPB bacteria. However, we do not know how it can affect the various PGPB properties. For this reason, the main goal of this study was to observe the evolution of soil biochemical activity and the changes in the PGPB population in an agricultural soil affected by oxyfluorfen. To that end, the microbial activity of soil treated with different doses of oxyfluorfen was determined and then the cultivable bacteria were isolated, estimating the different species and analysing their PGP properties at the different doses. To determine the microbial activity of the soil, the dehydrogenase enzymatic activity was used. This enzyme is related to oxidative phosphorylation, making it a good indicator of microbial activity (Tejada et al., 2023). Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 2 of 17 2. Materials and methods 2.1. Design of the experimental assay An Arenic Calcaric Regosol (WRB, 2014) agricultural soil from southwestern Spain was employed in this study. This soil contains 616 ± 62 g kg −1 sand, 123 ± 29 g kg −1 silt, and 260 ± 35 g kg −1 clay. The soil pH was 7.9 ± 0.2, and it contained 17.2 ± 1.8 g kg −1 organic matter, and 0.93 ± 0.07 g kg −1 N. The methodology used to determine these physico-chemical parameters is described in Rodríguez-Morgado et al. (2014). A 500-g sample of dried and sieved soil (<2 mm) was preincubated in 1-L glass bottles for 7 days at 30–40% of the maximum water-holding capacity, as per the methodology described by Tejada (2009). Subsequently, the soil was mixed with 4 L·ha −1 oxyfluorfen (commercial formulation Fenfen; Lainco, S.A., Spain) in the half of the bottles. Distilled water was then added to each glass bottle until 60% of the maximum water-holding capacity. In detail, the treatments carried out were: (1) S, control soil non-polluted with oxyfluorfen (2) S + Ox, soil polluted with oxyfluorfen In the S+Ox treatment, the addition of oxyfluorfen was repeated after 30 and 60 days from the first addition. Glass bottles containing polluted and non-polluted soil (n = 3) were deposited randomly and placed in an incubation chamber at 25 ± 1 °C. During the experiment (90 days), distilled water was added to maintain the soil at 60% of its maximum water-holding capacity. Samples were taken from each bottle at 30, 60, and 90 days from the start of the experiment and deposited in labelled bottles for the subsequent analysis (determination of oxyfluorfen concentration, dehydrogenase activity, and bacterial isolation). Soil samples were stored at −20º C until the analysis. 2.2. Soil analysis 2.2.1. Soil oxyfluorfen The soil oxyfluorfen concentration was determined by chromatography following the procedure described in Gómez et al. (2014), with previous extraction of the herbicide as per the protocol described in Anastassiades et al. (2003). 2.2.2. Soil dehydrogenase activity Soil dehydrogenase activity was determined by the reduction of 2-p-iodo-3-nitrophenyl-5-phenyl- tetrazolium chloride to iodonitrophenyl formazan according to the method of Trevors et al. (1982) as modified by García et al. (1993). 2.2.3. Isolation of cultivable bacteria Cultivable bacteria were isolated from the different samples collected at different times following the protocol described in Navarro-Torre et al. (2016). Briefly, soil was resuspended in sterile 0.9% saline solution for 10 min by shaking. Then, 10 −1 , 10 −2 , and 10 −3 dilutions were plated on TSA (Tryptic Soy Agar; Scharlau, Spain) plates in duplicate and incubated at 28 °C for 48 h. To determine the total bacterial population in each soil sample, colony forming units (CFU) were counted for the 10 −3 dilution. After that, different colonies from all dilutions were separated onto different TSA plates based on their colour, size, and morphology. Pure cultures were stored at −80°C in 15% glycerol. Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 3 of 17 2.2.4. Analysis of bacterial population by Box-PCR Bacterial population from the different levels of contamination was analysed by Box-PCR. Thus, DNA extraction was performed using the EZNA® Bacterial DNA kit (Omega Bio-Tek, United States) following the instructions supplied by the manufacturer. Box-PCR was performed using the Maxime™ PCR PreMix kit (i-Taq™) (Intron Biotechnology, Korea) and BOX A1R primer (Flores- Duarte et al., 2022). PCR conditions were the following: initial denaturation at 94 °C for 2 min, 30 cycles of denaturation at 94 °C for 20 s, annealing at 52 °C for 20 s, extension at 72 °C for 1 min, and final extension at 72 °C for 5 min. Electrophoresis was performed in a 1.5% (w/v) agarose gel and a voltage of 70 V for 2 h. After the electrophoresis, the gel was photographed and analysed using the PyElph software package (v. 1.4) (Pavel & Vasile, 2012). Finally, the similarities were presented as dendrograms using an unweighted pair-group method with arithmetic mean (UPGMA), as suggested by Pavel and Vasile (2012). 2.2.5. Determination of plant growth promoting properties in vitro Phosphate solubilisation, siderophore and indole-3-acetic acid (IAA) production, nitrogen fixation, and biofilm formation were the PGPB properties assayed in this work. Phosphate solubilisation was determined by growing the isolates in NBRIP (National Botanical Research Institute’s phosphate growth medium) plates at 28 °C for 5 days (Nautiyal, 1999). A transparent halo around the bacterial growth indicated that the bacteria solubilise phosphate. In positive samples, the halo was measured to semi-quantify the solubilisation. For siderophore production, bacteria were incubated in CAS (chrome azurol S) plates at 28°C for 5 days in the dark (Schwyn & Neilands, 1987). In this case, an orange halo confirmed that the bacteria produced siderophores, and this halo was measured to semi-quantify the production. The IAA production by the isolates was assayed using a colorimetric method. Thus, bacteria were incubated in TSB (Tryptic Soy Broth; Scharlau, Spain) supplemented with L-tryptophan (100 mg·L −1 ) at 28 °C for 3 days. After incubation, the culture was centrifuged and the supernatant mixed with Salkowski reagent (Gordon & Weber, 1951) for 20 minutes and IAA-producing bacteria showed a pink coloration. To quantify the IAA production by each strain, the absorbance was measured using a spectrophotometer (GeneQuant® 1300; Biochrom Ltd., United Kingdom) at 535 nm and the IAA quantification was performed using a pattern curve. IAA pattern curve was performed using 1 mL of different known IAA concentrations mixed with 4 mL Salkowski reagent. The absorbance was recorded at 535 nm. Different known IAA concentrations were prepared from an IAA stock solution (200 mg·L −1 ). Nitrogen fixation was tested on NFA (nitrogen-free agar) plates at 28 °C for 5 days (Ji et al., 2014). Bacterial growth after incubation indicated that the bacteria could fix nitrogen from the atmosphere. Finally, biofilm formation was studied in the isolates in a similar manner to Del Castillo et al. (2012). Briefly, biofilm formation was determined in a six-well plate using TSB as culture medium, incubating at 28 °C for 4 days. After that time, the position of the biofilm in the well was determined (surface or bottom), then wells were washed with distilled water and stained with 0.01% violet crystal for 20 minutes to detect the bacterial biomass adhered to the wall of the wells (ring formation). After the determination of these PGP properties, the quantification of the number of bacteria showing each property was analysed in order to observe changes in the PGP properties caused by oxyfluorfen. 2.2.6. Statistical analyses Statistical analyses were performed using Statistica software version 6.0 (StatSoft Inc.). The normality of the results was determined by applying the Kolmogorov—Smirnov test. Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 4 of 17 Dehydrogenase activity, number of colonies, and number of different isolated strains were compared using a one-way ANOVA to determine de effect of oxyfluorfen and the interaction. On the other hand, the evolution of the different PGP properties, the IAA and siderophores production, and the phosphate solubilisation were analysed using GLM. In both cases, Fisher test (LSD) was carried out as post hoc test. 3. Results 3.1. Effect of different oxyfluorfen concentrations on the microbial population in affected soil The oxyfluorfen concentration increased in each taken sample due to repeated application of the herbicide (Figure 1). Figure 2 shows the evolution of dehydrogenase activity in non-polluted and herbicidecontaminated soil during the experimental period. Successive application of oxyfluorfen to the soil caused a significant (p < 0.05) decrease in dehydrogenase activity throughout the incubation period. Compared with the non-polluted soil, this enzymatic activity decreased by 57.7% in the polluted soil at 30 days, 65.4% at 60 days and 69.9% at the end of the experimental period. After the negative effect of oxyfluorfen on the dehydrogenase activity had been confirmed, cultivable bacteria were isolated from each sample in order to observe the effect of oxyfluorfen on them. The number of CFU·mL −1 in both soils increased with the passage of time, although soils polluted with oxyfluorfen presented a lower value than control soils for each sample taken, 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 30 60 90 Oxyfluorfen (mg kg-1) Incubation days c b a Figure 1. Evolution of soil oxyfluorfen concentration (mean ± standard error, n = 3) during the experimental period. 0 0.5 1 1.5 2 2.5 3 3.5 30 60 90 Incubation days S S+Ox a a b ccc Figure 2. Evolution of dehydrogenase activity (μg INTF g −1 h −1 ) (mean ± standard error, n = 3) in control and oxyfluorfenpolluted soil during the experiment. Columns with the same letter(s) do not present significant differences (p > 0.05). INTF: 2-p-iodo-3-nitrophenyl formazan. Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 5 of 17 although the difference was not significant (Figure 3(a)). This result demonstrates that oxyfluorfen negatively affects the size of the cultivable microbial population. A total of 55 bacteria from control soil and 58 bacteria from oxyfluorfen-polluted soil, which were apparently different, were isolated. However, the Box-PCR results indicated that there were 49 different bacterial profiles in the control soil and 53 different bacterial profiles in the polluted soil (Figure 4). The number of different bacteria isolated from the soil increased at 60 and 90 days after the oxyfluorfen addition (Figure 3(b)), thus indicating a higher diversity of bacteria in the presence of the herbicide. 0 5 10 15 20 25 30 60 90 103CFU mL-1 Incubation days S S+Ox a a ab ab ab bA 0 5 10 15 20 25 30 60 90 Number of bacteria Incubation days S S+Ox ab a a aa b B Figure 3. Cultivable bacterial population (mean ± standard error, n = 3) in control and oxyfluorfen-polluted soil during the experiment. (a) Number of colony forming units (CFU) per mL; (b) Number of bacteria. Columns with the same letter(s) do not present significant differences (p > 0.05). Figure 4. Box-PCR dendrograms. (a) Strains isolated from control soils after 30 days; (b) Strains isolated from polluted soils with oxyfluorfen after 30 days; (C) strains isolated from control soils after 60 days; (d) Strains isolated from polluted soils with oxyfluorfen after 60 days; (e) Strains isolated from control soils after 90 days; (f) Strains isolated from polluted soils with oxyfluorfen after 90 days. Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 6 of 17 As can be seen Figure 5, the population of cultivable bacteria changed over time for both the control and oxyfluorfen-polluted soils, with the exception of two strains (SR2.17 and SR4.7) isolated from soil polluted with oxyfluorfen at days 30 and 60, which showed the same Box-PCR profile (Figure 5(b)). Similar results were observed when the cultivable bacterial populations from control and polluted soil were compared after 30, 60, and 90 days from the first treatment with oxyfluorfen (Figure 5). Thus, both treatments showed different bacterial populations for all samples taken. However, one species of bacteria was isolated from each soil treatment at day 90 (strain SR5.13 and SR6.7 for control and polluted soil, respectively; Figure 6(c)). 3.2. Evolution of the PGPB in soils with different oxyfluorfen concentrations In order to observe the possible effect of oxyfluorfen doses on the PGPB, all strains isolated were characterized to determine their PGP properties. All strains showed at least one PGP property, except strain SR4.5, which showed none of them (Table 1). The number of PGP properties per strain was maintained at the different oxyfluorfen doses, with no significant differences (Table 2; GLM, LSD test, p > 0.05). The effect of oxyfluorfen on each PGP property tested also was studied (Figure 7). The percentage of bacteria with biofilm formation capacity was very similar at 30 days from the first Figure 5. Cultivable bacterial populations during the experiment. (a) Dendrogram for all the strains isolated from control soils after 30 (strains SR1), 60 (strains SR3), and 90 (strains SR5) days; (b) Dendrogram for all the strains isolated from soils polluted with oxyfluorfen after 30 (strains SR2), 60 (strains SR4), and 90 (strains SR6) days. Figure 6. Cultivable bacterial populations in control and polluted soils at the same time points. (a) Dendrogram for all the strains isolated from control soils (strains SR1) and soils polluted with oxyfluorfen (strains SR2) after 30 days; (b) Dendrogram for all the strains isolated for control soils (strains SR3) and soils polluted with oxyfluorfen (strains SR4) after 60 days; (c) Dendrogram for all the strains isolated from control soils (strains SR5) and Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 7 of 17 Table 1. PGP properties of the strains isolated from control and those polluted with oxyfluorfen at different times Strains Biofilm IAA (mg·L −1 ) Siderophores (diameter of the halo in cm) Phosphate solubilization (diameter of the halo incm) Nitrogen fixation SR1.1 surface 1.388 3 − + SR1.2 bottom 6.428 4 − − SR1.3 bottom 7.068 4.5 1.2 + SR1.4 bottom and ring − 1.7 − + SR1.5 bottom 4.908 − − + SR1.6 surface 2.348 3 1.5 + SR1.7 bottom and ring 0.068 − − + SR1.8 − 0.428 − − + SR1.9 bottom − − − + SR1.10 surface and ring 4.388 − 1.4 + SR1.11 surface snd ring 1.868 − − + SR1.12 bottom 4.028 − − − SR1.13 bottom 6.028 − − + SR1.14 bottom 4.068 3.6 1.4 + SR1.15 bottom − − − + SR1.16 bottom and ring 0.188 − − + SR1.17 bottom andring 0.748 − − + SR1.18 surface and ring 2.228 − − + SR1.19 bottom 1.788 − − + SR1.20 bottom 6.148 2.9 1.6 + SR1.21 bottom 4.268 − − + SR1.22 bottom and ring 6.268 − − + SR2.1 bottom and ring 0.388 1.7 − + SR2.2 bottom 4.148 − − + SR2.3 bottom 8.268 − − + SR2.4 bottom 1.348 3.2 1.4 + SR2.5 − 48.748 3.2 1.5 + SR2.6 bottom 1.868 3.3 − + SR2.7 bottom and ring 3.308 2.8 1.1 + SR2.8 bottom 0.588 3 1.2 + SR2.9 surface 5.628 − − + SR2.10 bottom 4.028 3.1 1.5 + SR2.11 bottom 6.988 3.4 − + SR2.12 bottom 9.148 3.1 1.4 + SR2.13 bottom 6.468 − − + SR2.14 bottom 5.948 − − + SR2.15 bottom and ring 2.508 − − − SR2.16 bottom 6.468 − − + SR2.17 bottom and ring 2.828 − − + SR2.18 bottom and surface 6.708 − − + SR3.1 − 4.988 − − + (Continued) Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 8 of 17 partitioning and “Dispersive solid-phase extraction” for the determination of pesticide residues in produce. Journal of AOAC International, 86(2), 412–431. https://doi.org/10.1093/jaoac/86.2.412 Araujo, A. S. F., Pertile, M., Costa, R. M., Costa, M. K. L., de Aviz, R. O., Mendes, L. W., de Medeiros, E. V., da Costa, D. P., Melo, V. M. M., & Pereira, A. P. A. (2023). Short-term