Deciphering the Structural and Functional Diversity of Rhizobacteria from Stone Pine Inoculated with Plant Growth Promoting Rhizobacteria (PGPR) before and after Transplanted into Degraded Agricultural Soil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors used PGPR to improve pine seedlings in the reforestation of degraded agricultural soil and they also used a metagenomic approach to study bacterial rhizosphere structure and function. The following corrections are required before accepting the manuscript.

Rephrasing of line 78-84

Did the authors perform a soil microbial count of the abandoned agricultural land?

What amount/concentration/CFU-mL is added each month?

Check the complete manuscript where italics are required, e.g. line 140 in vitro.

Line 164-165, In general, tricalcium phosphate is used to evaluate phosphate solubilization, please mention the reference from where the recipe was adopted.

Line 208. Check if it is 10 mL or 10 uL.

Line 207, check if it is ufc or CFU?

Mention that the inoculations applied each month were of the same concentration used initially.

 

Figure 1 is it quitinses or chitinase, check?

Author Response

First, we would like to thank you for your constructive criticisms, which have substantially improved the quality of the manuscript. We have reviewed and modified all changes suggested by the reviewer.

The authors used PGPR to improve pine seedlings in the reforestation of degraded agricultural soil and they also used a metagenomic approach to study bacterial rhizosphere structure and function. The following corrections are required before accepting the manuscript.

Rephrasing of line 78-84.

Following the referee's instructions, we have rephrased this line (new lines 69-75).

Did the authors perform a soil microbial count of the abandoned agricultural land?

We have not done a count of microorganisms in abandoned agricultural soil. It is well known that the biodiversity in these soils is very poor and our hypothesis is that the growth of roots through the soil will integrate the rhizosphere bacteria into the soil ecosystem, enriching it in biodiversity. Thus, we have demonstrated it in the results, in which we have been able to observe a different microbial diversity in the rhizosphere depending on the inoculations received.

What amount/concentration/CFU-mL is added each month?

We have specified it in line 213 (100 mL of 10⁷ CFU/mL in each inoculation).

Check the complete manuscript where italics are required, e.g. line 140 in vitro.

We have reviewed the entire manuscript and have italicized all the terms that required it.

Line 164-165, In general, tricalcium phosphate is used to evaluate phosphate solubilization, please mention the reference from where the recipe was adopted.

The protocol used is described in the reference number 24, cited at the beginning of the paragraph.

 

De Freitas, J.; Banerjee, M.; Germida, J. Phosphate-solubilizing rhizobacteria enhance the growth and yieldbut not phosphorus uptake of canola (Brassica napus L.). Biol. Fertil. Soils 1997, 24, 358–364.

Line 208. Check if it is 10 mL or 10 uL.

Indeed, the correct unit is microliters (mL).  It has been corrected in the manuscript (line 200).

Line 207, check if it is ufc or CFU?

It is CFU. We have changed it twice, on lines 209 and 210.

Mention that the inoculations applied each month were of the same concentration used initially.

This information has been included in the text line 213.

 

Figure 1 is it quitinases or chitinase, check?

It is indeed chitinases, we have modified it in Figure 1.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the benefits of inoculating tree seedlings with plant-promoting microorganisms. Saplings of stone pine were inoculated several times with three different bacterial strains and a mixture of these strains (consortium). Subsequently, the size and thickness growth of stone pine seedlings before and after planting on a degraded soil area was investigated on two dates. In parallel, the microbiome in the rhizosphere of the seedlings was investigated.

The work is based on the comprehensive analytical repertoire of current molecular biology and bioinformatics evaluation methods. Using the latter, the authors have produced various data analyses, which are presented in 13 figures.

The manuscript is not suitable for publication.

This is primarily due to the fact that the objectives are weak and inadequate. The "search for beneficial bacteria" is a methodological aspect, but not a scientific question that corresponds to the experimental setup described. Accordingly, the results of this work are presented in great detail but rather as loose collections of different data sets. The subsequent discussion gets lost in generalities and considerations of the methods used. However, no overarching connections and insights are gained.

The text loses itself in generalities in many places. These are often not sufficiently related to the work presented or contexts are so abbreviated that statements are oversimplified and therefore incorrect.

·       Introduction: Omit the first section (lines 29-49) and start with the text from line 50. Lines 93-97 are aspects that belong in the material and methods section.

