Effects of Glomus iranicum Inoculation on Growth and Nutrient Uptake in Potatoes Associated with Broad Beans Under Greenhouse Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study presented in this manuscript was designed to assess the effects of arbuscular mycorrhizal inoculation on growth responses of potato (Solanum tuberosum) and broad bean (Vicia faba; also referred to as fava bean in the manuscript, and faba bean is another common name) under intercropped conditions in pots. As the authors note, intercropping and AMF both are increasingly being used to improve sustainable farming practices, yet many questions remain from the research to date about the benefits of these approaches. As a result, this study is timely and has the potential to be of broad interest. I do have several comments and suggestions about the study, as well as a couple of concerns. In particular, no data are provided on the degree of mycorrhizal colonisation of either potato or broad bean, and no data are provided comparing AMF treatment effects on broad bean root modulation. My other comments and suggestions are outlined below.


Abstract

The initial part of the Abstract is straightforward and the rationale for the study is clear. I do have some suggestions about the results presented in this section.

I would drop the values after the decimal for the % data; these values go beyond the level of resolution possible for the underlying measurements.

Also, there seems to be a typo in the phrase ‘Consequently, the interaction of G. iranicum…’ (l. 24), given that that there does not appear to be any interactive treatments. The study is also described as an interactive design in the Results/Discussion (l. 236), but the study design doesn’t interactively assess the effects of broad bean and AMF on potato growth.

The information about treatment effects on potato (e.g., the response to 3 g of AMF inoculum) isn’t correct. While this section indicates that AMF treatment affected foliar dry weight (mass) and tuber dry weight (mass), the effect on foliar dry mass peaked in the T3 (2 g AMF) treatment, and tuber dry mass didn’t statistically differ among AMF treatments.

Also, the Results presented in the Abstract don’t clearly distinguish effects on potato plants and effects on broad bean plants.

And, the Results feel somewhat finessed here, in that for potatoes the largest AMF treatment (4 g) generally was not statistically significantly different than the 0 g treatment, indicating that the largest growth enhancements for potato plants were in the ‘moderate’ treatments. In contrast, increasing AMF inoculum up to 4 g did increase some measures of broad bean growth, but the differential responses of the two plant species isn’t made clear in this section.


Introduction

This section generally provides an adequate background to frame and justify the study. However, it's-very lean on citations to support statements. The first two and last two paragraphs in particular would be more impactful if supported by more citations.

Also, the paragraph on AMF effects has a lot of redundancy.

And, it’s unclear why potassium, rather than nitrogen, was assessed in the tubers. The Intro notes the role of AMF in increasing nitrogen uptake, and legumes typically are intercropped because they increase soil N, so nitrogen seems like an obvious nutrient to measure. In contrast, potassium isn’t mentioned in the Intro until the last sentence.


Methods

For soil phosphorus (l. 97, Table 1), it’s not clear why the value is referred to as ‘available phosphorus’, particularly given the many forms in which phosphorus may occur in soils. Somewhat along those lines, what specific analytical tests were used to measure P and K both in the soil (recognising that these data are from the INIA) and in the tubers?

What specific cultivars of potato and broad beans were used?

Why was MycoUp used as the AMF source (l. 111)? Also, it would be helpful if information about the manufacturer was provided.


Results/Discussion

Figure 3 legend: Vicia haba should be Vicia faba.

In Figure 4, the y-axis is labelled as K for all four graphs, but a) and c) present data on P.

Most treatment effects on potato growth were non-significant, and where there were significant treatment effects, they tended to peak at moderate AMF inoculation, with the highest AMF treatment not differing statistically from the 0 AMF treatment.

Also, some of the fresh mass/dry mass data suggest that the primary effect of AMF on tissue mass was through effects on plant water status.

The PCAs presented in the Results/Discussion (starting at l. 203) aren’t described in the Statistical Analysis section (2.4, l. 142). Also, it’s unclear why PCAs were run at all. Based on how they’re presented in this section, they indicate the extent to which plant variables co-varied within and across the AMF treatments. However, because no rationale for these analyses is presented, it’s not clear how these results fit into the overall study. Further, the way these analyses are presented at the end of the Results/Discussion (l. 285) isn’t consistent with either what the PCAs represent or how they should be interpreted. For example, the PCA results cannot be used to infer specific treatment effects, as PCAs aren’t useful for hypothesis testing (unlike the ANOVA results), and if the intent is to look at the effects of AMF treatments across plant growth variables, then either MANOVAs (multivariate analysis of variance) or covariate approach appear to be more appropriate. (Or a Bayesian approach could be used, which would sidestep the whole issue of statistical significance.)