responses of plant growth-promoting bacterial community to the herbicides imazethapyr and flumioxazin. Chemosphere, 328, 138581. https:// doi.org/10.1016/j.chemosphere.2023.138581 Ávila-Pozo, P., Parrado, J., Caballero, P., Díaz-López, M., Bastida, F., & Tejada, M. (2021). Use of slaughterhouse sludge in the bioremediation of an oxyfluorfen-polluted soil. International Journal of Environmental Health Research, 15(4), 723–731. https://doi.org/10.1007/s41742-021-00351-z Backer, R., Rokem, J. S., Ilangumaran, G., Lamont, J., Praslickova, D., Ricci, E., Subramanian, S., & Smith, D. L. (2018). Plant growth-promoting rhizobacteria: Context, mechanisms of action, and roadmap to commercialization of biostimulants for sustainable agriculture. Frontiers in Plant Science, 871, 1473. https://doi.org/10.3389/fpls.2018.01473 Barba, S., Villaseñor, J., Rodrigo, M. A., & Cañizares, P. (2017). Effect of the polarity reversal frequency in the electrokinetic-biological remediation of oxyfluorfen polluted soil. Chemosphere, 177, 120–127. https://doi. org/10.1016/j.chemosphere.2017.03.002 Bhagat, N., Raghav, M., Dubey, S., & Bedi, N. (2021). Bacterial exopolysaccharides: Insight into their role in plant abiotic stress tolerance. Journal of Microbiology and Biotechnology, 31(8), 1045–1059. https://doi.org/10.4014/jmb.2105.05009 Castillo Diaz, J. M., Delgado-Moreno, L., Núñez, R., Nogales, R., & Romero, E. (2016). Enhancing pesticide degradation using indigenous microorganisms isolated under high pesticide load in bioremediation systems with vermicomposts. Bioresource Technology, 214, 234–241. https://doi.org/10.1016/j. biortech.2016.04.105 Chamkhi, I., El Omari, N., Balahbib, A., El Menyiy, N., Benali, T., & Ghoulam, C. (2022). Is —— the rhizosphere a source of applicable multi-beneficial microorganisms for plant enhancement? Saudi Journal of Biological Sciences, 29(2), 1246. https://doi. org/10.1016/j.sjbs.2021.09.032 Das, A. C., & Debnath, A. (2006). Effect of systemic herbicides on N 2 -fixing and phosphate solubilizing microorganisms in relation to availability of nitrogen and phosphorus in paddy soils of West Bengal. Chemosphere, 65(6), 1082–1086. https://doi.org/10. 1016/j.chemosphere.2006.02.063 Das, A. C., Debnath, A., & Mukherjee, D. (2003). Effect of the herbicides oxadiazon and oxyfluorfen on phosphates solubilizing microorganisms and their persistence in rice fields. Chemosphere, 53(3), 217–221. https://doi.org/10.1016/S0045-6535(03)00440-5 Del Castillo, I., Hernández, P., Lafuente, A., Rodríguez- Llorente, I. D., Caviedes, M. A., & Pajuelo, E. (2012). Self-bioremediation of cork-processing wastewaters by (chloro)phenol-degrading bacteria immobilised onto residual cork particles. Water Research, 46(6), 1723–1734. https://doi.org/10.1016/j.watres.2011.12. 038 EPA. (2002) . Office of pesticide programs. U. US EPA - Pesticides - Fact Sheet for Oxyfluorfen. Filimon, M. N., Roman, D. L., Bordean, D. M., & Isvoran, A. (2021). Impact of the herbicide oxyfluorfen on the activities of some enzymes found in soil and on the populations of soil microorganisms. Agronomy, 11(9), 1702. https://doi.org/10.3390/agronomy11091702 Flores-Duarte, N. J., Mateos-Naranjo, E., Redondo-Gómez, S., Pajuelo, E., Rodriguez-Llorente, I. D., & Navarro- Torre, S. (2022). Role of nodulation-enhancing rhizobacteria in the promotion of Medicago sativa development in nutrient-poor soils. Plants, 11, 1164. https://doi.org/10.3390/plants11091164 Franco-Andreu, L., Gómez, I., Parrado, J., García, C., Hernández, T., & Tejada, M. (2016). Behavior of two pesticides in a soil subjected to severe drought. Effects on soil biology. Applied Soil Ecology, 105, 17–24. https://doi.org/10.1016/j. apsoil.2016.04.001 García, C., Hernández, T., Costa, F., Ceccanti, B., & Masciandaro, G. (1993). The dehydrogenase activity of soils and ecological marker in processes of perturbed system regeneration. In J. Gallardo-Lancho (Ed.), XI