The Phenolic Composition of Hops (Humulus lupulus L.) Was Highly Influenced by Cultivar and Year and Little by Soil Liming or Foliar Spray Rich in Nutrients or Algae

Round 1

Reviewer 1 Report

Dear authors, please see report in attached document.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer

You show a very good knowledge of the crop and will know that harvesting the cones is very time-consuming. It seems inhumane that you consider that more plants should be used to improve the statistics, 6 plants were used as replicates, even though we all recognize that the more plants sampled, the better.

About the critic regarding the field installation 20 years ago with rhizomes. Just to clarified: This study did not take place on an experimental station, this is a commercial field (on-farm research), and all commercial fields were installed with rhizomes. And, as you may well know, homogeneity in the field is achieved by annual pruning and by putting up an adequate number of shoots.

Moreover, when you refer the contamination by diseases and viruses (Pseudoperonospora humuli or Downy mildew of hop) we can also point out the possibility of Apple Mosaic Virus infection. However, all plants, especially perennials, are somehow contaminated with viruses, diseases and pests. We have to believe that sampling multiple plants incorporates this variability. This crop is not different from any other. Even if twenty years ago the farmer had used plants free from viruses and diseases, what guarantee would we have that the crop remained completely healthy?

We truly respect your opinion, but we proceed to a profound restructuring of the manuscript as the others reviewers suggested. Hope you can take a second look to the manuscript and, somehow, change your opinion.

Reviewer 2 Report

The present study is a systematic essay of the effect of several agronomical practices or parameters on the phenolic composition of hop cones. In general, the study is interesting to a specialized audience but not to the average reader.  The main problem is that the experimental design is complex and thus difficult to follow. However, there are some strong points and I would recommend the authors to address some comments and corrections to improve the manuscript.

1) An attempt should be made to improve the description of the experimental design, although some good effort has already been done (figure 2).

2) There is a problem with the plant vigor issue. Although it is stated as a problem in hops cultivation, which, normally, the study could help to resolve, no such reference is included in the discussion section. Therefore, readers keep wondering if some evidence came up from the study concerning the cause of low plant vigor or not.

3) Some parts of the Materials and Methods and some parts of the Discussion sections should be rearranged between sections of the manuscript. I have included specific comments in the attached pdf.

4) I wonder if including a graphical representation of the PCA could help the readers identify important factors affecting phenolic composition of hop cones.

5) Some minor corrections also appear in the attached pdf file.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer #2

We would like to thank your competent revision on this manuscript. Our responses were included in blue in this cover letter. Changes in the manuscript were marked in red. In the cover letter we specify all the lines where changes were made.

In addition, if any aspect of the manuscript was not to your liking, we will quickly make further corrections.

The present study is a systematic essay of the effect of several agronomical practices or parameters on the phenolic composition of hop cones. In general, the study is interesting to a specialized audience but not to the average reader.  The main problem is that the experimental design is complex and thus difficult to follow. However, there are some strong points and I would recommend the authors to address some comments and corrections to improve the manuscript.

To increase the available data on hop phenolic composition, we decided to add data from 4 trials. Of course it could be a little more confusing than a conventional one-single experiment, but we think it's worth it.

1) An attempt should be made to improve the description of the experimental design, although some good effort has already been done (figure 2).

We have separated the experiments into different paragraphs so that the reader can understand them better. We also have clarified better the experimental designs (lines 137-178).

2) There is a problem with the plant vigor issue. Although it is stated as a problem in hops cultivation, which, normally, the study could help to resolve, no such reference is included in the discussion section. Therefore, readers keep wondering if some evidence came up from the study concerning the cause of low plant vigor or not.

We add a new sentence in discussion section on this topic (lines 390-393).

3) Some parts of the Materials and Methods and some parts of the Discussion sections should be rearranged between sections of the manuscript. I have included specific comments in the attached pdf.

The specific comments were taken into account and several improvements in the manuscript were made. See lines 17, 23, 76-77, 91-92, 113, Table 1, 188, 209, 312, Table 4, 333, 374, 434-439, 497, 537-540.

Only one note was not fully addressed, which consisted in sending a large paragraph with many references from discussion two results section. Despite the high number of citations, this text seems very important to us in discussion section.

4) I wonder if including a graphical representation of the PCA could help the readers identify important factors affecting phenolic composition of hop cones.

This is a very long manuscript with six tables and seven figures. We feel that making the manuscript longer may not be better for readers. But if you think it's mandatory we can do it.

5) Some minor corrections also appear in the attached pdf file.

