Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, entitled "Eco-Efficient Intensification of Potatoes with Bacillus subtilis and Trichoderma viride under NPK Fertilization", publishes the results of a study on the effects of introducing bacterial and fungal inoculates into potato fertilizers in high-altitude areas. The research is of practical interest, as it is aimed at solving the applied issue of increasing yields and reducing fertilizer costs. The text is written correctly, the methods are generally suitable, the conclusions are confirmed by the results. The number of references to literary sources is sufficient, the graphic design is acceptable, but it is necessary to rewrite the captions to the figures in order to facilitate the perception of the material. It is recommended to accept the article after minor changes.

1) Formal remarks on the design of the article. Lines 81-82. The title is separated from the text of the chapter. Line 110. The contents of the table are divided into two pages. Pages 8 and 10. There is too much free space at the end of the page that is not occupied by the text. Lines 34, 327 and 328. Latin names are written in the usual font. Chapter numbering error: there is no sub-chapter number 3.7. The font of the captions to the figures differs from the font of the text of the article.

2) Lines 25-26. The annotation uses the symbol "m2n2", which has not been deciphered. This short designation is convenient when signing samples, but the text of the article should be written without abbreviations. The same goes for the numerous figures in the article. It is advisable to change these designations to more visual and well-perceived by readers.

3) Line 52. "Plant growth–promoting microorganisms (PGPM) offer a viable alternative". It is advisable to rephrase. Perhaps it would be better to start the sentence with the phrase: Using Plant growth–promoting microorganisms (PGPM) can be an alternative...

4) Lines 148-150. "Agricultural oil was added to each solu-148 tion to protect the microorganisms from solar radiation, and molasses was included as a 149 carbon source." In this case, an experimental variant was needed, in which spraying with the same oil and molasses solution was carried out, but without bacteria.

5) Lines 166-167. "Vigor was scored visually on a nine-point ordinary scale..." Are there any examples of using this indicator in the scientific literature? It is advisable to provide a link.

6) When the authors use the term "alternative", it seems that bacteria may replace fertilizers in the future. This idea seems to be quite controversial. Plants need sources of nitrogen, phosphorus and other elements. These needs cannot be met by microbial biomass. Bacteria can help assimilate some nutritional sources, but they themselves are not such sources. This was confirmed by the results – the best yield is achieved by combining bacterial inoculum with fertilizers. This suggests that bacilli can be effectively combined with fertilizers, but not replaced by one another. It can only be a partial replacement.

Author Response

Author´s Reponse to the Reviewer 1

Manuscript ID: applmicrobiol-3849622

Title: Eco-Efficient Intensification of Potato with Bacillus subtilis and Trichoderma viride under NPK Fertilization

Journal: Applied Microbiology

Dear Reviewer,

We sincerely thank you for your careful reading of our manuscript and for providing constructive comments and suggestions. Your feedback has greatly contributed to improving the clarity, reproducibility, and scientific rigor of our study. Below, we provide point-by-point responses to each of your comments. All changes have been incorporated into the revised manuscript, where they are highlighted for easy reference.

Comment 1: 1) Formal remarks on the design of the article. Lines 81-82. The title is separated from the text of the chapter. Line 110. The contents of the table are divided into two pages. Pages 8 and 10. There is too much free space at the end of the page that is not occupied by the text. Lines 34, 327 and 328. Latin names are written in the usual font. Chapter numbering error: there is no sub-chapter number 3.7. The font of the captions to the figures differs from the font of the text of the article.

Answer 1: We have checked carefully all suggestions and we have modified the document with the corrections.

Comment 2: 2) Lines 25-26. The annotation uses the symbol "m2n2", which has not been deciphered. This short designation is convenient when signing samples, but the text of the article should be written without abbreviations. The same goes for the numerous figures in the article. It is advisable to change these designations to more visual and well-perceived by readers.

Answer 2: We have checked carefully all suggestions and we have modified the document with the corrections highlighted with turquoise color.

Comment 3: 3) Line 52. "Plant growth–promoting microorganisms (PGPM) offer a viable alternative". It is advisable to rephrase. Perhaps it would be better to start the sentence with the phrase: Using Plant growth–promoting microorganisms (PGPM) can be an alternative...

Answer 3: Thank you for the suggestion. We rephrased the sentence to begin with “Using plant growth–promoting microorganisms (PGPM) can be an alternative to conventional high-input management based on mineral fertilization and chemical crop protection” and lightly edited the paragraph for clarity and style while preserving the original meaning and citations.

