Morphophysiological, Enzymatic, and Elemental Activity in Greenhouse Tomato Saladette Seedlings from the Effect of Plant Growth-Promoting Rhizobacteria

REVIEWER

OBSERVATION

ANSWER

REVIEWER 2

1.        In introduction, rationale and novelty of study is not well clear. Likewise testing hypothesis is missing.

With the new version we consider that we will be complying with the required observations.

REVIEWER 2

2.        Materials and Methods section is very long and confusing. Moreover, procedure should be concisely described supported with relevant reference. Study design is unclear. From treatments given in Table 1, it looks completely randomized design while the authors stated factorial design. Moreover, how authors manage three inoculation methods regarding distilled water, nutrient solution and buffer. The authors should consider it completely randomized design and reanalyze the data taking 15 treatments as given in Table 1.

With the new version we will be complying with these observations…. Thank you very much for those fine insights indicated throughout the document….

Some sections were rewritten to be more explicit throughout the document.

REVIEWER 2

3.        In methodological section the authors claimed total 15 treatments but in Fig 2-4 there are 21 treatments given. In Fig 6 treatments are different from rest of the data and even without SD. The authors should reanalyze data following RCBD to solve all such issues.

We appreciate these fine observations because in this way we enrich the document and thus make us understand better.

REVIEWER 2

4.        After reanalyzing the data, the authors should concisely write results as the trend is almost similar.

Thanks for each and every one of the observations… we attach two documents (1.- the new version with registered changes and the pdf version, showing each and every one of the changes made).

REVIEWER 2

5.        Results and discussion sections should be separated. Currently logical explanation of results is totally missing.

We appreciate these fine observations because in this way we enrich the document and thus make us understand better.

REVIEWER 2

6.        In table 4 the authors presented data of bacterial types for different nutrients but ignored the 2^nd factor (methods of application) without any reason. By analyzing data according to completely randomized design such issues will be solve.

Thanks for each and every one of the observations… we attach two documents (1.- the new version with registered changes and the pdf version, showing each and every one of the changes made).

REVIEWER 2

7.        The authors should concise the Materials and Methods, results, discussion and conclusion section.

We considered this observation

REVIEWER

OBSERVATION

ANSWER

REVIEWER 3

1.        The results are not sufficiently discussed compared with data from other publications either in the form of a separate section "discussion" or in "results and discussion". I would recommend making a separate "discussion" section. This would help to generalize the results obtained.

We have separated discussion results.

REVIEWER 3

2.        In conclusion section, there are no approval about which treatment options (spraying, seed treatment) were successful.

Thanks for this observation…. we included this section required.

REVIEWER 3

3.        According to the Abstract, the goal was to isolate bacteria capable, among other things, of solubilizing phosphate (SF) and synthesizing organic acids (PS), which in principle can be related. But the manuscript contains no data on the quantity of phosphates dissolved by bacteria or organic acids synthesized by bacteria

These results are appreciated in section 3.1.

First the total of isolates obtained from the soil is indicated and then from the root.

Next the results of the salinity test, of temperature.

Next of fixation of nitrogen.

Of solubilization of phosphates and then of production of siderophores. Finally, in that same section it is indicated that of the 19 isolates, only 4 were those that showed the highest values.

REVIEWER 3

4.There is no single structure for interpreting the results that were obtained. For example, the representation of 19 isolates in either percentage or quantitative form is not easy to understand.

It was conssidering and was changed to an exact number of bacterial isolates.

REVIEWER 3

5.        It is not entirely clear why in the section "materials and methods" refers to the ideality of the selected tomato variety for the world market.

In section 2.4 you can see why this hybrid is selected by the producers:

The hybrid produces uniform fruits of good quality, with soft shoulders, shine, and firmness throughout the season, even in adverse conditions. Extra-large to large fruit are of intermediate maturity, and are ideal for national and export markets [25].

REVIEWER 3

6.        The purpose of introducing three controls in a greenhouse experiment with tomato seedlings is not explained. What role does each of them play? For what purpose is Steiner Solution selected? Why were the plants treated with distilled water?