Comments on the Quality of English Language

There are a fair number of typos that need to be tidied up. More pressing, the word selection in some areas doesn't seem consistent with the intended meaning, such as how 'interactive' is used in some places in the text.

Author Response

Response to Reviewer 1

Thank you very much for taking the time to review this manuscript. Below you will find detailed responses and any relevant revisions or corrections highlighted or tracked in the forwarded files.

 

We sincerely appreciate your comment on the possible confusion between the effects of fava beans (Vicia faba) and arbuscular mycorrhizal fungi (Glomus iranicum) on growth and nutrients of potatoes (Solanum tuberosum). His commentary is very valuable and allows us to reflect on the scope and interpretations of the study.

The main goal of the study was to evaluate how inoculation with G. iranicum influences the growth and nutritional composition of potatoes in a mixed cropping system with broad beans, simulating real agricultural conditions in Andean systems. We know that, in these systems, plant-plant and plant-microorganism interactions are common and are part of sustainable management. Therefore, our intention was not to separate the individual effects, but to explore how the combination of these factors affects potato crop yield.

We recognize that the interaction between broad beans and mycorrhizal fungi may influence the observed results, since broad beans have the ability to fix atmospheric nitrogen and may indirectly promote potato growth. However, the results of our study show clear and significant differences in the variables measured according to the inoculation doses of G. iranicum. For example:

• With 3 g of G. iranicum, a significant increase in plant height (24.35%), leaf dry weight (90.76%) and tuber dry weight (57.12%) was observed compared to the control.
• With 4 g of G. iranicum, the greatest increases were recorded in leaf fresh weight (115.79%), root length (124.53%), and root fresh and dry weight (159.93% and 243.48%, respectively).

These results indicate that the arbuscular mycorrhizal fungus had a significant effect on potatoes, even in a co-cultivation system.

 

Review 1 (olive drab)

Coment 1. I would drop the values after the decimal for the % data; these values go beyond the level of resolution possible for the underlying measurements.

Response 1. Done

Coment 2. Also, there seems to be a typo in the phrase ‘Consequently, the interaction of G. iranicum…’ (l. 24), given that that there does not appear to be any interactive treatments. The study is also described as an interactive design in the Results/Discussion (l. 236), but the study design doesn’t interactively assess the effects of broad bean and AMF on potato growth.

Response 2. Line 24

Change, In particular for consequenthy

Line 247

Change, …”Crop association coupled with inoculation”…, for …”The interaction between associated crop and G. iranicum”…

Coment 3. The information about treatment effects on potato (e.g., the response to 3 g of AMF inoculum) isn’t correct. While this section indicates that AMF treatment affected foliar dry weight (mass) and tuber dry weight (mass), the effect on foliar dry mass peaked in the T3 (2 g AMF) treatment, and tuber dry mass didn’t statistically differ among AMF treatments.
Response 3.

the results paragraph was reorganized.

Line 23-28

Coment 4. Also, the Results presented in the Abstract don’t clearly distinguish effects on potato plants and effects on broad bean plants.

Response 4.

sentence changed for clarification

line 24-27

Coment 5. And, the Results feel somewhat finessed here, in that for potatoes the largest AMF treatment (4 g) generally was not statistically significantly different than the 0 g treatment, indicating that the largest growth enhancements for potato plants were in the ‘moderate’ treatments. In contrast, increasing AMF inoculum up to 4 g did increase some measures of broad bean growth, but the differential responses of the two plant species isn’t made clear in this section.

This section generally provides an adequate background to frame and justify the study. However, it's-very lean on citations to support statements. The first two and last two paragraphs in particular would be more impactful if supported by more citations.

Response 5. A new revision was made and the citations were increased and the paragraphs were reorganized and quoted.

Coment 6. Also, the paragraph on AMF effects has a lot of redundancy.

Response 6.

