Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Keywords: Delete biofertilizer potential, add root colonization and growth-promoting properties.

2. The aesthetic issue of Figure 2 should comply with the requirements of this journal (whether the grid lines are retained in the drawing area).

3. The inter group significance test conducted in Figures 3 and 4 should include the mean and intra group repeatability for each data group (as shown in Table 2). Additionally, the choice of uppercase or lowercase letters to indicate whether the inter group differences are significant should comply with the requirements of this journal.

4. L277-279 “Suggesting suitability for soils with low to moderate salinity stress”, this statement is not scientific,the experimental results obtained from the cultivation medium cannot represent the conclusions drawn under complex soil conditions.

Author Response

Manuscript ID horticulturae-3410664

“Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue”

 

 

Response to Reviewer 1

 

We sincerely thank you for the valuable comments and observations. We have carefully reviewed the entire manuscript, addressed all minor grammatical issues, and incorporated the suggested corrections. A detailed point-by-point response to the comments has been prepared to ensure all concerns are addressed. We appreciate your thorough review and trust that the revisions enhance the manuscript's quality.

 

Reviewer 1

1. Keywords: Delete biofertilizer potential, add root colonization and growth-promoting properties.

We have revised the keywords as suggested. The term "biofertilizer potential" has been removed, and "root colonization" and "growth-promoting properties" have been added to better align with the content of the manuscript

2. The aesthetic issue of Figure 2 should comply with the requirements of this journal (whether the grid lines are retained in the drawing area).

Thank you for the observation. The aesthetic elements of Figure 2 have been revised. The grid lines have been removed, and the colors and legends have been updated to comply with the journal's requirements.

3. The inter group significance test conducted in Figures 3 and 4 should include the mean and intra group repeatability for each data group (as shown in Table 2). Additionally, the choice of uppercase or lowercase letters to indicate whether the inter group differences are significant should comply with the requirements of this journal.

Thank you for the observation. We have addressed it.

4. L277-279 “Suggesting suitability for soils with low to moderate salinity stress”, this statement is not scientific,the experimental results obtained from the cultivation medium cannot represent the conclusions drawn under complex soil conditions.

The statement has been revised to address this concern. The revised wording avoids overgeneralization and acknowledges the limitations of our experimental conditions

Reviewer 2 Report

Comments and Suggestions for Authors

 

The manuscript explains “Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue." After a careful review, I found this work worthy to publish in the Horticulturae. I propose this work can be published after major revision as follows:

·         In the title, the authors should mention a specific Rhizobium name instead of native Rhizobia.

·         In the abstract, the authors should explain their results with numerical values and also explain the best strain in comparison of overall their results.

·         Line 43: In the introduction, the authors should give examples of inulin-type fructans and bioactive compounds and also add references in this line.

·         Line 45-46: Which functional foods? Kindly give few examples

·         In introduction, once used abbreviation in one paragraph, then authors should use only abbreviation in whole manuscript.

·         Line 81-89: The authors should give proper reference for whole statements.

·         Line 95, “shorten the growth cycle of Agave tequilana” Kindly give reference and explain how it will shorten the growth cycle.

·         Line 105-106: in materials and methods, have the authors studied/tested for these statements? “These bacterial strains are non-pathogenic and non-toxic to humans, animals." Give reference if authors have studied these aspects in their previous studies.

·         Line 113-115: The authors should give the reference.

·         Line 129-131: The authors should explain the methodology for analysis of aluminum, copper, zinc, and lead with reference.

·         Line 227-231: Kindly explain the whole methodology for carbohydrate contents in HPLC-IR.

·         Line 233: Kindly write this line in English.

·         In results, Line 236-243: kindly mention figure 1 (A), Line 244-250: kindly mention figure 1 (B), and Line 251-254: kindly mention figure 1 (C).

·         In table 2, kindly also give statistically significant letters for each strain.

·         In figure 2, kindly add SI unit instead of absorbance leve

·         In discussion, kindly add more discussion with respect to your results.

·         In conclusion, only focus on main results and key findings of your studies.