In section 2.5 this response to the reviewer's observation is appreciated:

Distilled water was considered an additional treatment because it does not contain minerals. The water from Lagunera Comarca is rich in minerals; according to the classification of water for agricultural irrigation in Manual 60 of the US Department of Agriculture [27], the water is classified C2S1 of medium salinity [28].

Phosphate buffered saline (PBS) is a pH buffer solution commonly used for biochemical procedures due to its osmolarity and ion concentration (Cl^-, Na⁺, and K⁺). This solution is isotonic and non-toxic to cells, and is prepared from sodium chloride, sodium phosphate, and, in some formulations, potassium phosphate (Table 2). PBS is used as a neutral vehicle for cells because it does not modify the expression profile and normal cell functioning. It can be used as a diluent for methods of drying biomolecules because the water molecules present in PBS adhere around the biomolecule allowing immobilization on a solid surface.  

REVIEWER 3

7.        If it is planned to grow tomatoes in saline conditions, why were the properties of bacteria and their effect on tomato seedlings not tested in saline conditions? Why is peat chosen as the seed substrate? Under these conditions, the plants do not experience stress caused by high salinity. There are no studies on how well seedlings with introduced bacteria survive in saline soil. Without this, this work looks unfinished.

The second part, which consisted in evaluating the bacteria with their types of inoculation in tomato seedlings, was under conditions that the nurseryman and seedling producer developed in a production system. And that when showing the producer the results, he equates them with the System that he develops.

Peat moss is a sterile commercial product that is widely used by seedling-producing nurserymen (it is organic moss).

REVIEWER 3

8.        Why were tomatoes grown in greenhouses? How do Lagunera Comarca and greenhouse water relate to each other?

In the introductory section it was rewritten. We consider that the new version is better supported in this aspect

REVIEWER 3

9.        The activity of nitrate reductase is used as an indirect indicator of nitrogen nutrition, the nitrogen content is also an indirect indicator of nitrogen nutrition. Why were two indicators investigated? It is not discussed how they relate. The activity of nitrate reductase was studied by three different methods. Why? Do these methods complement each other?

This explanation is indicated in section 3.2.6. in each paragraph of each enzyme the following can be seen:

Regarding nitrate reductase, the endogenous reaction quantifies the contribution of nitrate naturally assimilated by the plant to later reduce it to nitrite.

Regarding the nitrate reductase enzyme, the reaction induced with NO3- as a substrate quantifies the contribution of nitrate assimilated by the plant and subsequently reduced to nitrite.

In the determination of the enzyme nitrate reductase in reaction with molybdenum (Mo) as a cofactor, Mo serves as a bridge to bring the substrate and the enzyme together. In this case, it helps to make the reaction more stable for the NR enzyme.

REVIEWER 3

10.     The authors write that the studied bacteria fix nitrogen, although they do not quantify this process. Its correlation with nitrate reductase activity is not discussed

In section 3.1 results, each result of each test was rewritten and sectioned with paragraphs:

First the total of isolates obtained from the soil is indicated and then from the root.

Next the results of the salinity test, of temperature.

Next of fixation of nitrogen.

Of solubilization of phosphates and then of production of siderophores. Finally, in that same section it is indicated that of the 19 isolates, only 4 were those that showed the highest values.

REVIEWER 3

11.     In the introduction, the need to search for local strains is justified by the fact that they will greater survive in saline soil. But there is no evidence that author's strains survive in saline soil and are not displaced by other bacteria.

In the introductory section it was rewritten. We consider that the new version is better supported in this aspect.

REVIEWER 3

12.     The plant experiment was evaluated 40 days after sowing. What is the reason for this period?

It was evaluated at 40 days because it is the period in which a seedling to be manipulated in a transplant, stops being sensitive and vulnerable to mechanical damage, and consequently the entry of pathogens that could be in the soil where it will be developed until production.

REVIEWER 3

13.     Do these bacteria survive during seed germination under conditions of osmotic stress caused by distilled water?

The literature review and experiments based on this type of microorganisms indicate that it is an alternative that may be successful.

