Phenology, Nitrogen Status, and Yield of Red Clover (Trifolium pretense L.) Affected by Application of Vitamin B12, Humic Acid, and Enriched Biochar

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments

Abstract

-       The authors should mention the data (numbers ) that showed increasing the values of all measured features (Total number of stems, flowers, trichome thickness and flowering time and other measurements). To give the readers the clear idea about the significant and not significant. 

Keywords:

-       Add Phenology and delete (Root dry matter, Clover yield) because this is not significant important to added this in Keywords.

Introduction

-       Lines (65 to 73) move to first paragraph in introduction part, and add more information and references need to add about red clover, such as (planting area, growing areas and suitable conditions) this need to be added as locally and internationally, to give the readers clear idea about the background of this plants in the article.

-       Next paragraph talk about vitamin B12, and humic acid both and the previous studies.

-       Last paragraph will be about  Biochar and their interaction between the study factors.

-       The aim of this work is not mentioned in the introduction part, so the authors should add the scientific aim for this manuscript in the end of the introduction.

Materials and Methods

-       This part not organized so more work need to be done.

-       How many times the authors repeat the experiment ?

-       All the information about the instruments are not mentioned correctly such as, An n-hexane (95%) was purchased from Sigma-Aldrich (catalogue number 270504-2L; Castle Hill, NSW, Australia). This should be mentioned in this way for whole Materials and Methods.

-       Line 115 there is no information about the a stereo microscope, please  add the manufacture and made as above.

-       Clovers were harvested 100 days, according to what the authors used this time after 100 days if there is a reference please provide here.

-       ground into a fine powder (line 124) by which instrument the authors did the ground ?

-       for the statistical analysis, how many plants are used in this experiment, so the authors should mentioned that. 

Results

-       All the tables in this part should mentioned (n=4 or 3) that is the number of replicates

-        Figure 2. The authors should explain in full what is meaning of (ns and **) under the figure.

-       Figure 3. What is the meaning of (P) in this figure

-       3.1.4. Total number of stems, what is the unit of the used number in this factor (vitamin B12 (73.0) is it stem per plant or something else.

-       Figure 5, again as figure 2

-       3.3.1. SPAD reading, line 300-313, should move with table 2 and 3 because the authors talked about these tables in the end of this result and this is not scientific writing.

Discussion

-       This part must be organized according to results part for systematic work to avoid the complicated issues and confusing.

Conclusions

-       In this part, the authors should give just a good findings because there is many repeated information for the results part and this is not a good way to write the conclusion. The reason for write conclusion is to give a brief idea about the authors work, thus this part need to rewrite.

 

Author Response

Reviewer 1:

Abstract

-       The authors should mention the data (numbers) that showed increasing the values of all measured features (Total number of stems, flowers, trichome thickness and flowering time and other measurements). To give the readers the clear idea about the significant and not significant. It was done.

Response:

Thank you for your constructive comments. We have included the quantitative factors that showed significant influence in the abstract section.

Keywords:

-       Add Phenology and delete (Root dry matter, Clover yield) because this is not significant important to added this in Keywords. It was done.

Response:

The keywords were selected based on being representative of the key concept of the context, being descriptive, and most repetitive words in the text. We have added the phenology to the keyword as suggested by the reviewer. However, the authors believe that root dry matter is often overlooked and should be considered as one of the important keywords. Therefore, with all due respect, we decided to keep the root dry weight in the keywords.

Introduction

-       Lines (65 to 73) move to first paragraph in introduction part, and add more information and references need to add about red clover, such as (planting area, growing areas and suitable conditions) this need to be added as locally and internationally, to give the readers clear idea about the background of this plants in the article. The next paragraphs talk about vitamin B12, and humic acid both and the previous studies. The last paragraph will be about Biochar and their interaction between the study factors. It was rearranged.

 

Response:

The information about red clover that was suggested by the reviewer was added to the introduction. However, we had to separate the vitamin B12 and humic acid because vitamin B12 was the main focus and was the only factor that was added to all growing media (potting soil, humic acid, enriched biochar, and field soil). Therefore we allocated one paragraph specifically for the role of Vitamin B12 as the main factor. Per the reviewer's suggestion, we moved the biochar section to the last part of the introduction.

 

-       The aim of this work is not mentioned in the introduction part, so the authors should add the scientific aim for this manuscript in the end of the introduction.

It was done.

 

Response:

Based on the reviewer’s recommendation, we rewrote the end of the introduction to make the major aim of the study easier to follow.

Materials and Methods

-       This part not organized so more work need to be done.