Line 63-69,

paragraphs were edited

Coment 7. And, it’s unclear why potassium, rather than nitrogen, was assessed in the tubers. The Intro notes the role of AMF in increasing nitrogen uptake, and legumes typically are intercropped because they increase soil N, so nitrogen seems like an obvious nutrient to measure. In contrast, potassium isn’t mentioned in the Intro until the last sentence.

Response 7. Potassium is a nutrient that is improved by inoculations of arbuscular fungi. This is why it is included in this study.

Methods

Coment 8. For soil phosphorus (l. 97, Table 1), it’s not clear why the value is referred to as ‘available phosphorus’, particularly given the many forms in which phosphorus may occur in soils. Somewhat along those lines, what specific analytical tests were used to measure P and K both in the soil (recognising that these data are from the INIA) and in the tubers?

Response 8.

Line, 106-116

The methodology section describes the methods used to calculate the variables described in Table 1.

Coment 9. What specific cultivars of potato and broad beans were used?

Response 9.

 we use variety and not specific cultivars for this research.

Coment 10. Why was MycoUp used as the AMF source (l. 111)? Also, it would be helpful if information about the manufacturer was provided.

Response 10.

the commercial inoculant was chosen

Results/Discussion

We added a section to recognize the possible influence of beans on the observed results and how these interactions reflect real agricultural conditions.

Coment 11. Figure 3 legend: Vicia haba should be Vicia faba.

Response 11. Done

Coment 12. In Figure 4, the y-axis is labelled as K for all four graphs, but a) and c) present data on P.

Response 12.

corrections made

Coment 13. Most treatment effects on potato growth were non-significant, and where there were significant treatment effects, they tended to peak at moderate AMF inoculation, with the highest AMF treatment not differing statistically from the 0 AMF treatment.

Also, some of the fresh mass/dry mass data suggest that the primary effect of AMF on tissue mass was through effects on plant water status.
Response 13.

modifications were made

Line 165-167           

Coment 14. The PCAs presented in the Results/Discussion (starting at l. 203) aren’t described in the Statistical Analysis section (2.4, l. 142). Also, it’s unclear why PCAs were run at all. Based on how they’re presented in this section, they indicate the extent to which plant variables co-varied within and across the AMF treatments. However, because no rationale for these analyses is presented, it’s not clear how these results fit into the overall study. Further, the way these analyses are presented at the end of the Results/Discussion (l. 285) isn’t consistent with either what the PCAs represent or how they should be interpreted. For example, the PCA results cannot be used to infer specific treatment effects, as PCAs aren’t useful for hypothesis testing (unlike the ANOVA results), and if the intent is to look at the effects of AMF treatments across plant growth variables, then either MANOVAs (multivariate analysis of variance) or covariate approach appear to be more appropriate. (Or a Bayesian approach could be used, which would sidestep the whole issue of statistical significance.)

Response 14.

The analysis carried out in the methodology was added.

Line 162-163

The description of the results with respect to the principal components and analyses was reorganized.
Line 214-234

 

We appreciate your comments and consider that your suggestion is an excellent opportunity to deepen this line of research. However, we believe that the current findings provide valuable information on the positive interactions between G. iranicum and associated crops in mixed farming systems, which are representative of agricultural practices in Andean regions.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Contreras-Pino et al. investigated the effects of Glomus iranicum inoculation on growth and nutrient uptake in potatoes associated with broad beans under greenhouse conditions. This work is interesting and has practical significance. I have two major comments. First, there is a lack of discussion on relevant studies. For example, in the Introduction, the authors provide a great deal of general background information, but previous studies closely related to their work are not mentioned at all. Second, the uncertainties of the results (or limitations of this study) should be analyzed in the Discussion section. After all, this is only a three-month experiment and a specific type of potato and broad bean was used.

I also have some specific comments which are listed below:

1.      L132: It should be ‘December 2023’.

2.      Figures: The y-axis label should be revised. For example, the y-axis label for Figure 2a should be ‘Plant height (cm)’.

3.      Many numbers are too precise. Too many decimal places (such as the percentage in the Abstract) are completely unnecessary.

4.      There is a mixed use of p-value, p and P. Please be consistent.

5.      L223: ‘the variables evaluated’ is too general. Please rephrase this sentence.

6.      A grammar check is needed, as there are too many errors.

Comments on the Quality of English Language

Must be improved.