REVIEWER 3

14.     Comparison of the elemental composition of plants obtained as a result of a series of experiments with different types of treatment with salt solutions and distilled water is incorrect because when spraying with salt solutions, plant nutrition can go through the leaves and roots.

It is not properly as indicated by the reviewer… .we remember that the tomato plant is a plant that is semi-tolerant to salinity… ..it is a physiological strategy that this particular plant presents …… despite being semi-caloric, it requires a nutritional source to comply totally your metabolism and is reflected in the organoleptic characteristics that the consumer demands.

REVIEWER 3

15.     The study of the properties of bacteria is one of the main goals of the work. However, generally accepted quantitative methods are not used to study their properties. The authors limit themselves to indirect (but rather qualitative) research methods. Measuring the diameter of colonies cannot be considered a quantitative method for assessing the biochemical activity of bacteria or their quantity, since the diameter of colonies, in addition to the growth rate, is influenced by many factors, such as bacterial motility the ability, the amount of intercellular matrix. I believe that the authors should obtain additional experimental data on the properties of bacteria and add them to the manuscript before considering publishing it.

The properties of microorganisms were studied according to the Manual AND tests for the Identification of Medical Bacteria [16, 17], and not according to generally accepted books, for example, Bergey’s Manual of Systematic Bacteriology.

The methods applied in this research (section 2.3.) Are based on the method used in various experiments… .for this case we reference 12 references ……. But there are many many more !!!

For the characterization of bacterial microorganisms, although it is true that biochemical characteristics can be considered for their identification, FOR THIS STUDY WE DO NOT CONSIDER THESE BIOCHEMICAL TESTS…. WE DIRECTED OUR STUDY TO A CHARACTERIZATION OF THE 16Sribosomal GENE. …… ..that is why we do not refer to the Bergey Manual of Systematic Bacteriology.

REVIEWER 3

16.     How do you recognize your bacteria from others when analyzing the microorganisms of the rhizosphere (chapter 2.6.4)?

This fine observation is appreciated, and we consider that with the new wording the method developed will be more explicit.

REVIEWER 3

17.     The strains are poorly described as PGPR, it is recommended to evaluate their nitrogenase activity, ability to synthesize phytohormones ore ACC deaminase.

Based on studies of published articles, and on the experience as a work team in this line, the tests developed can be considered sufficient. We did not omit that the phytohormones and deaminase tests were considered, the study may be more complete ………… however, we reiterate, in previous publications this observation had not been made to us… .in addition, the characterized microorganisms indicating that they DO BELONG TO MICROORGANISMS PROMOTERS OF PLANT GROWTH.

REVIEWER 3

18.     What method was used to measure the nitrogen concentration?

In section 2.6.3. we added the reference "[32]".

REVIEWER 3

19.     30% of the 19 bacteria are 5.7 bacteria. The exact number is required (line 282).

We believe that with the new wording there will be a better understanding considering the fine observations of the reviewers.

REVIEWER 3

20.     Initially, strains were selected from the roots of halophytic plants. Why was the first passage performed in media with zero NaCl content ?

For the isolation of the bacteria, 4 concentrations of salt were used (0, 0.25, 0.5 and 0.75 M of Nacl) ... ... for us as a team that works this line, we are interested in knowing the faculty that these isolates have and making them known to the public interested (about the ability of these bacteria to grow without and with salt)

REVIEWER 3

21.     There are significant problems in the bacteria identification. Probably, the data does not correspond with those in fact. Сombination of short sequence and a 96% similarity does not allow you to accurately determine the species. On the contrary, a short sequence shows 100% similarity with many other sequences and then you should use additional methods for identification, including sequencing of other genes. It is not specified that the sequences are deposited somewhere or placed in databases. This does not allow for independent verification and clarification of this data. There are no phylogenetic trees.

Thanks for this observation ... we consider again that with the new explanation in the method used, we can be more explicit.