Please, see our response to point 3)

Reviewer 3 Report

Authors of the manuscript presented four combinations of field trials and the influence of different factors (year, algae- and nutrient- rich foliar sprays, liming and cultivars) on the content of polyphenols in Humulus lupulus L. The field trials were performed in years 2016-2018 in north-eastern Portugal.

It is really interesting work, the experiments were well designed and performed.

I have only a few comments:

line 102: “figure 1” should be with capital letter “F”

line 112: “table 1” should be also with capital letter “T”

line 177: “Figure 2.” should be bolded

2.5. HPLC analysis – what kind of eluents were used ? What was the HPLC analysis program?

Author Response

Dear Reviewer 3

We would like to thank your competent revision and the positive comments. Our responses were included in blue in this cover letter. Changes in the manuscript were marked in red. In the cover letter we specify all the lines where changes were made.

In addition, if any aspect of the manuscript was not to your liking, we will quickly make further corrections.

Reviewer 3 report:

General comments:

Authors of the manuscript presented four combinations of field trials and the influence of different factors (year, algae- and nutrient- rich foliar sprays, liming and cultivars) on the content of polyphenols in Humulus lupulus L. The field trials were performed in years 2016-2018 in north-eastern Portugal.

It is really interesting work, the experiments were well designed and performed.

I have only a few comments:

Thank you for your nice comments.

line 102: “figure 1” should be with capital letter “F”

The correction was made (line 106).

line 112: “table 1” should be also with capital letter “T”

The correction was made (line 117).

line 177: “Figure 2.” should be bolded

The correction was made (line 183).

2.5. HPLC analysis – what kind of eluents were used? What was the HPLC analysis program?

The solvents used for phenolic compounds analysis were: (A) 0.1% formic acid in water, (B) acetonitrile. The elution gradient established was isocratic 15% B (5 min), 15% B to 20% B (5 min), 20–25% B (10 min), 25–35% B (10 min), 35–50% B (10 min), and re-equilibration of the column, using a flow rate of 0.5 mL/min. Data acquisition was carried out with Xcalibur® data system (ThermoFinnigan, San Jose, CA, USA).

This information is described in the reference Nº 28: Bessada, S.M.F.; Barreira, J.C.M.; Barros, L.; Ferreira, I.C.F.R.; Oliveira, M.B.P.P. Phenolic profile and antioxidant activity of Coleostephus myconis (L.) Rchb.f.: An underexploited and highly disseminated species. Ind Crop Prod 2016, 89, 45-51, doi:10.1016/j.indcrop.2016.04.065.

The decision not to put all this information completely described in the manuscript was to avoid plagiarism situations. We have a vast curriculum of manuscripts using this technique (article with more than 200 citations) and therefore the information described has to be carefully chosen.

Reviewer 4 Report

Comments for author File: Comments.pdf

Author Response

Dear Reviewer #4

We would like to thank you the competent review of this manuscript. Our responses were included in blue in this cover letter. Changes in the manuscript were marked in red. In the cover letter we specify all the lines where changes were made.

In addition, if any aspect of the manuscript was not to your liking, we will quickly make further corrections.

_ Line 17: In abstract should be mentioned, that investigation was made on 20 years old plantation.

This data was provided (line 20).

_ Line 19: We advise: Adaptation to local growing conditions.

Ok, done (line 19).

_ Line 30: It should be added, that total content is related to variety and add the answer if there are any differences among varieties in composition of polyphenols.

A new sentence was added (lines 32-33).

_ Line 61: Where greater knowledge should be applied (in Portugal)?

Yes, we added Portuguese (line 63)

_ Line 64: Reference about fresh edible shoots could be added for Slovenia: (VIDMAR, Mateja, ABRAM, Veronika, ČEH, Barbara, DEMŠAR, Lea, POKLAR ULRIH, Nataša. White hop shoot production in Slovenia : total phenolics, microelements and pesticides content from five commercial cultivars. Food technology and biotechnology : journal of the Faculty of Food Technology and Biotechnology University of Zagreb. 2019, vol. 57, no. 4, str. 525-534. ISSN 1330-9862. DOI: 10.17113/ftb.57.04.19.6269.)

The reference was added with number 14 (lines 67, 574-575)

_ Line 105: The size of plots is missing.

Data was provided (lines 108-109)

_ Lines 113-116: Numbers in Table 1 are not readable as they are too close together. Explain the description of the vigour classes.

Changes were made in Table 1. The people managing final formulation usually made improvements. We think data on vigour classes lies in lines 142-144.

_ Line 119: Which variety of hop was grown? Maybe you could add this data to Table 1, which variety was in each plot.

