Crop Response to Leaf and Seed Applications of the Biostimulant ComCat^® under Stress Conditions

Round 1

Reviewer 1 Report

1. Introduction - If possible, I recommend supplementing the results of research into the use of the bio-stimulant ComCat  in the cultivation of other crops. 

2. Materials and Methods

• It is not clear from the methodology whether the field experiments as well as the experiments in pots were one-year-old or two-year-old.
• 2.1.1. Field experiments - high plant density per ha; I recommend supplementing the agrotechnical analysis of the soil 
• 2.1.2 - 2.1.4. Pot experiments - It is not clearly defined in the methodology - which is experiment A and experiment B, which are compared in the results. 

4. Discussion

• I recommend supplementing the discussion with studies that focus on the use of biostimulants from the point of view of reducing the application of synthetic fertilizers. 

Author Response

Dear reviewer,

thanks for your valuable comments and suggestions to our manuscript.

We have revised the manuscript according to the comments and suggestions of all reviewers. Please for enclosed our revised manuscript and the point-by-point response to your points.

1. Introduction - If possible, I recommend supplementing the results of research into the use of the bio-stimulant ComCat  in the cultivation of other crops.

Yes, we mentioned the use of ComCat® in different crops.

2. Materials and Methods

• It is not clear from the methodology whether the field experiments as well as the experiments in pots were one-year-old or two-year-old.
• 2.1.1. Field experiments - high plant density per ha; I recommend supplementing the agrotechnical analysis of the soil 

We corrected the maize density. I am sorry that we do not have the capacity to conduct soil analysis in Benin.

• 2.1.2 - 2.1.4. Pot experiments - It is not clearly defined in the methodology - which is experiment A and experiment B, which are compared in the results. 

We clarified this.

4. Discussion

• I recommend supplementing the discussion with studies that focus on the use of biostimulants from the point of view of reducing the application of synthetic fertilizers. 

Yes, we included another study on biofertilizer in the discussion.

Reviewer 2 Report

To whom it may concern,

This research is composed by 8 different studies dealing with 4 different stresses (drought, nutritional, weed competition and herbicide injury), on 2 crops (maize and barley), 2 situations (Field and greenhouse) and 2 locations (Benin and Germany). There are too many factors involved for such a low number of experiments as half of them are replicates, only 4 experiments in total. The only common factor is ComCat. I think, authors should have focus on one stress and one crop, and then perform greenhouse plus field experiments. The present manuscript is a mixture of non-linked experiments.

Abstract:

When reading the abstract, one have the feeling that both crops have been tested under 4 stresses and as both, seed and leaf treatment (Lines 20-22) and this is not true. The same for yield determination (it was just determined in one field in Benin) (Lines:23-24) not in all conducted experiments.

In line 24-25 authors say “ComCat did not promote maize and barley biomass production of the unstressed plants”. Afterwards in lines 29-29, the same  idea is expressed (perhaps a more generalist approach). I think authors should have joined these 2 ideas in just one sentence.

Keywords:

There are too many keywords. Some of them (novel secondary pathways or plant strengthener) would have been useful if there have would been a good discussion in the introduction or discussion sections, but this was not the case.

Introduction:

Line 46: I don´t think biostimulants are inexpensive products. They would be inexpensive if they could replace fertilizers and/or pesticides but in my opinion, they can´t substitute them, perhaps they can reduce them. But they are normally expensive products above all if their effect is less evident to occur as the authors mention.

Line 49-51: Again, I don´t agree with the idea of complete mineral fertilization replacing by biostimulants and I don´t see the link to the mung bean use.

Line 70: Authors should report those numerous reports of positive impact of ComCat, unless they are anecdotal and not scientific, in that case (anecdotal) you shouldn´t mention it as this is a scientific publication.

In which crops have ComCat been useful during its 20-year life-span? Is there any scientific ComCat report on Maize or Barley? If so, please report them and discuss.

Material and methods:

Line 94: The authors don´t explain that the experiments were replicated, I had to guess it as I didn´t understand letters “A” and “B” in the graphics. Need to explain it at the beginning of this section.

Which was the maize cultivar in the Benin experiment?

Type of soil in Benin? Soil analysis?