REVIEWER 3

22.     Bring the name of the variants of the experiments to a single form. Somewhere the code name of the treatment is used, somewhere the name of the strains.

we standardize what is requested

REVIEWER 3

23. Not every growth-stimulating activity improves seedling quality. For example, lengthening stems without developing roots reduces resistance to drought. Formulate and justify any morphological or physiological criteria (a strong root system, a large number of leaves, or something else) do you consider the most important for high-quality tomato seedlings. Does bacterial treatment help to achieve better performance on these criteria?

That is correct …… the results were variable.

Separating results and discussion, these sections can be better appreciated

REVIEWER 3

24.     Quite a lot of indicators are used (nitrate reduction, elemental composition of plants, dry and wet weight, etc.). In each case, the results of one or the other bacteria are better. The authors do not provide an algorithm by which they chose two strains as the best. Do they give to all the studied parameters the same importance, or do they consider some of them more important?

Thanks for this observation… we consider once again that with the new version we will be complying with the requested changes.

REVIEWER 3

25.     There is no table summarizing the properties of the selected four strains. What specific properties does each strain have? In general, the section about bacteria is difficult to understand. None of the properties of the new strains that characterize them are given.

They are indicated in writing, because we consider that other variables require these tables to better understand the study.

REVIEWER 3

26.     In Figure 2 the arrangement of letters looks doubtful. Very close values are marked with different letters. In the captions under the drawings, it is not written what is represented by markers. Confidence interval? Standard error? Standard deviation? This is also not available in the methods section.

We apologize, we changed figure 2 and correct this error and plot accordingly.

REVIEWER 3

27.     In Table 4, it is necessary to indicate the significance of the statistical differences between the experimental variants.

We apologize, we corrected this error and added the comparison of means.

REVIEWER 3

28.     In Table 2, unit of measurement is not written.

We corrected this error and added units.

REVIEWER 3

29.     What are the ″leaves″ and ″roots″ in Figure 6 mean? Is this the location of the microorganisms (chapter 2.6.4 describes only the counting of bacteria from the roots)? These are the treatment options (previously they were designated "inoculation of seeds" and "spraying of shoots")? In the text, it is written that double processing is better, but it is not shown in the figure.

This fine observation is appreciated, and we consider that with the new wording the method developed will be more explicit.

30.     In lines 1291, 1292, the authors write "without appreciable significance compared to the control microorganism (Azospirillum halopraeferens)" Is it worth continuing to work with these 4 strains?

Of course, yes, since Azospirillum is considered worldwide as a reference microorganism with a high capacity to fix nitrogen, solubilize phosphates and produce siderophores.

31.     Aeromonas caviae is a facultative pathogen for humans. Have the toxicological and hygienic properties of the strain been studied?

There are bacteria that according to the name they have, confuse the reader that you could be a pathogenic bacterium of humans. However, there are reports where there are bacteria that also play an important role in the rhizospheres of plants. example Klebsiella pneumoniae, which is a plant growth promoting bacteria but is not pathogenic to humans

Ie: https://0-doi-org.brum.beds.ac.uk/10.1046/j.1439-037X.2003.00051.x

REVIEWER 3

In text Aeromona caviae à Aeromonas caviae

Considered this observation,

we changed all.

REVIEWER 3

In line 518, it is stated that Aeromonas caviae is an anaerobic microorganism, is it really so?

Considered this observation,

we changed by facultative anaerobic bacterium.

REVIEWER 3

In the "Introduction" section, the presented volumes of tomato production do not correspond to each other, in China (62,869,502 t) they produce more than in the whole world (4,271,914 t)?

With the new version, we attend to this observation ... thank you!

REVIEWER 3

In line 22, replace "planting dates" with "growing period".

Considered this observation,

we changed this.

REVIEWER 3

In line 198 "however, pests, fertilization, and losses during transplantation are the main challenges to growth-transplantation and challenges" (which means "challenge", here we mean the problem), the word chosen in this key is not correct.

With the new version, we attend to this observation ... thank you!

