Debaryomyces hansenii Enhances Growth, Nutrient Uptake, and Yield in Rice Plants (Oryza sativa L.) Cultivated in Calcareous Soil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

View letter

• The title is clear but could be more conciseto emphasize the primary findings.
• Line 18  Why emphasize phosphorus acquisition?
• Abstract  The relevant results lack concrete data support.
• Line 24The statement "Dh stimulated acid phosphatase activity in both P-sufficient and P-deficient treatments" lacks quantitative data. Specify whether the increase was statistically significant and provide relative activity levels.
• Line 52The sentence "rice belongs to the graminaceous group and partially relies on Strategy II Fe acquisition" is incomplete. Clarify the distinction between Strategy I and II mechanisms and why rice is vulnerable despite this.
• Line 123The root inoculation method mentions "1.5 L of a 107 cells/mL yeast suspension" but does not specify whether this volume was per pot or per plant. Clarify to ensure reproducibility.
• Table 1  Physical-chemical properties for the plant in the calcareous soil used is OK.
• Figure 1  1a maintains the same scale for ease of comparison.The statistical analysis mentions "repeated-measures ANOVA" but does not specify the post-hoc test used (e.g., Tukey’s HSD). The abbreviation used can be explained in the Materials and Methods section.
• Figure 2 Why not directly use mass for dry matter but instead use a ratio, and what does this ratio signify? The significance difference labeling in Figure 2b is not clearly understood.
• Figure 4  The significance labeling for Fe is missing letters. The significance labeling for Zn, Mn, and P does not seem entirely correct, please verify. Additionally, does this content data represent a single sampling period or the average across all sampling periods?
• Figure 5 The acid phosphatase activity assay uses BCIP but does not specify the incubation time or temperature. Can the concentration of this color be quantified and compared? Legend 7  9   11 data is 7  9  11 day?
• The significance difference labeling in Figure 6 is not entirely clear.
• Line 404Discussion section states that "Dh may act synergistically with the native soil microbiota," but the sterilized vs. non-sterilized soil data (Figure 2) show no significant difference in controls.
• Line 413Conclusion mentions "Dh upregulated the expression of genes associated with phosphorus acquisition" but does not specify whether this was observed under both P-sufficient and deficient conditions.
• This study investigates Debaryomyces hansenii as an inducing agent of growth, development and production in rice plants (Oryza sativa L.) grown in calcareous soil. However, flooding after rice planting can improve soil nutrient availability and buffer soil pH. The study would be strengthened by supplementing data on the availability of major nutrients during the cultivation process, as aboveground data alone are insufficient to support the relevant findings.
• Are there any data on soil microbial activity after vaccination?
• The experimental design of the manuscript is relatively complex, and the presentation of data and figures should emphasize logical consistency to facilitate readers' comprehension and understanding. Select soil and plant data closely related to this manuscript, while less important data can serve as supporting materials.
• The Conclusion need to be concise and streamlined.

Author Response

Dear Editor,

We would like to express our sincere gratitude for the opportunity to revise our manuscript and for the additional time granted to complete the revision process. We truly appreciate your support and understanding throughout the review.

We are also especially thankful for the thorough and constructive feedback provided by the reviewers. Their detailed comments and suggestions have significantly contributed to improving the quality and clarity of our work. We have carefully addressed each point raised, and we hope that the revised version meets the expectations of the journal.

Thank you again for your time and efforts.

Best regards,

Carlos Lucena (on behalf of all authors)

 

Reviewer 1

 

The title is clear but could be more concist to emphasize the primary findings.

Authors’ Response: We thank the reviewer for this suggestion. Following your advice, we have modified the title to make it more concise and outcome-oriented. The revised title now reads: “Debaryomyces hansenii enhances growth, nutrient uptake, and yield in rice cultivated in calcareous soil”, which more directly reflects the core findings of the study.

 

Line 18  Why emphasize phosphorus acquisition?

Authors’ Response: Thank you for your observation. We have reworded the sentence to better reflect the scope of the findings, highlighting the induction of acid phosphatase activity and related gene expression, rather than implying a clear increase in P uptake. The new sentence is: “Although leaf phosphorus levels were not significantly increased, Dh stimulated acid phosphatase activity and upregulated genes involved in phosphorus acquisition under both P-sufficient and P-deficient condition”

 

Abstract: The relevant results lack concrete data support.

Authors’ Response: Thank you for pointing this out. We have corrected the statement in the Abstract to reflect the actual observed increase in dry matter content (~4%) under sterilized soil conditions, ensuring alignment with the reported data.

 

Line 24The statement "Dh stimulated acid phosphatase activity in both P-sufficient and P-deficient treatments" lacks quantitative data. Specify whether the increase was statistically significant and provide relative activity levels.

Authors’ Response: Thank you for your comment. We have revised the Abstract to specify that the increase in acid phosphatase activity was qualitatively observed using BCIP staining, rather than measured through quantitative assays.

 

Line 52. The sentence "rice belongs to the graminaceous group and partially relies on Strategy II Fe acquisition" is incomplete. Clarify the distinction between Strategy I and II mechanisms and why rice is vulnerable despite this.

Authors’ Response: We appreciate this insightful comment. We have revised the sentence to include a brief explanation of both Strategy I and Strategy II mechanisms of iron acquisition, and highlighted the specific limitations of rice in this context. The updated text explains that rice produces lower amounts of phytosiderophores compared to other graminaceous species and has a limited Strategy I-like response, which together contribute to its susceptibility to Fe deficiency in calcareous soils.

 

Line 123. The root inoculation method mentions "1.5 L of a 107 cells/mL yeast suspension" but does not specify whether this volume was per pot or per plant. Clarify to ensure reproducibility.

