Effects of Zinc Oxide and Zinc–Silica-Based Nanofertilizers with Yeasts on Selected Components of Soybean in the Central European Agronomic Region: A Short-Term Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Generally, the science is sound. However, the novelty of your science is not well articulated, as you do not put it in the context of the literature in the introduction, and therefore do not articulate the knowledge gap that it fills clearly. Also, the claims you make are not always backed up by the data, and the results are not discussed in enough depth and detail. The writing is sometimes unclear, and difficult to read.

 

Lines  40-42 You write: Due to their variable  crystallinity, diverse morphology with a nanoporous-specific reactive surfaces [3], and  plant-essentiality, they exhibit various physicochemical and biological effects [4]. Please can you expand on this? You need to provide details about the physicochemical and biological effects that they make, and give examples

Lines 42-44: You write: When these nanoporous-silica hybrid biomaterials are combined with different type of microorganisms and micronutrient-based NPs, it should reflect in potential unpredictable agronomic response. Could you please expand what you mean in ‘it should reflect in potential unpredictable agronomic response’ – why is it unpredictable? Surely, we know enough about plant mineral nutrition to make some good predictions? This should be expanded upon, and explained much further.

Line 54: What do you mean by ‘energy nutrition’? Please expand on the uses of soybean (e.g. human nutrition and animal feed)

Line 56: Please explain further what you mean by ‘unexpected plant stress responses’- why are we not expecting them? There are plenty of papers about the effect of Zn on plant stress…

Lines 61 and 62: You need to include information about the effect of Zn NPs on seed accumulation of Zn- this has been the main thrust of research thus far in wheat, for example.

Your introduction does not cover any of the previous work completed regarding foliar application of Zn NPs on soybean (e.g. Dimpka et al (2017) 10.1007/s13593-016-0412-8 and Dola and Mannan (2023) https://doi.org/10.3329/baj.v25i2.65940). This makes the introduction a little disingenuous, as it implies that no previous work exists. As such, you need to include previous work (there are more papers than the examples I have given), and you need to explain clearly what the differences are between your work and theirs, to explain the knowledge gap. The fact that you are in central Europe is not novel enough to justify this as ‘novel work’, unless you give good reasons to why there are likely to be differences in plant uptake and translocation of Zn NPs in soybean grown in your location.

84: Please either reword this, or explain why you are looking at the effect of soybean on NPs, and not the effect of NPs on soybean

Line 138: Please give more information about how you measured bulk density of grain

Line 165: Why were sunflower leaves measured? Please check this is correct

Lines 151-152: Please provide more information about your AAS methods- did you use a certified reference material? How did you digest the samples. Please provide an explanation of what F-AAS stands for

.ine 175- Please explain what the vegetation season is

Line 188-189: You point out that there was a significant difference in bulk density of grain between treatments. However, on of your treatments caused greater bulk density than the control, and the other reduced bulk density. More discussion is needed as to why you think this is, as this is missing at the moment

Lines 197-198 You write that These factors positively impacted the number of seeds per plant – however, your data did not show significant differences in number of seeds per plant, and therefore this claim is unfounded and either needs to be qualified or removed

Line 205-206 You write that NFs acted as a soybean stimulant. However, your data do not support this claim

Line 207-208: Please explain by ‘indicative’ level

Lines 228-230: You need to qualify this by also discussing that although the differences between your control and treatment plants is significant, that amount of difference in energy content is so small that you are unlikely to see a difference in growth of pigs/other livestock

Line 263: You mention macronutrients, but not micronutrients, therefore making this point of very little relevance for Zn, which is a micronutrient

;Line 310: Please expand as to what you mean by ‘positive physological effect’, as this vagueness does not allow you to discuss your work in the context of the literature optimally

Ine 317: What do you mean by ‘highlights’?

Comments on the Quality of English Language

The paper is hard to read in places, and could be more clear. 