Author Response

Response to Reviewer 2

Thank you very much for taking the time to review this manuscript. Below you will find detailed responses and any relevant revisions or corrections highlighted or tracked in the forwarded files.

 

Reviewer 2 (red)

Coment 1. L132: It should be ‘December 2023’.

Response 1.

Done, “…December 2023…”

Coment 2. Figures: The y-axis label should be revised. For example, the y-axis label for Figure 2a should be ‘Plant height (cm)’.

Response 2.

Done, the suggested changes were made to the titles of the figure axes.

Coment 3. Many numbers are too precise. Too many decimal places (such as the percentage in the Abstract) are completely unnecessary.

Response 3.

Done        

Coment 4. There is a mixed use of p-value, p and P. Please be consistent.

Response 4.

the use was standardized to p-value

Coment 5. L223: ‘the variables evaluated’ is too general. Please rephrase this sentence.

Response 5.

change in sentence

L 223-224: “ … evaluated (plant leigth, Foliar dry weigth, root dry weigth, root fresh weigth), although in many cases they were not significant…”

Coment 6. A grammar check is needed, as there are too many errors.

Response 6.

A general check-up was performed, as recommended.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

I have read a study that is very important from the point of view of looking for opportunities in agriculture to reduce the negative impact on the natural environment. The use of biofertilisers, which reduce the use of synthetic fertilisers but also have the ability to increase soil organic matter, is certainly a great opportunity. The topic of researching the impact of biosurfactants on the yield of agricultural crops, in this case potatoes, is important in terms of the relevance of this crop to agriculture globally. I consider the research methods used to be well used, as evidenced by the free and substantive description used by the authors. The objectives in the paper were set and met. I congratulate the interesting discussion of the results. However, I have a remark on the structure of the paper, it lacks a separate chapter with a literature review. In this topic such a theoretical chapter is essential. In it, please describe the importance of biofertilisers in agricultural crops in other countries, including the European Union, and please refer to the experience of other authors. Many studies on this subject are available in the international literature. Without this chapter, the study is too technical. Please note that in journals with Impact Factors, a theoretical chapter is necessary to enhance the quality of the paper. The strong point is the good illustration of the research results, well prepared graphs.

Author Response

Response to Reviewer 3

Thank you very much for taking the time to review this manuscript. Below you will find detailed responses and any relevant revisions or corrections highlighted or tracked in the forwarded files.

 

Reviewer 3 (yellow)

Response  1. A couple of paragraphs were added to the introduction and the studies were reviewed to support our findings.

 

We sincerely appreciate your comment on the possible confusion between the effects of fava beans (Vicia faba) and arbuscular mycorrhizal fungi (Glomus iranicum) on growth and nutrients of potatoes (Solanum tuberosum). His commentary is very valuable and allows us to reflect on the scope and interpretations of the study.

The main goal of the study was to evaluate how inoculation with G. iranicum influences the growth and nutritional composition of potatoes in a mixed cropping system with broad beans, simulating real agricultural conditions in Andean systems. We know that, in these systems, plant-plant and plant-microorganism interactions are common and are part of sustainable management. Therefore, our intention was not to separate the individual effects, but to explore how the combination of these factors affects potato crop yield.

We recognize that the interaction between broad beans and mycorrhizal fungi may influence the observed results, since broad beans have the ability to fix atmospheric nitrogen and may indirectly promote potato growth. However, the results of our study show clear and significant differences in the variables measured according to the inoculation doses of G. iranicum. For example:

• With 3 g of G. iranicum, a significant increase in plant height (24.35%), leaf dry weight (90.76%) and tuber dry weight (57.12%) was observed compared to the control.
• With 4 g of G. iranicum, the greatest increases were recorded in leaf fresh weight (115.79%), root length (124.53%), and root fresh and dry weight (159.93% and 243.48%, respectively).