Authors’ Response: We thank the reviewer for this observation. The method has been clarified to state that the bare roots of the plants were immersed in a 1.5 L suspension of yeast (10⁷ cells/mL in deionized water) under constant agitation for 30 minutes prior to transplanting. We hope this change improves clarity in the manuscript.

 

Table 1.  Physical-chemical properties for the plant in the calcareous soil used is OK.

Authors’ Response: thank the reviewer for this positive comment regarding Table 1.

 

Figure 1.  1a maintains the same scale for ease of comparison.The statistical analysis mentions "repeated-measures ANOVA" but does not specify the post-hoc test used (e.g., Tukey’s HSD). The abbreviation used can be explained in the Materials and Methods section.

Authors’ Response: We thank the reviewer for this helpful observation. We have revised the Statistical Analysis section to specify that Tukey’s HSD post-hoc test was used for agronomic, physiological, and biochemical parameters, while Dunnett’s test was used for gene expression analysis when comparing each treatment to the control. Additionally, we have clarified that different letters in the figures indicate statistically significant differences among treatments.

 

Figure 2 Why not directly use mass for dry matter but instead use a ratio, and what does this ratio signify? The significance difference labeling in Figure 2b is not clearly understood.

Authors’ Response: We thank the reviewer for this observation. Dry matter content was expressed as a percentage (dry weight/fresh weight × 100) because it serves as a physiological indicator of plant water status and allows comparisons across treatments with different initial biomass or water content. This approach helps to avoid potential biases introduced by absolute dry mass differences. We have clarified this rationale in the Materials and Methods section. Additionally, we have reviewed and corrected the significance labeling in Figure 2b for clarity and accuracy.

 

Figure 4  The significance labeling for Fe is missing letters. The significance labeling for Zn, Mn, and P does not seem entirely correct, please verify. Additionally, does this content data represent a single sampling period or the average across all sampling periods?

Authors’ Response: We thank the reviewer for this detailed observation. Following your suggestion, and in line with a similar comment from another reviewer, we carefully reviewed and corrected the significance lettering in Figure 4 for Fe, Zn, Mn, and P to ensure full consistency with the statistical results. Additionally, we have clarified in material and methods that these data correspond to a single sampling conducted at the end of the experimental period.

 

Figure 5 The acid phosphatase activity assay uses BCIP but does not specify the incubation time or temperature. Can the concentration of this color be quantified and compared? Legend 7  9   11 data is 7  9  11 day?

Authors’ Response: We thank the reviewer for these valuable suggestions. We have revised the Materials and Methods section to include the BCIP incubation conditions: 2 h at 22 °C (growth chamber temperature). As the assay was conducted for qualitative visualization, color intensity was evaluated visually and comparatively against the control plants. Additionally, the figure legend has been updated.

 

The significance difference labeling in Figure 6 is not entirely clear.

Authors’ Response: We appreciate this comment. We have clarified in the figure legend that gene expression values are presented relative to day 0, and that asterisks indicate statistically significant differences with respect to day 0, based on Dunnett’s test (p < 0.05). This clarification improves the accuracy and interpretability of Figure 6.

 

Line 404. Discussion section states that "Dh may act synergistically with the native soil microbiota," but the sterilized vs. non-sterilized soil data (Figure 2) show no significant difference in controls.

Authors’ Response: Thank you for pointing this out. We acknowledge that the current data do not provide evidence of synergistic effects between D. hansenii and native microbiota, as no significant differences were observed between sterilized and non-sterilized controls. We have revised the discussion to reflect this and now suggest that such interactions remain hypothetical and should be evaluated in future studies.

 

Line 413. Conclusion mentions "Dh upregulated the expression of genes associated with phosphorus acquisition" but does not specify whether this was observed under both P-sufficient and deficient conditions.

Authors’ Response: We appreciate the reviewer’s suggestion. In response to this comment and the next comment below, we have rewritten the Conclusion section to improve conciseness and clarity while retaining all key findings.

 

This study investigates Debaryomyces hansenii as an inducing agent of growth, development and production in rice plants (Oryza sativa L.) grown in calcareous soil. However, flooding after rice planting can improve soil nutrient availability and buffer soil pH. The study would be strengthened by supplementing data on the availability of major nutrients during the cultivation process, as aboveground data alone are insufficient to support the relevant findings.

Authors’ Response: We thank the reviewer for this important observation. To clarify the cultivation conditions, we have added a sentence in the Materials and Methods section indicating that pots were placed in trays with a constant water level to maintain the moisture requirements of rice without inducing full soil flooding. This clarification helps distinguish our experimental conditions from typical field flooding, and explains the expected limited effect on soil pH or nutrient solubility.

 

Are there any data on soil microbial activity after vaccination?

Authors’ Response: We thank the reviewer for this pertinent question. Although soil microbial activity was not presented in this manuscript, we would like to clarify that we have evaluated the persistence of D. hansenii in calcareous soils under various conditions in this and related studies. Our findings indicate that the yeast remains viable in the soil environment for approximately 21 days post-inoculation. However, we chose not to include this data here as they fall outside the main focus of the present work, which was centered on plant physiological and molecular responses to D. hansenii. Including detailed microbial survival dynamics would have required additional methodological description and discussion, potentially detracting from the manuscript’s central objectives.

 

The experimental design of the manuscript is relatively complex, and the presentation of data and figures should emphasize logical consistency to facilitate readers' comprehension and understanding. Select soil and plant data closely related to this manuscript, while less important data can serve as supporting materials.

Authors’ Response: We appreciate the reviewer’s observation. We acknowledge that the experimental design includes multiple treatments and conditions to capture both physiological and molecular responses in rice. However, we believe that each dataset presented provides complementary and essential insight into the role of D. hansenii. We have carefully revised the structure and flow of the Results and Figures to enhance clarity and logical progression, ensuring that all included data directly support the study’s objectives. We have also verified that figure legends are detailed and informative to help guide interpretation.

