Macronutrient Fertilization and Cadmium Absorption in Two Cocoa Clones

Round 1

Reviewer 1 Report

The task considered in this paper is very important. The work of the authors was well organized and executed. The experimental design is accurate and complete. The figures are significant and very clear. The data statistical treatment is correct and complete. This research will be of great usefulness for the scientific community.

I have only two recommendations: 1) the authors should show more clearly the innovation of their study which can also be identified in the greater number of variables taken into consideration and, therefore, in the search to evaluate the entire complexity of the environmental situation examined; 2) It would be very useful to the readers if the authors add a table reporting the bibliographic information used by us for data discussion. This table would report citation number, plant type, macronutrient/s added, positive or negative effect on Cd adsorption in root and shoot. In this way it would be easier to compare the results obtained in this study with bibliographic data. I think that this table would be an added value to the paper.

I recommend the paper for publication with only further minor corrections as indicated below:

Line 21, page 1: please, change “y” with “and” between “CCN-51” and “EET-103”.

Line 30, page 1: It doesn't seem correct to me to report "heavy metals" as a keyword since only Cd is considered. I therefore propose to change this keyword taking into account the most important results obtained in this study which are not highlighted by the title of the paper.

Line 47, page 2: I suggest eliminating “contamination” because a high concentration of Cd already represents a contamination, and it is not necessary to specify this further.

Line 66, page 2: I suggest writing “E” in brackets as “A”.

Line 80, page 2: I suggest writing the full name of “WUE” and not only the acronym.

Line 119, page 3: it is necessary to add the ammonium charge, i.e., NH₄⁺.

Line 120, page 3: please, write “-1” instead of “1” for S 6 mg kg¹.

Line 163, page 3: are you sure you used toilet paper to dry the mill? Why didn't you use absorbent paper?

Line 166, page 3: it is not present “λ” between the “at” and “=228.8 nm”.

Figure 1: Why the letters above bars are only represented in B, I and J plot? The statistical evaluation of the significant differences among the treatments should be performed for all conditions under consideration.

Line 280, page 8: I suppose that there is a missing data because graph F must also be considered where the behaviour of Ca absorption as a function of the nutrients added is shown.

Lines 290-291, page 9: In my opinion, it is better to specify the pH range observed for each clone. In the sentence this information is not clear.

Line 293, page 9: the EC concentrations reported are not correct. I think there is a mistake of an order of magnitude in 0.34 ±0.01 - 0.43 ± 0.01 dS m^-1 range. Also in this case it is necessary to better specify the EC range it refers to that clone.

Lines 356 and 361, page 11: please, write “CI” in place of “IC”.

Line 412, page 13: “values” is repeated twice. Please, correct this typing mistake.

The manuscript is written in a correct English in almost all its parts.

Author Response

I have only two recommendations: 1) the authors should show more clearly the innovation of their study which can also be identified in the greater number of variables taken into consideration and, therefore, in the search to evaluate the entire complexity of the environmental situation examined;

Page 2- 3  Lines 98-101

Cadmium uptake in cocoa is extremely complex. This study addressed trying to decrease Cd uptake by fertilizing with macronutrients while measuring a large number of physiological variables to assess more closely the complexity of the environmental situation studied.

2) It would be very useful to the readers if the authors add a table reporting the bibliographic information used by us for data discussion. This table would report citation number, plant type, macronutrient/s added, positive or negative effect on Cd adsorption in root and shoot. In this way it would be easier to compare the results obtained in this study with bibliographic data. I think that this table would be an added value to the paper.

Page 15-16 We added a new session and table 5  Line 347-348

Table 5. Plant species, purpose of the research, nutrient interactions with Cd and references of bibliographic information used by us for data discussion, in which assessed the interactions between macro and micronutrients with Cd uptake of different species.

Page 13  Line 335-340

3.8. Nutrient interactions with Cd

            No unique effect was observed in the interaction with nutrient fertilization and its effect on shoots and root Cd uptake (Table 5). Regardless of the species, in some studies a positive effect was found, observing that fertilization with nutrients caused a reduction in Cd absorption; while in other studies the effect was negative, i.e. the application of some nutrients increased Cd accumulation.