 

Response:

We sincerely thank the reviewer for catching the missing part. Unfortunately, for unknown reasons, one paragraph of the M&M was deleted when we used the journal’s format. We have added the missing part to the M&M.

 

How many times the authors repeat the experiment?

 

Response:

The study was conducted only one time. However, we had three biological observations (3 plants per pot) and four replications (12 plants in total). We revised the statistical analysis section.

 

-       All the information about the instruments are not mentioned correctly such as, An n-hexane (95%) was purchased from Sigma-Aldrich (catalogue number 270504-2L; Castle Hill, NSW, Australia). This should be mentioned in this way for whole Materials and Methods.

 

Response:

The requested information was added to the M&M.

 

-       Line 115 there is no information about the a stereo microscope, please add the manufacture and made as above.

 

Response:

The suggested information was added.

 

-       Clovers were harvested 100 days, according to what the authors used this time after 100 days if there is a reference please provide here.

Response:

The harvested time was selected based on the early flowering stage of clover which is a tradeoff of forage quality and biomass. Generally, the quality of forages (grasses and legumes) declines as plants mature and reach the flowering stage. Now, adding one reference to highlight this well-known fact would have changed the numbering of all the references  

-       ground into a fine powder (line 124) by which instrument the authors did the ground

Response:

The grinder information Foss Cyclotec 1093, Hilleroed, Denmark was added to the M&M.

 

-       for the statistical analysis, how many plants are used in this experiment, so the authors should mention that.

 

Response:

The total plants (3 Plants per pot × 4 Replications) for each treatment (4 different growing media × two levels of vitamin B12) were considered. This was added to the M&M.

Results

-       All the tables in this part should mentioned (n=4 or 3) that is the number of replicates

 

Response:

Thank you for catching the missing part. It was added.

 

-        Figure 2. The authors should explain in full what is meaning of (ns and **) under the figure.

 

Response:

Explantation was added.

-       Figure 3. What is the meaning of (P) in this figure.

Response:
The P (potting soil) abbreviation was mentioned in all figures.

-       3.1.4. Total number of stems, what is the unit of the used number in this factor (vitamin B12 (73.0) is it stem per plant or something else.

Response:
It is the number of stems per plant in each pot.

-       Figure 5, again as figure 2

 

Response:

Corrected

-       3.3.1. SPAD reading, line 300-313, should move with table 2 and 3 because the authors talked about these tables in the end of this result and this is not scientific writing.

 Response:
Table 2 demonstrates the influence of vitamin B12 on several traits, including SPAD values. However, the results section begins with phenological traits, including flowering time. Thus Table 2 must be presented with this section so the readers can more easily follow the results of the changes in phenology.  

Discussion

-       This part must be organized according to results part for systematic work to avoid the complicated issues and confusing.

 

Response:

The discussion section was rearranged based on the reviewers’ comments.

Conclusions

-       In this part, the authors should give just good findings because there is many repeated information for the results part and this is not a good way to write the conclusion. The reason for writing conclusion is to give a brief idea about the authors work, thus this part need to rewrite.

Response:
With all due respect, the authors do not agree with the reviewer’s suggestion. The authors strongly believe that the conclusion should not be repetitive in the result section. Instead, it should demonstrate the overall influence (positive or negative) of the treatments without mentioning numbers/values. Therefore, we left the conclusion section almost unchanged (made some minor changes in wording). Let’s not forget, that we enthusiastically accepted the constructive comments from the respected reviewer about revising the abstract and have added numeric results to the abstract.

Reviewer 2 Report

Comments and Suggestions for Authors

The study investigated the impact of vitamin B12, humic acid, and enriched biochar application on the phenology, nitrogen status, and yield of red clover. While the research is interesting, several issues need addressing:

1. The workload appears to be insufficient. Since the assessment considered the influence of these factors, the evaluation of nutritional components should not be limited to nitrogen (N) alone. Other nutritional factors should be considered for a comprehensive assessment, as there may be variations in nutritional substances even when total nitrogen shows no significant differences.

2. Line 118 The abbreviation " SPAD" needs clarification.

3. The botanical Latin name should be consistently used for red clover from its first mention, rather than in the methods and materials.

4. The methodology for determining gland density using a stereo microscope needs clarification. The images lack a scale, and differences in gland density are not clearly discernible. Additionally, gland density is known to be significantly correlated with leaf developmental stages, with substantial differences between flesh and old leaves. How did the study account for and eliminate the influence of leaf developmental stages in this context?

