Round 1

Reviewer 1 Report

This manuscript explored the effect of L-α-amino acid-based biostimulant (Terramin® Pro) on phenotype of different lettuce cultivars and soil microbial community and structure under different soil types and conditions. The results suggested although the application changed soil microbial community and structure, the effects of this biostimulant on lettuce phonotype and soil microbial community and structure differed from soil type and conditions. It was still a sustainable alternative to modulate soil microbiome. This study will guide the application of biostimulants in crop growth and different soil types.

 There are some details needed to be improved.

For introduction part:

1. The author introduced the general situation of biostimulant in agriculture system, but it lacks the information of the progresses of the effect of L-α-amino acid-based biostimulant on soil microbial community and structure under different crops, soil types and conditions. Since this experiment concerned about the crop type, soil type and condition.

2. Please add the reason for choosing lettuce as the test plant.

For results part:

1. In Figure S1 and Figure 1(c), there is no meaning to compare the different among the different time series under the same soil type. It should be compared the different among the three soil types in the same time.

2. there is big different of soil nutrient among the three soil types, e.g. the soil texture, pH, M.O.total, P, Fe, Mn, Cu, Zn, NO₃- in table 1. And the soil properties are also the main factor affect the soil microbial community and structure. Hence, I suggest the author add the analysis of the effect of soil nutrient on soil microbial community and structure.

 For discussion part:

1. Line 204-212, it duplicated with the information in introduction part, or this description should be moved to the introduction part.

2. In the discussion part, the effect of soil properties on crop growth, soil microbial community and structure should be explained.

  3. The information of comparison of previous studies of different crops or soil types is lacked.

Comments for author File: Comments.docx

Author Response

Reviewer 1

This manuscript explored the effect of L-α-amino acid-based biostimulant (Terramin® Pro) on phenotype of different lettuce cultivars and soil microbial community and structure under different soil types and conditions. The results suggested although the application changed soil microbial community and structure, the effects of this biostimulant on lettuce phonotype and soil microbial community and structure differed from soil type and conditions. It was still a sustainable alternative to modulate soil microbiome. This study will guide the application of biostimulants in crop growth and different soil types.

There are some details needed to be improved.

For introduction part:

1. The author introduced the general situation of biostimulant in agriculture system, but it lacks the information of the progresses of the effect of L-α-amino acid-based biostimulant on soil microbial community and structure under different crops, soil types and conditions. Since this experiment concerned about the crop type, soil type and condition.

We have updated the information depending on  protein hydrolysates and free amino acids including other soil types, crops (corn, maize) and different conditions. (Line 52 - 87)

2. Please add the reason for choosing lettuce as the test plant.

We have specified the reasons why lettuce was used in the manuscript (Line 90-92). This was mainly due to their economic importance worldwide, as it has relatively short cultivation cycles and fits a wide variety of soils.

For results part:

1. In Figure S1 and Figure 1(c), there is no meaning to compare the difference among the different time series under the same soil type. It should be compared the different among the three soil types in the same time.

Note that plant performance indicators, such as harvest yield, were determined only at T1. Following your comment, we updated the analysis and  we analyzed, at the same time, the difference among means of treatment levels in the three soil types both for Figure 1 (Panel C) and Figure S1. To facilitate results description and discussion we showed statistical differences among factor levels (e.g. treated versus control), within the same soil type (described in the Figure 1 and S1 caption). Figure 1 (Panel A) may provide a better overview of the general differences among soil types.

2. there is big different of soil nutrient among the three soil types, e.g. the soil texture, pH, M.O.total, P, Fe, Mn, Cu, Zn, NO3- in table 1. And the soil properties are also the main factor affect the soil microbial community and structure. Hence, I suggest the author add the analysis of the effect of soil nutrient on soil microbial community and structure.

Assessing the effect of external factors on microbial communities, such as physicochemical properties in soil, is one of the current challenges in microbiome research, including diet on human microbiome (Weiss et al., 2016). Oftenly, we can only assess  relationships through correlation analysis, which does not imply a relationship of cause-effect (Röttjers & Faust, 2018). We have added a comment about that in the discussion section (Line 298-240) and remarked it in the conclusion section (Line 339-341). For this reason, we performed correlation of soil physicochemical properties with microbial composition. We had to introduce pseudoreplication on microbiome samples, as we only had one measurement for soil properties in each soil type, at time 0 for the control condition. 