Missing Nozzle and pressure in Benin as it has been reported for Germany experiments like soil or maize cultivar

Line 107: Need to explain the acronym DAS (I guess: Days after sowing)

Line 110: I don´t understand why the Materi plot was weeded at 45 DAS if it was destroyed by a storm. If it was not flooded before 45 DAS, why don´t authors report fresh biomass at 42 DAS?

Line 134: Figure 1 is not interesting to be reported for the manuscript as it is only showing the effect of drought which is obvious. I can´t see Figure 1 written in the text.

Line 143: Phenological stage of weeds on the transplant day? Perhaps is the one shown in figure 2? if so, please indicate.

I guess ComCat was also applied on the weeds, therefore you are also improving their growth. Discuss this. I would have applied ComCat as a maize seed treatment.

Perhaps, 43 days after sowing is not enough to see effects on weed competition.

From line 158 to 169 everything is very confusing (The third factor…). If you speak about factors, why don´t you conduct multifactorial ANOVAs?

The same for Barley: is 21-27 DAS enough time to see weed competition?

Results

General: Authors should have edited all graphics equally in terms of letter size, or statistical symbols reporting (in one graphic (Figure 3) you say “ns” and in others you put the same letter in all columns which is ns)

Lines 189-191: The ComCat applications did not generate higher yields in comparison to control.

Due to heavy rainfall events at Materi, one of the experiments just had results at 28 DAS which is too early to look for differences. There is a need to have 2 equal experiments repeated in time or place.

Paragraph (206-212) and figure 5: Need to explain what are experiment A and B. I Know now each one is a different study (2 replicate studies). Need to edit the graphics in figure 5 equally and with the same Y axis scale in order to better compare both experiments. Why was the effect stronger (lines: 206-207) in one experiment compared to the other?

Figure 6: Why there was so much maize growth in replicate A in comparison to B? Why there was so much weed competition in replicate B? Need to explain if both replicate experiments were done differently in terms of management or under different greenhouse climatic conditions.

Figure 7: I would use the same scale for the Y axis in both graphs: maximum 35 in both graphs.

Figure 8: Please use a bigger letter size in the graphs.

 Discussion

Lines 254-255: Authors say that the hypothesis that early post-emergence applications of ComCat increased maize biomass under drought, nutrient deficiency and weed competition has to be rejected. I think they should add “in the present experimental conditions”, as perhaps another dose, biomass harvesting time, etc… would have changed the situation.

Lines 260 to 266: This idea is neither well linked to the previous paragraph nor the present research, as the authors did not conduct any physiological determination.

Paragraph 272-287: Not clear discussion on the fact the biostimulant detoxified the herbicide. Why it just happened once? Were both experiments equally conducted?

Paragraph 288 to 300: I think the objective of this research was not the beneficial effect of mixing a legume crop with another one in terms of nitrogen fixing. There is no need to discuss this issue.

Paragraph 301-309: I think the biostimulant was applied in general at the same time as the stress but not after stress.

Conclusions:

They are too long.

Conclusions of this manuscript are: None of the hypothesis have been demonstrated. There was just one positive effect of ComCat when applied on barley seeds before an herbicide stress induction but it could not be replicated.

The manuscript has a lot of typography mistakes:

 Line 3: Need to wright ComCat®? Or is ComCat without the ® OK?

Line 4: Different letter sizes

Line 25 and 95: change “barely” for “barley”.

Line 45: Change fertilizer for fertilizers

Line 53: Please add the scientific name of Maize

Line 55 and others: Change Mung for mung

Line 64: Please add the author letter in the scientific name of Lychnis viscaria

Line 118: missing “a” in “Germany”

Line 141: Please all weeds in the same paragraph.

Line 157 and 165: Please express the herbicide dose in g/ha not in l/ha (if required by the Journal).

Line 196: change NKP for NPK

Line 202: change “within a graph” by “above columns”

Line 215: Please change g/pot by g pot^-1, check other similar mistakes.

Line 224: delete the dot after “B”

Line 225: add “g” after “25”

Line 228: change “und” for “and”.

Line 236: add a comma after 2020.

Line 258: I don´t understand the word “to”, do the authors mean “two”?

Line 259: Please add the scientific names of both legume species.

Line 260: Change “this” for “that”

Author Response

Dear reviewer,

thanks for your valuable comments and suggestions to our manuscript.