 

Comments on the Quality of English Language

There are minor English grammar mistakes in the whole manuscript. Kindly check it and correct it in the revised version

 

Author Response

Manuscript ID horticulturae-3410664

“Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue”

 

 

Response to Reviewer 2

 

We sincerely thank you for the valuable comments and observations. We have carefully reviewed the entire manuscript, addressed all minor grammatical issues, and incorporated the suggested corrections. A detailed point-by-point response to the comments has been prepared to ensure all concerns are addressed. We appreciate your thorough review and trust that the revisions enhance the manuscript's quality.

Reviewer 2

The manuscript explains “Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue." After a careful review, I found this work worthy to publish in the Horticulturae. I propose this work can be published after major revision as follows:

• In the title, the authors should mention a specific Rhizobium name instead of native Rhizobia.

Thank you for the observation. We have decided to keep "Native Rhizobia" in the title instead of including the specific names of the strains (Rhizobium sp. ACO-34A and Sinorhizobium mexicanum ITTG R7T) because we are working with more than one rhizobial species (including one Rhizobium and two Sinorhizobium). Moreover, we consider that keeping the term "Native Rhizobia" is more inclusive and better reflects the broader focus of the study on the diversity of native strains used as probiotics in agave.

 

• In the abstract, the authors should explain their results with numerical values and also explain the best strain in comparison of overall their results.

Thank you for the observation. We have revised the abstract to include quantitative results of the effects of the inoculation

• Line 43: In the introduction, the authors should give examples of inulin-type fructans and bioactive compounds and also add references in this line.

Thank you for the observation. We have revised the text to include specific examples.

• Line 45-46: Which functional foods? Kindly give few examples

Thank you for the observation. We have revised the text to include specific examples

• In introduction, once used abbreviation in one paragraph, then authors should use only abbreviation in whole manuscript.

Thank you for the observation. We have revised the abbreviations

• Line 81-89: The authors should give proper reference for whole statements.

Thank you for the observation. The corresponding references have been included

• Line 95, “shorten the growth cycle of Agave tequilana” Kindly give reference and explain how it will shorten the growth cycle.

Thank you for the observation. We have revised line 95 and made the adjustments. Additionally, we have added the corresponding reference.

• Line 105-106: in materials and methods, have the authors studied/tested for these statements? “These bacterial strains arenon-pathogenic and non-toxic to humans, animals." Give reference if authors have studied these aspects in their previous studies.

Thank you for the observation. To avoid confusion and ensure accuracy, we have removed this statement from the manuscript, as it was not directly evaluated in this study

• Line 113-115: The authors should give the reference.

Thank you for the observation. We have revised lines 113–115 and included a relevant reference to support the statement.

• Line 129-131: The authors should explain the methodology for analysis of aluminum, copper, zinc, and lead with reference.

Thank you for the observation. We have provided more details about the methodology used to evaluate heavy metal tolerance in the rhizobial strains.

• Line 227-231: Kindly explain the whole methodology for carbohydrate contents in HPLC-IR.

Thank you for the observation. We have provided more details about the methodology used for carbohydrate analysis

• Line 233: Kindly write this line in English.

Thank you for the observation. The modification has been made.

• In results, Line 236-243: kindly mention figure 1 (A), Line 244-250: kindly mention figure 1 (B), and Line 251-254: kindly mention figure 1 (C).

Thank you for the observation. The modification has been made.

• In table 2, kindly also give statistically significant letters for each strain.

Thank you for the observation. However, statistical analysis was not considered for this table, as the objective was exclusively to evaluate the activity of the strains in terms of the reported parameters. This table aims to highlight the obtained values descriptively and does not intend to perform statistical comparisons between treatments.

• In figure 2, kindly add SI unit instead of absorbance level

Thank you for the observation. We have reviewed and updated Figure 2.

• In discussion, kindly add more discussion with respect to your results.

Thank you for the observation. We have modified the discussion

 

• In conclusion, only focus on main results and key findings of your studies.