Comment 4: 4) Lines 148-150. "Agricultural oil was added to each solution to protect the microorganisms from solar radiation, and molasses was included as a carbon source." In this case, an experimental variant was needed, in which spraying with the same oil and molasses solution was carried out, but without bacteria.

Answer 4: Thank you for the thoughtful comment. In our protocol, agricultural oil and molasses were used exclusively as adjuvants in the inoculated suspensions to enhance microbial viability and facilitate application (sticker/spreader function, improved wetting, and early carbon supply). Because there are no propagules to protect or deliver in the uninoculated treatment, we did not apply the oil–molasses carrier to that control. We agree that including a carrier-only spray would help isolate any potential adjuvant effects; due to plot and logistical constraints in this field season, such a variant was not implemented. We have revised the Methods to explicitly clarify the selective use of adjuvants with inoculants. We plan to incorporate this variant in future trials.

Comment 5:  Lines 166-167. "Vigor was scored visually on a nine-point ordinary scale..." Are there any examples of using this indicator in the scientific literature? It is advisable to provide a link.

Answer 5: Thank you for the thoughtful comment. Yes, Chang et al. (2022) used an ordinal scale from 1 to 9 to evaluate plant vigor by focusing on plant size (plant width and height), with 1 being the least vigorous and 10 the most. It could be revised in the following link: https://doi.org/10.1007/s11032-022-01296-7

Comment 6:  When the authors use the term "alternative", it seems that bacteria may replace fertilizers in the future. This idea seems to be quite controversial. Plants need sources of nitrogen, phosphorus and other elements. These needs cannot be met by microbial biomass. Bacteria can help assimilate some nutritional sources, but they themselves are not such sources. This was confirmed by the results – the best yield is achieved by combining bacterial inoculum with fertilizers. This suggests that bacilli can be effectively combined with fertilizers, but not replaced by one another. It can only be a partial replacement.

Answer 6: Thank you for this important clarification. We agree that plant growth–promoting microorganisms (PGPM) are not nutrient sources and should not be presented as stand-alone replacements for mineral fertilizers. Our intention was to emphasize their role in improving fertilizer-use efficiency and enabling partial substitution of mineral inputs under specific conditions. We have revised the manuscript to replace the term “alternative” with “complementary strategy” and we now explicitly state that the highest yields were obtained when inoculants were combined with mineral fertilizers, while inoculants with 50% of the recommended NPK sustained comparable performance for several traits in our field context. We also added text in the Discussion and Conclusions underscoring that PGPM function by enhancing nutrient availability and plant performance, not by supplying nutrients themselves, and thus are best deployed within integrated nutrient management frameworks.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

Thank you for your great work, please consider the following comments:

1- Soil physicochemical properties section (2.2):

The description of the soil analyses is useful and well supported by citations. However, to ensure transparency and reproducibility, it is strongly recommended that the authors provide more detail on the equipment used (brand and model) for each parameter measured (e.g., pH meter, conductivity meter, spectrophotometer/ICP for P and K, Kjeldahl apparatus or CHN analyzer for total nitrogen).

Including this information will allow readers to better evaluate the accuracy of the measurements and

2- The inoculation procedure is well described in terms of strain identity, formulation, dosage, and application method. However, I recommend the authors to clarify whether the microbial dose (20 g Bacillus and 30 g Trichoderma per 15 L) was chosen based on manufacturer recommendations, previous experimental trials, or local practice. This would help readers understand the rationale behind the selected inoculum rates and whether they represent optimal, standard, or experimental dosages.

Additionally, since the inoculation was performed only once, it would be useful to briefly justify this choice (e.g., constraints, cost, or evidence that a single application is effective).

 

3- The methodology clearly states that agricultural oil and molasses were added to the inoculum suspension to protect microorganisms and provide a carbon source. However, it is important to specify the exact type and commercial source (company/brand) of both the agricultural oil and the molasses used.

Such details are essential for reproducibility, as different formulations or sources may vary in composition and could influence microbial viability and performance.

4- The methodology indicates that microbial inoculants (Bacillus subtilis and Trichoderma viride) were applied only once, immediately before covering the seed tubers with soil. Could the authors clarify why they chose to apply the inoculants only at this stage, and not at later growth stages (e.g., during vegetative growth or tuber bulking)?

A brief justification would help readers understand whether this decision was based on:

• previous experimental evidence,
• manufacturer recommendations,
• cost/resource constraints, or
• the assumption that a single pre-plant application is sufficient.