 

The Conclusion need to be concise and streamlined.

Authors’ Response: We appreciate the reviewer’s suggestion. In response, we have rewritten the Conclusion section to improve conciseness and clarity while retaining all key findings. The revised version merges and simplifies the final paragraphs to provide a more fluid summary of the main outcomes and the biotechnological potential of D. hansenii.

Reviewer 2 Report

Comments and Suggestions for Authors

General comments

The authors investigated the effects of Debaryomyces hansenii inoculation in calcareous soil and hydroponic system on the growth and physiology of rice plants. The following parameters were measured: chlorophyll content, weight, trace metal uptake, phosphatase activity, and gene expression regarding the activity. The results indicated that D. hansenii inoculation had a positive effect on rice growth and physiology under specific conditions.

Although various experiments were conducted in this study, it was concluded that the manuscript is not yet at a stage where they can be published as a scientific paper due to the following reasons.

 

Major comments

Comment 1.

The authors should reconsider the overall structure of the manuscript and revise it to ensure that it is suitable for publication as a scientific paper. The following points should be addressed (however, as it is not possible to list all points here, the authors should confirm that there are no similar points in the manuscript):

The order in which the content of the Materials and Methods section did not correspond to the results. The authors should describe the methods used in the experiments with calcareous soil (including measurements) first, followed by the methods used in the experiments with hydroponic system. In both experiments, the number of replicates (both the number of plants grown and the number of measurements taken, if different) should be explained.

 

P2: either a hydroponic system or calcareous soil

“calcareous soil” should be first (“calcareous soil or a hydroponic system”)

 

P3: capacity was reached.

2.4-2.6 should be written after this sentence.

 

The Results section includes discussion. (e.g., P6: “This increase in biomass … the rhizosphere level, P6: highlighting the…the yeast., P6: suggesting that the…as calcareous soils., P8: suggesting that this application … Fe uptake. P8: suggesting a possible … phosphorus dynamics.) These parts should be moved to the Discussion section.

 

On the other hand, the Discussion section contained little discussion. (The contents from “Rice plants  … as a biofertilizer” (P12) should be included in the Introduction. Most of the content on “Indeed, our results (P12) ...conditions (Figures 6), (P13)” was a repetition of the results. Additionally, there was an overinterpretation of the results (ignoring results that showed no effect). (See below and Comment 2)

 

P12: our results demonstrated … grain production (Figures 1, 2, and 3).

Results showing no effect were ignored and the results were overinterpreted the results. The results need to be discussed and explained along with the individual conditions under which significant effects were observed.

 

P12: Although no clear … in inoculated plants (Figure 4,

The figure for P content in Fig. 4 does not match the text.

 

The Conclusion section contained information that is not mentioned in the Discussion section.

 

 

Comment 2.

In the Results section, the authors need to completely revise their explanation of the results. There were instances of overinterpretation of results (ignoring results that showed no effect) and inconsistencies between figures and text. The authors should objectively interpret the results and explain them clearly in the text.

 

P5: Statistical analysis revealed significant differences between treatments. 

The authors need to provide specific details of the results (e.g., between non-sterile and sterile soil, between Control and Dh inoculation, and between Dh irrigation and Dh immersion). Additionally, the authors cannot ignore conditions where no significant differences were found. Furthermore, the authors must consider whether the changes were significantly increased or significantly decreased.

 

P6: Fig1b

I expected that each value would be the average SPAD value for the entire period under each condition. However, it is not reasonable to summarize the entire period. The authors should compare SPAD values between conditions with the same cultivation period. Information showing variability (such as standard deviation) is necessary. An explanation of the meaning of labels such as “NSS-Control” is needed in the legend of Fig 1 (like in the legend of Fig2).

 

P6: Fig. 1 onwards 

“inmersion” should be “immersion.” 

 

P6: The inoculation of ... calcareous soil. 

This sentence did not match those shown in Fig. 2. Based on Fig. 2, D. hansenii had no effect on rice growth under most conditions. Similar overinterpretations of results were found throughout the manuscript.

 

P6, This trend ... per plant (Figure 2b),

Since the results differ from those shown in Fig. 2a, the authors should explain the results individually.

 

P6, highlighting the … of the yeast.

Based on Fig. 2b, grain production decreased even with immersion in sterilized soil (SS-Control and SS-Dh Immersion). Therefore, this discussion is incorrect, and the authors need to correctly interpret the results.

 

P7, the tallest plants … calcareous soils.

In Fig. 3, Dh Immersion in NSS did not appear to be higher than Dh Irrigation in NSS. The authors should present quantitative data and perform statistical analysis.

 

P7,Fig3

The authors need to present quantitative data rather than photographs.

Based on photograph in Fig3, rice in Dh Immersion in SS was the tallest. The discrepancy between this result and the result in Fig. 2a (NSS-Dh immersion was large) needs to be explained in the Discussion section.

 

P8, A similar trend

P8, closely mirrored

The authors' recognition of “similar” and “closely mirrored” were unclear. Based on Fig. 2, the results for Fe, Zn, and Mn (patterns of effects according to conditions) were different. Therefore, the authors need to describe the results individually in clear sentences.

 

P8, Phosphorus (P) content … D. hansenii inoculation.

Based on Fig. 4, this statement was incorrect. SS-Dh-Irrigation and SS-Dh-immersion had significantly higher P content than the other four treatments.

 

P8, However, no significant … respective controls.

Based on Fig. 4, this statement was also incorrect. SS-Dh-Irrigation and SS-Dh-immersion had significantly higher P content than SS-Control.

 

P8, the lowest P concentrations  … with D. hansenii,

Based on Fig. 4, this statement was also incorrect. SS-Dh-Irrigation and SS-Dh-immersion have significantly higher P content than SS-Control. Referring to the P content graph in Fig. 4, I was unable to understand any of the statements in this paragraph regarding phosphorus (P) content. I may have fundamentally misread the graph or the text.