Thank you for the observation. We have modified the conclusion

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

All my comments are in pdf-document.

Please address ALL of my comments/questions. If some of them you would not like to accept please explain why. Also please make list of responses so that I could follow your responses easily. 

 

Comments for author File: Comments.pdf

Author Response

Manuscript ID horticulturae-3410664

“Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue”

 

 

Response to Reviewer 3

 

We sincerely thank you for the valuable comments and observations. We have carefully reviewed the entire manuscript, addressing all minor grammatical issues, and incorporated the suggested corrections. A detailed point-by-point response to the comments in the provided PDF document has been prepared to ensure all concerns are addressed. We appreciate your thorough review and trust that the revisions enhance the manuscript's quality.

 

Reviewer 3

L54_This is also definition of plant growth promoting bacteria (PGPB) or plant growth promoting rhizobacteria (BGPR). Please make comparison of PPB vs. PGPB. Are PGPB subgroup of PPB etc. Only sentence or two just to orient readers what is what. Thank you.

 

Response:  

We appreciate the reviewer’s insightful comment. Plant probiotic bacteria (PPB) and plant growth-promoting bacteria (PGPB) are closely related; however, PPB encompass a broader range of benefits, including enhanced nutritional value, improved plant resilience, and support for biofortification. PGPB, including plant growth-promoting rhizobacteria (PGPR), represent a subgroup of PPB that primarily focus on growth enhancement and stress mitigation through mechanisms such as nutrient solubilization and phytohormone production.

To address the reviewer’s observation, we have clarified this distinction in Line 54 by revising the following text for better comprehension of the topic: “Plant probiotic bacteria (PPB) are beneficial microbes that colonize plant roots, promoting growth and health through mechanisms such as phytohormone production, nitrogen fixation, phosphate solubilization, and secretion of antibacterial and antifungal metabolites. By positively interacting with their host plants, PPB enhance crop yields and quality while reducing the need for synthetic fertilizers and pesticides. Additionally, PPB contribute to biofortified foods by boosting bioactive compounds beneficial to human health, such as antioxidants and fructans in A. tequilana, positioning these bacteria as promising biofertilizers. PPB differ from plant growth-promoting bacteria (PGPB) by their strict requirement for non-pathogenicity to humans and plants, a criterion critical for food safety. This restricts PPB to specific bacterial genera such as Rhizobium, Azotobacter, Azospirillum, and a few species of Bacillus, which exhibit both safety and efficacy in plant growth promotion. PGPB, including plant growth-promoting rhizobacteria (PGPR), are a subgroup of PPB that primarily focus on growth enhancement and stress mitigation”.

 

Reviewer 3

L130_Please add to which antibiotics/concentrations and please add method used (reference).

Response:  We thank the reviewer for their insightful comment. To address the observation, we have added the requested details to the manuscript in L130. Specifically, the information now includes the antibiotics tested, their respective concentrations, and the methodology used, as follows: “Additionally, heavy metal tolerance was tested for aluminum (Al³⁺), copper (Cu²⁺), zinc (Zn²⁺), and lead (Pb²⁺) by culturing the rhizobial strains on PY medium supplemented with varying concentrations 500 μM Al³⁺ and 100 μM for Cu²⁺, Zn²⁺, and Pb²⁺. The minimum inhibitory concentrations (MIC) for each metal were determined following the protocol described by Ajayi and Adekanmbi (2020) [30]. Intrinsic antibiotic resistance was evaluated using the disk diffusion method on PY-Ca²⁺ medium. Strains were evenly inoculated, and commercial antibiotic disks (PT-36 Multibac® (JAFS, Mexico) targeting Gram-positive and Gram-negative bacteria were applied. The antibiotics tested included Netilmicin (10 mg), Penicillin (10 mg), Chloramphenicol (30 mg), Gentamicin (10 mg), Ciprofloxacin (5 mg), Cefalexin (30 mg), Amikacin (30 mg), and Ampicillin (10 mg). Plates were incubated at 28°C for 48 hours, and resistance (+) or susceptibility (-) was determined based on the presence or absence of inhibition zones [17]”.

