Influence of N, K, and Seaweed Extract Fertilization on Biomass, Photosynthetic Pigments, and Essential Oil of Thymus vulgaris: Optimization Study by Response Surface Methodology
Round 1
Reviewer 1 Report
The author in this study was aimed to explore the possibility of reducing fertilizer application rates while maximizing the biomass of thyme and reducing the risk of environmental pollution,which is very interesting and meaningfull. However, there are some mistakes, and I give some comments as follows:
(1) The author should add more discussion to introduce why different concentrations of fertilization affect the synthesis of chemical substances. The discussion is not deep enough.
(2) The author should add more discussion to introduce why different concentrations of fertilizer will affect the suitability of some chemical components, and whether its mechanism is clear
(3) Has the author verified the RSM results? If not, please give reasons and how to verify the reliability of the model
（4）Please describe in detail why the author chose Box-Behnken design instead of other models
（5）How did the author determine the appropriate fertilization interval? Before the RSM analysis, was the orthogonal test used？
（6）Why did the author use RSM instead of the classic 3414 experiment？
Author Response
Reviewer 1
The author in this study was aimed to explore the possibility of reducing fertilizer application rates while maximizing the biomass of thyme and reducing the risk of environmental pollution,which is very interesting and meaningfull. However, there are some mistakes, and I give some comments as follows:
(1) The author should add more discussion to introduce why different concentrations of fertilization affect the synthesis of chemical substances. The discussion is not deep enough.
Answer: More information and content about the effects of fertilizers concentrations and levels were added to the discussion section.
(2) The author should add more discussion to introduce why different concentrations of fertilizer will affect the suitability of some chemical components, and whether its mechanism is clear
Answer: Additional information and citations were added to the discussion describing how fertilizer levels affect the chemical components of the essential oil.
(3) Has the author verified the RSM results? If not, please give reasons and how to verify the reliability of the model
Answer: The results of RSM technique in this study are verified and approved by the authors. In RSM studies, there are appropriate indicators to determine the validity of the answers, including “R square”, “Adequate precision”, “CV”, and “Lack of fit”, which were all within an acceptable limits (Tables 3 and 4).
（4）Please describe in detail why the author chose Box-Behnken design instead of other models
Answer: Box-Behnken is one of the efficient and capable designs in the RSM technique which can be used for analyzing the experiments with quantitative factors (independent variables). Box-Behnken design is used for comprehensive analysis such as fitting the empirical models and optimizing the resources (such as fertilizers, irrigation, plant density, pesticides, temperature, light intensity, etc.). The analysis used in this study cannot be done through classical experimental designs.
（5）How did the author determine the appropriate fertilization interval? Before the RSM analysis, was the orthogonal test used？
Answer: Determining the levels and intervals of the fertilizers in this study was based on an extensive review of literature as well as soil test results. Another important point in Box-Behnken design is that fertilizer levels should be chosen with equal intervals for example 0-200-400 kg/ha N fertilizer.
（6）Why did the author use RSM instead of the classic 3414 experiment？
Answer: RSM is a more comprehensive and advanced multivariate technique in empirical modeling and optimizing resources and experimental conditions. The superiority of the RSM technique over the classic methods is that useful operations such as modeling, determining the trend of changes in a specific range, and forecasting and optimization are not possible through classical methods. In addition, with the RSM technique, the effects of factors can be presented in the form of powerful diagrams including response surface and contour plots.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Title look little confusing
Introduction part must be little elaborated to get idea about the current practice of cultivation and what new author wants to address
How author identified and quantified essential oil percentage??
In t ex, why author has not studied interaction of different soil parameters as soil fertility gradient and soil nutrient compositions varies.
The experiment was conducted using three fertilizer and three levels each and results were examined using statistical tool. It is mere representation only as with Sem or SD or PCA can also be used to represent the data.
Line number 58..growth stimulation and fertilizer is different …fertliser provide essential elements while growth stimulation enhances uptake of nutrient and assist in metabolism
How did author confirm the application of foliar spray and not the drenching or soil application in method section 2.1
Change the percentage :- terpinene (%22.77) as the second major essential oil compound
Line 113-115 what was oil recovery in this process.