·       Discussion: L. 424-432: Belongs to material and methods.

·       L. 433-443; 500-507: These are generalities.

·       L. 511 ff: The basic properties and functions of Proteobacteria, Bacteroidetes, Acidobacteria and Actinobacteria are widely known textbook knowledge.

·       L. 570-599: Here the authors get lost in the discussion of the bioinformatics tools they have chosen. This discussion is irrelevant for the interpretation of the data presented in the results section.

Material and methods: The site used in the study is not characterized in any way. There is no information on the site (relief, climate and weather, existing vegetation, etc.), the soil (soil type, texture, pH, nutrient content, etc.) and the sampling (depth, sample preparation, etc.). In the introduction and discussion, very prominent mention is made of site disadvantages such as erosion and nutrient depletion; however, there is no indication of the extent to which this applies to the specific site.

In many places in the results section, the authors show the usual composition of the microbial community of soils or rhizosphere soils. Differences between the investigated variants, however, remain weak. The Principal Component Analyses presented in Fig. 12 and 13 explain 25.5% and less of the variance. This means that the data distribution in the point cloud is largely random. In contrast, the authors reveal that the seasonal effect (sampling in fall vs. spring, Fig. 6) has a very large influence. Only on closer consideration of the information in the material and methods section does it become apparent that the fall sampling was carried out before planting in the nursery, while the spring sampling was carried out at the recultivated site. This means that two fundamentally different pairs of samples are compared and thrown into the same pot.

Figures: The number of 13 figures is excessive. The content of Fig. 3 and 4 can be summarized in a table. The content of Figures 5 and 6 can be summarized in one figure. Figures 7, 8 and 9 are only mentioned very briefly in the results section. These should be moved to an appendix (Supporting Material). Figures 12 and 13 show the results of a PCA which, due to the low explained variance, shows that the tested parameters do not approximately explain the distribution of the data points in the point cloud. A brief mention of this result in the text would suffice; the Figs. should be deleted.

Comments on the Quality of English Language

See the ticked answer in recommendations for authors

Author Response

First, we would like to thank you for your constructive criticisms, which have substantially improved the quality of the manuscript. We have reviewed all your comments and modified or provided a rebuttal.

 

Below we present a summary of all the actions carried out based on your comments and suggestions:

 

1. We have eliminated the introduction and discussion paragraphs that were about material and methods.
2. We have summarized the first paragraph of the introduction.
3. We have summarized the paragraphs that are considered generalities to maintain the idea without doing it too long.
4. In the paragraph about the Phyllum, on the one hand, we have summarized the information that can be considered textbook knowledge, and on the other hand, we have added figures 7S, 8S and 9S in supporting material to give this explanation the necessary scientific relevance, emphasizing the differences that inoculations with PGPR and the consortium causes the composition of the rhizosphere microbiome. This aspect has been included as a new idea in conclusions section.
5. We have converted figures 3 and 4 into a table.
6. We have merged figures 5 and 6 into one.
7. We have included figures 7, 8 and 9 in supporting material, adding 3 more that reinforce the objectives of the work.
8. We have included in material and methods a paragraph with the description of the climatology of the site and the characteristics of the abandoned agricultural soil where the pines were transplanted.
9. We have eliminated figures 12 and 13 leaving only a small comment on those results.

 

This study investigates the benefits of inoculating tree seedlings with plant-promoting microorganisms. Saplings of stone pine were inoculated several times with three different bacterial strains and a mixture of these strains (consortium). Subsequently, the size and thickness growth of stone pine seedlings before and after planting on a degraded soil area was investigated on two dates. In parallel, the microbiome in the rhizosphere of the seedlings was investigated.

The work is based on the comprehensive analytical repertoire of current molecular biology and bioinformatics evaluation methods. Using the latter, the authors have produced various data analyses, which are presented in 13 figures.

The manuscript is not suitable for publication.

This is primarily due to the fact that the objectives are weak and inadequate. The "search for beneficial bacteria" is a methodological aspect, but not a scientific question that corresponds to the experimental setup described. Accordingly, the results of this work are presented in great detail but rather as loose collections of different data sets. The subsequent discussion gets lost in generalities and considerations of the methods used. However, no overarching connections and insights are gained.