 

Reviewer 3

L191_Please add here appropriate reference. As I know Christiansen et al. was first to employ such method.

Response: We appreciate the reviewer’s suggestion. In response, we have added the appropriate reference as indicated. Additionally, the full citation has been included in the References section as entry No. 30:

 

Christensen, G.D.; Simpson, W.A.; Younger, J.J.; Baddour, L.M.; Barrett, F.F.; Melton, D.M.; Beachey, E.H. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol. 1985, 22, 996–1006. https://doi.org/10.1128/jcm.22.6.996-1006.1985.

 

Reviewer 3

L191_Please add in which plates (round or flat bottom, 96 wp or...?), type and manufacturer. Please add in which medium biofilm was grown. How it was stained with crystal violet, destained, in which plate absorbance at 570 nm was measured etc. Please describe this test in more detail as you did for other analyses conducted.

 

Response: We appreciate the recommendation. In line 191, we revised the methodology for the Assessment of biofilm formation ability and added further details about the procedure and analyses conducted: "The biofilm-forming capacity of native rhizobial strains was quantitatively assessed using a 96-well flat-bottom polystyrene microtiter plate (Costar^®, Corning Inc., NY, USA) and crystal violet staining [30]. Strains were cultured in PY-Ca²⁺ medium at 28 °C until reaching an optical density (OD600) of 0.2 (approximately 1 × 10⁸ CFU mL⁻¹). Aliquots of 200 μL were inoculated into individual wells containing minimal medium and incubated statically at 28 °C for 48 h. After incubation, unbound cells and growth media were carefully removed, and wells were rinsed three times with sterile phosphate-buffered saline (PBS, pH 7.4) to eliminate loosely adherent cells. The biofilms were then stained with 200 μL of 0.3% (w/v) crystal violet solution (Sigma-Aldrich^®, USA) for 10 min at room temperature. Excess stain was removed by rinsing the wells three times with deionized water, followed by air-drying for 15 min. The adhered crystal violet was solubilized with 200 μL of 80% ethanol–20% acetone solution, and absorbance was measured at 570 nm using a microplate reader (Bio-Rad^® Model 680, Bio-Rad Laboratories, USA). Biofilm production was expressed as relative absorbance units, normalized to account for variability in the initial inoculum. Statistical analysis of biofilm formation across strains was conducted using one-way ANOVA, followed by Fisher’s post hoc test (p < 0.05) in StatView 5.0 software (Abacus Corporation, USA). This standardized approach ensured reproducibility and accuracy in evaluating the biofilm-forming capabilities of the rhizobial strains."

 

Reviewer 3

L230_Instead of "mean" I think it should be written "post-hoc". Please correct.

Response: We appreciate the reviewer’s observation and have revised the text for clarity and accuracy. The corrected text now reads: “Data were analyzed using analysis of variance (ANOVA), followed by post-hoc comparisons with Tukey’s test (p < 0.05)."

 

Reviewer 3

L234_Please in English.

 

Response: We apologize for the oversight and appreciate the reviewer’s observation. The section title has been revised to English for clarity. It now reads: "3.1. Biosynthetic gene clusters in native rhizobial strains with PGP potential." Thank you for highlighting this correction.

 

Reviewer 3

L241_I do not see that in legend on the right side of Figure 1. Under which subsystem feature count is biofilm formation. Please explain in the text or add in the legend.

 

L258_Also could you please specify which genes are responsible for biofilm formation. Here and in line 258.

 

L248_Please explain on what biosynthesis you think. Biosynthesis of what.

 

L254_Again please add in which subsystem feature MDR genes belongs.

Response:

 

We appreciate the reviewer’s thoughtful observations and constructive feedback. The following responses detail how each point has been addressed in the revised manuscript:

 

L241: "I do not see that in legend on the right side of Figure 1. Under which subsystem feature count is biofilm formation. Please explain in the text or add in the legend."