REVIEWER 3

In line 109 (Rennie) – this is the name of the environment (then you need to give a reference) or a reference to the source (then you need to write it correctly). It is also worth paying attention to the phrase "cultivation in an environment with a lack of a nitrogen source", perhaps it is worth writing "cultivation in an environment with a lack of a nitrogen source".

With the new version, we attend to this observation ... thank you!

In section 2.2. you can appreciate that request.

REVIEWER 3

In line 119 in the reference [15] there is no declared method.

In line 161 in the reference [15] there is no declared method.

The wrong references were verified in this section (2.4.)… .. We are providing the references in case there is someone interested in using these methods….

REVIEWER 3

In line 285, the word «experienced» is not quite the right word. Perhaps you should replace it with «show»?

Considering your observation, we changed this word by showed.

REVIEWER 3

The notation of the ordinate axis in Fig. 4, 5 should be translated into English.

We apologize, correct this error and plot accordingly

REVIEWER 3

Link [24] BlAST à BLAST

We correct this mistake.

REVIEWER 3

In line 23 PGPR means ″Plant growth promoting rhizobacteria″ while ″Plant growth promoting bacteria″ is PGPB.

Considering your observation, we changed this by Plant growth promoting rhizobacteria.

2

My major concern during 1^st evaluation was about treatments, statistical analysis, data interpretation and rewriting of results section. In this revised manuscript these issues are not solved.

Now the authors stated that they have 21 treatment combination (7 × 3) but actually there are 15 treatments because in case of three control treatments i.e., DW, NS and BS there is no bacterial application then what does means by seed inoculation, seedling spray of their combination. Actually, there are 15 treatments (4 strains applied by three methods) and three controls as was mentioned in earlier submitted manuscript. The authors should use RCBD instead of factorial then all these issues will be resolved.  In all Figs three application methods in case of DW, NS and BS have same value. Likewise, still in Table 4 no information about application methods is given. Therefore, the authors should reanalyze data following RCBD to solve all such issues. After reanalysis rewrite whole results section concisely.

In its current form manuscript cannot be accepted for publication. After reanalyzing data, results interpretation and modification of discussion accordingly the manuscript should be submitted for evaluation.

Dear reviewer, considering the observations of two reviewers and yours, we decided to change the statistical analysis. In section 2.4 (paragraph 2), you can identify how the treatments were distributed; a control treatment (buffer) was eliminated and only the distilled water and the nutrient solution remained. The first in order to highlight the bacteria under study vs nutrient solution. It should be noted that the nutritive solution is one of the conventional forms used by the seedling producer; that is why he got involved in the study.

Also, in section 2.7. you will appreciate the experimental design. It should be noted that the results are modified and also the tables and figures in results.

We consider that with these changes we comply with the request, avoiding questioning the buffer-based control treatment. We take this opportunity to apologize for all these changes carried out in this phase.

3

Dear authors, after the revision, the quality of the article improved. But there are still serious shortcomings. The main ones are incorrect microorganisms´ identification, inaccurate and outdated methods of studying microorganisms, no saline variants of experiments. The unresolved issues are described in more detail below.

The answer to the earlier question was not received (question №3 in the previous review). Organic acids are only mentioned in the Abstract but the manuscript contains no data on the quantity of phosphates dissolved by bacteria or organic acids synthesized by bacteria. The authors ' response states that organic acids are discussed in section 3.1 but there are no quantitative values expressed in moles or other dimensions. The authors indicate only an indirect sign in millimeters of the zones of enlightenment – this is a qualitative indicator and it is not quantitative. Thus, the data is incomplete and does not allow us to draw any conclusions.

Dear reviewer, considering the observations of two reviewers and yours, we decided to change the statistical analysis. In section 2.4 (paragraph 2), you can identify how the treatments were distributed; a control treatment (buffer) was eliminated and only the one with distilled water and the nutritive solution remained. The first in order to highlight the bacteria under study vs nutrient solution. It should be noted that the nutritive solution is one of the conventional forms used by the seedling producer; That is why he got involved in the study.

Also, in section 2.7. you will appreciate the experimental design. It should be noted that the results are modified and also the tables and figures in results.