The text loses itself in generalities in many places. These are often not sufficiently related to the work presented or contexts are so abbreviated that statements are oversimplified and therefore incorrect. 

We do not agree with this statement: “The "search for beneficial bacteria" is a methodological aspect, but not a scientific question that corresponds to the experimental setup described.” and we are going to argue it.

It questionless that the degradation of abandoned agricultural land is one of the most serious environmental problems we have today, in fact, it is one of the priority lines EU's Common Agricultural Policy (CAP). This is because the soil is probably the most important heritage that humanity has and abandoned agricultural soils are the main focus of erosion that impoverishes them in biodiversity, in nutrients and leads them almost to sterility in a short time. We will also all agree that reforestation programs are one of the main ways to avoid this. But it is well known that the introduction of tree species into this type of soil is not easy, because the trees encounter very complicated physical-chemical and biological conditions and the survival rates can be low. The use of PGPR and mycorrhizae in these trees prior to transplant is a practice that has been scientifically proven to be very valid to improve the survival of these seedlings at the time of transplant. But obviously, not all bacteria are valid for this purpose, and therefore, an enormous research effort is needed to find those bacteria that with a high probability may be suitable. In our opinion, this is a key aspect of the work, because it has been carried out methodically, scientifically and with a clear objective. The chosen bacteria come from the rhizosphere of plants of the same species growing in a soil similar to which the seedlings are transplanted. This is something that may be decisive due to the selective pressure that the plant exerts on the bacteria in order to achieve a microbiome that facilitates its adaptation to the soil in which it develops.

Therefore the screening process is a scientifically complex process that requires the management of a series of criteria based on biological tests that allow the selection of strains specially prepared to exert a favorable effect on the adaptation and survival of the plant. It is widely described in the literature and is well known by the scientific community that there are many metabolic capacities that serve as markers of PGPR capacity. The capacities that we have looked for in the strains (auxin production, phosphate solubilization, chitinase capacity and siderophore production) have not been chosen at random but are capacities that can help plants to improve their ability to acclimatize to those degraded soils. Moreover, PGPRs have a wide spectrum of effects on plants through direct and indirect mechanisms. In general, these effects usually produce better fitness and adaptation capacity of the plant. But the PGPR-plant interaction has a specificity component, whereby not all bacteria will have the same effects on a specific plant species in a soil with specific characteristics. For this reason, in this work the search for native PGPR that can help plants adaptation to the degraded soil is of special importance. That´s why the PGPR screening has been described and discussed in the work, because it seems very important to us as the central core of one of the objectives of the work.

However, we have reformulated the objective as follows: “The main objective is to determine a procedure to improve plant adaptation to degraded agricultural soils improving soil biodiversity by delivering PGPR inocula to seedlings during the nursery process. To achieve this purpose, a screening of PGPR from stone pine growing in low nutrient soils will be carried out, isolates will be characterized according to genetic diversity and putative beneficial traits. Selected strains will be delivered to stone pine seedlings in the nursery. After transplant to agricultural soil, effects of inoculum on rhizosphere from a structural and functional perspective and plant growth will be determined.” 

Regarding de statement: “The subsequent discussion gets lost in generalities and considerations of the methods used.”

The biodiversity of the rhizosphere microbiome is currently understood as a good indicator of the capacity of the root environment to provide the plant with adaptive advantages and everything related to the discussion around biodiversity could be considered speculative due to the enormous complexity of the aspects discussed and the lack of solid pillars (frame of reference) on which to support the different arguments, but this is a general aspect for all works in which the concept of biodiversity is handled. Our purpose is to study what is the effect of the PGPR bacteria introduced into the rhizosphere of the seedlings on the microbial biodiversity of this type of soil.

New omics techniques and bioinformatics tools are helping us begin to understand this enormous complexity. Within this complex system we have structurally and functionally characterized the rhizosphere communities and their biodiversity under the effect of the different treatments, finding differential effects. The presence, for example, of unique taxa, demonstrates the enriching effect of biodiversity. We therefore consider that a very comprehensive analysis of the communities has been carried out, so we do not agree that we are lost in generalities. What's more, the discussion points are so broad that it makes the discussion seem diffuse, but the discussion is perfectly threaded to explain the effect of the selected PGPR in the improvement of plant adaptation and soil biodiversity.

