the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 11 Feb 2026
Unraveling the plant growth promotion potential of Pseudomonas species isolated from the rhizosphere of Lotus creticus grown in the Mediterranean coastal regions of Morocco
Abstract. Rhizosphere-associated bacteria play a key role in improving plant performance in saline and nutrient-poor soils. In this study, the plant growth-promoting potential of rhizobacteria isolated from the rhizosphere of Lotus creticus naturally growing in Mediterranean coastal regions of northwestern Morocco was evaluated. Out of 30 isolates, five bacterial strains R125, P79, R8, R150, and R15 were selected based on their plant growth-promoting rhizobacteria (PGPR) traits. These strains were identified through 16S rRNA gene sequencing as Pseudomonas protegens, Pseudomonas sesami, Pseudomonas versuta, Pseudomonas helleri, and Pseudomonas trivialis, respectively. Phenotypic characteristics, including IAA production, phosphate solubilization capacity, cellulase and protease activities, and tolerance to salinity and temperature were evaluated. Additionally, a pot experiment was conducted to assess the impact of inoculation on the growth of Lotus creticus. Significant variation was observed among the selected strains. Pseudomonas protegens P79 stood out for its strong indole-3-acetic acid (IAA) production, high phosphate solubilization capacity (150.5 mg·L⁻¹), and notable cellulase and protease activities. It also demonstrated high tolerance to salinity (up to 13 % NaCl) and elevated temperatures (up to 45 °C). Conversely, Pseudomonas sesami R8 exhibited a broad spectrum of antifungal activity, including strong inhibition of Aspergillus ochraceus. The pot experiment revealed that inoculation with Pseudomonas versuta R15 and Pseudomonas helleri R125 significantly enhanced the aerial dry biomass and shoot length of Lotus creticus under controlled chamber conditions. This study highlights five Pseudomonas strains, particularly Pseudomonas protegens P79, as promising biostimulants for sustainable agriculture and the rehabilitation of saline marginal lands, due to their diverse plant growth-promoting traits.
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2026-298', Anonymous Referee #1, 20 Mar 2026
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AC1: 'Reply on RC1', Imane Achkouk, 22 Mar 2026
We would like to sincerely thank the referee for the careful evaluation of our manuscript and for the constructive and insightful comments. We highly appreciate the time and effort devoted to reviewing our work.
Please find attached our detailed, point-by-point response to the reviewer’s comments in the supplementary PDF.
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AC1: 'Reply on RC1', Imane Achkouk, 22 Mar 2026
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RC2: 'Comment on egusphere-2026-298', Anonymous Referee #2, 24 Mar 2026
Comments on Egusphere-2026-298 R2
Manuscript: “Unraveling the plant growth promotion potential of Pseudomonas species isolated from the rhizosphere of Lotus creticus grown in the Mediterranean coastal regions of Morocco”
General Comments
This manuscript presents a relevant and promising study linking rhizosphere microbiology, plant–microbe interactions, and soil constraints in Mediterranean coastal ecosystems. The isolation and characterization of Pseudomonas strains associated with Lotus creticus contribute valuable data for soil fertility restoration, saline soil resilience, and sustainable bioinoculation strategies.
Overall, the manuscript is clear, technically solid, and well articulated. The dataset is rich, the methodological effort is commendable, and the work addresses a biologically and agronomically significant legume species in an understudied environmental context. The study fits well within the scope of soil science journals dealing with soil–plant–microbe interactions in marginal environments.
However, several key aspects—particularly regarding the pedological context, sampling design transparency, and depth of mechanistic interpretation—should be improved to strengthen the scientific impact and reproducibility.
I therefore recommend: Minor Revision.
Major Comments
- Pedological context needs deeper integration into the scientific narrative
The study aims at using rhizobacteria for the restoration of saline marginal soils, but currently lacks a sufficiently detailed pedological framework describing what makes these soils limiting, and how the identified traits mitigate these limitations.
Although Table 1 provides essential soil chemical parameters, the study would greatly benefit from a more explicit and comprehensive description of the soil system in which L. creticus thrives.
The manuscript does not mention the soil order, WRB classification, or granulometry. Given coastal Mediterranean systems, identifying whether the soils are Arenosols, Regosols, or Calcisols, for example, would strengthen ecological interpretation.