We have revised the manuscript according to the comments and suggestions of all reviewers. Please for enclosed our revised manuscript and the point-by-point response to your points.

Reviewer 2:

This research is composed by 8 different studies dealing with 4 different stresses (drought, nutritional, weed competition and herbicide injury), on 2 crops (maize and barley), 2 situations (Field and greenhouse) and 2 locations (Benin and Germany). There are too many factors involved for such a low number of experiments as half of them are replicates, only 4 experiments in total. The only common factor is ComCat. I think, authors should have focus on one stress and one crop, and then perform greenhouse plus field experiments. The present manuscript is a mixture of non-linked experiments.

This is true. But we clarified it in the text.

Abstract:

When reading the abstract, one have the feeling that both crops have been tested under 4 stresses and as both, seed and leaf treatment (Lines 20-22) and this is not true. The same for yield determination (it was just determined in one field in Benin) (Lines:23-24) not in all conducted experiments.

This is true. We clarified in the abstract.

In line 24-25 authors say “ComCat did not promote maize and barley biomass production of the unstressed plants”. Afterwards in lines 29-29, the same  idea is expressed (perhaps a more generalist approach). I think authors should have joined these 2 ideas in just one sentence.

 We deleted the second statement.

Keywords:

There are too many keywords. Some of them (novel secondary pathways or plant strengthener) would have been useful if there have would been a good discussion in the introduction or discussion sections, but this was not the case.

We agree and reduced it.

Introduction:

Line 46: I don´t think biostimulants are inexpensive products. They would be inexpensive if they could replace fertilizers and/or pesticides but in my opinion, they can´t substitute them, perhaps they can reduce them. But they are normally expensive products above all if their effect is less evident to occur as the authors mention.

 We deleted inexpensive and share your opinion.

Line 49-51: Again, I don´t agree with the idea of complete mineral fertilization replacing by biostimulants and I don´t see the link to the mung bean use.

 We agree and modified the text accordingly.

Line 70: Authors should report those numerous reports of positive impact of ComCat, unless they are anecdotal and not scientific, in that case (anecdotal) you shouldn´t mention it as this is a scientific publication.

In which crops have ComCat been useful during its 20-year life-span? Is there any scientific ComCat report on Maize or Barley? If so, please report them and discuss.

 We agree and deleted the speculative effects.

Material and methods:

Line 94: The authors don´t explain that the experiments were replicated, I had to guess it as I didn´t understand letters “A” and “B” in the graphics. Need to explain it at the beginning of this section.

 We clarified this.

Which was the maize cultivar in the Benin experiment?

Type of soil in Benin? Soil analysis?

Missing Nozzle and pressure in Benin as it has been reported for Germany experiments like soil or maize cultivar

Line 107: Need to explain the acronym DAS (I guess: Days after sowing)

 Experimental details are included.

Line 110: I don´t understand why the Materi plot was weeded at 45 DAS if it was destroyed by a storm. If it was not flooded before 45 DAS, why don´t authors report fresh biomass at 42 DAS?

Yes, this is a mistake. This is only true for Banikoara We corrected this.

Line 134: Figure 1 is not interesting to be reported for the manuscript as it is only showing the effect of drought which is obvious. I can´t see Figure 1 written in the text.

 Figure 1 is included to demonstrate the extend of drought stress.

Line 143: Phenological stage of weeds on the transplant day? Perhaps is the one shown in figure 2? if so, please indicate.

 Growth stage is included.

I guess ComCat was also applied on the weeds, therefore you are also improving their growth. Discuss this. I would have applied ComCat as a maize seed treatment.

This is a good point. We agree, but we did not know before the experiments 4 A and B.

Perhaps, 43 days after sowing is not enough to see effects on weed competition.

 No, weed competition was visible 43 DAS.

From line 158 to 169 everything is very confusing (The third factor…). If you speak about factors, why don´t you conduct multifactorial ANOVAs?

 Yes, we did multifactorial ANOVA in this experiment.

The same for Barley: is 21-27 DAS enough time to see weed competition?

 There were no weeds in this experiment.

Results

General: Authors should have edited all graphics equally in terms of letter size, or statistical symbols reporting (in one graphic (Figure 3) you say “ns” and in others you put the same letter in all columns which is ns)

We agree and changed the graphs accordingly.