We consider that with these changes we comply with the request, avoiding questioning the buffer-based control treatment. We take this opportunity to apologize for all these changes carried out in this phase.

We apologize for not having been clear in our proposed methodology and the results presented.

This version has highlighted this application for biological tests of bacteria with N fixation (NF), phosphate solubilization (SF) and siderophore production (PS).

Sorry for that organic acid production variable that was raised earlier ……… ..we omitted it in the document. (Forgiveness!!!)

3

A detailed answer to question №6 of the previous review was not received. Why distilled water, phosphate buffer, and Steiner solution are chosen. What is the purpose of this choice? In addition, what is the difference between PBS and Steiner Solution? In the soil of the Comarca Lagunera region, according to the data given in the article, the content of calcium is 275 g / kg, sodium 195 g/kg of soil (Line 496), which in terms of moles of matter is 6,975 and 8,478 mol, respectively. In experiments, the Steiner Solution is used where the concentrations of these substances are either lower or completely absent. The article focuses on salinity and salt resistance. However, the concentrations of the selected salts are not very salty, for example, 12 mol of nitrate ion per cubic meter in Steiner Solution (0.012 mol per liter). The article does not disclose the role of isolates for stress reduction in salinity conditions. Still, what kind of water is used for watering greenhouses? Probably, the study is aimed at reducing osmotic stress in plants and increasing salt resistance? In this case, why is there no comparison with the usual water for this region? Thus, the answer is not given how bacteria affect the resistance of tomatoes to salinity, since such conditions have not been created in the work.

Dear reviewer, considering the observations of two reviewers and yours, we decided to change the statistical analysis. In section 2.4 (paragraph 2), you can identify how the treatments were distributed; a control treatment (buffer) was eliminated and only the distilled water and the nutrient solution remained. The first in order to highlight the bacteria under study vs nutrient solution. It should be noted that the nutritive solution is one of the conventional forms used by the seedling producer; that is why he got involved in the study.

Also, in section 2.7. you will appreciate the experimental design. It should be noted that the results are modified and also the tables and figures in results.

We consider that with these changes we comply with the request, avoiding questioning the buffer-based control treatment. We take this opportunity to apologize for all these changes carried out in this phase.

3

Although it is true that the document is responsible for isolating halotolerant bacteria, it is important to indicate that seedling producers demand these biological alternatives (Plant growth promoting bacteria) not only to use them in this phase of seedling production (a productive stage and highly demanded by farmers) but so that these microorganisms can be used by the complete organic production system.

It is justified on line 60 to 70.

Remember that this study is only in the seedling stage. Of course, the study is being considered under salinity conditions, but it is for the next stage (after transplanting to production) which is under field conditions.

3

The answer (question №7 of the previous review) why tomato plants were not tested for resistance to salinity was not given. Based on the Introduction (lines 66-68), it is assumed that tomatoes will be subjected to such stress. Lines 77-79 refer to the ability of native salt-resistant microorganisms to increase the stability and adaptability of these cultivated plants. However, the article does not provide evidence for this thesis and the work does not contain studies on the salt resistance of tomatoes in the soil and climate conditions of the Comarca Lagunera region.

In the previous answer this observation is answered:

However, we include it again:

The study is directed only at the seedling stage.

It is justified on line 60 to 70.

The study that you mention will be developed considering the field conditions that the producer lives.

Although it is true that the document is responsible for isolating halotolerant bacteria, it is important to indicate that seedling producers demand these biological alternatives (Plant growth promoting bacteria) not only to use them in this phase of seedling production (a productive stage and highly demanded by farmers) but so that these microorganisms can be used by the complete organic production system.

It is justified on line 60 to 70.

Remember that this study is only in the seedling stage. Of course, the study is being considered under salinity conditions, but it is for the next stage (after transplanting to production) which is under field conditions..

3

The study is directed only at the seedling stage.

It is justified on line 60 to 70.

The study that you mention will be developed considering the field conditions that the producer lives

We are not evading we are just realizing that you as a reviewer want results that are not for this investigation to be offered.