Only pH and nutrient levels are described. Salinity characteristics (electrical conductivity, cation composition, sodicity indices) are missing, even though saline stress is mentioned as a key driver of plant–microbe interactions. Including at least EC and exchangeable sodium percentage (ESP) would contextualize why these PGPR traits matter physiologically.
Processes such as P mobilization in alkaline/sandy soils, nutrient leaching, or cation–anion imbalances in salt-affected soils should be linked more directly to the microbial traits measured (phosphate solubilization, ACC deaminase, siderophores). This would anchor the microbiological results more firmly within the soil science domain.
2. Sampling, Replicates, and Soil Background:
The high number of assays is impressive, but clearer descriptions would enhance transparency. While the methods are generally well written, several clarifications would strengthen reproducibility and scientific robustness.
a) Sampling Design, the manuscript would benefit from clarification on:
- the exact number of sampled plants,
- the number of rhizosphere soil samples,
- whether isolates represent distinct plants or repeated subsampling of one plant’s rhizosphere.
Explicitly defining replicate structure is essential.
b) Soil Analyses, some parameters require clarification:
- Was organic matter expressed as Walkley–Black oxidizable C or loss on ignition? Both methods are mentioned (Walkley–Black and incineration at 550°C).
- Specify whether P₂O₅ values are available P or total P.
3. Interpretation of Results:
While the Discussion is rich in citations and provides solid biological contextualization, several improvements would increase its value for soil science readership.
Such patterns are scientifically interesting and could provide deeper insights into strain-specific modes of action if more explicitly discussed.
Interpretation would be strengthened by directly linking each PGPR trait to the pedological constraints identified:
- Phosphate solubilization should be discussed in the context of high CaCO₃ and alkaline pH in coastal sandy soils.
- ACC deaminase should be connected with osmotic stress typical of coastal saline systems.
- Siderophores could be better related to Fe limitation in high-pH environments.
The results show strong heterogeneity across Pseudomonas species.
The Discussion could more explicitly interpret:- why P. protegens performs better enzymatically,
- why P. sesami exhibits high antifungal activity,
- why P. helleri and P. trivialis promote shoot biomass more strongly.
Currently, these differences are stated but not mechanistically explored.
A few patterns warrant explanation (clarify plant response inconsistencies):
- R15 increases shoot length substantially but not biomass.
- P79 increases root dry weight despite moderate performance in other traits.
- R8 increases root dry weight but is among the least stress-tolerant strains.
4. Technical Corrections
- Ensure all abbreviations (CFU, SI, ACCD) are defined at first appearance.
- Some numerical values in tables should be harmonized to avoid differing precision (e.g., 150.5 vs 59.70).
- The Discussion sometimes repeats results instead of interpreting them—condensation and synthesis would improve flow.
- Figures 4 and 5: The error bars appear highly asymmetrical at times; verify whether they represent SD or SE.
The figures do not specify the value of n (number of individuals per treatment). This information should be added for clarity and reproducibility.
Inconsistent spacing before units:
Some concentrations are written with or without a space before the unit (e.g., “150.5 mg·L⁻¹” vs “150.5mg·L⁻¹”). This should be standardized throughout the manuscript.Incorrect capitalization of pH:
The term pH appears at times as “PH” in uppercase. Please correct these occurrences.Incorrect formatting of Latin binomials:
Latin species names must always appear in italics, with the genus capitalized and the species in lowercase.- Pseudomonas Helleri (species name must not be capitalized).
- Lotus creticus is occasionally written without italics.
Citation: https://doi.org/10.5194/egusphere-2026-298-RC2
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General Comments
This manuscript focuses on five Pseudomonas strains isolated from the rhizosphere of Lotus creticus and evaluates their plant growth-promoting potential and possible biostimulant functions. The topic itself has a certain degree of potential value.
However, the manuscript does not yet sufficiently clarify its novelty, and the soil-science focus is not prominent enough. In addition, the scientific hypothesis in the Introduction, the methodological descriptions, and the background information on the soils in the study area (e.g., sampling locations, soil type, and texture) all require substantial strengthening.
Therefore, I consider that the manuscript still requires major revision. In particular, it should be comprehensively improved with respect to the positioning of its novelty, methodological transparency, and the systematic development of a clear soil-science focus.