Data was provided in lines 148, 164-165, 170, 174-175

_ Line 126: A graph for irrigated water would be needed.

This manuscript already has six tables and seven figures. These are commercial fields where irrigation water is not quantified, neither the number of irrigation events nor the amount of water in each irrigation event. The figure would have poor quality information, and would unnecessarily increase the size of the manuscript.

_ Lines 129-130: In which growth stages the fertilization was performed?

Data was provided (lines 131-135)

_ Line 131: Put experiment data into table, for each experiment we need clear explanation what was happening (which plot, variety, how many replications, treatments, how many samples at sampling…) Like this it is a bit confused; we do not know what was happening where.

The text has been rearranged into different paragraphs at the request of another reviewer (We are trying to manage the requests of five reviewers). On the other hand, there is already a figure showing details of the experiments. We are avoiding over-increasing the number of tables and figures.

_ Line 132: Apparatus is strange word, maybe design? Replace it everywhere in article.

The word apparatus was changed in three situations (lines 135, 177 and 182).

_ Line 147: 20 years old plantation is very old, in hop growing countries we dig hop plantation out after 15 years.

These are commercial fields, most of them producing more than 20000 kg/ha of dry cones. With the exception of the aforementioned plots, in normal fields the plants look very good.

_ Line 232: Results should be presented separately for experiment 1, 2, 3 and 4. Make subtitles and put all results of one experiment under one subtitle.

We have 5 reviewers on this manuscript (You are reviewer #4). We are trying to manage the comments of all. We respect everyone, but the general recommendations for the different sections of the manuscript differ from each other. We cannot make such radical changes to the manuscript because this compromises our ability to respond to other reviewers' suggestions. On the other hand, arranging by experiment we would have to repeat the subjects within each experience, which is not clear to us that would simplify the manuscript.

_ Line 238: Was the variety Nugget? Make clear in Materials.

Data was provided in lines 148, 164-165, 170, 174-175

_ Line 259: Liming was done with quite low amount of limestone. Add in materials, if it was mixed in the soil after application or not. February is late for liming in such dry conditions ...

This is on-farm research. We usually respect farmer practices. They usually use a small amount of lime to avoid antagonism between nutrient or other kind of problems. Lime was incorporated into the soil (line 169).

_ Lines 304-307: The data in Table 4 is not clearly seen, numbers are too close together.

We removed an unnecessary column to create space.

_ Line 309: There is no Table 7.

Yes, table 6 (line 323)

_ Line 310: Include chromatogram of other two varieties as well.

The chromatogram shown in Figure 7 was chosen because it is the most representative of all the samples. As you can see, in the identification and quantification of phenolic compounds, all samples showed the same peaks, so the presentation of other chromatograms would not add any information to the manuscript. If the dear reviewer really wants the presentation of the chromatograms, we can do it as supplementary material.

_ Line 318: Use “plants on limed soil had…” instead of “plants, limed presented…”.

Done (line 332).

_ Line 323-327: Legend for Table 5 is missing. We should know what are the abbreviations in the first column.

Full names and abbreviations were provided.

_ Line 337: Legend for Table 6 for first column is missing.

Full names and abbreviations were provided.

_ Line 340: Discussion would be clearer with more paragraphs. I suggest you to make more paragraphs.

The entire section was divided into much more paragraphs.

_ Line 346: We advise: phenols in cones in plants of different vigour…

Done (line 369).

_ Line 349: We advise: …of hop plants grown in different locations (Slovenia, Austria, CZ).

Done (line 373).

_ Line 360: In references could also include nr. 28. [11,13,28,30-33].

Done (line 384).

_ Line 363: Content of total phenols did not vary significantly between the plots of different plant vigour. At which varieties? On average?

See line 388.

_ Line 364: Which stress affects plant growth and yield? The sentence is not understandable.

See lines 391-393.

_ Line 419: It would be good to add citation that phenols are secondary metabolites that form in such and such conditions.

See lines 347-449.

_ Line 420: Please, reorganize the chapter to more shorter paragraphs, because it is difficult to follow the content this way.

The entire section was divided into much more paragraphs.

_ Line 449: Hop cones from less vigorous plants of which variety?

See line 480.

_ Line 460: We advise: Hop cones of plants on limed soil.

See line 492.

_ Line 477: Most of experimental factors under study- list factors in brackets.

See line 509.

_ Line 478: the measured variables- list them in brackets. ~

See line 511.

_ Line 480-482: Sentence is not understandable.

See line 516.