 

P10 Three sampling points … limiting conditions.

The evaluation of acid phosphatase activity was unclear, and I was unable to determine whether the differences described in the text were significant or not. The authors should present acid phosphatase activity as quantitative data rather than photographs.

 

P10 3.5. Effect of Debaryomyces … to Dunnett’s test (p < 0.001).

Due to insufficient information in the Materials and Methods section regarding gene expression analysis (P4-5 2.8 Gene Expression Analysis by qRT-PCR), I was unable to comment on the content described.

 

 

Comment 3.

In the Discussion section, the authors should discuss the conditions (non-sterilization and sterilization, Dh inoculation and non-inoculation, irrigation and immersion under the same soil conditions) under which positive or negative effects were observed individually (including conditions where no differences were observed, if applicable), along with the presumed causes. It is also necessary to clearly state what new discoveries were made in this study.

 

Minner comments

Abstract and entire manuscript

The author should standardize the notation of Debaryomyces hansenii to either “Dh” or “D. hansenii”. In my opinion, “D. hansenii” (initially “Debaryomyces hansenii”) is preferable in the main text. Since “Dh” was not used in the abstract, the “(Dh)” in “yeast Debaryomyces hansenii (Dh) on the growth” is unnecessary.

 

P1, …under certain conditions,

The authors should provide specific details.

 

P2, Similarly, certain strains

The authors should provide specific details (genus, species, and strain name).

 

P2, In addition … tolerance [24,25].

The authors should specify which plants they are referring to.

 

P2, a tray

P2, moist perlite

The authors should add information necessary for others to reproduce the experiment, such as manufacturer name, model number, and size.

 

P3, the role of Debaryomyces hansenii on crop yield.

Italics, “D. hansenii”

 

P3, For yeast inoculation … 2% peptone [28].

The authors should specify the culture temperature.

 

P5, RNA extraction … in Table 2.

The authors need to provide more details for others to replicate the results. This includes the RT-PCR program, internal standards, calculation method using day 0 as 1, method for calculating relative expression, and whether or not there was a control without D. hansenii infection (I understood from the results that there was a control without D. hansenii infection).

 

P8, As shown in Figures 8 and 9,

The authors need to correct the figure numbers.

 

P8, the highest concentrations … sterilized soil.

The authors compared two conditions (NSS-Control and SS-Control). Therefore, “highest” is grammatically incorrect.

 

P8, Fig4 Fe

Some of the labels indicating the statistical analysis were missing.

 

P9, As extensively described in the literature

References are required.

 

P10, “(data)” (many other places)

The authors' intent is unclear. The manuscript appears to be in progress.

Author Response

Dear Editor,

We would like to express our sincere gratitude for the opportunity to revise our manuscript and for the additional time granted to complete the revision process. We truly appreciate your support and understanding throughout the review.

We are also especially thankful for the thorough and constructive feedback provided by the reviewers. Their detailed comments and suggestions have significantly contributed to improving the quality and clarity of our work. We have carefully addressed each point raised, and we hope that the revised version meets the expectations of the journal.

Thank you again for your time and efforts.

Best regards,

Carlos Lucena (on behalf of all authors)

Reviewer 2

Comments and Suggestions for Authors

General comments

The authors investigated the effects of Debaryomyces hansenii inoculation in calcareous soil and hydroponic system on the growth and physiology of rice plants. The following parameters were measured: chlorophyll content, weight, trace metal uptake, phosphatase activity, and gene expression regarding the activity. The results indicated that D. hansenii inoculation had a positive effect on rice growth and physiology under specific conditions.

Although various experiments were conducted in this study, it was concluded that the manuscript is not yet at a stage where they can be published as a scientific paper due to the following reasons.

 

Major comments

Comment 1.

The authors should reconsider the overall structure of the manuscript and revise it to ensure that it is suitable for publication as a scientific paper. The following points should be addressed (however, as it is not possible to list all points here, the authors should confirm that there are no similar points in the manuscript):

The order in which the content of the Materials and Methods section did not correspond to the results. The authors should describe the methods used in the experiments with calcareous soil (including measurements) first, followed by the methods used in the experiments with hydroponic system. In both experiments, the number of replicates (both the number of plants grown and the number of measurements taken, if different) should be explained.

P2: either a hydroponic system or calcareous soil

“calcareous soil” should be first (“calcareous soil or a hydroponic system”)

P3: capacity was reached.

2.4-2.6 should be written after this sentence.

Authors’ Response: line 160 page 4. We fully agree with the reviewer and appreciate this valuable observation. In the revised version of the manuscript, we have reordered the Materials and Methods section. This change aligns the methodology section with the sequence of the results and improves the overall readability of the manuscript. Line 132 page 3 and Line 188 page 5. In addition, we have explicitly included the number of biological replicates and individual plants analyzed for each treatment in both experimental systems.

 

The Results section includes discussion. (e.g., P6: “This increase in biomass … the rhizosphere level, P6: highlighting the…the yeast., P6: suggesting that the…as calcareous soils., P8: suggesting that this application … Fe uptake. P8: suggesting a possible … phosphorus dynamics.) These parts should be moved to the Discussion section.

Authors’ Response: lines from 464 page 16 to line 601 page 19. We thank the reviewer for this valuable observation. As suggested, we have carefully reviewed the Results section and removed all interpretative or speculative statements that were previously included. These sentences have now been appropriately relocated to the Discussion section. Furthermore, we have taken this opportunity to significantly improve the Discussion, incorporating a more critical and detailed interpretation of the results, as recommended by both Reviewer 3 and the other reviewers.