International Symposium Environmental Biogeochemistry (pp. 89–100). Gómez, I., Rodríguez-Morgado, B., Parrado, J., García, C., Hernández, T., & Tejada, M. (2014). Behavior of oxyfluorfen in soils amended with different sources of organic matter. Effects on soil biology. Journal of Hazardous Materials, 273, 207–214. https://doi.org/ 10.1016/j.jhazmat.2014.03.051 Gordon, S. A., & Weber, R. P. (1951). Colorimetric estimation of indoleacetic acid. Plant Physiology, 26(1), 192–195. https://doi.org/10.1104/pp.26.1.192 Goswami, D., Parmar, S., Vaghela, H., Dhandhukia, P. C., Thakker, J. N., & Moral, M. T. (2015). Describing Paenibacillus mucilaginosus strain N3 as an efficient plant growth promoting rhizobacteria (PGPR). Cogent Food & Agriculture, 1(1), 1000714. https://doi.org/10. 1080/23311932.2014.1000714 Goswami, D., Thakker, J. N., Dhandhukia, P. C., & Tejada Moral, M. (2016). Portraying mechanics of Plant Growth Promoting Rhizobacteria (PGPR): A review. Cogent Food & Agriculture, 2(1), 1127500. https://doi. org/10.1080/23311932.2015.1127500 Ji, S. H., Gururani, M. A., & Chun, S. C. (2014). Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars. Microbiological Research, 169(1), 83–98. https://doi.org/10.1016/j.micres.2013.06.003 Kang, S. M., Bilal, S., Shahzad, R., Kim, Y. N., Park, C. W., Lee, K. E., Lee, J. R., & Lee, I. J. (2020). Effect of ammonia and indole-3-acetic acid producing endophytic Klebsiella pneumoniae YNA12 as a bio-herbicide for weed inhibition: Special reference with evening primroses. Plants, 9(6), 761. https://doi. org/10.3390/plants9060761 Khalid, S., Shahid, M., Murtaza, B., Bibi, I., Naeem, M. N. A., Niazi, N. K., & Niazi, N. K. (2020). A critical review of different factors governing the fate of pesticides in soil under biochar application. Science of the Total Environment, 711, 134645. https://doi.org/10.1016/j. scitotenv.2019.134645 Lewis, K. A., Tzilivakis, J., Warner, D. J., & Green, A. (2016). An international database for pesticide risk assessments and management. Human & Ecological Risk Assessment, 22(4), 1050–1064. https://doi.org/10. 1080/10807039.2015.1133242 Liu, Y. C., Qu, R. Y., Chen, Q., Yang, J. F., Cong-Wei, N., Zhen, X., & Yang, G. F. (2016). Triazolopyrimidines as a new herbicidal lead for combating weed resistance associated with acetohydroxyacid synthase mutation. Journal of Agricultural & Food Chemistry, 64(24), 4845–4857. https://doi.org/10.1021/acs.jafc. 6b00720 Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 15 of 17 Maeda, H., & Dudareva, N. (2012). The shikimate pathway and aromatic amino acid biosynthesis in plants. Annual Review of Plant Biology, 63(1), 73–105. https://doi.org/10.1146/annurev-arplant -042811-105439 MAPA. (2022). Lista comunitaria de sustancias activas aprobadas, excluidas y en evaluación comunitaria, sustancias de bajo riesgo, sustancias candidatas a la sustitución y lista de sustancias básicas. Pub. L. No. Regalmento (CE) N o 1107/2009 (2022). Ministerio de Agricultura, Pesca y Alimentación. Mesa, J., Mateos-Naranjo, E., Caviedes, M. A., Redondo- Gómez, S., Pajuelo, E., & Rodríguez-Llorente, I. D. (2015). Scouting contaminated estuaries: Heavy metal resistant and plant growth promoting rhizobacteria in the native metal rhizoaccumulator Spartina maritima. Marine Pollution Bulletin, 90(1–2), 150–159. https://doi.org/10.1016/j.marpolbul.2014. 11.002 Mohamed, A. T., El Hussein, A. A., El Siddig, M. A., & Osman, A. G. (2011). Degradation of oxyfluorfen herbicide by soil microorganisms biodegradation of herbicides. Biotechnology, 10(3), 274–279. https://doi. org/10.3923/biotech.2011.274.279 Nautiyal, C. S. (1999). An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters, 170(1), 265–270. https://doi.org/10.1111/j.1574-6968.1999. tb13383.x Navarro-Torre, S., Bessadok, K., Flores-Duarte, N. J., Rodríguez-Llorente, I. D., Caviedes, M. A., & Pajuelo, E. (2020). Helping legumes under stress situations: Inoculation with beneficial microorganisms. In M. Hasanuzzaman (Ed.), Legume crops. https://doi. org/10.5772/INTECHOPEN.91857 Navarro-Torre, S., Mateos-Naranjo, E., Caviedes, M. A., Pajuelo, E., & Rodríguez-Llorente, I. D. (2016). Isolation of plant-growth-promoting and metal-resistant cultivable bacteria from Arthrocnemum macrostachyum in the Odiel marshes with potential use in phytoremediation. Marine Pollution Bulletin, 110(1), 133–142. https://doi.org/10. 