5. The lack of explanations for the images and the term "**" "ns" needs clarification in legend.

6. In the case of plant flowering, it is generally recognized that flowering entails a significant energy cost for the plant. For forage grasses intended for livestock consumption, flowering is typically considered a disadvantage as it can diminish the overall biomass of the plant. Therefore, if early flowering is deemed a merit trait for red clover, it should be explicitly addressed. Furthermore, these considerations should be introduced in the introduction section.

7. How to eliminate factors related to differences in plant genotype that may cause variations in flower color.

8. There are multiple treatment groups, but why does Table 2 only display two?

9. Where is the data for the determination of chlorophyll content of leaves mentioned in line 304, and the methodology for this measurement is not provided.

10. 377-379 "In a multiple-cutting forage system, earlier flowering may provide the opportunity to have an extra harvest, which enhances growers' income, especially in short-growing sea-son areas. ". This may seem perplexing when considering that the forage serves as fodder for livestock.

11. The citation format for Reference 2 is incorrect. 

 

12. The overall structure of the text appears disorganized and requires further clarification for improved coherence.

Author Response

Reviewer 2:

The study investigated the impact of vitamin B12, humic acid, and enriched biochar application on the phenology, nitrogen status, and yield of red clover. While the research is interesting, several issues need addressing:

1. The workload appears to be insufficient. Since the assessment considered the influence of these factors, the evaluation of nutritional components should not be limited to nitrogen (N) alone. Other nutritional factors should be considered for a comprehensive assessment, as there may be variations in nutritional substances even when total nitrogen shows no significant differences.

Response:

We have not added any fertilizer to the growing media, thus measuring other nutritional elements seems meaningless. The reason for studying N status was the fact that red clover as a legume can fix atmospheric N.

 

2. Line 118 The abbreviation "SPAD" needs clarification.

 

Response:
Thank you. We explained the abbreviation of SPAD and added the manufacturing information.

3. The botanical Latin name should be consistently used for red clover from its first mention, rather than in the methods and materials.

Response:

The rule of the majority of journals requires to inclusion of the botanical Latin name of plant species when they are mentioned for the first time in the manuscript. After that, the common name can be used. The Latin name of red clover was mentioned the very first time we used its name. Therefore the (Trifolium pretense.L) was added to the title, abstract, and keywords, in addition to the introduction.

4. The methodology for determining gland density using a stereo microscope needs clarification. The images lack a scale, and differences in gland density are not clearly discernible. Additionally, gland density is known to be significantly correlated with leaf developmental stages, with substantial differences between flesh and old leaves. How did the study account for and eliminate the influence of leaf developmental stages in this context?

Response:

The methodology for determining gland density was added to the M&M. “A cohort of leaves was selected by choosing the middle leaf of the tri foliate (7-week-old leaves) of the second node from the apex for all three plants in a pot. Trichome numbers were counted in each specific area of the virtual ruler on images under the microscope to keep uniformity.”

For your information, numerous images were collected to record trichome density data, however, we demonstrated the difference between those who received vitamin B12 and those that not receive vitamin B12.
The authors propound the probable reason for trichome density differences in the discussion part. As the pictures show, apart from the number, plants with vitamin B12, had thinner trichomes.

5. The lack of explanations for the images and the term "**" "ns" needs clarification in legend.

 

Response:

The footnotes were added to all figures.

 

6. In the case of plant flowering, it is generally recognized that flowering entails a significant energy cost for the plant. For forage grasses intended for livestock consumption, flowering is typically considered a disadvantage as it can diminish the overall biomass of the plant. Therefore, if early flowering is deemed a merit trait for red clover, it should be explicitly addressed. Furthermore, these considerations should be introduced in the introduction section.

Response:
The authors cannot agree more with the reviewer’s comments. In the introduction, a sentence was added to highlight this topic. In the current study, we discussed accelerating flowering due to the application of vitamin B12 especially in conjunction with humic acid resulting in early flowering which provides the opportunity for having one extra harvest. 

7. How to eliminate factors related to differences in plant genotype that may cause variations in flower color.

Response:
The authors do not intend to exclude any factor that may cause variations in flowers’ color. We just focused on observing some changes in flowers’ color and shape as a result of the application of vitamin B12, without even knowing if the observation carries any significant merit with it. We assumed the observation may be useful or interesting to some researchers. A photo of the plants with flowers is attached to this response.

 

 

8. There are multiple treatment groups, but why does Table 2 only display two?

Response:

 

Vitamin B12 was the main effect and our primary interest factor.  The other main effect (growing media) is presented in Table 3. The interactive effects of the main effects are illustrated in the figures.