Lines 189-191: The ComCat applications did not generate higher yields in comparison to control.

 We agree.

Due to heavy rainfall events at Materi, one of the experiments just had results at 28 DAS which is too early to look for differences. There is a need to have 2 equal experiments repeated in time or place.

We cannot repeat the experiment again.

Paragraph (206-212) and figure 5: Need to explain what are experiment A and B. I Know now each one is a different study (2 replicate studies). Need to edit the graphics in figure 5 equally and with the same Y axis scale in order to better compare both experiments. Why was the effect stronger (lines: 206-207) in one experiment compared to the other?

Figure 6: Why there was so much maize growth in replicate A in comparison to B? Why there was so much weed competition in replicate B? Need to explain if both replicate experiments were done differently in terms of management or under different greenhouse climatic conditions.

Figure 7: I would use the same scale for the Y axis in both graphs: maximum 35 in both graphs.

Figure 8: Please use a bigger letter size in the graphs. We canot explain the differences between 5A and B and 7A and B. Maybe, plants reacted differently on transplanting.

 All graphs have been rescaled.

Discussion

Lines 254-255: Authors say that the hypothesis that early post-emergence applications of ComCat increased maize biomass under drought, nutrient deficiency and weed competition has to be rejected. I think they should add “in the present experimental conditions”, as perhaps another dose, biomass harvesting time, etc… would have changed the situation.

 We agree and modified the text accordingly.

Lines 260 to 266: This idea is neither well linked to the previous paragraph nor the present research, as the authors did not conduct any physiological determination.

 We made a link to this paragraph.

Paragraph 272-287: Not clear discussion on the fact the biostimulant detoxified the herbicide. Why it just happened once? Were both experiments equally conducted?

Paragraph 288 to 300: I think the objective of this research was not the beneficial effect of mixing a legume crop with another one in terms of nitrogen fixing. There is no need to discuss this issue.

 We thought it is worth mentioning that there is also an alternative to mineral fertilizers and biostimulants.

Paragraph 301-309: I think the biostimulant was applied in general at the same time as the stress but not after stress.

 We agree and modified the sentence.

Conclusions:

They are too long.

Conclusions of this manuscript are: None of the hypothesis have been demonstrated. There was just one positive effect of ComCat when applied on barley seeds before an herbicide stress induction but it could not be replicated.

 We agree and changed the conclusion accordingly.

The manuscript has a lot of typography mistakes:

Line 3: Need to wright ComCat®? Or is ComCat without the ® OK?

Line 4: Different letter sizes

Line 25 and 95: change “barely” for “barley”.

Line 45: Change fertilizer for fertilizers

Line 53: Please add the scientific name of Maize

Line 55 and others: Change Mung for mung

Line 64: Please add the author letter in the scientific name of Lychnis viscaria

Line 118: missing “a” in “Germany”

Line 141: Please all weeds in the same paragraph.

Line 157 and 165: Please express the herbicide dose in g/ha not in l/ha (if required by the Journal).

Line 196: change NKP for NPK

Line 202: change “within a graph” by “above columns”

Line 215: Please change g/pot by g pot^-1, check other similar mistakes.

Line 224: delete the dot after “B”

Line 225: add “g” after “25”

Line 228: change “und” for “and”.

Line 236: add a comma after 2020.

Line 258: I don´t understand the word “to”, do the authors mean “two”?

Line 259: Please add the scientific names of both legume species.

Line 260: Change “this” for “that”

We made all corrections.

Reviewer 3 Report

The researchers are presenting data which should warn the regulatory bodies in a country before the product is registered.  It appears it is free-for-all for companies with good sales team, even though product is not much field-evidence of efficacy. 

Were the experiments repeated with same protocols? Could the trial have been repeated in another year or site?

Row13: Many products are being introduced and marketed as bio-stimulants, with clear evidence for benefit to the crop production in field.

Row 25: exposed to stress

Row 134, Fig1.  Must also include photo of ComCat treated pot plants under similar conditions of watering/irrigation.

Fig 5 and others:  The fonts are quite small to read clearly. 

Row 242: What is different between Exp A and B?
Why inconsistent result for herbicide response?