Here below we reported the resulting squared correlation coefficients and p-values for both 16S and ITS microbiome (Table X). Generally, almost all physicochemical properties were highly correlated with microbiome composition, both for ITS and 16S. This is in line with the expected outcome, since beta diversity analysis clearly clustered samples according to their soil type, but we can not assure if physicochemical properties affect soil microbiome or the opposite cause. Altogether, we are afraid to mislead the scope of the manuscript with a not conclusive statement about the microbiome vs physicochemical properties. However, we have modified the discussion section to include our hypothesis with putative changes in soil physical properties (see comment 2, discussion, 267-271). Soil physicochemical properties table has been moved to supplementary material.

Table X Correlation analysis of microbiome composition with soil physicochemical properties, , using envfit function from vegan R package.  

Röttjers, L., & Faust, K. (2018). From hairballs to hypotheses–biological insights from microbial networks. FEMS Microbiology Reviews, 42(6), 761–780. https://doi.org/10.1093/femsre/fuy030

Weiss, S., Van Treuren, W., Lozupone, C., Faust, K., Friedman, J., Deng, Y., Xia, L. C., Xu, Z. Z., Ursell, L., Alm, E. J., Birmingham, A., Cram, J. A., Fuhrman, J. A., Raes, J., Sun, F., Zhou, J., & Knight, R. (2016). Correlation detection strategies in microbial data sets vary widely in sensitivity and precision. The ISME Journal, 10(7), Article 7. https://doi.org/10.1038/ismej.2015.235

 For discussion part:

1. Line 204-212, it duplicated with the information in introduction part, or this description should be moved to the introduction part.

We excluded this section since some of the information was already in the introduction part. We included some of this information in the introduction section (Line 54-57).

2. In the discussion part, the effect of soil properties on crop growth, soil microbial community and structure should be explained.

How soil properties related to the most important plant nutrients (NPK) could be linked to crop growth and biostimulant supplementation were outlined in lines 267-271. Next, iron content was 2-fold lower in soils deviating from standard conditions. Deficit of iron was linked to the reduced content of chlorophyll in those soils and how Actinobacteria promotion through the use of the biostimulant could help to overcome this (Line 323-333).

3. The information of comparison of previous studies of different crops or soil types is lacked.

We have included several comparisons of PHs biostimulant application in lettuces but also in other crops, as well as included studies which address PHs impact on crop performance in contrasting soil types (Line 268-282).

Reviewer 2 Report

The manuscript “Lettuce soil microbiome modulated by an L-α-amino acid-based
biostimulant”  demonstrated that L-α-amino acid-based biostimu-
lant promotes plant performance and modulates soil microbiome community and structure
depending on the agronomical condition of lettuce crops. The results reported in manuscript is justified the topic, but few remarkable input must require to strengthen this manuscript.

1] Abstract portion is well written, but author unable to reflect hypothesis of present work, so author must be write perfect hypothesis that also justified the statistics applied in this research.

2] In introduction Line 23 to 25 “Hence, management of soil microbiome using environmen
tally friendly biostimulants is an appealing complement to chemical synthetic fertilisers
to improve crop yield and meet upcoming demand, while preserving and restoring soil
health status [3,4]. Author must strength with more suitable and justified references, author can outline from https://doi.org/10.1007/s10653-022-01433-3; https://doi.org/10.1080/02648725.2022.2131958.

3] Line 39 to 44 author should be explain abiotic stress that really address and connect to this study, author also add few line in details about biostimulants etc

4] Result section is perfectly design and impressive

5] In the discussion section the nematodes and its related study are not discussed properly , the author must be addressed with suitable justification, author can also justify this issue by connecting other concepts also.

6] Conclusion section look like as abstract, so please restructure, and write about future perspective

Author Response

Reviewer 2

The manuscript “Lettuce soil microbiome modulated by an L-α-amino acid-based biostimulant”  demonstrated that L-α-amino acid-based biostimu- lant promotes plant performance and modulates soil microbiome community and structuredepending on the agronomical condition of lettuce crops. The results reported in manuscript is justified the topic, but few remarkable input must require to strengthen this manuscript.

1] Abstract portion is well written, but author unable to reflect hypothesis of present work, so author must be write perfect hypothesis that also justified the statistics applied in this research.

We specified the hypothesis of our work in the abstract before briefly mentioning and linking our experimental approach (Line 6-10).