 

Thank you for pointing this out. We have clarified in the text that genes related to biofilm formation are categorized under the "Cell Wall and Capsule" subsystem. Additionally, the updated legend of Figure 1 now specifies this information, ensuring clarity for readers.

 

L258: "Also could you please specify which genes are responsible for biofilm formation. Here and in line 258."

 

We have specified in the revised text that genes such as bcsA, bcsB, bcsC (for cellulose production), and genes involved in exopolysaccharide biosynthesis are responsible for biofilm formation. Furthermore, regulatory genes such as expR and luxR from the "Regulation and Cell Signaling" subsystem, which govern exopolysaccharide production and quorum sensing, were highlighted.

 

L248: "Please explain on what biosynthesis you think. Biosynthesis of what?"

 

To address this, we have detailed that the biosynthesis mentioned refers to the production of exopolysaccharides and cellulose, which are critical for the extracellular matrix of biofilms and structural development.

 

L254: "Again please add in which subsystem feature MDR genes belongs."

 

We have included in the revised text that MDR (multidrug resistance) genes, such as MdtA-N, are categorized under the "Stress Response" subsystem. Their role in providing resilience against environmental and chemical stressors has been emphasized.

The updates and clarifications are now reflected in the new section: "3.1. Biosynthetic gene clusters in native rhizobial strains with PGP potential." The revised text provides a comprehensive explanation of how subsystems and specific genes contribute to biofilm formation, MDR, and other plant probiotic traits in the analyzed strains. 

We hope these modifications adequately address your comments and enhance the manuscript's clarity and scientific value.

 

 

 

 

Reviewer 3

L269_How those concentrations were chosen? Are these concentration environmentally relevant?

 

Response: We appreciate the reviewer’s insightful comment. The concentrations of antibiotics selected for this study reflect standard dosages widely used in antibiotic resistance assays for rhizobial strains. These concentrations are designed to evaluate intrinsic and acquired resistance mechanisms while ensuring ecological relevance to environments such as agricultural soils and plant rhizospheres. Previous studies, including those on Rhizobium species (Hagedorn, 2018) and antibiotic impacts on microbial communities (Le Page et al., 2017), have utilized similar concentrations to assess resistance patterns in ecologically representative conditions. Thus, these concentrations allow robust evaluation of resistance traits, ensuring experimental rigor and relevance to the ecological context of sustainable agriculture.

 

Reviewer 3

L270 The same question as for antibiotics. Please I would like to see your response why those concentrations were chosen?

 

L271_This explanation is misleading. What does it mean, e.g. that Netilmicin stimulate development of R7T? Please reformulate explanation what (+) and (-) mean.

 

L282_According to table 1 ACO-34A was also resistant to ampicillin. Please check what is correct, table or text.

 

L282_Well I would not say broader as it was (-) to gentamicin. Please be here a little bit more precise.

 

Response: We sincerely thank the reviewer for the observations, specifically regarding L270, L271, and L282.

The section 3.2. Phenotypic, genomic, and tolerance characteristics of rhizobial strains has been revised to address each of the questions and comments. “The results highlight significant physiological, morphological, and adaptive traits of the rhizobial strains Rhizobium sp. ACO-34A, S. mexicanum ITTG R7^T, and S. chiapasense ITTG S70^T, underscoring their potential as plant probiotic bacteria for Agave tequilana cultivation (Table 1). Morphological analysis revealed that all strains are rod-shaped, with dimensions of 0.4 × 1.1 µm for Rhizobium sp. ACO-34A, 0.7 × 1.2 µm for S. mexicanum ITTG R7^T, and 0.6 × 1.4 µm for S. chiapasense ITTG S70^T, and all possess peritrichous flagella, facilitating motility and root colonization. Genomic analysis indicated similar GC content across the strains: Rhizobium sp. ACO-34A (61.1%), S. mexicanum ITTG R7^T (62.0%), and S. chiapasense ITTG S70^T (61.8%), reflecting moderate genetic stability.