I recommend the paper for publication with only further minor corrections as indicated below:

Line 21, page 1: please, change “y” with “and” between “CCN-51” and “EET-103”.

Done  “y” was change by and

Line 30, page 1: It doesn't seem correct to me to report "heavy metals" as a keyword since only Cd is considered. I therefore propose to change this keyword taking into account the most important results obtained in this study which are not highlighted by the title of the paper.

Done “heavy metals” was changed by gas exchange

Line 47, page 2: I suggest eliminating “contamination” because a high concentration of Cd already represents a contamination, and it is not necessary to specify this further.

Done, contamination was left out

Line 66, page 2: I suggest writing “E” in brackets as “A”

Done, brackets were added

Line 80, page 2: I suggest writing the full name of “WUE” and not only the acronym.

Done full name “water use efficiency” was added

Line 119, page 3: it is necessary to add the ammonium charge, i.e., NH₄⁺.

Done ammonium charge was added

Line 120, page 3: please, write “-1” instead of “1” for S 6 mg kg¹.

Done, the unit was corrected

Line 163, page 3: are you sure you used toilet paper to dry the mill? Why didn't you use absorbent paper?

We used absorbent paper, that was corrected in the manuscripts

Line 166, page 3: it is not present “λ” between the “at” and “=228.8 nm”.

Done l Was added.

Figure 1: Why the letters above bars are only represented in B, I and J plot? The statistical evaluation of the significant differences among the treatments should be performed for all conditions under consideration.

The statistics were performed for all conditions, no letters appear in the other graphs because there were no significant differences and it was to place the same letters on the "a" above bars which made the graph more loaded with unnecessary information. in legend we say that the absence of letters in those panels is because there were no significant differences.

Line 280, page 8: I suppose that there is a missing data because graph F must also be considered where the behaviour of Ca absorption as a function of the nutrients added is shown.

 Done,   Figures. 3A, B, and D was changed by   Figures. 3A, B, C and D; C was added. There is not a figure F. 

Lines 290-291, page 9: In my opinion, it is better to specify the pH range observed for each clone. In the sentence this information is not clear.

Agree, the sentences was rewrite The minimum and maximum pH values went from 4.9 ± 0.1 to 7.0 ± 0.1 (EET-103) and 5.0 ± 0.1 to 6.8 ± 0.3 (CCN-51), respectively

Line 293, page 9: the EC concentrations reported are not correct. I think there is a mistake of an order of magnitude in 0.34 ±0.01 - 0.43 ± 0.01 dS m^-1 range. Also in this case it is necessary to better specify the EC range it refers to that clone.

There is no calculation error, the low values correspond to the control where no salt was applied. The ranges for each type of clone have been redrawn. see below.

Page 9 line 292-295

Low and high values of electrical conductivity were found: 0.34 ± 0.01 to 4.67 ± 0.20 dS m^-1 (EET-103) and 0.43 ± 0.01 to 4.49 ±0.16 dS m^-1 (CCN-51). The low values of electrical conductivity correspond to the control plants, where no fertilization was applied.

Lines 356 and 361, page 11: please, write “CI” in place of “IC”.

Done

Line 412, page 13: “values” is repeated twice. Please, correct this typing mistake.

Done, values was deleted.

Author Response File: Author Response.docx

Reviewer 2 Report

Using the actual needs encountered in the commercialization of cocoa as a guide, the manuscript systematically investigates whether exogenous application of macronutrients slows down the absorption, transport and accumulation of Cd by plants. However, a number of issues needed to be resolved before the manuscript could be received：

1) In the Introduction section (L59-64), the damage caused by Cd to plants is introduced, including chloroplast metabolism imbalance, etc. The effect of Cd on mitochondrial homeostasis is introduced in the latest study (https://doi.org/10.1016/j.jhazmat.2022.129930). Please integrate it into the Introduction content and relate it to oxidative damage and ROS that have been introduced in the manuscript.

2) Figures in the manuscript need to be adjusted as required. The text is deformed as a result of the image being stretched.

3) The content of the figure legend needs to be more detailed, such as clarifying how many sets of parallel experiments were performed.

4) The results section (L345-365) calculates the Pearson`s correlation matrix. Please provide the detection results of the data distribution type.