These results indicate that the arbuscular mycorrhizal fungus had a significant effect on potatoes, even in a co-cultivation system.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The author explored the effects of adding arbuscular mycorrhizal fungi on the growth and nutrients of potatoes through greenhouse experiments. The results of this study have certain application value for guiding agricultural production. Although the author emphasized the new combination of potatoes and broad beans in the experimental design, it is inevitable to confuse the promoting effects of broad beans and arbuscular mycorrhizal fungi on potatoes.  Therefore, in current experiments, the growth and nutrients of potatoes are regulated by the interaction between fava beans and arbuscular mycorrhizal fungi, unless the author has potted potatoes separately and added arbuscular mycorrhizal fungi. In this way, the theme of this study will be clearer and more focused, rather than scattered on the effects of arbuscular mycorrhizal fungi on both potatoes and broad beans. This manuscript also has flaws, including a lot of grammatical problems, inconsistent image styles, and poorly readable wording. The entire manuscript should be redone and polished. Please refer to the manuscript annotations for detailed revision suggestions.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

It is recommended to polish the manuscript to enhance its quality, including readability and correction of grammatical errors.

Author Response

Response to Reviewer 4

Thank you very much for taking the time to review this manuscript. Below you will find detailed responses and any relevant revisions or corrections highlighted or tracked in the forwarded files.

 

Reviewer 4 (green)

Response 1.

Line 22, Done

Line 23: done

Line 24: done

Line 26: done

Line 28: done

Line 48. Done

Line 50. Done

Line 70. Done

Line 74-79. Change

Line 93. Delete

Line 98. Done

Line 100. Done

Line 103: Done

Line 107. Done

Line 108. Done

Line 112. Done

Line 126-27. Done

Line 151. Done

Line 152.

Coment 2. What does the error bar represent? Why are the error bars in Figure 2g and Figure 2i identical? Please check the data. Unit error in Figure 2i.

Response 2.

We regret the error, it has been corrected.

Line 162-64. Done

Line 172, done

Line 177. Done

Line 179. Done

Line 189-190. Done

Author Response File: Author Response.docx

Reviewer 5 Report

Comments and Suggestions for Authors

In manuscript mikrobiolores-3398712, the authors presented research aimed at determining the effect of the mycorrhizal fungus Glomus iranicum on the yield of potatoes and broad beans and on the levels of phosphorus and potassium in the plants and available phosphorus and available potassium in the soil. The main crop was potato and the secondary crop was broad bean. The source of Glomus iranicum was the commercial biostimulant Glomus MycoUp ®. As the authors actually tested the effect of the biostimulant, it can be assumed that the effect of the fungus Glomus iranicum was tested before it was patented.

In the reviewed manuscript, there is also a tendency towards a positive effect of the tested fungus on the tested plants. However, one cannot overuse percentage data, as was done in the abstract and discussion of the results, because after taking into account the experimental error, this effect will not be as large.

 

Detailed comments

Abstract - the last sentence does not follow from the research carried out. There was no series of experiments without the stimulator, but with optimal mineral fertilisation.

Several times in the paper Kg was written instead of kg (e.g. Table 1).

Figure 1 is very carelessly drawn up. There is also no indication of the research objects.

In chapter 2.2. Experimental design, the authors wrote that the experiment included five levels of G. iranicum: T1 (0 g G. iranicum); T2 (1 g G. iranicum); T3 (2 g G. iranicum), T4 (3 g G. iranicum) and T5 (4 g G. iranicum), but these were not doses of the fungus but doses of the biostimulant. This comment applies to the whole manuscript. Please describe the stimulator and the procedure for its application in the studies in more detail.

The methods of chemical analysis should be described in more detail. It is not sufficient to refer to the service provider.

Chemical analyses should be statistically developed.

The soil preparation procedure for experiments and chemical analyses should be described in detail.

Chapter 3. Results and discussion, was incorrectly titled because the manuscript contains Chapter 4. Discussion.

Figure 2 - Figure 2i gives the number of tubers in grams. This is clearly an error.

 The sentence in lines 168-170 is an oversimplification because not all doses had a significant positive effect.

Figure 4 - There are no homogeneous groups, so the description of the results in this figure cannot be clearly evaluated.

 Chapter 5. Conclusions need to be corrected. It is not true that the increase in dose led to an increase in the variables analysed. The conclusions should be more balanced and unambiguously derived from the research conducted.

 

Author Response

Response to Reviewer 5

Thank you very much for taking the time to review this manuscript. Below you will find detailed responses and any relevant revisions or corrections highlighted or tracked in the forwarded files.

 

Reviewer 5 (sky blue)

Coment 1. Abstract - the last sentence does not follow from the research carried out. There was no series of experiments without the stimulator, but with optimal mineral fertilisation.