Physiological assessments demonstrated their moderate salinity tolerance, with all strains growing effectively at 1% and 2% NaCl but inhibited growth at 5% NaCl, under laboratory conditions, indicating their potential for further evaluation in different soils under real field management. The antibiotic resistance profiles varied significantly, with ACO-34A showing resistance to penicillin, chloramphenicol, gentamicin, and ciprofloxacin, while ITTG R7^T and ITTG S70^T exhibited broader resistance, including to netilmicin and ampicillin. Regarding heavy metals, all strains tolerated aluminum (Al³⁺) and copper (Cu²⁺); however, only ITTG R7^T and ITTG S70^T were resilient to zinc (Zn²⁺), a trait advantageous in zinc-contaminated soils, while none tolerated lead (Pb²⁺). The concentrations of heavy metals used in this study (Al³⁺ 500 μM, Cu²⁺ 100 μM, Zn²⁺ 100 μM, and Pb²⁺ 100 μM) were selected based on previous studies evaluating rhizobial tolerance to environmental stressors. These concentrations represent levels commonly found in contaminated or naturally metal-rich soils, where rhizobia often coexist with other microbial communities. For example, Cu²⁺ and Zn²⁺ concentrations of 100 μM align with thresholds widely reported for evaluating microbial tolerance in agricultural and industrial contexts, while Al³⁺ at 500 μM reflects its solubility and bioavailability in acidic soils, typical of A. tequilana cultivation regions. Lead (Pb²⁺) at 100 μM corresponds to sublethal concentrations observed in anthropogenically impacted soils.

All strains thrived across a broad pH range (5.0–8.0) and demonstrated growth at temperatures up to 37 °C, supporting their application across diverse soil types and climates. These findings establish Rhizobium sp. ACO-34A as a promising biofertilizer for A. tequilana, with S. mexicanum ITTG R7^T and S. chiapasense ITTG S70^T offering additional benefits, particularly in zinc-enriched soils, contributing to sustainable agricultural practices under challenging environmental conditions.”.

 

This updated text provides additional details on the criteria for selecting heavy metal concentrations, clarifies the interpretation of the symbols (+) and (-) regarding resistance/susceptibility, and offers precise information on antibiotic resistance profiles as outlined in Table 1. We trust these revisions adequately address the reviewer’s concerns and enhance the clarity of the manuscript. Thank you once again for your valuable feedback, which has greatly improved the quality of our work.

 

Reviewer 3

L363_You stated in line 195 that biofilm formation was quantified across serial dilutions. What does this image represents? Diluted or undiluted bacterial suspensions, and if diluted which dilution is represented by Figure 2?

 

Response:

Thank you for your observation. Figure 2 represents the biofilm formation quantified from undiluted bacterial suspensions. While the experimental protocol involved assessing biofilm production across serial dilutions, the data presented in this figure specifically corresponds to measurements obtained from the original, undiluted bacterial suspensions at three distinct time points (24, 48, and 72 hours). This approach was chosen to highlight the maximum biofilm-forming capacity of each strain under the conditions tested. We will ensure that this clarification is incorporated into the manuscript for better transparency.

 

 

Reviewer 3

L375_Please add Image of these EPS layers. I find it very important to show such result.

 

Response: We sincerely thank the reviewer for this valuable observation. To address the request, we have included Figure S1 in the supplementary material, which provides detailed microscopy images illustrating the exopolysaccharide (EPS) layers formed by Rhizobium sp. ACO-34A during colonization of Agave tequilana roots. These images, captured at various time points (7, 12, and 15 dpi) and magnifications, showcase the progressive bacterial accumulation and EPS development, enhancing the comprehension of this critical trait. We believe this addition significantly enriches the discussion of our findings.

 

 

Reviewer 3

L387_This variability in EPS production should be supported with results.

 

Response:

We appreciate the comment. We honestly do not have any images to support what is mentioned in the paragraph. What we have is our data from the reports made in our laboratory notebooks and theses that have been done. We will be attentive to your opinion and we will be careful in our future work to try to have complete information.