5) In the materials and methods section, there is no sufficient reason for choosing two Cocoas for research. In addition to considering the commercial value, the scientific value should also be taken into consideration.

Author Response

Reviewer # 2

Using the actual needs encountered in the commercialization of cocoa as a guide, the manuscript systematically investigates whether exogenous application of macronutrients slows down the absorption, transport and accumulation of Cd by plants. However, a number of issues needed to be resolved before the manuscript could be received

• In the Introduction section (L59-64), the damage caused by Cd to plants is introduced, including chloroplast metabolism imbalance, etc. The effect of Cd on mitochondrial homeostasis is introduced in the latest study (https://doi.org/10.1016/j.jhazmat.2022.129930). Please integrate it into the Introduction content and relate it to oxidative damage and ROS that have been introduced in the manuscript.

Page 2 Line 65 This reference was added

Page 20 Line 560-561

[14] Liu M., Huang Z., Xie K., Guo C., Wang Y., Wang X., Mitostasis is the central biological hub underlying the response of plants to cadmium stress, Journal of Hazardous Materials Volume 441, 2023, 129930 doi.org/10.1016/j.jhazmat.2022.129930.

Line 63-68

However, plants can increase the activity of antioxidant enzymes, thereby reducing the cellular damage induced by ROS. Recently [14] reported a conserved set of genes involved in the response to Cd stress in Arabidopsis thaliana and Oryza sativa in which effectors in the mitochondrial protein quality control networks were significantly activated in the shoot and root under exposure to Cd allowing the maintenance of mitochondrial homeostasis

• Figures in the manuscript need to be adjusted as required. The text is deformed as a result of the image being stretched.

All figures were adjusted correctly

• The content of the figure legend needs to be more detailed, such as clarifying how many sets of parallel experiments were performed.

The content of the figures legend were improved,  we added "The vertical bars represent the mean (n = 6) ± SE"

• The results section (L345-365) calculates the Pearson`s correlation matrix. Please provide the detection results of the data distribution type.

Page 5 Line 202-205

To ensure reliability in the parametric statistical tests, such as ANOVA and Pearson's correlation, the data were subjected to a normality test using the Shapiro-Wilk Test and the Bartlett test was applied to check the assumption of homogeneity of variance

5) In the materials and methods section, there is no sufficient reason for choosing two Cocoas for research. In addition to considering the commercial value, the scientific value should also be taken into consideration.

Apologize, we disagree that there is no sufficient reason in the choice of the two cocoa clones used.   We believe that the quoted paragraph " Lines 108-111 “The CCN-51 and EET-103 clones originate from Ecuador: EET-103 is a national cocoa resistant to ¨machete disease´´ (Ceratocystis cacaofunesta) while CCN 51 is susceptible, they were chosen because they are highly commercial, productive genotypes and CCN 51 is currently being generalized as rootstock [18,19]” gave the reasons for the decision to choose these clones.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript with the title “Macronutrient Fertilization and Cadmium Absorption in two Cocoa Clones” reports some fertilizers gradients influence on Cd uptake in cocoa. The research aimed to identify potential decrease of Cd uptake by the plants to reduce the food chain accumulation risk in cocoa beans. Cd is a contaminant (of geogenic + anthropogenic source) in regions of high potential for this crop, hence the high practical relevance of this study. As a non-essential element for the terrestrial life forms, Cd is highly toxic to plants, animals and humans alike. Latest updates on admissible limit of Cd in chocolate prompts feasible solutions to be found, which otherwise would threaten the exports.  

Abstract is informative and representative for the study carried out.

Introduction provides a good background for the research.

Material and Method is overall sufficiently detailed. Chapter 2.5.4. is the first formula used for calculating root and shoot content from a standard protocol? Because is no source given.
For the formula 1 and 2 from chapter 2.5.5 (which should be formula 2 and 3 in fact). I suppose source 23 was used?