 

9. Where is the data for the determination of chlorophyll content of leaves mentioned in line 304, and the methodology for this measurement is not provided.

Response:
The SPAD measurement is included in the MM sections. Tables 2 and 3 are demonstrating the changes in SPAD values (indirect assessment of chlorophyll content) as affected by the main effects.

10. 377-379 "In a multiple-cutting forage system, earlier flowering may provide the opportunity to have an extra harvest, which enhances growers' income, especially in short-growing sea-son areas.". This may seem perplexing when considering that the forage serves as fodder for livestock.

Response:
The following sentence was added to the introduction. Also, the merit of earlier flowering was mentioned in the discussion section:

“Generally, the quality of forages (grasses and legumes) declines as plants reach the flowering stage. Therefore accelerating flowering time is considered positive and may provide an opportunity for increasing the frequency of harvesting.”

11. The citation format for Reference 2 is incorrect. 

Response:
The citation format was corrected.  

12. The overall structure of the text appears disorganized and requires further clarification for improved coherence.

Response:
The Introduction, Materials and methods, and discussion parts were rearranged.

Reviewer 3 Report

Comments and Suggestions for Authors

This paper explored the impact of vitamin B12 on red clover in a greenhouse experiment. Vitamin B12, applied alone and with humic acid and biochar enriched with compost, was studied across two dosage levels (0, 20 mg pot-1) and four growing media. Results showed a reduced flowering time, increased root dry weight, flower count, and decreased leaf trichome density with vitamin B12. Biochar had minimal impact. The study emphasizes the importance of manure application, rich in vitamin B12 and humic acid, for enhancing forage crop characteristics, particularly in red clover.

The work presented in this paper makes a significant contribution to the field and is commendably well-organized and well-written. However, there are a few minor suggestions that can be addressed. The discussion section can be strengthened by highlighting the insights gained from the findings and their applicability to future research. Addressing important questions, such as the research gaps addressed by this study, the beneficiaries of the improvements, and potential future directions, will greatly enhance the discussion and allow for a clearer statement of the paper's contribution. For example, exploring the synergistic effects of combining vitamin B12, humic acid, and biochar in more detail might provide a comprehensive understanding of their interactions. Long-term studies might be conducted to assess the lasting impacts of these treatments on soil health, including microbial activity and nutrient cycling. Economic feasibility studies might be valuable for evaluating the viability of implementing these strategies on a larger scale in agricultural practices. Additionally, investigating the potential influence of vitamin B12 on other physiological aspects of red clover might contribute to a more holistic understanding. Further exploration into the variations in dosage and application timing of vitamin B12 might be conducted to determine their specific effects on outcomes.

 

Author Response

Reviewer 3:

This paper explored the impact of vitamin B12 on red clover in a greenhouse experiment. Vitamin B12, applied alone and with humic acid and biochar enriched with compost, was studied across two dosage levels (0, 20 mg pot-1) and four growing media. Results showed a reduced flowering time, increased root dry weight, flower count, and decreased leaf trichome density with vitamin B12. Biochar had minimal impact. The study emphasizes the importance of manure application, rich in vitamin B12 and humic acid, for enhancing forage crop characteristics, particularly in red clover.

The work presented in this paper makes a significant contribution to the field and is commendably well-organized and well-written. However, there are a few minor suggestions that can be addressed. The discussion section can be strengthened by highlighting the insights gained from the findings and their applicability to future research. Addressing important questions, such as the research gaps addressed by this study, the beneficiaries of the improvements, and potential future directions, will greatly enhance the discussion and allow for a clearer statement of the paper's contribution. For example, exploring the synergistic effects of combining vitamin B12, humic acid, and biochar in more detail might provide a comprehensive understanding of their interactions. Long-term studies might be conducted to assess the lasting impacts of these treatments on soil health, including microbial activity and nutrient cycling. Economic feasibility studies might be valuable for evaluating the viability of implementing these strategies on a larger scale in agricultural practices. Additionally, investigating the potential influence of vitamin B12 on other physiological aspects of red clover might contribute to a more holistic understanding. Further exploration into the variations in dosage and application timing of vitamin B12 might be conducted to determine their specific effects on outcomes.

Response:
The authors thank the reviewer for all the positive comments and encouragement. The authors loved all the suggested topics and ideas highlighted by the reviewer. However, the current study should be considered as an introductory approach to the use of vitamin B12 and soil amendment on the growth and yield performance of red clover. The wonderful research areas mentioned by the reviewer should be considered and perused in future studies.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have no other comment.