 

Reviewer 3

L390_These SEM images are not of the same magnification. Please adjust them so that they represent the same magnification. That would make comparison much more easy for your readers.

Also add information after how many days these images were taken. I would like to see here 15 dpi.

 

Response:

Thank you for the observation. The SEM images were taken at 15 dpi, we added the information in the foot image. Regarding the image magnification, we were unable to obtain images with the same magnification because the image data was provided to us by an external collaborator, and it was difficult to obtain the images. We offer our most sincere apologies

 

 

Reviewer 3

L400_How so, in Table 3 there are different letters:            852.45 A and 604.50 B. Please correct.

 

Response:

Thank you for pointing this out. Table 3 have been reviewed and corrected to ensure that the annotations are consistent and align with the statistical analysis.

 

Reviewer 3

L414_Please delete "often" and "or matching".

 

Response:

Thank you for the suggestion. The terms "often" and "or matching" have been removed for clarity and precision.

 

Reviewer 3

L419_Please add here standard deviations of those four measurements. You could add them below mean values e.g. in brackets.

 

Response:

Thank you for the suggestion. Standard deviations have been added for clarity and completeness.

 

Reviewer 3

L421_tequilana

 

Response:

We agree, and the corrected term "tequilana" has been applied consistently throughout the manuscript.

 

Reviewer 3

L434_Delete "and fructose". It was different as it was not significantly different.

 

Response:

Thank you for the observation. The phrase "and fructose" has been removed to reflect that it was not significantly different.

 

Reviewer 3

L441_Please add here standard deviations of those four measurements. You could add them below mean values e.g. in brackets.

Response:

Thank you for the suggestion. Standard deviations have been added

 

Reviewer 3

L485_You did not measure biofilm forming ability under variable soil conditions. As matter in fact you did not measure biofilm formation in soil at all but in plastic well plate. Please delete that sentence as it does not reflect what was measured.

 

Response:

We agree with the reviewer. The sentence referencing biofilm formation under variable soil conditions has been removed to accurately reflect the experimental setup.

 

Reviewer 3

L486_Delete "fluorescence" as there was no results regarding fluorescence microscopy.

 

Response:

Thank you for the observation. The term "fluorescence" has been removed as it was not supported by experimental results.

 

Reviewer 3

L487_If you are regarding to figure 3 I must disagree with you. Please provide pictures which support such statement (15 dpi) or change the statement. Figure 3 certainly does not present dense bacterial clusters. That are still solitary cells in 2D arrangement. That is why I asked you to give 15 dpi pictures and that these must be of the sama magnification. From those images you could see how much cells was colonizing the surface and in combination with the Fig 2 make conclusion does the Crystal violet was adhered to cells or to EPS.

 

Response:

Thank you for the observation. We have change the statement

 

Reviewer 3

L494_Yes but you investigated pure cultures biofilms, so I do not see the point here. Maybe you could place that sentence in the Introduction which does not give any introduction to importance of biofilms although it seems biofilm forming ability of your ACO strain is important in for your work.

 

Response:

We appreciate the suggestion. The sentence have been edited for better comprehension

 

 

 

 

Reviewer 3

L497_ You mean "fertilizer". You reported results for only one chemical fertilizer (Triple 17). Please correct.

 

Response:

Thank you for the correction. We have corrected the term to "fertilizer".

 

 

Reviewer 3

L507_That is true if Agave production could be conducted in sterile mixture of peat and perlite. If instead native, nonsterile soil would be used the results could be very different. How different is not measured in this study. Hence there should be caution regarding usability of ACO-34A in Agave production.

I doubt Agave is produced in sterile substrate, so I must ask you why you did not conduct green-house experiment in real conditions of Agave production?

 

Response:

We appreciate the comment. Thank you for your valuable feedback. Conducting experiments in real agave production conditions would indeed provide additional insights and applicability to our findings. However, for this study, our primary objective was to establish a controlled environment to accurately assess the effects of the variables being tested (e.g., microbial inoculants, soil properties, etc.) without the interference of external, uncontrollable factors such as weather, pests, or variability in field conditions. The greenhouse setting allowed us to isolate and better understand the specific mechanisms and interactions at play. We acknowledge that field experiments represent an important next step in validating our findings under real-world conditions, and we intend to address this in future research to ensure the practical relevance of our results for agave production systems.