It exists in the work plan, for all those studies that you comment, but here it is only in one stage (in seedling) and under certain conditions.

It is justified on line 60 to 70.

 The results invite us to evaluate in other studies (not this one), the specificity of bacteria for other phenological stages of tomato and under various conditions.

We certainly understand and appreciate your comments; We commented that the microorganisms from which they were isolated are from a different-alien plant Distichlis spicata, and in this context, specificity plays an important role. Let us remember that the exudates and the chemotactive effect of each phenological stage of plants respond differently for bacteria… .in this case, we are only testing only at the seedling stage.

Without a doubt it is important to carry out more studies! 1

3

The previously asked 10 question remained unanswered. In line 220 the given reference [10] has contain no data on the medium for measuring nitrogen fixers. If we return to the reference [9], the authors who proposed this nitrogen-free nutrient medium found that 75% of the strains had the ability to fix nitrogen (from which it follows that 25% did not have such ability). Thus, the measurement of colony diameters is a preliminary and qualitative indicator and requires refinement by instrumental methods (for example, gas chromatography methods).

We apologize for not having been clear in our proposed methodology and the results presented.

This version has highlighted this application for biological tests of bacteria with N fixation (NF), phosphate solubilization (SF) and siderophore production (PS).

Sorry for that organic acid production variable that was raised earlier ……… ..we omitted it in the document. (Forgiveness!!!)

3

No answer to the question posed (№11 in the previous review) It is not experimentally proven that the isolated strains survive in saline soil and are not displaced by other bacteria.

Totally in agreement with your appreciation question number 11. The results invite us to evaluate in other studies (not in this one), the specificity of the bacteria for other phenological stages of tomato and in different conditions, since it is a different plant-alien from where they were isolated. obtained Therefore it is important to carry out studies

3

To the previously asked question №13 is given an evasive answer. Experimental data on the survival of isolated bacteria under these conditions is not given.

We disagree with this assessment of yours… ..in an investigation it is not possible to meet all the parameters that may arise… ..

We are not evading we are just realizing that you as a reviewer want results that are not for this investigation to be offered.

There is in the work plan, all those studies that you comment, but here it is only in a stage and under certain conditions.

It is justified on line 60 to 70.

 The results invite us to evaluate in other studies (not in this one), the specificity of the bacteria for other phenological stages of tomato and under various conditions, since it is a different plant-alien from which they were isolated. obtained Therefore it is important to carry out studies

3

About question 14 of the previous review. The question was not about the organoleptic characteristics of the tomato vegetable. In addition, no vegetables were taken in this study. The question was in the design of the experiment. Namely, that when treated with salt solutions, including when spraying, the plant will receive them through the leaves, too. Thus, the experiment is not correct when studying the number of micro-and macro elements.

We appreciate and thank you for your insightful comments.

The data has been run again.

The design has been modified and therefore the results are different. We have modified the tables and figures.

Regarding your expression that (the experiment is not correct when studying the number of micro and macro elements), your appreciation surprises us.

For each element we justify its importance; i.e. for N, P, and Ca on lines 1070 to 1080. On subsequent lines, justify for each item.

3

On question 15 of the previous review. As mentioned earlier, all the indicators given by the authors regarding nitrogen fixation and phosphate solubilization are qualitative. They are not quantified anywhere. Thus, we did not get an answer in the form of numbers (activities of the enzyme nitrate reductase). For 16S RNA there are a number of questions (more on this below).

In the previous version (cover letter) we responded to this modification. However, we noticed that it was omitted in that version. We appreciate such a fine observation that we apologize.

In results and in discussion, it has been considered to include an additional section

3

17th issue of the previous review. It is postulated that the isolated microorganisms belong to PGPR without studying the properties as PGPR (nitrogenase activity, ability to synthesize phytohormones or ACC deaminase).

We apologize for not having been clear in our proposed methodology and the results presented.

This version has highlighted this application for biological tests of bacteria with N fixation (NF), phosphate solubilization (SF) and siderophore production (PS).

Sorry for that organic acid production variable that was raised earlier ……… ..we omitted it in the document. (Forgiveness!!!)