_ Line 483: We advise: Cascade or Colombus varieties…

See line 517.

_ Line 483: The high levels of Zn are associated with high phenol content? Improve the sentence.

See line 518.

_ Line 488: The plants treated with foliar sprays... (What do foliar sprays contain? List in brackets.)

See line 522.

_ Line 489: We advise: Plants on limed soil were notably higher…

See line 523.

_ Line 495: What would be the main conclusion? What would be recommended to grow to get the most convenient hop cones (which variety, what vigour, what agrotechnic …)?

A main conclusion for the study was provided (lines 529-531)

_ Line 583: Surnames correction in reference 33:

MacKinnon, D.; Pavlovič, V.; Čeh, B.; Naglič, B.; Pavlovič, M. The impact of weather conditions on alpha-acid content in hop (Humulus lupulus L.) cv. Aurora. Plant Soil Environ 2020, 66, 519-525, doi:10.17221/344/2020-PSE.

Done (line 617)

Reviewer 5 Report

The proposed manuscript is acceptable in present form.

Author Response

Dear reviewer, thank you so much for your approval to our manuscript.

Round 2

Reviewer 1 Report

Dear Authors,

Some improvements are visible, but I can see you didn't accept my collegial and in fact, very friendly advice I gave you in my previous report, which I really gave you in my best intentions(!). Nevertheless, it seems to me you didn't read my review report at all, or you didn't read in good intention. However, let us clarify something. 

1. Please, change the word 'plantion' in Abstract, Line 20 page 1 of 18, with the word hop garden, which is in official use. (By the way in corrected version you wrote plantion not plantation!)
2. You still didn't clear whether the hop rhizomes, page 4 of 18, line 124-125. Please, what do you think under the term rhizomes?! Morphologically, rhizomes are the underground parts of hop plant which are growing horizontally in phyllotaxis of 180^o from the underground tree of hop plant on depth of 12-18 cm below the soil surface. The rhizomes are evolutionary adaptation of wild hop to survive the competition of the other plants in spontaneous phytocenosis, because on the end of each rhizome from meristem tissue the new clone of hop plant will be developed from the mother plant.  The rhizomes could be used for propagation but very rarely nowadays, particularly not in commercial hop production(!). So, I am quite sure that some 20 years the farmer, in whose hop garden you set up your field trial, planted the hop seedlings of so called 'B' certificate,  which are the seedlings prepared after the pruning of the 'crown' of last year old underground tree, and the seedlings of 'B' certificate are collected and prepared from transplantation only(!) from hop garden planted with seedlings of 'A' certificate, which is 99 % virus-free (because the seedlings of 'A' certificate are produced by tissue culture from previously examined hop plants on plant viruses and viroids infections). If this is your situation, please, replace the word rhizomes with the word seedlings. 
3. In 31 years of my research career I didn't find any commercial  hop garden planted with any hop cultivar which is not infected with plant viruses, particularly with AMV (Apple Mosaic Virus) and/or some viroids. Thus I am 100 % positive that your hop garden, which is 20 years old (as you mentioned) is also infected. All plant viruses decrease vigor of hop plants and, consequently the yield and quality of hop cones. So, the plant - vigor interaction of hop cultivar Nugget considering the compounds of secondary metabolites, is the most valuable part of your research, but you MUST emphasize that explicitly in the text in chapter 5. Conclusions.

Thank you, your reviewer

Author Response

Dear Reviewer #1

We would like to thank your competent revision on this manuscript. Our responses were included in blue in this cover letter. Changes in the manuscript were marked in red. In the cover letter we specify all the lines where changes were made.

In addition, if any aspect of the manuscript was not to your liking, we will quickly make further corrections.

Best regards

Lillian Barros

Comments and Suggestions for Authors

Dear Authors,

Some improvements are visible, but I can see you didn't accept my collegial and in fact, very friendly advice I gave you in my previous report, which I really gave you in my best intentions(!). Nevertheless, it seems to me you didn't read my review report at all, or you didn't read in good intention. However, let us clarify something. 

Dear reviewer

We know that you made your suggestions with genuine intentions to improve the manuscript, and we greatly appreciate your effort. However, we had to manage the opinion of four more reviewers, who suggested only minor interventions in the manuscript. You must understand that your suggestions implied a profound restructuring of the manuscript and the elimination of a lot of its parts. We ask that you understand that, based on the opinion of other reviewers, we have tried to avoid removing part of the data of the manuscript. 

1. Please, change the word 'plantion' in Abstract, Line 20 page 1 of 18, with the word hop garden, which is in official use. (By the way in corrected version you wrote plantion not plantation!)