Author Response

Dear reviewer, Thank you for your time and valuable comments on our manuscript. We have incorporated your helpful suggestions. Our responses to your comments can be found in the enclosed cover letter. Changes made are noted in the manuscript. Thank you again for your review.
# Comment 1
Lines 40-42 You write: Due to their variable crystallinity, diverse morphology with a nanoporous-specific reactive surfaces [3], and plant-essentiality, they exhibit various physicochemical and biological effects [4]. Please can you expand on this? You need to provide details about the physicochemical and biological effects that they make, and give examples.
# Answer 1
I agree, the specific information has been added (L 41 – 43) “and plant-essentiality, they exhibit various physicochemical and biological effects, such as improved photosynthesis, abiotic stress tolerance, productivity, and microbial community structure”…
# Comment 2
Lines 42-44: You write: When these nanoporous-silica hybrid biomaterials are combined with different type of microorganisms and micronutrient-based NPs, it should reflect in potential unpredictable agronomic response. Could you please expand what you mean in ‘it should reflect in potential unpredictable agronomic response’ – why is it unpredictable? Surely, we know enough about plant mineral nutrition to make some good predictions? This should be expanded upon, and explained much further.
# Answer 2
We agree, the text has been modified (L 46-47) “The synergistic effects of these hybrid formulations, such as those combining zinc oxide nanoparticles (ZnO-NPs) with biosilica and yeast, may enhance nutrient delivery and plant uptake.”
# Comment 3
Line 54: What do you mean by ‘energy nutrition’? Please expand on the uses of soybean (e.g. human nutrition and animal feed)
# Answer 3
We agree, the term “energy nutrition” has been modified (L73) “particularly regarding energy value”…
# Comment 4
Line 56: Please explain further what you mean by ‘unexpected plant stress responses’- why are we not expecting them? There are plenty of papers about the effect of Zn on plant stress…
# Answer 4
The idea of the context of missing information, including the physiological response, has been involved into the manuscript (L48) …“This combination may not only improve plant growth and stress resistance but also significantly boosts the nutritional profile and physiological parameters, leading to increased yield and quality”…
# Comment 5
Lines 61 and 62: You need to include information about the effect of Zn NPs on seed accumulation of Zn- this has been the main thrust of research thus far in wheat, for example.
# Answer 5
This information has been added along with a relevant reference, (L 56-57) “ZnO-NPs were also used for Zn biofortification in seeds, e.g. in wheat [10].”
# Comment 6 Your introduction does not cover any of the previous work completed regarding foliar application of Zn NPs on soybean (e.g. Dimpka et al (2017) 10.1007/s13593-016-0412-8 and Dola and Mannan (2023) https://doi.org/10.3329/baj.v25i2.65940). This makes the introduction a little disingenuous, as it implies that no previous work exists. As such, you need to include previous work (there are more papers than the examples I have given), and you need to explain clearly what the differences are between your work and theirs, to explain the knowledge gap. The fact that you are in central Europe is not novel enough to justify this as ‘novel work’, unless you give good reasons to why there are likely to be differences in plant uptake and translocation of Zn NPs in soybean grown in your location.
# Answer 6
This information has been added along with a relevant reference (L 69 -70) “From a global-nutritional perspective, soybeans are a significant model crop, used also in experiments with ZnO-NPs [16,17], but there is a lack of academic studies on the positive effects of hybrid silica based NFs.”
# Comment 7 84: Please either reword this, or explain why you are looking at the effect of soybean on NPs, and not the effect of NPs on soybean
# Answer 7
The information has been completely rewritten in the manuscript, (L 76 -83) “This study addresses this gap by investigating the foliar application of ZnO-NPs combined with a nanoporous hybrid bio-SiO₂ material and yeast (ZnSi-bio), in comparison to ZnO-NPs alone and a nanoparticle-free control. The research evaluates the impact of these treatments on soybean production, nutritional components, and the potential zinc biofortification, under real-world conditions in the Central European agronomic region.”
# Comment 8
Line 138: Please give more information about how you measured bulk density of grain
# Answer 8
Essentially, it is the measurement of the quantity of seeds in a specific container, which has also been added to the explanation (L 166-167) “…and bulk density according to STN standard 461011 which involves filling a standard container with grain, leveling the surface, and weighing it were used a Kern PCB3500-2 laboratory scale…”
# Comment 9
Line 165: Why were sunflower leaves measured? Please check this is correct
# Answer 9
I agree, it has been changed to (L 138) “soybean leaves.”
# Comment 10
Lines 151-152: Please provide more information about your AAS methods- did you use a certified reference material? How did you digest the samples. Please provide an explanation of what F-AAS stands for