Response 1.

The study included treatment with an inoculant. Therefore, we believe that the paragraph should be kept.

Coment 2. Several times in the paper Kg was written instead of kg (e.g. Table 1).

Response 2.

corrections were made throughout the document.

Coment 3. Figure 1 is very carelessly drawn up.

Response 3.

Change line 125-128

Coment 4. There is also no indication of the research objects.

Response 4.

Presented in line 84-87

Coment 5. In chapter 2.2. Experimental design, the authors wrote that the experiment included five levels of G. iranicum: T1 (0 g G. iranicum); T2 (1 g G. iranicum); T3 (2 g G. iranicum), T4 (3 g G. iranicum) and T5 (4 g G. iranicum), but these were not doses of the fungus but doses of the biostimulant. This comment applies to the whole manuscript. Please describe the stimulator and the procedure for its application in the studies in more detail.

Response 5.

Thank you for your observation. The clarification was made throughout the document.

Coment 6. The methods of chemical analysis should be described in more detail. It is not sufficient to refer to the service provider.

Response 6.

 Line 95-105. the description of the methodology used by the laboratory for soil analysis has been added.

Coment 7. The soil preparation procedure for experiments and chemical analyses should be described in detail.

Response 7.

A description of the methods used for soil analysis was included.

Coment 8. Chapter 3. Results and discussion, was incorrectly titled because the manuscript contains Chapter 4. Discussion.

Response 8.

Change

Coment 9. Figure 2 - Figure 2i gives the number of tubers in grams. This is clearly an error.

error corrected

Coment 10. The sentence in lines 168-170 is an oversimplification because not all doses had a significant positive effect.

Coment 10. the sentence was rewritten

Line 180-181

Coment 11. Figure 4 - There are no homogeneous groups, so the description of the results in this figure cannot be clearly evaluated.

Coment 11. For this reason, a general description of the results is presented.

Coment 12. Chapter 5. Conclusions need to be corrected. It is not true that the increase in dose led to an increase in the variables analysed. The conclusions should be more balanced and unambiguously derived from the research conducted.

Coment 12. The conclusions were reformulated taking into account their recommendations.

Line 337-346

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Before I add in my comments about this draft, I’d like to thank the authors for their very thorough consideration and reply to my first set of comments (I was Reviewer 1). I do have some concerns about the revised manuscript, and i’m sorry if the following comes across as overly negative - I think there is merit in this manuscript, but still disagree with some of the assertions.

Perhaps most importantly, I disagree with the assertion that mycorrhizal inoculation has clear effects on potato growth. Across four mycorrhizal treatments, the highest rate of addition (4 g; T5) had no statistically significant effects on any measures of potato growth. And, the next-highest treatment (3 g; T4) was the only one of the four inoculation treatments that had a clear effect on plant height and tuber fresh mass (but no significant effect on tuber dry mass, suggesting that the effect on tuber fresh mass was due to treatment differences in water content). T3, the second lowest addition rate (2 g), was the only treatment that had an effect on foliar dry mass. (The reply letter indicates that this was a T4/ 3 g effect, but that doesn’t match the figure.) So, of nine plant growth measures, only three exhibited treatment effects, and the treatment effects were inconsistent even among those three variables.

Also, the reply is inaccurate about the effects of the T5 (4 g) treatment. The reply indicates that the T5 treatment affected potato leaf fresh mass, root length, and root fresh and dry mass. However, these effects were observed in broad bean, not in potatoes. Further, while the mean effect size was large, there was a lot of variation, which is why none of the other treatments statistically differed from the control, and why there was no clear effect on broad bean height, leaf dry mass, or number of flower nods. (Also the effect on leaf fresh mass likely was due to differences in water content, given no treatment effects were observed for leaf dry mass.)

For both of these issues, I disagree with the rephrasing in the Results. For potato (starting l. 160), it seems more accurate to say that inoculation exhibited an inconsistent effect on growth variables, and that most growth variables exhibited no clear response to inoculation. For broad bean (starting l. 174), the phrasing in this paragraph suggests all seven measured variables differed with mycorrhizal treatment; however, three of the variables (height, leaf dry mass, and number of flower nodes) did not clearly differ among the treatments.