Row 304: The product was applied as seed treatment also, but that did not show an impact.  Were the recommendations of the label followed for the treatments?

Author Response

Dear reviewer,

thanks for your valuable comments and suggestions to our manuscript.

We have revised the manuscript according to the comments and suggestions of all reviewers. Please for enclosed our revised manuscript and the point-by-point response to your points.

Reviewer 3:

The researchers are presenting data which should warn the regulatory bodies in a country before the product is registered.  It appears it is free-for-all for companies with good sales team, even though product is not much field-evidence of efficacy. 

Were the experiments repeated with same protocols? Could the trial have been repeated in another year or site?

Yes, the experiments were repeated with same protocols. We stated that in the text.

Row13: Many products are being introduced and marketed as bio-stimulants, with clear evidence for benefit to the crop production in field.

We modified our sentence.

Row 25: exposed to stress

Thanks, we corrected this sentence.

Row 134, Fig1.  Must also include photo of ComCat treated pot plants under similar conditions of watering/irrigation.

ComCat had no effect.

Fig 5 and others:  The fonts are quite small to read clearly. 

We revised the figures.

Row 242: What is different between Exp A and B?
Why inconsistent result for herbicide response?

We explained in the text.

Row 304: The product was applied as seed treatment also, but that did not show an impact.  Were the recommendations of the label followed for the treatments?

It was the first time used as seed treatment. We had good experience with seed treatment of a safener in a different study.

Reviewer 4 Report

To add coordinate of each experimental site.

It is necessary to specify the number, materials and methods of each experiment. Review the Figures (A and B).

With the aim of to get better conclutions, physiological variables must be measured.

It is necessary to follow the rules of the Journal for each bibliographic reference.

Author Response

Dear reviewer,

thanks for your valuable comments and suggestions to our manuscript.

We have revised the manuscript according to the comments and suggestions of all reviewers. Please for enclosed our revised manuscript and the point-by-point response to your points.

Reviewer 4:

o add coordinate of each experimental site.

We included the coordinates.

It is necessary to specify the number, materials and methods of each experiment. Review the Figures (A and B).

This was clarified in the text.

With the aim of to get better conclutions, physiological variables must be measured.

We expected differences in biomass production. Physiological studies were not intended in this study. But, that is planned to be done in a different project.

It is necessary to follow the rules of the Journal for each bibliographic reference.

We checked the rules.

Round 2

Reviewer 2 Report

Authors have made a great effort to improve the manuscript, however, I still believe this is not appropriate for a Q1 journal. This is a mixture of experiments without no clear connection, too much types of stressses. The discussion is partially based on several findings made on other authors´ work not correlated with the present experimentation and conclusions are not clear.

Material and Methods

Spraying pressure and type of nozzles are still missing.

Line 116: I don´t think Figure 1 is useful and above all, don´t call figure one when talking about testing factors as figure 1 should go in the results section.

Results

Lines 186 and 191: Figures 3 and 4 are surrounded by a black frame but the other figures not, please homogenize format.

Line 197: Drought did not reduce dry biomass in the B experiment.

Lines 199-200: There wasn´t a biomass increase with ComCat (in well-watered plants)

Discussion

Lines 256-265: Authors are discussing several findings from literature that are not under experimentation in the present research.

Lines 280 to 293: In my opinion most of it should go to the introduction section.

Line 294: If there is sufficient evidence, please report.

Conclusion

Lines 307-309: this is not a conclusion of the experiment

Minor mistakes

Lines: 38, 188, 230, and 257 and reference list: change red color into black

Line 41: Join the numbers with the percentage symbol: 13-94%

Line 65: delete the underline style

Lines 89-92: Equal the interspacing between lines with the rest of the text.

Line 107: explain here the acronym DAS, not in the line109.

Line 251: Write Barley instead of Barely.

Line 245: Separate “ha^-1at” into “ha^-1 at”

Author Response

Dear reviewer,

we apologize that we missed your comments in the R2-version of the mansucript. We have follwoed all your comments in the R3 version of the mansucript.

Reviewer 4 Report

Follow the recomendations of the document.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

thanks for your corrections.

We made all corrections in the manuscript attached.

Two suggested corrections were not included:

1. Scientific name for mung bean has been mentioned earlier in the text.
2. Two brackets seem correct.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.