# Answer 10
We agree, a comment on the phytomass digest and the mineral nutrients analysis has been added along with a relevant citation, (L 180 - 185) “The seed samples weighing 0.15–0.30 g was digested in a mixture of HNO3 and HClO4 (2:1, v/v). For soybean zinc analyzation was used the flame atomic absorption spectrometry (F-AAS) (Perkin-Elmer Model 1100, Waltham, MA, USA) according to the method described by Losak et al. [27] while phosphorus content was determined colorimetrically using a spectrophotometer at a wavelength of 660 nm.”
# Comment 11
Line 175- Please explain what the vegetation season is
# Answer 11 For clarification, the year of the growing season has been added (L 191) “in the growing season 2023”…
# Comment 12
Line 188-189: You point out that there was a significant difference in bulk density of grain between treatments. However, on of your treatments caused greater bulk density than the control, and the other reduced bulk density. More discussion is needed as to why you think this is, as this is missing at the moment
# Answer 12 We agree, an explanation regarding the bulk density results has been added, (L 207-210) “…This result can be attributed to a lower number of pods per plant, yet a relatively larger number of smaller seeds, leading to reduced seed size and a lower thousand-seed weight (Table 1). This presents an interesting agronomic outcome, as it suggests a higher potential for seed quality despite reduced seed size…”
# Comment 13
Lines 197-198 You write that These factors positively impacted the number of seeds per plant – however, your data did not show significant differences in number of seeds per plant, and therefore this claim is unfounded and either needs to be qualified or removed
# Answer 13 We agree, the text has been rewritten to more logical form, (L 215 – 219) “The statistically non-significant negative results of number of pods per plant and weight of thousand seeds associated with ZnO-NPs application can be explained by the higher number of
inflorescences and pollen favorable development as reproductive organs [4, 5]. These the bulk density of seeds, rather than their weight and yield (Table 1)”
# Comment 14
Line 205-206 You write that NFs acted as a soybean stimulant. However, your data do not support this claim
# Answer 14 We agree, the information regarding the soybean stimulant has been removed from the manuscript.
# Comment 15
Line 207-208: Please explain by ‘indicative’ level
# Answer 15 We agree, it has been revised in the manuscript (L 226 – 227) “Regarding production parameters, the ZnSi-bio application produced highest yield, albeit at a statistically non-significant level (Table 1)”
# Comment 16
Lines 228-230: You need to qualify this by also discussing that although the differences between your control and treatment plants is significant, that amount of difference in energy content is so small that you are unlikely to see a difference in growth of pigs/other livestock
# Answer 16 i) …qualify this by also discussing that although the differences between your control and treatment plants is significant –information bioenergetic value has been already written in the text with relevant references (L 259-262) “The most likely, zinc plays a direct role as a cofactor in carbon and nitrogen metabolism, such as enhancing carbohydrate formation, facilitating atmospheric N-fixation during amino acid synthesis, glycerol utilization, and the biosynthesis of growth hormones and nucleic acids, thereby intensify their bioenergetic value [6, 7]; ii) ... that amount of difference in energy content is so small that you are unlikely to see a difference in growth of pigs/other livestock, this information was added into manuscript “”
# Comment 17
Line 263: You mention macronutrients, but not micronutrients, therefore making this point of very little relevance for Zn, which is a micronutrient
# Answer 17
We agree, this information has of course been reformulated, (L 322 -323) “Principally, ZnO-NPs in soybean spray applications may act as Zn2+ micronutrient or photocatalytic agents.”
# Comment 18
Line 310: Please expand as to what you mean by ‘positive physological effect’, as this vagueness does not allow you to discuss your work in the context of the literature optimally
# Answer 18
We agree; the discussion on physiology was expanded to include the potential application of our nano-fertilizers as a strategy to mitigate the impact of climate change with relevant citations, (L 300-305) “…In this context, our applied NFs can be considered as relative functional tool for mitigating climate changes, as the seasonal thermal requirements for soybeans aligned with a relative optimum around 2500°C (Fig. 1a) with interactive effect of CO2 on plant growth [8]. However, the water precipitation management supply (Fig. 1b) were non-ideal during the growing season [9], with NFs-treated variants showing better physiological adaptation in our agronomical region (Table 4, Figure 3)…”
# Comments 19
Line 317: What do you mean by ‘highlights’?
# Answer 19 We agree, “context” has been replaced with a more logical term in the manuscript (L 350-351)“Our study in Central Europe agronomical region found interesting results in the foliar application of environmentally-friendly concentration of NFs on soybeans”
# Comments 20
Comments on the Quality of English Language & The paper is hard to read in places, and could be more clear.
# Answer 20
We agree, the reviewers’ comments have been incorporated into several parts of the manuscript, along with extensive editing of the English language.