1016/j.marpolbul.2016.06.070 Ogura, A. P., Lima, J. Z., Marques, J. P., Sousa, L. M., Rodrigues, V. G. S., & Espíndola, E. L. G. (2021). A review of pesticides sorption in biochar from maize, rice, and wheat residues: Current status and challenges for soil application. Journal of Environmental Management, 300, 113753. https://doi.org/10.1016/j. jenvman.2021.113753 Olanrewaju, O. S., Glick, B. R., & Babalola, O. O. (2017). Mechanisms of action of plant growth promoting bacteria. World Journal of Microbiology & Biotechnology, 33(11), 197. https://doi.org/10.1007/ s11274-017-2364-9 Park, J. M., Radhakrishnan, R., Kang, S. M., & Lee, I. J. (2015). IAA producing Enterobacter sp. I-3 as a potent bio-herbicide candidate for weed control: A special reference with lettuce growth inhibition. Indian Journal of Microbiology, 55(2), 207. https://doi. org/10.1007/s12088-015-0515-y Pavel, A. B., & Vasile, C. I. (2012). PyElph - a software tool for gel images analysis and phylogenetics. BMC Bioinformatics, 13(1), 9. https://doi.org/10.1186/ 1471-2105-13-9 Rodríguez-Morgado, B., Gómez, I., Parrado, J., & Tejada, M. (2014). Behaviour of oxyfluorfen in soils amended with edaphic biostimulants/biofertilizers obtained from sewage sludge and chicken feathers. Effects on soil biological properties. Environmental Science and Pollution Research, 21(18), 11027–11035. https://doi. org/10.1007/s11356-014-3040-3 Rossi, F. (2020). Beneficial biofilms for land rehabilitation and fertilization. FEMS Microbiology Letters, 367(21), fnna184. https://doi.org/10.1093/femsle/fnaa184 Schwyn, B., & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry, 160(1), 47–56. https://doi.org/10.1016/0003-2697(87)90612-9 Sheeba, P. S. V., Kumar, S. P., Mohan, P. S., & Mohan Prasad, S. (2011). Differential physiological and biochemical responses of two cyanobacteria Nostoc muscorum and Phormidium foveolarum against oxyfluorfen and UV-B radiation. Ecotoxicology & Environmental Safety, 74(7), 1981–1993. https://doi. org/10.1016/j.ecoenv.2011.07.006 Sheng, E., Lu, Y., Tan, Y., Xiao, Y., Li, Z., & Dai, Z. (2020). Oxidase-mimicking activity of ultrathin MnO 2 nanosheets in a colorimetric assay of chlorothalonil in food samples. Food Chemistry, 331, 127090. https://doi.org/10.1016/j.foodchem.2020. 127090 Shen, X., Hu, H., Peng, H., Wang, W., & Zhang, X. (2013). Comparative genomic analysis of four representative plant growth-promoting rhizobacteria in Pseudomonas. BMC Genomics, 14(1), 1–20. https:// doi.org/10.1186/1471-2164-14-271 Singh, M. K., Singh, N. K., & Singh, S. P. (2020). Impact of herbicide use on soil microorganisms. In P. Singh, S. K. Singh, & S. M. Prasad (Eds.), Plant responses to soil pollution (pp. 179–194). Springer. Sondhia, S. (2010). Persistence and bioaccumulation of oxyfluorfen residues in onion. Environmental Monitoring and Assessment, 162(1–4), 163–168. https://doi.org/10.1007/s10661-009-0784-1 Starke, R., Jehmlich, N., & Bastida, F. (2019). Using proteins to study how microbes contribute to soil ecosystem services: The current state and future perspectives of soil metaproteomics. Journal of Proteomics, 198, 50–58. https://doi.org/10.1016/j. jprot.2018.11.011 Tejada, M. (2009). Application of different organic wastes in a soil polluted by cadmium: Effects on soil biological properties. Geoderma, 153(1–2), 254–268. https://doi.org/10.1016/j.geoderma.2009.08.009 Tejada, M., Gómez, I., Franco-Andreu, L., & Benitez, C. (2016). Role of different earthworms in