Results

Authors shall present first a table with ANOVA results: p values for factors and their interactions.  Then, further in figures below authors could expand with figures only for the influence of factors or interactions for which p values were significant. Normally, post hoc is  applied only to the gradients of factors and/or interaction which showed significant p after ANOVA.
 In text authors can present these but I see no point in figures exploring post hoc for non-significant interaction for the most of the figures 1, figure 2A, figure 3E, figure 5A. The figures are clouding the manuscript with redudant information and makes the main finding to be difficult to extract. Focus on most important findings and insist on those which are most relevant.

Figures 1, 2, 3, 4 and 5 are squeezed either laterally or vertically. Make sure you place them with “insert image” not with copy-paste, so they retain their original shape. Authors MUST explain under the figure what whiskers are standing for: SD, SE, CI?

Chapter 3.2. Table 2 – why was Tukey applied only for 90 DAS? I find so surprising that Mg application was not associated with consistently increased chlorophyll levels. However, authors shall make clearer in the text if there is a consistent trend found for any of the fertilizers regimes. Can they rank the treatments and see if any pattern emerges?  

Chapter 3.7. Lines 344-365 to a great extent relates what is seen in table 3. Only the most important trends shall be given in text.

General remarks

The authors wanted to see if fertilization with macronutrients in different gradients can influence cocoa plants under Cd stress. The main findings are difficult to extract because authors paid similar attention to non-significant as well as significant findings alike. They should find a way to solve this.
 
I would have liked to see a clearer justification why macronutrients in particular were explored. Are mineral fertilizers preferably used in cocoa plantations? There are known antagonistic cation interactions which could help, which authors briefly mentioned in the introduction, relatively well-documented for some time: Zn2+ presence in soil solution can decrease Cd absorbance by roots, while Fe3+, Zn 2+, Mn+ during Cd uptake can decrease phyto-toxicity. But since these are also heavy metals, it is not wise to enrich the soil with them. Therefore, for food crops other mechanisms are needed to be explored.


Best regards.

some grammar and syntax issues

Author Response

.- Material and Method is overall sufficiently detailed. Chapter 2.5.4. is the first formula used for calculating root and shoot content from a standard protocol? Because is no source given.
For the formula 1 and 2 from chapter 2.5.5 (which should be formula 2 and 3 in fact). I suppose source 23 was used?

Page 4-5  Line 177-183

Yes, the first formula used for calculating root and shoot content was from a standard protocol. For that reason, no source gave.  The number of the equations were corrected

.-Results

Authors shall present first a table with ANOVA results: p values for factors and their interactions.  Then, further in figures below authors could expand with figures only for the influence of factors or interactions for which p values were significant. Normally, post hoc is  applied only to the gradients of factors and/or interaction which showed significant p after ANOVA.

Done we shown a  new table with ANOVA results: p values for factors and their interactions. Only the influence of factors or interactions for which  p-values were significant are shown in the figures.

Page 6 Line 222-224

Table 2. Results of the ANOVAs on the effect of clone, fertilization, and the interaction  effect clone  x fertilization on the different physiological traits studied. Red values indicate statistically significant effect of the factor at p < 0.05

In text authors can present these but I see no point in figures exploring post hoc for non-significant interaction for the most of the figures 1, figure 2A, figure 3E, figure 5A. The figures are clouding the manuscript with redudant information and makes the main finding to be difficult to extract. Focus on most important findings and insist on those which are most relevant.

Figures were corrected

.
Figures 1, 2, 3, 4 and 5 are squeezed either laterally or vertically. Make sure you place them with “insert image” not with copy-paste, so they retain their original shape. Authors MUST explain under the figure what whiskers are standing for: SD, SE, CI?

Thank you for suggestion. We changed of figures and were Insert as image, so, Now the figures maintain the original shape.  We also included on the legend  figures  “The vertical bars represent the mean (n = 6) ± SE “.

.- Chapter 3.2. Table 2 – why was Tukey applied only for 90 DAS? I find so surprising that Mg application was not associated with consistently increased chlorophyll levels. However, authors shall make clearer in the text if there is a consistent trend found for any of the fertilizers regimes. Can they rank the treatments and see if any pattern emerges?  

Tukey was applied only for the 90-day period, since this date showed significant differences in the interaction. On the other dates (45, 60 and 75 day) there were no significant differences.