Moreover we have included a paragraph (and 7S, 8S and 9S in supporting material) emphasizing the differences that inoculations with PGPR and the consortium causes the composition of the rhizosphere microbiome.

• Introduction: Omit the first section (lines 29-49) and start with the text from line 50. Lines 93-97 are aspects that belong in the material and methods section.

Lines 29-49 have been summarized in a single paragraph, but we have not eliminated it because we believe that we introduced some important ideas in the context of the work (new lines 29-39).

We have removed lines 93-97 because it is, indeed, information that is about material and methods section.

• Discussion: L. 424-432: Belongs to material and methods.

Indeed this paragraph corresponds to material and methods and we have eliminated it.

433-443; 500-507: These are generalities.

1. 433-443. We have rewritten this paragraph, trying to synthesize the important ideas, trying to avoid generalities, but we have not eliminated it since it seems necessary for the discussion of the work (new lines 422-431).
2. 500-507. We have rewritten this paragraph, trying to synthesize the important ideas, trying to avoid generalities, but we have not eliminated it since it seems necessary for the discussion of the work (490-497).

• L. 511 ff: The basic properties and functions of Proteobacteria, Bacteroidetes, Acidobacteria and Actinobacteria are widely known textbook knowledge.

Indeed, we agree with you that in this paragraph contains information widely known in the literature. To give it the value it has in the discussion of the work and in the achievement of the objectives, we have summarized the information and we have added to supporting material 3 new graphs in which the variations of the 4 Phyllum indicated at the level of Class and Order are shown. It can be clearly seen the changes caused by the different treatments in the taxonomic composition of these Phyllum at these taxonomic levels. This aspect is key to understanding the objectives of the work, since it is clearly seen how each treatment has a differential impact on the biodiversity of the rhizosphere microbiome (lines 498-526).

• L. 570-599: Here the authors get lost in the discussion of the bioinformatics tools they have chosen. This discussion is irrelevant for the interpretation of the data presented in the results section.

We believe that it is not irrelevant since the structure-function relationship is fundamental to understanding the role of microbial communities. That is why we have summarized it to maintain the idea without explaining in detail bioinformatics tools and getting lost in the discussion (lines 552-565).

.  Material and methods: The site used in the study is not characterized in any way. There is no information on the site (relief, climate and weather, existing vegetation, etc.), the soil (soil type, texture, pH, nutrient content, etc.) and the sampling (depth, sample preparation, etc.). In the introduction and discussion, very prominent mention is made of site disadvantages such as erosion and nutrient depletion; however, there is no indication of the extent to which this applies to the specific site.

Indeed, there was a lack of information regarding the soil, climate, etc. as the referee indicates. Thus, we have included information on the soil and climate of the area in section 2.7, lines 219-231.

In many places in the results section, the authors show the usual composition of the microbial community of soils or rhizosphere soils. Differences between the investigated variants, however, remain weak. The Principal Component Analyses presented in Fig. 12 and 13 explain 25.5% and less of the variance. This means that the data distribution in the point cloud is largely random. In contrast, the authors reveal that the seasonal effect (sampling in fall vs. spring, Fig. 6) has a very large influence. Only on closer consideration of the information in the material and methods section does it become apparent that the fall sampling was carried out before planting in the nursery, while the spring sampling was carried out at the recultivated site. This means that two fundamentally different pairs of samples are compared and thrown into the same pot.

As suggested below, Figures 12 and 13 have been removed. On the other hand, in relation to Figure 6, we have not attempted to reveal a seasonal effect, we simply present diversity data at the two sampling moments. We have emphasized this fact in lines 479-479.

Figures: The number of 13 figures is excessive. The content of Fig. 3 and 4 can be summarized in a table. The content of Figures 5 and 6 can be summarized in one figure. Figures 7, 8 and 9 are only mentioned very briefly in the results section. These should be moved to an appendix (Supporting Material). Figures 12 and 13 show the results of a PCA which, due to the low explained variance, shows that the tested parameters do not approximately explain the distribution of the data points in the point cloud. A brief mention of this result in the text would suffice; the Figs. should be deleted.

We have replaced Figures 3 and 4 with a table (Table 1) and summarized Figures 5 and 6 in one Figure (Figure 3 a) and b)).