5- The monitoring of crop performance is generally well described. However, I would encourage the authors to clarify the following points:

• Why were the phenological checkpoints limited to 30, 60, 75, and 120 DAS? Including a justification for these choices (e.g., alignment with cultivar growth stages or resource limitations) would be helpful.
• For plant height and root biomass, the number of sampled plants varied (n = 7–10). Could the authors explain the reason for this variability and whether a fixed sample size was considered?
• Since measurements were taken from the central row only, did the authors consider potential border effects? It would be helpful if the authors could clarify whether border plants were excluded from sampling in order to avoid edge-related growth advantages. This would strengthen the reliability of the reported plant height, vigor, and root biomass measurements.
• For the vigor scoring, was the assessment carried out by one or multiple evaluators to reduce observer bias?

6- Regarding plant vigor, you indicate that it was visually scored on a nine-point ordinal scale (3 = weak, 5 = moderate, 7 = vigorous, 9 = very vigorous). Could you please provide a reference for this scale (e.g., a standard agronomic evaluation protocol or previous studies using this method)? And why you choose 60 and 75 days ?

7- for result section add table show the numbers

8- I recommend that the authors include a short “Future Recommendations” section in the conclusion or discussion.

regards

Author Response

Author´s Reponse to the Review Report

Manuscript ID: applmicrobiol-3849622

Title: Eco-Efficient Intensification of Potato with Bacillus subtilis and Trichoderma viride under NPK Fertilization

Journal: Applied Microbiology

Dear Reviewer,

We sincerely thank you for your careful reading of our manuscript and for providing constructive comments and suggestions. Your feedback has greatly contributed to improving the clarity, reproducibility, and scientific rigor of our study. Below, we provide point-by-point responses to each of your comments. We have checked carefully all suggestions and we have modified the document with the corrections highlighted with turquoise color.

Comment 1. - Soil physicochemical properties section (2.2):

The description of the soil analyses is useful and well supported by citations. However, to ensure transparency and reproducibility, it is strongly recommended that the authors provide more detail on the equipment used (brand and model) for each parameter measured (e.g., pH meter, conductivity meter, spectrophotometer/ICP for P and K, Kjeldahl apparatus or CHN analyzer for total nitrogen).

Including this information will allow readers to better evaluate the accuracy of the measurements and

Response 1. We appreciate this suggestion. We have added details of the equipment (brand and model) used for each soil parameter  

Change in manuscript: Section 2.2 (Soil physicochemical properties, page 4, paragraph 2, lines 114-125).

“To ensure accuracy, analyses were performed with certified equipment. Soil pH and electrical conductivity were measured using a pH meter (WTW INOLAB, model pH 7310) and conductivity meter (WTW INOLAB, model Cond 7310), respectively. Organic matter content was quantified with a digital burette (BDeco, model DCB5000). Soil texture was determined using a hydrometer (THERMCO ASTM, model 152H) and a mechanical dispenser (Hamilton Bech, model HMD400). Total nitrogen was measured employing a digestion system (BERT, model Heizblack K24) and distillation equipment (Wasserdam, model Destillere 52). Available phosphorus was quantified colorimetrically using a spectrophotometer (Thermo Scientific, model Genesys 150), while exchangeable bases and potassium were determined with an atomic emission spectrophotometer (Agilent Technologies, model 4210). Mass determinations were carried out with an analytical balance (Bell, model M214Ai) (Table 1).”

Comment 2. The inoculation procedure is well described in terms of strain identity, formulation, dosage, and application method. However, I recommend the authors to clarify whether the microbial dose (20 g Bacillus and 30 g Trichoderma per 15 L) was chosen based on manufacturer recommendations, previous experimental trials, or local practice. This would help readers understand the rationale behind the selected inoculum rates and whether they represent optimal, standard, or experimental dosages.

Additionally, since the inoculation was performed only once, it would be useful to briefly justify this choice (e.g., constraints, cost, or evidence that a single application is effective).

Response 2. Thank you for pointing this out. We confirm that the inoculum rates correspond to the manufacturer’s recommended doses. We have clarified this in the revised manuscript and added a justification for the single application, highlighting that this approach is effective according to previous studies and represents a practical and sustainable strategy.