2] In introduction Line 23 to 25 “Hence, management of soil microbiome using environmentally friendly biostimulants is an appealing complement to chemical synthetic fertilizers to improve crop yield and meet upcoming demand, while preserving and restoring soil health status [3,4]. Author must strengthen with more suitable and justified references, author can outline from https://doi.org/10.1007/s10653-022-01433-3 ; https://doi.org/10.1080/02648725.2022.2131958.

Thank you for suggesting such useful references. We outlined some of the mechanisms microbiome may increase yields and aid soil restoration after these sentences (Line 28-30).

3] Line 39 to 44 author should be explain abiotic stress that really address and connect to this study, author also add few line in details about biostimulants etc

We specified some examples of abiotic stress in Line 49. In addition, we provided how PHs derived biostimulants mitigate some abiotic stressors, such as extreme temperature waves, and salinity in chickpea and lettuce cultivars (Line 62-65).

4] Result section is perfectly design and impressive

Thank you for the positive feedback.

5] In the discussion section the nematodes and its related study are not discussed properly , the author must be addressed with suitable justification, author can also justify this issue by connecting other concepts also.

We recaped nematode content of sick soil (Line 301-319). Sick soil has historically a track of nematode infestation. However, we do not have qualitative data regarding nematode content.

6] Conclusion section look like as abstract, so please restructure, and write about future perspective.

We have rewritten the conclusion section providing future perspective for this study line.

Reviewer 3 Report

In this Manuscript the authors evaluated the “Lettuce soil microbiome modulated by an L-α-amino acid-based biostimulant” using a commercial product from the company Bioiberica S.A.U. with whom they have a working relationship.

The approach of introduction, materials and methods, and the results look solid. I Consider important clarified the next points in the discussion:

1.- Are the beneficial effects on the plant due to the biostimulant or are they due to the effect of the microbiome on the plants?

2.- Is the modulation of the microbiome directly related to the biostimulant or is it due to changes in the compounds secreted by the roots of the plants?

3.- For the entomopathogenic fungi Beauveria and Metarhizium, there are several articles where it is reported that they have the ability to increase plant growth through their root association. I think it is important to discuss this in the Manuscript.

Author Response

Reviewer 3

In this Manuscript the authors evaluated the “Lettuce soil microbiome modulated by an L-α-amino acid-based biostimulant” using a commercial product from the company Bioiberica S.A.U. with whom they have a working relationship.

The approach of introduction, materials and methods, and the results look solid. I Consider important clarified the next points in the discussion:

1.- Are the beneficial effects on the plant due to the biostimulant or are they due to the effect of the microbiome on the plants?

This would be an interesting point. Unfortunately, we cannot claim this since it is the study of a causal relation and we just saw "correlations", we can hypothesize the observed effect or at least part of the effect is due to microbiome but further studies should be done in order to confirm this. 

This question is related to an above question by reviewer 1 (see result part/question 2), where we provided additional references and comments about causal vs correlation associations between microbiome and additional factors. In addition, we have added a comment about that in the discussion section (Line 339-341).

2.- Is the modulation of the microbiome directly related to the biostimulant or is it due to changes in the compounds secreted by the roots of the plants?

This would also be a great idea for future perspectives of the study. However, we could not address this in our study because we do not have metabolomic data regarding root exudates. In order to make this present, we pointed this concept out as a future perspective in the conclusion section (Line 343-344).

3.- For the entomopathogenic fungi Beauveria and Metarhizium, there are several articles where it is reported that they have the ability to increase plant growth through their root association. I think it is important to discuss this in the Manuscript.

Thank you for the suggestion. Indeed, these fungi genus have a wide range of beneficial applications. We focused more on the entomopathogenic impact during our discussion. Following your suggestion, we outlined other beneficial effects, such as plant growth promotion through their root association and heavy metal detoxification (Line 310-315).

Round 2

Reviewer 1 Report

The comment was demonstrated in the attachment.

Comments for author File: Comments.pdf

Author Response

Thank you for checking it carefully. We have made the changes requested. However, we cannot change the bibliography format since we are already using the MDPI official Latex template. We have informed the Editor and he said it is fine.

Reviewer 2 Report

The revised manuscript is substantially adress the suggestions and remarks

Author Response

Thank you for your feedback

Reviewer 3 Report

Considered that the authors have made the recommendations to the manuscript.

Author Response

Thank you for your feedback