 

We have moved Figures 7, 8 and 9 to Supporting Material section as Figures 4S, 5S and 6S.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

You integerated the required changes to the manuscript. 

The only point is that the initial cfu count was not performed on known knowledge of poor biodiversity of that soil, still cfu would have further authenticated the results.

Author Response

Dear reviewer, once again we want to thank you for your efforts valuing our work, your comments are very valuable to continue improving the quality of the manuscript

The only point is that the initial cfu count was not performed on known knowledge of poor biodiversity of that soil, still cfu would have further authenticated the results.

We are not sure if you refer to the isolation of bacteria in the ten areas chosen for PGPR isolation and screening or to the functional and structural study performed in the rhizosphere of pines inoculated with the selected strains.

In the first case (screening), indeed we did not know the existing biodiversity, but the objective of that part of the work was to isolate strains that could potentially be used as PGPR in pine trees that would later be transplanted to abandoned agricultural land. An initial count may have given us interesting information, but we believe that it was not very important for achieving the objectives. We intended to isolate bacteria from pine trees that years ago had to face the same situation as the pine trees in our experiment. The hypothesis was that the selection capacity of the plants would have configured optimal biodiversity in the rhizosphere of these pines to acclimate well to those conditions, and that would improve the capacity of these strains to colonize and influence the physiology of the pines improving their acclimatization capacity when transplanted.

If you refer to the cfu that were in the field where the pines were transplanted, it would undoubtedly have been an interesting information, although counting the cultivable bacteria does not give a clear idea of the biodiversity of the soil. From the point of view of the objective of the work, which was to study the biodiversity of culturable and non-culturabe bacteria (metagenomic approach)  that is generated in the rhizosphere of the pines inoculated with the different PGPR chosen, we do not quite see the interest of having the cfu number of culturable bacteria.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have responded to my comments and substantially improved the manuscript.

With my comments, I did not intend the relevance of the work but to ask the authors for much clearer objectives and with this a much sounder and deeper data evaluation and discussion.

Still some revision and additional data is required.

The title should be changed, since two subsequent samplings at different times hardly cover microbial dynamics. “Deciphering the structural diversity and functions of rhizobacteria from stone pine inoculated with PGPR before and after transplanted into degraded agricultural soil”.

L 24: How did your study biomarkers that can be generally applied?

L. 47: Reword this part of the sentence.

L. 82-83: Change to “…  one-year-old pine trees were inoculated that were going to be transplanted … .”

L. 84: Here and throughout the manuscript: Write everything you did and found out in past tense. Only information taken from references is written in present tense.

L. 100. Here and throughout the manuscript: Write out numbers smaller than 10 that come without a unit.

L. 102: Correct to “Three hundred and sixty one-year-old pine trees”.

L. 138. Here and throughout the manuscript: Write stoichiometric numbers as suffix.

L. 172: Correct to “Dimethyl”.

L. 204: In section 2.7 information is in part repeated that was already presented in 2.1. Reorganize and omit redundancies.

L. 219: The information on the soil is still insufficient. Due to this, the authors have to speculate in the discussion, e.g. about the P an other nutrients’ status in the soil.

L. 329. Here and throughout the manuscript: Change the terms used for the samplings. The major difference was not the time of sampling but that the samplings were before (nursery, spring) and after transplanting the trees to the degraded soil (autumn). Just mentioning the sampling time gives a wrong impression.

Fig. 10 and 11. Correct the numbering of the figures to 4 and 5. The legends to the figures with many abbreviations and the explanation of these abbreviations in the subtitle makes the understanding of the figures very inconvenient. To make them much more easy to read, it is suggested to write the names of the treatments to the right of the figure, in the respective position. The legend can be even deleted instead.

L. 415. Correct the wording of “samples can be stablished in Autumn”.

L. 438. Here and elsewhere in the text, the authors claim that the abandoned agricultural field site used for recultivation with pine was low in nutrients. They cite various references as proof. However, this could be valid or not. Especially high phosphate contents originating from previous agricultural use can persist for very long times (up to centuries). The authors should rely on own analytical data instead. Results of an analysis of the plant available nutrients in that soil would avoid the many unproven speculations in the discussion, concerning nutrients and microbial nutrient cycling.

L. 440-444: Here the authors drift off into the very general or global. Instead, they should refer to the specific conditions at the site under investigation.