Change in manuscript: Section 2.6 (Microbial inoculation procedure, page 6, paragraph 1, lines 182-186):

 

“This application was performed only once, immediately prior to covering the rows with soil, following the manufacturer’s recommendation for both inoculants. A single pre-sowing treatment was chosen because previous studies have shown that seed or tuber inoculation with PGPR such as Bacillus subtilis and biocontrol fungi such as Trichoderma viride can be effective with a single intervention”

Comment 3. The methodology clearly states that agricultural oil and molasses were added to the inoculum suspension to protect microorganisms and provide a carbon source. However, it is important to specify the exact type and commercial source (company/brand) of both the agricultural oil and the molasses used.

Such details are essential for reproducibility, as different formulations or sources may vary in composition and could influence microbial viability and performance.

Response 3. We agree with this valuable suggestion. We have specified the brand and supplier of the agricultural oil and molasses.

Change in manuscript: Section 2.6 (Microbial inoculation procedure, page 6, paragraph 1, lines 175 -179):

 

“Agricultural oil (Wett Oil; Comercial Andina Industrial S.A.C., Peru) was added to each solution as a spreader/sticker to improve adhesion and reduce desiccation/UV exposure, and molasses (PROBIOLSUR; Productos Biológicos del Sur S.A.C., Peru) was included as a carbon source only to the treatments containing microbial propagules.”

Comment 4. The methodology indicates that microbial inoculants (Bacillus subtilis and Trichoderma viride) were applied only once, immediately before covering the seed tubers with soil. Could the authors clarify why they chose to apply the inoculants only at this stage, and not at later growth stages (e.g., during vegetative growth or tuber bulking)?

A brief justification would help readers understand whether this decision was based on:

• previous experimental evidence,
• manufacturer recommendations,
• cost/resource constraints, or
• the assumption that a single pre-plant application is sufficient.

Response 4. We thank the reviewer for this comment. We have clarified that the decision was based primarily on the manufacturer’s recommendation. In addition, we highlighted supporting literature indicating that a single pre-sowing inoculation of seed tubers with PGPR and Trichoderma can be effective. We also note that this approach reflects resource-efficient practices suitable for highland farming systems.

Change in manuscript: Section 2.6 (page 6, paragraph 1, lines 182-188):

This application was performed only once, immediately prior to covering the rows with soil, following the manufacturer’s recommendation for both inoculants. A single pre-sowing treatment was chosen because previous studies have shown that seed or tuber inoculation with PGPR such as Bacillus subtilis and biocontrol fungi such as Trichoderma viride can be effective with a single intervention [43]. This approach also reflects the objective of reducing production costs while promoting sustainable agriculture and enhancing the natural fertility of soils [44,44,46].

Comment 5.  The monitoring of crop performance is generally well described. However, I would encourage the authors to clarify the following points:

• Why were the phenological checkpoints limited to 30, 60, 75, and 120 DAS? Including a justification for these choices (e.g., alignment with cultivar growth stages or resource limitations) would be helpful.
• For plant height and root biomass, the number of sampled plants varied (n = 7–10). Could the authors explain the reason for this variability and whether a fixed sample size was considered?
• Since measurements were taken from the central row only, did the authors consider potential border effects? It would be helpful if the authors could clarify whether border plants were excluded from sampling in order to avoid edge-related growth advantages. This would strengthen the reliability of the reported plant height, vigor, and root biomass measurements.
• For the vigor scoring, was the assessment carried out by one or multiple evaluators to reduce observer bias?

 

Response 5. We agree with this valuable suggestion. We have added details of the experimental design

Change in manuscript: Section 2.7 (Agronomic Measurements, page 6, paragraph 2, lines 195-200):

“These time points (30, 60, 75, and 120 days after sowing, DAS) were selected because they coincide with key growth stages of potato and thus allowed for timely and representative data collection. At 30 DAS, field emergence exceeded 95%, making this stage appropriate for counting established plants. At 60 DAS, early flowering and the appearance of initial stolons were recorded, while 75 DAS corresponded to the tuber bulking stage. The final observation occurred at 120 DAS, when both cultivars had reached physiological maturity and senescence”

Change in manuscript: Section 2.7 (Agronomic Measurements, page 6, paragraph 3, lines 210-212):

The number of sampled plants varied because in some treatments, particularly the uninoculated control, only 7–10 plants in the central row grew vigorously enough to be considered representative

Change in manuscript: Section 2.7 (Agronomic Measurements, page 6, paragraph 3, lines 206-207):

To minimize border effects, the first and last plants of each row were excluded from sampling

Change in manuscript: Section 2.7 (Agronomic Measurements, page 6, paragraph 3, lines 215-216):

To reduce observer bias, the assessment was performed simultaneously by three independent evaluators, and consensus scores were recorded for each plant

 

Comment 6. Regarding plant vigor, you indicate that it was visually scored on a nine-point ordinal scale (3 = weak, 5 = moderate, 7 = vigorous, 9 = very vigorous). Could you please provide a reference for this scale (e.g., a standard agronomic evaluation protocol or previous studies using this method)? And why you choose 60 and 75 days ?