In 2.2.4 how author has quantified the oil percentage using mentioned method as GC MS gives non quantitative value?
What is recommended package of practices for this crop?? Fertilizer application , time ..etc.,
Regarding Table 2 …what else author has used for experiment (fertilizer)
Why these parameters and their measurement was not mentioned in the method section thymol, γ-terpinene, p-cymene, and carvacrol …these are not there in response variable in table 6
Significance of such metabolites is not mentioned in the introduction and discussion .
Essential oil content and essential oil yield ???how author measure
The discussion give more emphasize on essential compositions thymol, γ-terpinene, p-cymene, and carvacro but tite does not describe the same
The use of nitrogen fertilizer had no significant effect on the percentage of essential oil (EO)….this line is confusing as nitrogen enhances the biomass as per your results but oil content showed negative trends …this could be due to the method used for oil content analysis or other factors..
Author Response
Reviewer 2
- Title look little confusing
Answer: The title was slightly modified.
- Introduction part must be little elaborated to get idea about the current practice of cultivation and what new author wants to address
Answer: The introduction was modified in lines 70 and 71.
- How author identified and quantified essential oil percentage?
Answer: As mentioned in section 2.2.3, a 50 g dried leaf sample from each experimental plot was randomly selected, powdered, and subjected to hydro-distillation in a Clevenger apparatus. Distillation was done for 3 hours, and the essential oil (EO) from the sample was collected and weighed. Let’s suppose that the amount of EO obtained from a 50-gram plant sample was 0.3 gram, by multiplying by 2, the amount of EO was obtained in grams per 100 grams of the plant sample, which is actually the same percentage of EO in the plant sample (i.e., 0.6%).
- In t ex, why author has not studied interaction of different soil parameters as soil fertility gradient and soil nutrient compositions varies.
Answer: This study aimed to optimize the amounts of N, K, and SW fertilizers to maximize dry matter, and essential oil yield by using a modeling approach. However, the comment made by the reviewer is very important and hopefully it will be addressed in our future studies.
- The experiment was conducted using three fertilizer and three levels each and results were examined using statistical tool. It is mere representation only as with Sem or SD or PCA can also be used to represent the data.
Answer: The mentioned criteria and methods (SEM, SD, PCA) are very important in classical designs and are very helpful in interpreting the results, however, in RSM designs, other criteria such as: Lack of fit, R square, Adequate precision, and CV are used to determine the validity of the models. In addition, the plant parameters can be presented well, in the form of response surface and contour plots.
- Line number 58..growth stimulation and fertilizer is different …fertlian well presentedser provide essential elements while growth stimulation enhances uptake of nutrient and assist in metabolism.
Answer: Thank you for the comment. You are right. In this study, seaweed extract (as a biostimulant) was applied to determine its efficiency in stimulating the growth and yield traits of thyme.
- How did author confirm the application of foliar spray and not the drenching or soil application in method section 2.1
Answer: Foliar spraying is an effective method for faster absorption of many nutrients and growth-stimulators into the plant. Additionally, the foliar application does not confront issues such as nutrient fixation in the soil and thus being unavailable to crops, and loss to leaching and runoff. Nevertheless, the effect of foliar and soil application on thyme can be compared in another study.
- Change the percentage :- terpinene (%22.77) as the second major essential oil compound
Answer: The percentage sign (%) for gama-terpinene was corrected (line 301, section 3.5).
- Line 113-115 what was oil recovery in this process.
Answer: The minimum and the maximum values of EO% obtained through Clevenger in the current study were 0.39% and 1.24%, respectively.
- In 2.2.4 how author has quantified the oil percentage using mentioned method as GC MS gives non quantitative value?
Answer: The quantity of essential oil components was determined through calculating the “GC peak area percentage” for each component. This explanation was added to the end of section 2.2.4.
- What is recommended package of practices for this crop?? Fertilizer application , time ..etc.,
Answer: According to the optimization results (Table 6) the best recommended package for thyme production was consuming 162 kg/ha urea, 12 kg/ha FSP, and 4 L/ha SW. when Urea is applied in two equal split; foliar application of potassium at stem elongation stage, and Seaweed extract as foliar spray three times with 14-day intervals from the stem elongation stage onwards.