Reviewer 2 Report

Comments and Suggestions for Authors

The topics of the submitted article are very interesting and of contemporary importance. I would love to see this work published in Agronomy. In my opinion, however, it needs several improvements before that can happen.

My concerns and comments are as follows:

-            The aim of the study should be more clearly stated. Was it the application of the nanofertilizers (NFs) itself or the effects of these formulations? It also raises the question of comparing the two NFs.  It should also be more clearly indicated what specific parameters were the measures of these effects; for example, list them in brackets in lines 73-74, respectively.

-            It is necessary to formulate a research hypothesis. What effects were expected?

-            In lines 73-74 you wrote: “…potential hazardous translocation of zinc to soybean were assessed…”. This is confusing to me. Were NFs used to enrich soybean seeds with Zn, or were you concerned about too high levels of this element in the seeds? I suggest that the issue of desirable and dangerous levels of Zn in seeds should be discussed in the paper.

-            In Section 2.4, the information on the experimental design and the number of replicates should be completed.

-            In my opinion, both the presentation of the research stages in the Materials and Methods chapter and the order in which the parameters studied are discussed in the Results and Discussion chapter should be consistent with the timing of data collection. So, the information contained in Section 2.7 should be presented first, followed by the information in Section 2.5, and finally in Section 2.6. Present the results in the same order.

-            The information in Section 2.6. should be organized. First list what parameters were tested, and then what methods and procedures were used to determine them. Line 143 reports that the seeds were analyzed for nutritional parameters but does not list these parameters. It is not clear for what purpose F-AAS measurements were used (line 149).

-            In Section 2.7, the terms in which physiological parameters were determined need to be supplemented.

-            Section 2.8 reports that the data were subjected to a one-factor analysis of variance. However, it does not state how the differences between the averages for the treatments were evaluated, i.e., which post-hoc test was used. The data in the tables indicate that both treatments with the fertilizers in question were referred to the control, but the differences between their effects were not analyzed. I consider this a weakness of the work. I suggest using, for example, the Tukey test (or another post-hoc test) to evaluate differences between the three treatments and marking these differences with letters (averages marked with the same letter do not differ at the assumed p, and those marked with different letters do).

-            Once the statistical analysis is corrected, the discussion of the results should be revised.

-            Line 44: NPs - please define this abbreviation when first used (or is it NFs? Then verify throughout the manuscript).

-            Line 74: “translocation of zinc to soybean” - What do you mean by this term? What was the path of this translocation (from where to where) and how was it measured?

-            Line 171: Section 2.8. – Simplify the title to “Statistical analysis”.

-            Line 176: “…no negative stress reactions were observed” - What effects were expected: negative or positive?

-            Table 1: Title - I suggest simplifying the title to: “Analyzation of Selected quantitative parameters after...”; apply this to all tables and figures. Footer - significant or insignificant in relation to what? rather use letter symbols to mark differences (see earlier comment); apply this to all tables.

-            Line 248: “…no increased concentrations were observed (Table 3) in the case of ZnO-NPs variant” - Here even a significant reduction was noted. How do you explain this?