L. 451: Refer to corresponding rhizobacteria that were abundant in the samples of this study. Otherwise this discussion appears to be general and detached from the study presented.

L. 462: Also the discussion on root geometry should be based on own data. Otherwise it is general and detached.

L. 473. It remains unclear whether the inoculated rhizobacteria survived and contributed to the rhizosphere community.

L. 488. Add a line break. The change in topic to the “use of biofertilizers” requires a separate paragraph.

L. 504 and 510: correct to “phyla”.

L. 509. Here and in the following: Write class and order not with upper case letters.

L. 552-565. This is still very theoretical information that is not sufficiently linked with the present research of this study. Reduce the information to one or two sentences that introduce the following paragraph.

Comments on the Quality of English Language

see detailed comments to the authors.

Author Response

Once again, we would like to thank you for your constructive criticisms, which have substantially improved the quality of the manuscript. We have reviewed all your comments and modified or provided a rebuttal

The authors have responded to my comments and substantially improved the manuscript.

With my comments, I did not intend the relevance of the work but to ask the authors for much clearer objectives and with this a much sounder and deeper data evaluation and discussion.

Still some revision and additional data is required.

The title should be changed, since two subsequent samplings at different times hardly cover microbial dynamics. “Deciphering the structural diversity and functions of rhizobacteria from stone pine inoculated with PGPR before and after transplanted into degraded agricultural soil”.

Following your suggestion we have modified the title: " Deciphering the structural and functional diversity of rhizobacteria from stone pine inoculated with PGPR before and after transplanted into degraded agricultural soil”

L 24: How did your study biomarkers that can be generally applied?

The study of biomarkers was not an objective of the work. Although the observed changes could lead to find biomarkers, it is true that these could only be validated as biomarkers after comparing several works under similar conditions. Since this is not the case, we have considered to remove this statement from the work.

1. 47: Reword this part of the sentence.

Following your suggestion, this sentence has been rephrased as follows: “This approach is not free of difficulties, being one of the most important the problems the transplanted forest species face in trying to colonize these soils, which are sometimes severely degraded and have lost some of the qualities required for the plants to root.”

1. 82-83: Change to “…  one-year-old pine trees were inoculated that were going to be transplanted … .”

This sentence has been rephased: “Three strains were chosen to inoculate one-year-old pine trees that were going to be transplanted to abandoned agricultural land.”

1. 84: Here and throughout the manuscript: Write everything you did and found out in past tense. Only information taken from references is written in present tense.

Done

L. 100. Here and throughout the manuscript: Write out numbers smaller than 10 that come without a unit.

Done

L. 102: Correct to “Three hundred and sixty one-year-old pine trees”.

Corrected

L. 138. Here and throughout the manuscript: Write stoichiometric numbers as suffix.

Done

L. 172: Correct to “Dimethyl”.

Done

L. 204: In section 2.7 information is in part repeated that was already presented in 2.1. Reorganize and omit redundancies.

This sentence of 2.7. section: “Three hundred and sixty potted one-year-old pine plantlets were arranged in the nursery. There were 5 treatments: control, Z7.15, Z4.3, Z5.4 and consortium (Z7.15+Z4.3+Z5.4). Therefore, each treatment contained 72 plants.” Has been deleted because this information is indeed repeated in 2.1 section.

L. 219: The information on the soil is still insufficient. Due to this, the authors have to speculate in the discussion, e.g. about the P an other nutrients’ status in the soil.

We think that probably due to a poor drafting of the paragraph you refer to in the commentary on L. 438, we have failed to develop the idea we wanted to discuss. The information we have provided on the soil, refers to the soil where the pines were transplanted. However, the speculations you refer to are about the soils where the PGPR screening was done. To avoid such speculation as much as possible, we have modified that paragraph (see comment on line 438), in which we simply discuss why siderophore production and phosphate solubilization were the two major PGPR capacities among the isolated bacteria. With the input we provide, we believe that speculation has been reduced to a minimum. With regard to the data on the soil where the pines were transplanted, we believe that they are sufficient, because they clearly indicate that it is a soil with low fertility (very low % organic matter, low C/N ratio), probably with a low availability of certain nutrients due to high pH and poor structure. With this we simply want to highlight that the soil were pines are transplanted is not a high quality one and that the adaptive advantage that the PGPR provides can help pines to better acclimatize, in addition to the fact that the rhizosphere of these pines can improve the soil biodiversity accelerated its recovery.