 

Reviewer 3

L514_Here I want to again emphasize which genes are responsible for biofilm formation. Please add them (as you did some of nitrogen metabolism genes) in results. Thank you.

 

Response:

Thank you for the suggestion. The genes responsible for biofilm formation, including those involved in exopolysaccharide production (bcsA, bcsB, bcsC) and quorum sensing (expR, luxR), have been added to the Results section 3.1. for clarity.

 

Reviewer 3

L518_I would like to see in Conclusions focus on only ACO strain as it was superior to other two, so dont mention them. Also I think it is necessary to add in conclusions what is by your opinion most important trait of ACO that made it so much better inoculant compared to other two strains. That is important to conclude.

 

Response:

Thank you for the recommendation. The Conclusions section has been revised and edited highlighting ACO34A traits contributing to its effectiveness as an inoculant.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript explains “Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue." After a careful review, I found this work worthy to publish in the Horticulturae. The authors have modified the manuscript and improved the quality. I propose this work can be published in its current form. 

Comments on the Quality of English Language

The English language can be improved. 

Author Response

Dear Reviewer 2,

We sincerely thank you for your thoughtful and encouraging feedback on our manuscript. Your constructive comments throughout the review process were instrumental in refining our research presentation. We greatly appreciate your time, effort, and expertise in evaluating our manuscript, and we are grateful for your recommendation to publish it in its current form.

Thank you 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

Here are my comments:

Line 268. Please delete one dot.
Line 217. You state that biofilm was incubated for 48 h but in Figure 2 there is also 72 h. Please correct.
Line 462-463. For pina weight you state here that there is no difference between chemical fertilizer and ACO-34A inoculated plants. But in Table 3 in Pina weight column there are different letters for chemical fertilizer and ACO-34A inoculated plants which clearly indicate statistically significant difference. What is correct? Table or text? Please correct.

It is bad for your reputation if contradictions are presented in your paper. As I have managed to notice them so will your readers. Now you have opportunity to correct that so please do.

Author Response

Manuscript ID horticulturae-3410664

“Plant Probiotic Potential of Native Rhizobia to Enhance Growth and Sugar Content in Agave tequilana Weber var. Blue”

 Response to Reviewer 3

 We sincerely thank you for the valuable comments and observations. We have carefully reviewed the entire manuscript, addressed all the suggested corrections. A detailed point-by-point response to the comments has been prepared to ensure all concerns are addressed. We appreciate your thorough review and trust that the revisions enhance the manuscript's quality.

 

Reviewer 1

Here are my comments:

Line 268. Please delete one dot.

Thank you for the observation. We have deleted the extra dot.

Line 217. You state that biofilm was incubated for 48 h but in Figure 2 there is also 72 h. Please correct.

Thank you for the observation. The incubation hours were corrected in the text.

Line 462-463. For pina weight you state here that there is no difference between chemical fertilizer and ACO-34A inoculated plants. But in Table 3 in Pina weight column there are different letters for chemical fertilizer and ACO-34A inoculated plants which clearly indicate statistically significant difference. What is correct? Table or text? Please correct.

Thank you for the observation. Yes, we have corrected the sentences in the text as there is clearly statistically significant difference in the table.

It is bad for your reputation if contradictions are presented in your paper. As I have managed to notice them so will your readers. Now you have opportunity to correct that so please do.

Thank you for your candid feedback and for bringing potential contradictions in our manuscript to our attention. We sincerely appreciate your thorough review and the opportunity to address these issues before publication. We understand the importance of maintaining clarity and consistency in our work, as it directly impacts its credibility and our reputation as authors. We carefully reviewed the manuscript to identify and resolve the contradictions you have noticed.

Thank you once again for your constructive comments and for allowing us the chance to strengthen our manuscript.