-            Table 3 – “zinc translocation” - What do you understand by this term? Is it not the Zn content of the seeds?

-            Lines 263-266: “…the highest values were observed for the control and surprisingly for the ZnO-NPs variant, while the lowest value corresponded to the ZnSi-bio variant, likely due to the effects of nanoporous biosilica or applied food yeasts” That is, ZnSi-bio caused a reduction in P levels?  What is the explanation for this effect?

-            Table 4: “physiological components” or rather physiological indices?

-            Figure 2: V1, V2, … - These symbols of growth stages need to be explained

-            Conclusions should carry the answer to the stated goals and hypothesis.

-            Line 320: “Our study in Central Europe agronomical region found unexpected results…” - The expected results were not indicated (no research hypothesis).

Author Response

Dear reviewer, Thank you for your time and valuable comments on our manuscript. We have incorporated your helpful suggestions. Our responses to your comments can be found in the enclosed cover letter. Changes made are noted in the manuscript. Thank you again for your review.
# Comments 21
The aim of the study should be more clearly stated. Was it the application of the nanofertilizers (NFs) itself or the effects of these formulations? It also raises the question of comparing the two NFs. It should also be more clearly indicated what specific parameters were the measures of these effects; for example, list them in brackets in lines 73-74, respectively.
# Answer 21
i) … effects of these formulations? It also raises the question of comparing the two NFs… for this issue, please, check R1 # comment 2; ii) … It should also be more clearly indicated what specific parameters were the measures...and the objective of the work has been reformulated (L 81 - 83) “The research evaluates the impact of these treatments on soybean production,
nutritional components, and the potential zinc biofortification, under real-world conditions in the Central European agronomic region.”
# Comments 22
It is necessary to formulate a research hypothesis. What effects were expected?
# Answer 22
We agree, the research hypotheses have been incorporated into the introduction as well as the conclusion section, e.g. (L 356 – 358) “…Hypothetically, our work focused on optimizing the qualitative functionality of soybean seeds where our results has demonstrated improved outcomes at a statistically significant level for phosphorous content but without Zn biofortification with ZnO-NPs application…”; (L 364) “…Additionally, the hypothesis of seasonally intensified physiology was observed in stomatal conductance (Ig) and the crop water stress index (CWSI) compared to the control variant…”, etc.
# Comments 23
In lines 73-74 you wrote: “…potential hazardous translocation of zinc to soybean were assessed…”. This is confusing to me. Were NFs used to enrich soybean seeds with Zn, or were you concerned about too high levels of this element in the seeds? I suggest that the issue of desirable and dangerous levels of Zn in seeds should be discussed in the paper.
# Answer 23
We agree, in our case we assume biofortification with zinc, which has been discussed in several parts of the manuscript, L 24-27 “…Significant differences were found in the final quality components of soybeans with respect to phosphorus content without ZnO-NPs biofortification. In the case of ZnSi-bio variant, soybeans were biofortified with zinc…”; (L 56 -57) “…ZnO-NPs were also used for Zn biofortification in seeds, e.g. in wheat [1], what is hypothetically aimed to be confirmed for soybeans in the Central European agronomic region…”; (L 270 – 273) “…In the case of Zn-positive biofortification affecting the final quality of soybean seeds, no increased concentrations were observed (Table 3)…”; (L 356 – 358) “…Hypothetically, our work focused on optimizing the qualitative functionality of soybean seeds where our results has demonstrated improved outcomes at a statistically significant level for phosphorous content but without Zn biofortification with ZnO-NPs application…”, etc.
# Comments 24
In Section 2.4, the information on the experimental design and the number of replicates should be completed.