Response 6. Thank you for pointing this out. We have added one reference for the nine-point vigor scale and clarified that vigor was assessed at 60 and 75 DAS because these stages coincide with early flowering/stolon initiation and tuber bulking, respectively, which are critical for crop performance.

Change in manuscript: Section 2.7 (Agronomic Measurements, page 6, paragraph 3, lines 215):

Comment 7. For the results section add a table to show the numbers.

Response 7. We agree with this suggestion. A new table has been included in the Supplementary material section to present the numerical values clearly.

Comment 8. I recommend that the authors include a short “Future Recommendations” section in the conclusion or discussion.

Response 8. Thank you for this helpful suggestion. We have included a short “Future Recommendations” section in the Conclusions, highlighting the need for large-scale trials, evaluation across edaphoclimatic conditions, long-term impacts on soil fertility and rhizosphere microbiota, and optimization of formulations and application methods.

Change in manuscript: Section 4 (Discussions, page 16, paragraph 1, lines 556-562):

 

Further large-scale field trials across contrasting edaphoclimatic conditions are recommended to validate the consistency of the combined effects of Bacillus subtilis and Trichoderma viride. Future studies should also address their long-term impacts on soil fertility, rhizosphere microbial communities, and postharvest tuber quality. In addition, evaluating different formulations and application methods could help optimize their effectiveness under reduced fertilization schemes, thereby strengthening their adoption in sustainable potato production systems.

 

We are grateful to the reviewer for their thoughtful and constructive feedback. We believe that the revisions have significantly strengthened the clarity, reproducibility, and practical relevance of our manuscript.

 

Sincerely,

Dr. Daniel Matsusaka

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Author, 

Thank you for your valuable effort on this manuscript. However, in the PGPM paragraph, most of the cited references fall within 2020–2023. While relevant, the section would be further strengthened by incorporating a more recent reference to update and modernize the introduction. I highly recommend adding Seham M. Al Raish, et al. (2025). Plant Growth-Promoting Microorganisms as Biocontrol Agents: Mechanisms, Challenges, and Future Prospects. Appl. Microbiol. 5(2), 44. https://doi.org/10.3390/applmicrobiol5020044, which is published in the same journal. 

Good luck.

Author Response

Author´s Reponse to the Review Report

Manuscript ID: applmicrobiol-3849622

Title: Eco-Efficient Intensification of Potato with Bacillus subtilis and Trichoderma viride under NPK Fertilization

Journal: Applied Microbiology

Dear Reviewer,

We sincerely thank you for your careful reading of our manuscript and for providing constructive comments and suggestions. Your feedback has been very valuable in helping us to update and modernize the introduction. Below, we provide our detailed response to your comment. We carefully considered your suggestion and have modified the manuscript accordingly, with the corrections highlighted in turquoise.

Comment 1.

Thank you for your valuable effort on this manuscript. However, in the PGPM paragraph, most of the cited references fall within 2020–2023. While relevant, the section would be further strengthened by incorporating a more recent reference to update and modernize the introduction. I highly recommend adding Seham M. Al Raish, et al. (2025). Plant Growth-Promoting Microorganisms as Biocontrol Agents: Mechanisms, Challenges, and Future Prospects. Appl. Microbiol. 5(2), 44. https://doi.org/10.3390/applmicrobiol5020044, which is published in the same journal. 

Response 1. Thank you for pointing this out. We agree with this comment. Therefore, we have incorporated the suggested reference (Al Raish et al., 2025) into the PGPM section to strengthen and modernize the introduction. Additionally, we have updated references 17 and 18 with more recent studies to further support this section. These modifications are reflected in the reference list on page 17, lines 630–636 of the revised manuscript.

We are grateful to the reviewer for their thoughtful and constructive feedback. We believe that the revisions have significantly strengthened the clarity, reproducibility, and practical relevance of our manuscript.

 

Sincerely,

Dr. Daniel Matsusaka