 

 

On the other hand, the Discussion section contained little discussion. (The contents from “Rice plants  … as a biofertilizer” (P12) should be included in the Introduction. Most of the content on “Indeed, our results (P12) ...conditions (Figures 6), (P13)” was a repetition of the results. Additionally, there was an overinterpretation of the results (ignoring results that showed no effect). (See below and Comment 2)

Authors’ Response: line 93 page 2. We appreciate the reviewer’s critical feedback regarding the structure and content of the Discussion section. These changes and additional improvements have been carried out in accordance with the reviewer’s suggestions and, where applicable, the recommendations from the other reviewers, in order to enhance the scientific clarity, rigor, and structure of the manuscript.

 

P12: our results demonstrated … grain production (Figures 1, 2, and 3).

Results showing no effect were ignored and the results were overinterpreted the results. The results need to be discussed and explained along with the individual conditions under which significant effects were observed.

Authors’ Response: line 258 page 6, line 298 page 8 and line 334 page 10. We appreciate this important observation. In the revised version of the manuscript, the interpretation of growth-related results (Figures 1, 2, and 3) has been rewritten to clearly specify under which conditions significant effects were observed, and to avoid generalizations. Line 501 page 16. The discussion now explicitly states that the positive effects on physiological and yield parameters were particularly evident under non-sterilized soil conditions and when the yeast was applied via root immersion. Line 591 page 18. We have also incorporated discussion of the conditions where no significant differences were found, to provide a more balanced and objective interpretation of the data. These changes reflect the reviewer’s recommendations and aim to improve the scientific rigor and transparency of our conclusions.

 

P12: Although no clear … in inoculated plants (Figure 4, The figure for P content in Fig. 4 does not match the text.

Authors’ Response: line 551 page 18. Thank you for this helpful observation. We have now rewritten this part of the discussion to reflect the actual results more accurately.

 

The Conclusion section contained information that is not mentioned in the Discussion section.

Authors’ Response: line 608 page 19. We thank the reviewer for pointing out this inconsistency. In the revised version of the manuscript, we have ensured that all statements included in the Conclusion section are now fully supported by the content of the Discussion.

 

 

Comment 2.

In the Results section, the authors need to completely revise their explanation of the results. There were instances of overinterpretation of results (ignoring results that showed no effect) and inconsistencies between figures and text. The authors should objectively interpret the results and explain them clearly in the text.

Authors’ Response: line 258 page 6, line 299 page 8, 352 page 10. We thank the reviewer for this important observation. In response, we have carefully revised the entire Results section to ensure that all findings are described objectively and consistently with the data presented in the figures. Line 308 page 8. Interpretative statements previously included in this section have been removed or relocated to the Discussion.

 

P5: Statistical analysis revealed significant differences between treatments. 

The authors need to provide specific details of the results (e.g., between non-sterile and sterile soil, between Control and Dh inoculation, and between Dh irrigation and Dh immersion). Additionally, the authors cannot ignore conditions where no significant differences were found. Furthermore, the authors must consider whether the changes were significantly increased or significantly decreased.

Authors’ Response: line 258 page 6, line 299 page 8, 352 page 10. We fully agree with the reviewer’s suggestion and have revised the Results section accordingly.

 

P6: Fig1b

I expected that each value would be the average SPAD value for the entire period under each condition. However, it is not reasonable to summarize the entire period. The authors should compare SPAD values between conditions with the same cultivation period. Information showing variability (such as standard deviation) is necessary. An explanation of the meaning of labels such as “NSS-Control” is needed in the legend of Fig 1 (like in the legend of Fig2).

Authors’ Response: line 268 page 7. We thank the reviewer for this valuable observation. In response, we have removed Figure 1b from the manuscript. Instead, we now present the temporal dynamics of the SPAD index for each treatment throughout the experimental period in a single, integrated figure (now Figure 1). Line 281 page 8. In addition, the figure includes standard error bars to show variability, and the legend has been expanded to fully define all treatment abbreviations (e.g., NSS, SS, Control, Irrigation, Immersion), as previously recommended.

 

P6: Fig. 1 onwards “inmersion” should be “immersion.” 

Authors’ Response: line 268 page 7, line 322 page 9, line 328 page 9… etc. Thank you for your correction. We have revised this mistake in all figures and manuscript.

 

P6: The inoculation of ... calcareous soil. This sentence did not match those shown in Fig. 2. Based on Fig. 2, D. hansenii had no effect on rice growth under most conditions. Similar overinterpretations of results were found throughout the manuscript.

Authors’ Response: line 293 page 8. The sentence was corrected.

 

P6, This trend ... per plant (Figure 2b),Since the results differ from those shown in Fig.    2a, the authors should explain the results individually.

Authors’ Response: line 298 page 8. We modified the section according to reviewer´s suggestion.

 

P6, highlighting the … of the yeast. Based on Fig. 2b, grain production decreased even with immersion in sterilized soil (SS-Control and SS-Dh Immersion). Therefore, this discussion is incorrect, and the authors need to correctly interpret the results.

Authors’ Response: line 305 page 8. We modified the section according to reviewer´s suggestion.

 

P7, the tallest plants … calcareous soils. In Fig. 3, Dh Immersion in NSS did not appear to be higher than Dh Irrigation in NSS. The authors should present quantitative data and perform statistical analysis.

P7,Fig3 The authors need to present quantitative data rather than photographs. Based on photograph in Fig3, rice in Dh Immersion in SS was the tallest. The discrepancy between this result and the result in Fig. 2a (NSS-Dh immersion was large) needs to be explained in the Discussion section.

Authors’ Response: line 334 page 10. We thank the reviewer for this observation and apologize for the confusion. The sentence in question resulted from a translation error during the adaptation of the manuscript from Spanish to English. The intent was not to state a definitive conclusion about plant height, but rather to illustrate a general visual trend. However, we recognize that without supporting quantitative data, such statements are inappropriate. As indicated, no systematic measurements of plant height were recorded throughout the experiment; only a representative photograph (Figure 3) was taken at harvest to qualitatively document general differences in plant development across treatments.