The P and K data also suggest that inoculation had no consistent effect on potato or broad bean responses to inoculation, given that the means for both P and K didn’t differ either in potato or broad beans across inoculation treatments.

Following on that, for my comment regarding measuring K, while AM infection may increase K content in plants, AM infection may also increase N content (as well as other elements), but N wasn’t measured and no rationale regarding why K was measured is provided in the Intro.

Finally, I’m still concerned about the PCA analyses. Because PCA cannot be used for hypothesis testing or assessing cause and effect, it’s still not clear to me why these analyses are included.

Comments on the Quality of English Language

Some of the differences in how we present our interpretations of the study may be due to differences in how we interpret specific words and phrases.

Author Response

Response to Reviewer 1

Thank you very much for taking the time to review this manuscript. Below you will find detailed responses and any relevant revisions or corrections highlighted or tracked in the forwarded files.

 

Before I add in my comments about this draft, I’d like to thank the authors for their very thorough consideration and reply to my first set of comments (I was Reviewer 1). I do have some concerns about the revised manuscript, and i’m sorry if the following comes across as overly negative - I think there is merit in this manuscript but still disagree with some of the assertions.

 

Review 1 (yellow)

Comment 1. Perhaps most importantly, I disagree with the assertion that mycorrhizal inoculation has clear effects on potato growth. Across four mycorrhizal treatments, the highest rate of addition (4 g; T5) had no statistically significant effects on any measures of potato growth. And, the next-highest treatment (3 g; T4) was the only one of the four inoculation treatments that had a clear effect on plant height and tuber fresh mass (but no significant effect on tuber dry mass, suggesting that the effect on tuber fresh mass was due to treatment differences in water content). T3, the second lowest addition rate (2 g), was the only treatment that had an effect on foliar dry mass. (The reply letter indicates that this was a T4/ 3 g effect, but that doesn’t match the figure.) So, of nine plant growth measures, only three exhibited treatment effects, and the treatment effects were inconsistent even among those three variables.

 

Response 1.

Thanks for the suggestion, we have made the change to the statements in the results section, line 209-212. In the discussion section, line 286-289; line 294-300 and line 319-320. We apologize for the error in the letter, we have made the change.

 

Comment 2.  Also, the reply is inaccurate about the effects of the T5 (4 g) treatment. The reply indicates that the T5 treatment affected potato leaf fresh mass, root length, and root fresh and dry mass. However, these effects were observed in broad bean, not in potatoes. Further, while the mean effect size was large, there was a lot of variation, which is why none of the other treatments statistically differed from the control, and why there was no clear effect on broad bean height, leaf dry mass, or number of flower nods. (Also the effect on leaf fresh mass likely was due to differences in water content, given no treatment effects were observed for leaf dry mass.)

Response 2.

Thanks for the suggestion. If it is true that some authors attribute the difference between fresh leaf mass to inoculations, which is not necessarily the case with dry mass, we have clarified this by adding a paragraph on lines 343-346.

 
Comment 3.  For both of these issues, I disagree with the rephrasing in the Results. For potato (starting l. 160), it seems more accurate to say that inoculation exhibited an inconsistent effect on growth variables, and that most growth variables exhibited no clear response to inoculation. For broad bean (starting l. 174), the phrasing in this paragraph suggests all seven measured variables differed with mycorrhizal treatment; however, three of the variables (height, leaf dry mass, and number of flower nodes) did not clearly differ among the treatments.

Response 3.

Thanks for the suggestion. We have made the suggested changes: In the results section line 209-212; line 224-226; line 286-289; line 294-300; line 332-333

 

Comment 4.  The P and K data also suggest that inoculation had no consistent effect on potato or broad bean responses to inoculation, given that the means for both P and K didn’t differ either in potato or broad beans across inoculation treatments.

Following on that, for my comment regarding measuring K, while AM infection may increase K content in plants, AM infection may also increase N content (as well as other elements), but N wasn’t measured and no rationale regarding why K was measured is provided in the Intro.

Response 4.

These studies suggest that inoculation with mycorrhizal fungi and plant growth-promoting bacteria can improve the absorption of phosphorus and potassium in plants, while the measurement of nitrogen is not always relevant in these contexts due to their focus on phosphorus use efficiency and symbiotic nitrogen fixation. We have added an item Line 99-135, where we summarize the benefits of nitrogen fixers and phosphorus and potassium solubizers.