- Regarding Table 2 …what else author has used for experiment (fertilizer)
Answer: No other nutrient was used. Soil test analysis (Table 1) revealed that the available P in soil (16.1 mg/kg) was sufficient.
- Why these parameters and their measurement was not mentioned in the method section thymol, γ-terpinene, p-cymene, and carvacrol …these are not there in response variable in table 6
Answer: The names of the four mentioned compounds and their measurement were added to the end of section 2.2.4. Regarding not mentioning two compounds of γ-terpinen and p-cymene in Table 6, since the statistical models for these two compounds were not significant (Table 4), they were excluded from the optimization calculations.
- Significance of such metabolites is not mentioned in the introduction and discussion.
Answer: The significance of these compounds was added to the introduction section (lines 67-69), and also to the discussion.
- Essential oil content and essential oil yield ???how author measure
Answer: The measurement of EO content (EO percentage) was explained above. The EO yield was calculated simply by multiplication of the leaf dry weight by the EO content and presented as kg/ha.
- The discussion give more emphasize on essential compositions thymol, γ-terpinene, p-cymene, and carvacro but tite does not describe the same
Answer: The title is already long. Adding “essential oil composition” to the title makes it even longer.
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- The use of nitrogen fertilizer had no significant effect on the percentage of essential oil (EO)….this line is confusing as nitrogen enhances the biomass as per your results but oil content showed negative trends …this could be due to the method used for oil content analysis or other factors.
Answer: As explained in the discussion, the non-significance of the N effect on EO percentage could be attributed to the soil organic level and availability of soil nitrogen in the current study. Similar to our findings, other reports also demonstrated that N fertilizer did not affect the EO percentage of medicinal and aromatic plants (Baranauskiene et al., 2003; Arabaci and Bayram, 2004; Chrysargyris et al., 2016) (references number 40, 41, and 4, respectively).
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript is interestingly written and generally looks correct. However in order to draw unequivocal conclusions from field experiments, especially if two agrotechnical factors are taken into account (such as the nitrogen and potassium fertilization and saeweed extract used), it is necessary to repeat the experiment in at least two growing seasons (preferably for three seasons).
Author Response
Reviewer 3:
Thank you for your comment. The authors generally totally agree with your comments. However, thyme is a perennial crop, and repeating the experiments for the second year doesn't apply to this medicinal crop.
Round 2
Reviewer 1 Report
The manuscript can be aacepted in current form.
Author Response
Responses to the Academic Editor:
Thank you very much for your review and comments. The suggested changes were made according to your comments as follows:
- The mean air temperature and rainfall during the growing season of 2021 and the norm condition during the growing period were presented in a new table (table 1) and also noted in the Material and Methods section (highlighted in green, Lines 82 and 83).
- More detail about the irrigation was included (highlighted in green, Lines 95-97).
- The requested explanations were added to the end of the discussion (highlighted in green, Lines 499-503).
- The following statement (formerly placed within the first paragraph of the discussion) was deleted: “Thus, the current results indicated that medicinal plants may respond differently to the increased N rate compared with non-medicinal plants.”. In addition, in Line 405, the following statement was added: “the plant species”, and in Lines 497 and 497, the statement of “medicinal plants” was changed to “the medicinal plant of thyme”.
- The word “compositions” was replaced with “components”, throughout the manuscript (highlighted in green).
Reviewer 3 Report
The answer to my review is laconic. I know perfectly well that thyme is a perennial plant, because I have conducted research on it many times. And agronomic experiments are always carried out in at least two growing seasons (preferably three) - they should simply be repeated in the next year, establishing a new plantation, implementing all the same experimental factors. Biomass, photosynthetic pigments, and essential oil production is as dependent on agrometeorological conditions and as agrotechnical factors as the use of fertilizers and seaweed extract.
They maintain my opinion that this manuscript is not suitable for publication in such a respectable journal as Agronomy
Author Response
In light of your valuable and scientific opinion, the necessary explanation was added at the end of the discussion (highlighted in green). Surely, we should repeat this experiment in the next growing season and if possible in other locations as well, so that we can check the interaction of the effects of environmental factors with the effects of the fertilizers, so the modeling and optimization operations would be even more accurate.