 

P8, A similar trend, P8, closely mirrored. The authors' recognition of “similar” and “closely mirrored” were unclear. Based on Fig. 2, the results for Fe, Zn, and Mn (patterns of effects according to conditions) were different. Therefore, the authors need to describe the results individually in clear sentences.

Authors’ Response: line 360 page 11 and line 361 page 11. We fully agree with the reviewer’s observation. In the revised version of the manuscript, we have removed vague expressions such as “a similar trend” and now describe the foliar concentrations of Fe, Zn, and Mn individually and explicitly.

 

P8, Phosphorus (P) content … D. hansenii inoculation. Based on Fig. 4, this statement was incorrect. SS-Dh-Irrigation and SS-Dh-immersion had significantly higher P content than the other four treatments.

Authors’ Response: line 365 page 11. We have corrected according to reviewer´s comment.

 

P8, However, no significant … respective controls. Based on Fig. 4, this statement was also incorrect. SS-Dh-Irrigation and SS-Dh-immersion had significantly higher P content than SS-Control.

Authors’ Response: line 365 page 11. We have corrected according to reviewer´s comment.

 

P8, the lowest P concentrations  … with D. hansenii, Based on Fig. 4, this statement was also incorrect. SS-Dh-Irrigation and SS-Dh-immersion have significantly higher P content than SS-Control. Referring to the P content graph in Fig. 4, I was unable to understand any of the statements in this paragraph regarding phosphorus (P) content. I may have fundamentally misread the graph or the text.

Authors’ Response: line 365 page 11. We have corrected according to reviewer´s comment.

 

P10 Three sampling points … limiting conditions. The evaluation of acid phosphatase activity was unclear, and I was unable to determine whether the differences described in the text were significant or not. The authors should present acid phosphatase activity as quantitative data rather than photographs.

Authors’ Response: line 415 page 14. We thank the reviewer for this important observation. In this study, the evaluation of acid phosphatase activity was conducted using a qualitative colorimetric assay based on BCIP staining as previous publications did before us. As shown in Figure 5, the assay reveals differences in staining intensity among treatments, which visually reflect enzyme activity in the root zone under P-sufficient and P-deficient conditions at different time points. However, we acknowledge that no quantitative measurements (e.g., absorbance or enzyme units) were obtained. Line 219 page 5. This has now been clarified in the Materials and Methods and Results sections, and we have revised the text to ensure that no statistical claims are made based on these qualitative observations. We appreciate the reviewer’s comment and recognize this as a limitation of the current study.

 

P10 3.5. Effect of Debaryomyces … to Dunnett’s test (p < 0.001). Due to insufficient information in the Materials and Methods section regarding gene expression analysis (P4-5 2.8 Gene Expression Analysis by qRT-PCR), I was unable to comment on the content described.

Authors’ Response: lines from 227 to 245 page 6. We appreciate the reviewer’s observation and apologize for the lack of detail in the original submission. In response, we have substantially revised and expanded the Gene Expression Analysis section in the Materials and Methods to provide the necessary methodological information. This now includes additional details on the RNA extraction procedure, cDNA synthesis, qRT-PCR thermal cycling conditions, reference gene selection, and the method used for relative expression analysis. We hope this additional information will allow for a more thorough evaluation of our gene expression data and improve the reproducibility of the experiment.

 

Comment 3.

In the Discussion section, the authors should discuss the conditions (non-sterilization and sterilization, Dh inoculation and non-inoculation, irrigation and immersion under the same soil conditions) under which positive or negative effects were observed individually (including conditions where no differences were observed, if applicable), along with the presumed causes. It is also necessary to clearly state what new discoveries were made in this study.

Authors’ Response: Line from 464 page 16 to line 601 page 19. lWe have modified the discussion according to your suggestions and other reviewers’ suggestions.

Minner comments

Abstract and entire manuscript

The author should standardize the notation of Debaryomyces hansenii to either “Dh” or “D. hansenii”. In my opinion, “D. hansenii” (initially “Debaryomyces hansenii”) is preferable in the main text. Since “Dh” was not used in the abstract, the “(Dh)” in “yeast Debaryomyces hansenii (Dh) on the growth” is unnecessary.

 P1, …under certain conditions,

The authors should provide specific details.

Authors’ Response: line 17 page 1, line 2o page 20… etc. Thank you for your comment. We have standardized the abstract.

 

P2, Similarly, certain strains

The authors should provide specific details (genus, species, and strain name).

Authors’ Response: line 78 page 2. We have modified this sentence according to the references.

 

P2, In addition … tolerance [24,25] The authors should specify which plants they are referring to.

Authors’ Response: line 80 page 2. We have added the microorganisms that the references used. 

 

P2, a tray. P2, moist perlite. The authors should add information necessary for others to reproduce the experiment, such as manufacturer name, model number, and size.

Authors’ Response: We appreciate the reviewer’s suggestion regarding the need to specify the manufacturer, model, and characteristics of the perlite used. However, the perlite substrate was provided in bulk by the technical services of our university, as is standard practice in many agronomy departments. Unfortunately, no commercial packaging or labeling was available at the time of use, and we were unable to retrieve precise information regarding the brand, model, or grain size from the university’s supply records. Despite our efforts to obtain these details retrospectively, no specific documentation could be retrieved.

 

P3, the role of Debaryomyces hansenii on crop yield. Italics, “D. hansenii”

Authors’ Response: line 116 page 3. Thank you for your detail revision. We have modified this mistake.

 

P3, For yeast inoculation … 2% peptone [28]. The authors should specify the culture temperature.

Authors’ Response: line 126 page 3. We have added this information to the manuscript.