Comment 5.  Finally, I’m still concerned about the PCA analyses. Because PCA cannot be used for hypothesis testing or assessing cause and effect, it’s still not clear to me why these analyses are included.

Response 5.

Thank you for your suggestion. We have changed the sentence to avoid the confusion that it is being used as a hypothesis test. 

The reason for using PCA was to explore and identify key patterns in the agronomic traits of potatoes and beans across different microorganism treatments for the whole dataset. In addition, we have included a graphical representation to improve the interpretation of the results.

 

Comment 6.  Replace “ These percentages exceed 25% of the total variance, according to [71], which is sufficient for a good interpretation. PCA analysis showed that the most relevant variables in differentiating treatments are the fresh and dry weight of tubers and roots, as well as plant height and number of tubers. Which suggests that the use of HMA improves potato production in terms of biomass and root development.

by “ These percentages exceed 25% of the total variance, according to [71], which is sufficient for a good interpretation. PCA analysis showed that the most relevant variables in differentiating treatments are the fresh and dry weight of tubers and roots, as well as plant height and number of tubers“

Response 6.

Thank you for your suggestion. We have made the changes. Line 359-362,

Comment 7.  Replace “ Likewise, the PCA analysis for the beans showed that the most important variables in the differentiation of treatments are the fresh and dry weight of the root and the foliage and that these are related to high doses of the inoculant. The PAC confirm that HMAs have a positive impact on the aerial and root biomass of potato and fava bean plants.

By “Likewise, the PCA analysis for the beans showed that the most important variables in the differentiation of treatments are the fresh and dry weight of the root and the foliage and that these are related to high doses of the inoculant”

Response 7.

Thank you for your suggestion. We have made the changes. Line 362-365.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has been improved significantly. I recommend its acceptance in its current form.

Comments on the Quality of English Language

Acceptable but could be improved.

Author Response

Thank you for your thoughtful feedback and for recognizing the improvements made to the revised manuscript. We are pleased to hear that you recommend its acceptance in its current form.

We are excited about the possibility of contributing to the field through this publication and are grateful for your support.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you to the authors for the revisions so far. The authors have incorporated many comments. However, a separate chapter with a literature review on the topic addressed is still missing. Please do a solid literature review with consideration of international literature. I think that 1 page is sufficient. The strength of the paper is the choice of the research topic, the results, the discussion of the results and the conclusions. Other elements of the study are well prepared, still a separate chapter is missing - literature review! ( after the introduction). I think that some content from the introduction is worth moving to a new chapter with a literature review.

 

Author Response

Comment 1.

Thank you to the authors for the revisions so far. The authors have incorporated many comments. However, a separate chapter with a literature review on the topic addressed is still missing. Please do a solid literature review with consideration of international literature. I think that 1 page is sufficient. The strength of the paper is the choice of the research topic, the results, the discussion of the results and the conclusions. Other elements of the study are well prepared, still a separate chapter is missing - literature review! (after the introduction). I think that some content from the introduction is worth moving to a new chapter with a literature review.

Response 1

Thank you very much for the suggestion. We have added a review of the importance and applications of biofertilizers in crops, we focused on biofertilizers that fix nitrogen and solubilize phosphorus and potassium, which are the focus of our study.

The addition can be found on lines 90 to 135.         

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

  

Author Response

Thank you for your thoughtful feedback and for recognizing the improvements made to the revised manuscript. We are pleased to hear that you recommend its acceptance in its current form.

We are excited about the possibility of contributing to the field through this publication and are grateful for your support.

Reviewer 5 Report

Comments and Suggestions for Authors

The authors have revised the manuscript mikrobiolores-3398712 according to the suggestions of the reviewer. It is now much better and, in the opinion of the reviewer, ready for publication.

Author Response

Thank you for your thoughtful feedback and for recognizing the improvements made to the revised manuscript. We are pleased to hear that you recommend its acceptance in its current form.

We are excited about the possibility of contributing to the field through this publication and are grateful for your support.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Many thanks to the authors for separating an additional chapter with a literature review. As well as supplementing the discussion. These are 2 important strengths of the study. In this form, I accept the study for publication.  I would like to remind the Authors for the future that studies sent to periodicals with Impact Factor should have an appropriate sructure, including a literature review.