# Answer 24 We agree, the information on the experimental design and the number of replicates were completed (L 134) “…Each variant was applied in three replications.…”
# Comment 25
In my opinion, both the presentation of the research stages in the Materials and Methods chapter and the order in which the parameters studied are discussed in the Results and Discussion chapter should be consistent with the timing of data collection. So, the information contained in Section 2.7 should be presented first, followed by the information in Section 2.5, and finally in Section 2.6. Present the results in the same order.
# Answer 25
We agree, the order of methodological chapter were accepted according to reviewer suggestion but this context seems to be little confusing, especially when many parts of result and discussion was completely rewrite and reevaluated.
# Comments 26
The information in Section 2.6. should be organized. First list what parameters were tested, and then what methods and procedures were used to determine them. Line 143 reports that the seeds were analyzed for nutritional parameters but does not list these parameters. It is not clear for what purpose F-AAS measurements were used (line 149).
We agree, the methodology of the analysis has been revised and supplemented according to the reviewer's suggestion, (L 180 – 184) “…The seed samples weighing 0.15–0.30 g was digested in a mixture of HNO3 and HClO4 (2:1, v/v). For soybean zinc analyzation was used the flame atomic absorption spectrometry (F-AAS) (Perkin-Elmer Model 1100, Waltham, MA, USA) according to the method described by Losak et al. [27] while phosphorus content was determined colorimetrically using a spectrophotometer at a wavelength of 660 nm…”, also, check R1 # comment 10
# Answer 26
# Comment 27
In Section 2.7, the terms in which physiological parameters were determined need to be supplemented.
# Answer 27
We agree, detailed information regarding the number of measurements of physiological parameters is shown in the added diagram – an image for better graphical illustration and the extension of the manuscript's scope due to the editor's recommendations, (L 158).
# Comment 28
Section 2.8 reports that the data were subjected to a one-factor analysis of variance. However, it does not state how the differences between the averages for the treatments were evaluated, i.e., which post-hoc test was used. The data in the tables indicate that both treatments with the fertilizers
in question were referred to the control, but the differences between their effects were not analyzed. I consider this a weakness of the work. I suggest using, for example, the Tukey test (or another post-hoc test) to evaluate differences between the three treatments and marking these differences with letters (averages marked with the same letter do not differ at the assumed p, and those marked with different letters do).
# Answer 28 We agree, the results were evaluated by Tukey’s post-hoc test, (L 188 – 190), “The gained results were statistically evaluated using one-way analysis of variance (ANOVA) at a significance level of 95% with Tukey's Honestly Significant Difference (HSD) post-hoc test (TIBCO Statistica™ 14.0.0)…”and differences are indicated are shown in all tables (LXY)
# Comments 29
Once the statistical analysis is corrected, the discussion of the results should be revised.
# Answer 29 We agree, all results were statistically modified according to Tukey’s Significant Difference (HSD), but its evaluation did not significantly affect the context of the results. Regarding the discussion, we have incorporated almost all suggestions into the work; please see the proposals and comments above.
# Comments 30
Line 44: NPs - please define this abbreviation when first used (or is it NFs? Then verify throughout the manuscript).
# Answer 30 In the manuscript, this is mentioned with the first reference in the text, (L 17) “The action-to-reaction dynamics of next-generation nanofertilizers (NFs) towards” and (L 19), …” Therefore, our aim was to evaluate the effects of foliar application of ZnO nanoparticles (ZnO-NPs)”…
# Comments 31
Line 74: “translocation of zinc to soybean” - What do you mean by this term? What was the path of this translocation (from where to where) and how was it measured?