 

P5, RNA extraction … in Table 2. The authors need to provide more details for others to replicate the results. This includes the RT-PCR program, internal standards, calculation method using day 0 as 1, method for calculating relative expression, and whether or not there was a control without D. hansenii infection (I understood from the results that there was a control without D. hansenii infection).

Authors’ Response: lines from 227 to 245 page 6. We appreciate the reviewer’s request for further methodological details. We have revised Section 2.8 to include the full qRT-PCR amplification protocol, the method used to calculate relative expression (2^–ΔΔCt), and more clarifications to ensure the reproducibility and clarity of our gene expression methodology.

 

P8, As shown in Figures 8 and 9, The authors need to correct the figure numbers.

Authors’ Response: line 344 page 10. We apologize for these mistakes. We have corrected in the manuscript.

 

P8, the highest concentrations … sterilized soil. The authors compared two conditions (NSS-Control and SS-Control). Therefore, “highest” is grammatically incorrect.

Authors’ Response: line 347 page 10. Thank you for pointing out this grammatical issue. We agree that “highest” is not appropriate when comparing only two conditions.

 

P8, Fig4 Fe Some of the labels indicating the statistical analysis were missing.

Authors’ Response: line 376 page 12. Thank you for you detailed revision. We have corrected the missing labels.

 

P9, As extensively described in the literature References are required.

Authors’ Response: line 391 page 13. We have added references to this sentence.

 

P10, “(data)” (many other places) The authors' intent is unclear. The manuscript appears to be in progress.

Authors’ Response: line 396 page 14. We appreciate the reviewer’s observation. In the original manuscript, the term “(data)” was used as a shorthand to indicate “days after treatment application.” However, we understand that this notation was unclear and could be mistaken for a placeholder or incomplete statement. To improve clarity and readability, we have now explained the meaning of (data) with the explicit phrase “days after treatment application”.  

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, this manuscript is well presented. However, some concerns should be addressed before the acceptance for publication.

Abstract

“Foliar concentrations of Cu, Fe, Zn, and Mn also increased under certain conditions”. This sentence is unclear; the reader doesn't know under what conditions.

Introduction

It seems that there have already been many studies on the effect of plant growth-promoting yeasts on rice growth. What is the novelty of your research, or what new insights does it provide? More statements regarding the novelty should be added in the last paragraph.

As the yeast cultures contain a large of C and N that may promote microbial activity and nutrient availability, I think it is best to include a control with sterilized inoculum in the experimental design to distinguish between the effects of the nutrients and the microorganisms in the inoculum.

Figure 1, the rice plant diagram in the background looks like wheat. Please modify it or delete it.

Figure 2 and also others, the significance markers of the ANOVAs are incorrect. They are generally supposed to be labeled starting from the highest value.

Discussion

Paragraph 2, these statements such as plant growth-promoting bacteria (PGPB), arbuscular mycorrhizal fungi (AMF), are irrelevant to the results. Please conduct an in-depth analysis based on the present results.

Paragraph 3, these statements are not a discussion! They are the same as those in the introduction.

Paragraph 4, these statements are not a discussion either! The authors should explain the reasons.

As the POlsen is 13.4 in the presented soil, the reason why yeast did not increase the phosphorus content in rice may be that the available phosphorus in the soil is not deficient.

Author Response

Dear Editor,

We would like to express our sincere gratitude for the opportunity to revise our manuscript and for the additional time granted to complete the revision process. We truly appreciate your support and understanding throughout the review.

We are also especially thankful for the thorough and constructive feedback provided by the reviewers. Their detailed comments and suggestions have significantly contributed to improving the quality and clarity of our work. We have carefully addressed each point raised, and we hope that the revised version meets the expectations of the journal.

Thank you again for your time and efforts.

Best regards,

Carlos Lucena (on behalf of all authors)

Reviewer 2

 

Comments and Suggestions for Authors

Overall, this manuscript is well presented. However, some concerns should be addressed before the acceptance for publication.

Abstract

“Foliar concentrations of Cu, Fe, Zn, and Mn also increased under certain conditions”. This sentence is unclear; the reader doesn't know under what conditions.

Authors’ Response: We thank the reviewer for this helpful comment. To address the ambiguity, we have revised the sentence in the abstract to clearly specify the conditions under which the increases in foliar micronutrients were observed. This clarification enhances the accuracy and readability of the abstract.

 

Introduction

It seems that there have already been many studies on the effect of plant growth-promoting yeasts on rice growth. What is the novelty of your research, or what new insights does it provide? More statements regarding the novelty should be added in the last paragraph.

Authors’ Response: We thank the reviewer for pointing out the need to better highlight the novelty of our study. To address this, we have revised the last paragraph of the Introduction to clarify the original contributions of our research. Specifically, we emphasize that this is the first study to assess the effects of D. hansenii strain CBS767 on rice plants cultivated in calcareous soil, integrating physiological, biochemical, and molecular data. We also note that our study is the first to report the upregulation of the OsPAP3, OsPAP9, and OsPHT1;6 genes in rice following yeast inoculation.

 

As the yeast cultures contain a large of C and N that may promote microbial activity and nutrient availability, I think it is best to include a control with sterilized inoculum in the experimental design to distinguish between the effects of the nutrients and the microorganisms in the inoculum.

Authors’ Response: We appreciate the reviewer’s thoughtful suggestion. Indeed, we recognize that including a control with sterilized inoculum could help differentiate between the effects of the yeast’s metabolic activity and the potential contribution of nutrient residues in the inoculum. While this control was not included in the present study, we attempted to minimize potential nutrient carryover by thoroughly washing roots before inoculation and by using sterile deionized water to prepare the inoculum suspensions. Additionally, the yeast was cultured separately and diluted to 10⁷ cells/mL in water without nutrient medium prior to application. Therefore, we consider that the contribution of residual C or N from the original growth medium was negligible. Nonetheless, we agree that future experiments should consider this important control to further dissect the mechanistic contributions of microbial metabolites versus active colonization.