a soil polluted with oxyfluorfen herbicide. Short-time response on soil biochemical properties. Ecological Engineering, 86, 39–44. https://doi.org/10.1016/j.ecoleng.2015.09.058 Tejada, M., Gómez, I., Paneque, P., Del Toro, M., García-Quintanilla, A., & Parrado, J. (2023). Use of biostimulants obtained from sewage sludge for the restoration of soils polluted by diuron: Effect on soil biochemical properties. Agronomy, 13(1), 24. https:// doi.org/10.3390/agronomy13010024 Tejada, M., Macias-Benitez, S., Caballero, P., Gómez, I., Paneque, P., & Parrado, J. (2022). Bioremediation of an oxyfluorfen-polluted soil using biostimulants obtained by fermentation processes: Effect on biological properties. Applied Soil Ecology, 170, 929–1393. https://doi.org/10.1016/j.apsoil.2021.104270 Tejada, M., Morillo, E., Gómez, I., Madrid, F., & Undabeytia, T. (2017). Effect of controlled release formulations of diuron and alachlor herbicides on the biochemical activity of agricultural soils. Journal of Hazardous Materials, 322, 334–347. https://doi.org/ 10.1016/j.jhazmat.2016.10.002 Trevors, J. T., Mayfield, C. I., & Inniss, W. E. (1982). Measurement of electron transport system (ETS) Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 16 of 17 activity in soil. Microbial Ecology, 8(2), 163–168. https://doi.org/10.1007/BF02010449 Tudi, M., Ruan, H. D., Wang, L., Lyu, J., Sadler, R., Connell, D., Chu, C., & Phung, D. T. (2021). Agriculture development, pesticide application and its impact on the environment. International Journal of Environment Research Public Health, 18(3), 1112–1124. https://doi.org/10.3390/ijerph18031112 Turan, M., Kıtır, N., Alkaya, Ü., Günes, A., Tüfenkçi, S., Yıldırım, E., & Nikerel, E. (2016). Making soil more accessible to plants: The case of plant growth promoting rhizobacteria. In E. C. Rigobelo (Ed.), Plant growth (pp. 61–69). https://doi.org/10.5772/64826 Wang, B., Wen, X., & Xi, Z. (2016). Molecular simulations bring new insights into protoporphyrinogen IX oxidase/protoporphyrinogen IX interaction modes. Molecular Informatics, 35(10), 476–482. https://doi. org/10.1002/MINF.201600008 WRB. (2014). World reference base for soil resources 2014. In International soil classification system for naming soils and creating legends for soil maps (pp. 192). Food and Agriculture Organization of the United Nations. Yu, H., Zou, W., Chen, J., Chen, H., Yu, Z., Huang, J., Tang, H., Wei, X., & Gao, B. (2019). Biochar amendment improves crop production in problem soils: A review. Journal of Environmental Management, 232, 8–21. https://doi.org/10.1016/j. jenvman.2018.10.117 Zeng, Q., Ding, X., Wang, J., Han, X., Iqbal, H. M. N., & Bilal, M. (2022). Insight into soil nitrogen and phosphorus availability and agricultural sustainability by plant growth-promoting rhizobacteria. Environmental Science and Pollution Research, 29(30), 45089–45106. https://doi.org/10.1007/s11356-022-20399-4 Zhang, Q., Zhang, L., Wang, H., Jiang, Q., & Zhu, X. (2018). Simultaneous efficient removal of oxyfluorfen with electricity generation in a microbial fuel cell and its microbial community analysis. Bioresource Technology, 250, 658–665. https://doi.org/10.1016/j. biortech.2017.11.091 Zhao, H., Xu, J., Dong, F., Liu, X., Wu, Y., Wu, X., & Zheng, Y. (2016). Characterization of a novel oxyfluorfen-degrading bacterial strain Chryseobacterium aquifrigidense and its biochemical degradation pathway. Applied Microbiology and Biotechnology, 100(15), 6837–6845. https://doi.org/10. 1007/s00253-016-7504-x Zhou, Y., Bastida, F., Zhou, B., Sun, Y., Gu, T., Li, S., & Li, Y. (2020). Soil fertility and crop production are fostered by micro-nano bubble irrigation with associated changes in soil bacterial community. Soil Biology & Biochemistry, 141, 107663. https://doi.org/10.1016/j. soilbio.2019.107663 Navarro-Torre et al., Cogent Food & Agriculture (2023), 9: 2247171 https://doi.org/10.1080/23311932.2023.2247171 Page 17 of 17