Specific Comments
1. Novelty and Contribution
At present, the Introduction does not clearly define the knowledge gap that this study is intended to address. Therefore, the discussion of the study’s novelty and contribution remains insufficient. More specifically, the manuscript does not sufficiently place this study within the broader context of existing research on plant growth-promoting rhizobacteria (PGPR), and thus does not adequately explain why rhizobacteria associated with L. creticus represent a clearly novel and scientifically meaningful research target. At the same time, the review of previous PGPR-related studies remains rather limited, making it difficult for readers to accurately identify the added contribution of this work relative to the existing literature.
In addition, the manuscript does not yet sufficiently explain why the saline soils in the study area are representative, or whether their scientific significance extends beyond a single regional case. This somewhat weakens the broader relevance and applicability of the results. Meanwhile, the soil-science focus in the Introduction remains insufficiently developed. The manuscript does not yet clearly articulate the key rhizosphere soil processes involved in this study, nor does it fully explain the potential importance of these bacteria under saline soil conditions.
Finally, the manuscript lacks a clear scientific hypothesis. The authors are encouraged to further improve the Introduction by:
strengthening the systematic review of relevant studies, particularly those concerning the role of PGPR in promoting plant growth, so as to more clearly define the knowledge gap addressed by this study;
clarifying the representativeness of the saline soils in the study area and their broader scientific significance, while reinforcing the logical chain among the saline soil environment, rhizosphere microbial functions, and plant growth-promoting effects, so that the soil-science-related research questions can be more clearly framed; and
proposing a clear scientific hypothesis to enhance the logical coherence and academic contribution of the study.
2. Methodological Justification
The most important issue in the current Materials and Methods section is the insufficient description of the sampling design and soil background information, which clearly weakens both the robustness of the data and the reproducibility of the study. Specifically, key information such as the sampling time, plot locations, number of sampling points, and whether replicate samples were included has not been clearly stated. In addition, the soil type and texture, as well as the definition of rhizosphere soil and the procedure used to collect it, are not sufficiently clear. The exact depth range corresponding to the “surface layer” should also be specified. The authors are encouraged to provide a more detailed description of the sampling design, the location of the study area, and the procedures used to obtain soil samples in the Materials and Methods section.
Furthermore, additional details are still needed for the analytical methods. In particular, the absence of basic parameters such as electrical conductivity (EC) means that the manuscript does not yet adequately characterize the basic environmental features of the saline soils, even though such information is important for defining the saline soil context and interpreting the subsequent results. The authors should further clarify the material analyzed for each parameter, the source of the samples, the sample pre-treatment procedures, and the purpose of each analysis. Key information on the statistical analyses should also be added in order to improve methodological transparency, the reliability of result interpretation, and the overall reproducibility of the study. In addition, some details regarding the replicate settings, evaluation criteria, and data presentation in the molecular identification, functional assays, and stress-tolerance assays also require further clarification.
3. Discussion
The Discussion does not yet sufficiently explain why these strains exhibit the reported functions under saline soil conditions, and the overall mechanistic interpretation remains rather weak. At the same time, the soil-science focus in this section should be further strengthened, particularly through a clearer analysis of the relationships among nutrient limitation in saline soils, rhizosphere microbial promotion of nutrient mobilization, and plant stress resistance.
In addition, the current Discussion still tends, to some extent, toward a restatement of results and literature listing. The authors are advised to interpret the results more cautiously, clearly distinguishing between conclusions directly supported by the data and reasonable inferences based on the literature. The logical linkage among the soil background, the functional traits of the strains, and the observed plant growth responses should also be further strengthened.
Technical Corrections
In Figure 3, some values are close to zero, and under the current scale it is difficult to clearly distinguish the differences among treatments, which affects the readability of the results. The authors are advised to optimize the presentation of this figure.
Line 256: Table 5 reports that the salinity tolerance of strain R8 is 9%, whereas line 256 states 11%. Please verify this and ensure consistency.
Line 264: Figure 4 shows that the root length under the R150 inoculation treatment is significantly lower than that in the control and most other treatments, whereas the main text states that there is no significant difference in root length between the inoculation treatment and the control. Please verify this and ensure consistency.
Line 300: In Table 4, the chitinase activity of P79 is reported as “moderate activity”; therefore, the corresponding wording in the main text should be more cautious.