 

Figure 1, the rice plant diagram in the background looks like wheat. Please modify it or delete it.

Authors’ Response: We thank the reviewer for pointing out this mistake. You are absolutely right—the background illustration in Figure 1a was indeed more representative of wheat than rice. We have now corrected the figure by removing the background diagram to avoid any confusion. We appreciate your attention to detail, which helped us improve the accuracy and clarity of the visual presentation.

 

Figure 2 and also others, the significance markers of the ANOVAs are incorrect. They are generally supposed to be labeled starting from the highest value.

Authors’ Response: We appreciate the reviewer’s thoughtful correction. We have changed in figure´s manuscript.

 

Discussion

Paragraph 2, these statements such as plant growth-promoting bacteria (PGPB), arbuscular mycorrhizal fungi (AMF), are irrelevant to the results. Please conduct an in-depth analysis based on the present results.

Authors’ Response: We thank the reviewer for the helpful suggestion. We have revised the second paragraph of the Discussion to retain only the most relevant background on beneficial microorganisms, while integrating a detailed interpretation of our findings with D. hansenii. The revised version focuses on the physiological and nutritional improvements observed in the present study and relates them to known microbial mechanisms of action.

 

Paragraph 3, these statements are not a discussion! They are the same as those in the introduction.

Authors’ Response: We thank the reviewer for pointing this out. We have revised Paragraph 3 of the Discussion to remove redundant introductory content and to incorporate a more detailed analysis of the results. The new version discusses the observed improvements in growth and yield traits in rice inoculated with D. hansenii, and how these findings support the yeast’s biotechnological potential under calcareous and saline soil conditions.

 

Paragraph 4, these statements are not a discussion either! The authors should explain the reasons.

Authors’ Response: We thank the reviewer for pointing out the need for further analysis. We have revised Paragraph 4 of the Discussion to move beyond the summary of results and to explore the possible physiological and microbiological mechanisms behind the observed growth promotion. In particular, we now discuss how the application method, microbial interactions, and potential effects on nutrient uptake may explain the increase in chlorophyll content and yield.

 

As the POlsen is 13.4 in the presented soil, the reason why yeast did not increase the phosphorus content in rice may be that the available phosphorus in the soil is not deficient.

Authors’ Response: We thank the reviewer for this helpful interpretation. In accordance with the reviewer’s suggestion, we have added a new explanatory paragraph in the Discussion section.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

View letter

1. Line 39 challenging edaphic environments. What does this mean?
2. Fig.4 The ss.Dh Irrigation of the Zn diagram should exhibit significant differences. The NSS-Contro of the Mn diagram should show differences.
3. Fig.5The depth of blue can be quantitatively analyzed and characterized?
4. Fig.6 The labeling of significant differences remains consistent with the preceding figures.

Author Response

Reviewer 1

1. Comment: Line 39 challenging edaphic environments. What does this mean?

Author´s Response: Thank you for your observation. To improve clarity and avoid overly technical terminology, we have rephrased this sentence. The revised version now reads: “contributing to sustainable agricultural practices in calcareous soils and other nutrient-limiting environments.” This change specifies the type of soil studied and clarifies the meaning for a broader readership.

1. Comment: Fig.4 The ss.Dh Irrigation of the Zn diagram should exhibit significant differences. The NSS-Contro of the Mn diagram should show differences.

Author´s Response: Thank you very much for your valuable observation. We have carefully re-examined the statistical analyses for Zn and Mn content and found that some significance markers required correction. The figure has now been revised accordingly to reflect the accurate statistical differences. We appreciate your attention to detail.

1. Comment: Fig.5The depth of blue can be quantitatively analyzed and characterized?

Author´s Response: Thank you for this thoughtful suggestion. In this study, acid phosphatase activity was evaluated using BCIP staining as a qualitative method to visually compare treatment effects, as commonly reported in the literature. This approach allows the detection of relative changes in enzyme activity based on the presence and intensity of blue coloration. However, since the method was not designed for quantitative measurement, it is not appropriate to extract numerical data from it. In other words, it is not possible to accurately quantify a parameter derived from a qualitative assay

4. Comment:6 The labeling of significant differences remains consistent with the preceding figures.

Author´s response: Thank you for your comment. We are not entirely certain about the intent of this observation, but we would like to clarify the rationale behind the use of a different labeling style in Figure 6. Unlike Figures 2 and 4, which involve comparisons among multiple treatments and were analyzed using Tukey’s HSD test (with different letters indicating significant differences), Figure 6 focuses exclusively on comparing gene expression levels between each treatment and its respective control. For this reason, we used Dunnett’s test, which is specifically designed for such comparisons. To reflect this, we chose to indicate statistical significance using asterisks, which is a commonly accepted format for this type of analysis. We believe this approach ensures clarity for readers and aligns with standard practices in molecular data presentation.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded appropriately to the reviewers' comments and revised the manuscript thoroughly. Therefore, it was concluded that the current manuscript is acceptable for publication.

Author Response

Reviewer 2

Comment: The authors responded appropriately to the reviewers' comments and revised the manuscript thoroughly. Therefore, it was concluded that the current manuscript is acceptable for publication.

Author´s response: Thank you very much for your kind comment. It has been a pleasure to address your corrections and suggestions in order to optimize the manuscript.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The statistical label in Figure 1b is inconsistent with the others.

Author Response

Reviewer 3

Comment: The statistical label in Figure 1b is inconsistent with the others.

Author´s response: Thank you for your comment. We would clarify that Figure 1b is no longer included in the current version of the manuscript. In a previous review round, another reviewer suggested that showing only the temporal evolution of SPAD values would be more informative than presenting an average comparison. We agreed with this recommendation and accordingly removed Figure 1b.