Thank you very much, we are not native English speakers so these errors are more frequent. We accept the hop garden option (line 20).

1. You still didn't clear whether the hop rhizomes, page 4 of 18, line 124-125. Please, what do you think under the term rhizomes?! Morphologically, rhizomes are the underground parts of hop plant which are growing horizontally in phyllotaxis of 180^o from the underground tree of hop plant on depth of 12-18 cm below the soil surface. The rhizomes are evolutionary adaptation of wild hop to survive the competition of the other plants in spontaneous phytocenosis, because on the end of each rhizome from meristem tissue the new clone of hop plant will be developed from the mother plant.  The rhizomes could be used for propagation but very rarely nowadays, particularly not in commercial hop production(!). So, I am quite sure that some 20 years the farmer, in whose hop garden you set up your field trial, planted the hop seedlings of so called 'B' certificate,  which are the seedlings prepared after the pruning of the 'crown' of last year old underground tree, and the seedlings of 'B' certificate are collected and prepared from transplantation only(!) from hop garden planted with seedlings of 'A' certificate, which is 99 % virus-free (because the seedlings of 'A' certificate are produced by tissue culture from previously examined hop plants on plant viruses and viroids infections). If this is your situation, please, replace the word rhizomeswith the word seedlings. 

The first information we had from the producers was that at the time rhizomes were used. We went to talk to the farmers again and we concluded that maybe you are right; the fields will have been installed with seedlings (lines 132-134).

1. In 31 years of my research career I didn't find any commercial  hop garden planted with any hop cultivar which is not infected with plant viruses, particularly with AMV (Apple Mosaic Virus) and/or some viroids. Thus I am 100 % positive that your hop garden, which is 20 years old (as you mentioned) is also infected. All plant viruses decrease vigor of hop plants and, consequently the yield and quality of hop cones. So, the plant - vigor interaction of hop cultivar Nugget considering the compounds of secondary metabolites, is the most valuable part of your research, but you MUST emphasize that explicitly in the text in chapter 5. Conclusions.

We add a new sentence trying to include a probable effect of viruses’ contamination of the hop gardens (lines 539-543).

Thank you, your reviewer

Thank you again for your valuable comments.

Reviewer 4 Report

The new sentence in lines 32 and 33 is not understandable.  The quantitatively significant differences among samples were 32 found to be related with the cultivar of the samples studied. Did you mean: Significant differences among cultivars were found in quantity and composition of phenols in hop cones?

Lines 126-127: Add the data when the plots were irrigated and with how much water. It seems rather too dry for hop during summer, so this data would be necessary to understand the plant response. Make a separate paragraph for this important topic. And move it maybe after the weather section.

In Materials and methods we still miss the explanation in 2.1, how you define weak, fair, good and very good plants. I suggest to move the data from the lines 144 to 147 to the section 2.1 under the Table 1, because it is valid for all experiments. What was the share of each this group of plants at each plot is missing (maybe you can add this data in the Table 1).

I suggest a separate paragraph for fertilization, not all text in one long paragraph.

Author Response

Dear Reviewer #4

We would like to thank your competent revision on this manuscript. Our responses were included in blue in this cover letter. Changes in the manuscript were marked in red. In the cover letter we specify all the lines where changes were made.

In addition, if any aspect of the manuscript was not to your liking, we will quickly make further corrections.

Best regards

Lillian Barros

Reviewer #4

Comments and Suggestions for Authors

The new sentence in lines 32 and 33 is not understandable.  The quantitatively significant differences among samples were found to be related with the cultivar of the samples studied. Did you mean: Significant differences among cultivars were found in quantity and composition of phenols in hop cones?

Maybe: Significant differences in quantity and composition of phenolic compounds in hop cones were also found between cultivars (lines 30-21).

Lines 126-127: Add the data when the plots were irrigated and with how much water. It seems rather too dry for hop during summer, so this data would be necessary to understand the plant response. Make a separate paragraph for this important topic. And move it maybe after the weather section.

A new paragraph with irrigation data was added (lines 126-138).

In Materials and methods we still miss the explanation in 2.1, how you define weak, fair, good and very good plants. I suggest to move the data from the lines.

This data was moved to before table 1. (lines 110-118).

What was the share of each this group of plants at each plot is missing (maybe you can add this data in the Table 1).

We do not know if we understood. The areas of the plots were provided (Lines 109-110).

I suggest a separate paragraph for fertilization, not all text in one long paragraph.

We tried to put all the information of experiment 2 in the same paragraph. Now we separate in three paragraphs.