# Answer 31
Please, see #comment 23, and #comment 25
# Comments 32
Line 171: Section 2.8. – Simplify the title to “Statistical analysis”.
# Answer 32 We agree, title of this section was simplified (L 186) “Statistical Analysis”.
# Comments 33
Line 176: “…no negative stress reactions were observed” - What effects were expected: negative or positive?
# Answer 33 We agree, this part was modified (L 191 - 193) “During the initial NFs foliar application in the vegetation season at the Veľký Krtíš location, as was expected, no negative stress reactions were observed in soybeans at our eco-friendly concentration.”...
# Comments 34
Table 1: Title - I suggest simplifying the title to: “Analyzation of Selected quantitative parameters after...”; apply this to all tables and figures. Footer - significant or insignificant in relation to what? rather use letter symbols to mark differences (see earlier comment); apply this to all tables.
# Answer 34 We agree, all tables and figure titles were simplified according to reviewer suggestions.
# Comments 35
Line 248: “…no increased concentrations were observed (Table 3) in the case of ZnO-NPs variant” - Here even a significant reduction was noted. How do you explain this?
# Answer 35 In relation to the lower zinc content, we believe that it was redistributed within the plant rather than accumulated in the final quality of the soybean, which is discussed in the manuscript, (L 274 – 276), “…It is likely that Zn was redistributed throughout the plant, similar to what has been observed in tomatoes [10], and could have had a physiological-metabolic effect in the soybeans as well…”
# Comments 36
Table 3 – “zinc translocation” - What do you understand by this term? Is it not the Zn content of the seeds?
# Answer 36
We agree, this has been revised in the manuscript to biofortification, (L 278 – 279) “…Table 3. Comparison of final qualitative-nutritional parameters of soybeans with potential zinc biofortification after spray deposition of ZnO-NPs and ZnSi-bio nanofertilizers…”, Please, see #comment 23, and #comment 25, #comment 31
# Comments 37
Lines 263-266: “…the highest values were observed for the control and surprisingly for the ZnO-NPs variant, while the lowest value corresponded to the ZnSi-bio variant, likely due to the effects of nanoporous biosilica or applied food yeasts” That is, ZnSi-bio caused a reduction in P levels? What is the explanation for this effect?
# Answer 37
Theoretically, the positive effects of biosilica and applied food yeasts have already been mentioned in the manuscript (L 46 - 50) “The synergistic effects of these hybrid formulations, such as those combining zinc oxide nanoparticles (ZnO-NPs) with biosilica and yeast, may enhance nutrient delivery and plant uptake. This combination may not only improve plant growth and stress resistance but also significantly boosts the nutritional profile and physiological parameters, leading to increased yield and quality.”; Regarding phosphorus content, the discussion was directed towards a better physiological response and thus the stress that would trigger a higher content in the seeds, (L 285 – 287) “…In our case, the highest values were observed for the control and surprisingly for the ZnO-NPs variant, while the lowest value corresponded to the ZnSi-bio variant, likely due to the relative positive physiological effects of nanoporous biosilica or applied food yeasts…”
# Comments 38
Table 4: “physiological components” or rather physiological indices?
# Answer 38
We agree, in the entire manuscript we have modified this term to “physiological indices”
# Comments 39
Figure 2: V1, V2, … - These symbols of growth stages need to be explained
# Answer 39
We agree, the terms have been clarified in the soybean development diagram; # see comment 27.
# Comments 40 # 41
Conclusions should carry the answer to the stated goals and hypothesis. Line 320: “Our study in Central Europe agronomical region found unexpected results…” - The expected results were not indicated (no research hypothesis).
# Answer 40, 41
We agree, the hypotheses have been incorporated into both the introduction and conclusion sections according to the reviewer's suggestions, see # comment 22