 

L. 329. Here and throughout the manuscript: Change the terms used for the samplings. The major difference was not the time of sampling but that the samplings were before (nursery, spring) and after transplanting the trees to the degraded soil (autumn). Just mentioning the sampling time gives a wrong impression.

We have corrected this throughout the manuscript. We want to point out that the sampling carried out in the nursery was done in Autumn 2019 and the one after transplanting was done in Spring 2020.

Fig. 10 and 11. Correct the numbering of the figures to 4 and 5. The legends to the figures with many abbreviations and the explanation of these abbreviations in the subtitle makes the understanding of the figures very inconvenient. To make them much more easy to read, it is suggested to write the names of the treatments to the right of the figure, in the respective position. The legend can be even deleted instead.

The numbering of the figures has been corrected and the legends of figures 4 and 5 have been modified according to your suggestions.

L. 415. Correct the wording of “samples can be stablished in Autumn”.

We have rephased as follow: “Aware of this fact, three clusters of Autumn samples can be stablished”

L. 438. Here and elsewhere in the text, the authors claim that the abandoned agricultural field site used for recultivation with pine was low in nutrients. They cite various references as proof. However, this could be valid or not. Especially high phosphate contents originating from previous agricultural use can persist for very long times (up to centuries). The authors should rely on own analytical data instead. Results of an analysis of the plant available nutrients in that soil would avoid the many unproven speculations in the discussion, concerning nutrients and microbial nutrient cycling.

We believe that the paragraph was poorly written and this has probably caused you to misinterpret it. In the paragraph you indicate, we don’t refer to the soil in which the pines were transplanted. We trying to discuss and justify why most of the bacteria isolated in the ten zones, showed capacity of producing of siderophores and solubilize phosphates as the predominant activities. There is a lot of literature on the subject, and it is quite well demonstrated that the capabilities of the bacteria that inhabit the rhizosphere of plants are related to the nutritional needs of plants. If there is little availability of iron, plants are able to select bacteria capable of producing siderophores and the same happens with phosphorus and other nutrients.

We have modified that paragraph so that this idea is clear and there is no confusion or speculation (lines 425-440).

L. 440-444: Here the authors drift off into the very general or global. Instead, they should refer to the specific conditions at the site under investigation.

See the previous comment.

L. 451: Refer to corresponding rhizobacteria that were abundant in the samples of this study. Otherwise this discussion appears to be general and detached from the study presented.

We do not quite understand what you mean by this comment. In this sentence, with "the selected PGPR" we are referring to the strains selected in this study. In this sentence we cannot refer to the rhizobacteria abundant in the samples of this study because we want to refer to the three selected bacteria. To reinforce the idea we want to convey in this paragraph, which is the relationship between auxin production, growth promotion and root structure, we have modified it with previous work carried out by the group, in which we have already demonstrated this relationship. See above comments also (lines 450-454).

L. 462: Also the discussion on root geometry should be based on own data. Otherwise it is general and detached.

Please see the previous comment.

L. 473. It remains unclear whether the inoculated rhizobacteria survived and contributed to the rhizosphere community.

We do not intend, because we cannot, to prove the presence of the inoculum bacteria with the metagenomic technique used. This technique uses the 16s rRNA gene sequence, a technique that is not accurate enough to differentiate strains of the same species, sometimes not even species of the same genus, and therefore it is almost impossible to identify the inoculated strains among the thousands of sequences analyzed. What we are trying to say in the paragraph that the referee indicates, is that the different inoculated bacteria have differential effects on the rhizosphere microbiome specially during the nursery period. However, these differences are attenuated once transplanted to the field.

L. 488. Add a line break. The change in topic to the “use of biofertilizers” requires a separate paragraph.

We have made a line break where referee indicates.

L. 504 and 510: correct to “phyla”.

Done

L. 509. Here and in the following: Write class and order not with upper case letters.

Done

L. 552-565. This is still very theoretical information that is not sufficiently linked with the present research of this study. Reduce the information to one or two sentences that introduce the following paragraph.

Following your suggestion we have reduced the information in two sentences.