With an electrical conductivity higher than 4.6 dS m^-1 in the treatments where Mg salts were applied, I would not expect a positive response between chlorophyll and Mg content, since Mg fertilization increased the concentration of ions in the solution and caused disturbances. A reduction of chlorophyll observed, was more severe with the application of S, Mg and Ca probably due to the ionic stress produced by the high presence of exchangeable bases affects the functioning of PSI and PSII, an alteration in electron transfer, and degradation of chlorophyll biosynthesis as have been reported for other  researchs.

Chapter 3.7. Lines 344-365 to a great extent relates what is seen in table 3. Only the most important trends shall be given in text.

We narrowed down the information and described the trends that we consider most important.

Page 12-13 Line319-333.

Only the correlation coefficients (r²) with a significance level of p < 0.001 were considered (Table 3). Thus, root dry mass had positive relationships with shoot dry mass, soil pH and A at 80 DAS, but had inversely proportional effects with soil EC, Cd uptake efficiency Cd translocation efficiency, all macronutrients’ contents. Likewise, shoot dry mass showed positive associations with A at 80 DAS and soil pH, but negative associations with Cd extraction efficiency, soil EC, N, P and leaf Mg.

While shoot and plant Cd content showed directly proportional relationships with the efficiency of Cd absorption, EC of the soil and leaf concentrations of N, P, S and Mg also presented a significant negative relationship with soil pH.

There were negative and strong associations among soil pH and leaf concentrations of N, P, S and Mg. On the contrary, EC had directly proportional relationships with leaf concentrations of N, P, S, Mg and Ca, but also exhibited negative associations with A at 80 DAS. Values of A at 80 DAS showed negative correlations with the concentrations of N, P, S, Mg, Ca and soil EC; however, it had positive associations with soil pH and E at 40 DAS. 

General remarks

The authors wanted to see if fertilization with macronutrients in different gradients can influence cocoa plants under Cd stress. The main findings are difficult to extract because authors paid similar attention to non-significant as well as significant findings alike. They should find a way to solve this.

I would have liked to see a clearer justification why macronutrients in particular were explored. Are mineral fertilizers preferably used in cocoa plantations? There are known antagonistic cation interactions which could help, which authors briefly mentioned in the introduction, relatively well-documented for some time: Zn2+ presence in soil solution can decrease Cd absorbance by roots, while Fe3+, Zn 2+, Mn+ during Cd uptake can decrease phyto-toxicity. But since these are also heavy metals, it is not wise to enrich the soil with them. Therefore, for food crops other mechanisms are needed to be explored.

The purpose of the work is to simulate what the producer does in the field, applying nutrients through fertilizers and their effect on the absorption of the metal. We wonder if with the application of N (all cocoa producers who have their plantations in tropical soils carry out this activity) the Cd in the soil is mostly absorbed by the plant and accumulated in its tissues? The same is true for the other nutrients evaluated.

We do not try to manage fertilizer application as a soil amendment to decrease absorption. Of course, for example, if it turns out that the application of P causes a decrease in the absorption of Cd, it would be a recommendation to make to the producer; but as it was seen in the result of the research, applying N increases the absorption of Cd, I will not be able to recommend the producer not to apply N, since it would decrease the absorption of Cd, but it would also affect the production of the crop and therefore its profitability.

Round 2

Reviewer 3 Report

Dear authors,

the Results section of the paper is significantly better. I am fine with the statistics approach now. Just note that when a p is below 0.001 it is not necessary to add the whole number just write <0.001 (see in your ANOVA table values such as 0.0001). This permits you to limit the length of all your p numbers to 3 decimals after dot.

It is important to be careful with the fine details.

Best regards.

some fine syntax and grammar improvement would help

Author Response

Just note that when a p is below 0.001 it is not necessary to add the whole number just write <0.001 (see in your ANOVA table values such as 0.0001). This permits you to limit the length of all your p numbers to 3 decimals after dot.

 We agree with the reviewer and thank you very much for the suggestions,  throughout the manuscript this aspect was corrected  see for example table 2.

It is important to be careful with the fine details.

Some fine syntax and grammar improvement would help

we carefully checked small details throughout the text and figures and corrected them. Throughout the document we carefully checked  syntax and grammar and it was  improved

Author Response File: Author Response.docx