3

21 question of the previous review. There are no phylogenetic trees and no deposit numbers in manuscript yet. I consider the data on 16S unproven. As an answer, the authors provide the following links. In line 452, there is no data in given references [22, 23, 24] about isolated bacteria of their deposition, phylogenetic trees, etc. In the reference [22] methods for medical use. We ask you to follow the generally accepted methods. The techniques described in reference [23] are applicable to archaea. According to the available data, it is impossible to confidently identify the bacteria you have isolated. In the corrected version, there are no phylogenetic trees, catalog numbers in GenBank, or morphology. The question of whether the isolated strains belong to a particular taxonomic group remains open.

We consider that new form to present the results could be better

3

The question №25of the previous review was ignored.

In this new version, Table 2 can be identified with those characteristics that you are requesting

3

Question 30 of the previous review (the conditional pathogenicity of the strain). The presence of useful properties does not yet indicate the safety of the strain.

There are bacteria that according to the name they have, confuse the reader that you could be a pathogenic bacterium of humans. However, there are reports where there are bacteria that also play an important role in the rhizospheres of plants. example Klebsiella pneumoniae, which is a plant growth promoting bacteria but is not pathogenic to humans

Ie: https://0-doi-org.brum.beds.ac.uk/10.1046/j.1439-037X.2003.00051.x

3

In line 219 at the link [18] there are no studies and methods for studying bacteria.

In the second paragraph of the discussion section (4), this test is discussed.

In relation to the logarithmic growth curves of each bacterial isolate, Rueda and Barron (2009) indicate that they must be carried out before each inoculation, since each bacterial strain does not have the same cell maturation cycle; Some bacterial cells mature at 12 h, others at 16 h or more and if this difference in maturity is not detected in the growth phase, it influences at the time of inoculation and establishment (colonization), the young bacterial cells are displaced by saprophytic microorganisms or those that are in the soil.

Reference included

319 line states that the water of Comarca Lagunera is rich in salt. But the authors do not test the effect of this particular water on plants. Thus, the positive effect of isolated bacteria on the salt resistance of plants inoculated with bacteria has not been proven.

n the previous answer this observation is answered:

However, we include it again:

The study is directed only at the seedling stage.

It is justified on line 60 to 70.

The study that you mention will be developed considering the field conditions that the producer lives.

Although it is true that the document is responsible for isolating halotolerant bacteria, it is important to indicate that seedling producers demand these biological alternatives (Plant growth promoting bacteria) not only to use them in this phase of seedling production (a productive stage and highly demanded by farmers) but so that these microorganisms can be used by the complete organic production system.

It is justified on line 60 to 70.

Remember that this study is only in the seedling stage. Of course, the study is being considered under salinity conditions, but it is for the next stage (after transplanting to production) which is under field conditions.

3

In line 362, it is declared  that the composition of the Phosphate bufer solution is given in Table 2. However, the table indicates that it is a Steiner Solution. Line 369 already says that this is a Steiner nutrient solution. It is impossible to clearly understand where the solution is.

Dear reviewer, considering the observations of two reviewers and yours, we decided to change the statistical analysis. In section 2.4 (paragraph 2), you can identify how the treatments were distributed; a control treatment (buffer) was eliminated and only the distilled water and the nutrient solution remained. The first in order to highlight the bacteria under study vs nutrient solution. It should be noted that the nutritive solution is one of the conventional forms used by the seedling producer; that is why he got involved in the study.

Also, in section 2.7. you will appreciate the experimental design. It should be noted that the results are modified and also the tables and figures in results.

We consider that with these changes we comply with the request, avoiding questioning the buffer-based control treatment. We take this opportunity to apologize for all these changes carried out in this phase.

3

Lines 378,385, 389,391 do not describe the country of origin and the manufacturer of the equipment.

Considered this observations

3

In line 433 in the article referred to by the authors contains research on iron ions, not nitrogen content.

Considered this observations

3

In line 439, according to the link [34], there is no methodology for Na ions.

Considered this observations