Reviewer 3 Report

Comments and Suggestions for Authors

I have reviewed this manuscript, but unfortunately, after careful consideration, it cannot be published in this Journal in my opinion, as the research is relatively simple and is a short-term experiment that cannot draw a definitive conclusion.

Author Response

Dear reviewer, Thank you for your time and comments on our manuscript. Our responses to your comments can be found in the enclosed cover letter. Thank you again for your review.
Thank you for your feedback. However, i) this short-term type of agronomical results has already been published in MDPI with our academics, including Agronomy journal, with enormous potential-generous citation responses, for example [11-15]; ii) other reviewers recommend publishing the manuscript after incorporating the comments and suggestions; iii) the results and discussion section has been completely rewritten and reformulated, including the proposed hypotheses, statistical background and the added 10 of relevant references. Please see 41 comments and suggestion answered to Reviewer 1 and Reviewer 2.
References
[1] Du W, Yang J, Peng Q, Liang X, Mao H. Comparison study of zinc nanoparticles and zinc sulphate on wheat growth: From toxicity and zinc biofortification. Chemosphere. 2019;227:109-16.
[2] Dimkpa CO, Bindraban PS, Fugice J, Agyin-Birikorang S, Singh U, Hellums D. Composite micronutrient nanoparticles and salts decrease drought stress in soybean. Agronomy for Sustainable Development. 2017;37:5.
[3] Dola D, Mannan M. Foliar Application Effects of Zinc Oxide Nanoparticles on Growth, Yield and Drought Tolerance of Soybean. Bangladesh Agronomy Journal. 2022;25:73-82.
[4] Pandey N, Gupta B, Pathak GC. Foliar application of Zn at flowering stage improves plant’s performance, yield and yield attributes of black gram. 2013.
[5] Pandey N, Pathak GC, Sharma CP. Zinc is critically required for pollen function and fertilisation in lentil. J Trace Elem Med Biol. 2006;20:89-96.
[6] Ali W, Ahmad MM, Iftikhar F, Qureshi M, Ceyhan A. Nutritive potentials of Soybean and its significance for humans health and animal production: A Review. Eurasian Journal of Food Science and Technology. 2020;4:41-53.
[7] Cabrera-Orozco A, Jiménez-Martínez C, Dávila-Ortiz G. Soybean: Non-nutritional factors and their biological functionality. Soybean-bio-active compounds. 2013:387-410.
[8] Baker JT, Allen Jr. LH, Boote KJ, Jones P, Jones JW. Response of Soybean to Air Temperature and Carbon Dioxide Concentration. Crop Science. 1989;29:cropsci1989.0011183X002900010024x.
[9] Zanon AJ, Streck NA, Grassini P. Climate and Management Factors Influence Soybean Yield Potential in a Subtropical Environment. Agronomy Journal. 2016;108:1447-54.
[10] Raliya R, Nair R, Chavalmane S, Wang W-N, Biswas P. Mechanistic evaluation of translocation and physiological impact of titanium dioxide and zinc oxide nanoparticles on the tomato (Solanum lycopersicum L.) plant. Metallomics. 2015;7:1584-94.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was significantly improved. I have only one concern.

The formulation of the research aim has been corrected and now does not raise my concerns. However, I still miss the research hypothesis for this particular experiment. It should be placed after the aim and indicate what the authors expected from the research in the context of the stated aim. In this particular experiment, it should indicate whether it is assumed that the use of ZnSi-bio will result in the same or better effects as the use of ZnO-NPs. In turn, the conclusions should already contain the answer to the research aim and the research hypothesis. They are not a placeholder for the hypothesis itself.

I will be happy to recommend the article for publication after the indicated corrections are made.

Author Response

Reviewer 2

# Comment 1

The formulation of the research aim has been corrected and now does not raise my concerns. However, I still miss the research hypothesis for this particular experiment. It should be placed after the aim and indicate what the authors expected from the research in the context of the stated aim. In this particular experiment, it should indicate whether it is assumed that the use of ZnSi-bio will result in the same or better effects as the use of ZnO-NPs. In turn, the conclusions should already contain the answer to the research aim and the research hypothesis. They are not a placeholder for the hypothesis itself.

# Answer 1,

We agree, the academic hypothesis has been added to the manuscript after the aim of the manuscript according to suggestion (L 83 -85) “The hypothesis was designed to verify whether ZnSi-bio has a stronger synergistic effect than ZnO-NPs on improving seed quality, yield, and physiology.”…; And in the conclusion, we focused only on addressing the research hypotheses, for example (L 358 - 362) “Our work focused on optimizing the qualitative functionality of soybean seeds where our results have demonstrated improved outcomes at a statistically significant level for ether extract content and bulk density of seeds but without Zn biofortification with ZnO-NPs application.”.. or (L 366 - 368) “Additionally, the seasonally intensified physiology was observed in stomatal conductance (Ig) and the crop water stress index (CWSI) compared to the control variant.”

Reviewer 3 Report

Comments and Suggestions for Authors

I think the author has carefully revised the manuscript and I have no other comments.

Author Response

Reviewer 3

# Comment 2

I think the author has carefully revised the manuscript and I have no other comments.

# Answer 2

Thank you for your comment. We have made an effort to incorporate all the reviewers' suggestions, please check comment 1.