Composts Obtained by Mixing Hop Leaves with Wheat Straw or Farmyard Manure Improved Soil Properties and Increased Microbial Communities

Round 1

Reviewer 1 Report

The article presents useful information about effects of organic fertilization on soil properties. The manuscript is well structured and written. Some suggestions of improvements are described as follows.

Line 99. The process of composting should be described further since it could affect the compost characteristics. The origin and some characteristics of the raw materials used in composts must also be presented.

Line 104. I suggest to explicit that the treatments were applied only before the first growing cycles (I could get it only by reading line 392).

Line 106-108. I could not follow “all the composts were used (…) double rates (D2)”, since the composts used at double rate do not have “the lowest C/N ratios”.

Line 108. More details on the soil used as substrate are recommendable (texture, horizon). It seems a soil with low levels of fertility and organic matter.

Line 109. I am not familiar with the units P2O5 and K2O for presenting soil analysis.

Line 112. The C/N ratios in Table 1 are from the final composts? How they were measured?

Line 199. I think that it was not “double rate of leaves”.

Line 324. I suggest “abundance of fungi and bacteria genus”.

Line 363. By Figure 2, it is supposed that the results of diversity indexes were submitted to analysis of variance. It seems an unusual procedure, that is not described in methodology. The index of each pot was considered as replication?

Line 372. This paragraph could be split.

Lines 406-417 describe again the results and could be synthesized, as well in other parts of Discussion.

Author Response

Manuscript ID: horticulturae-2740290

Manuscript Title: Composts obtained by mixing hop leaves with wheat straw or farmyard manure improved soil properties and increased microbial communities

Reviewer #1 report:

The article presents useful information about effects of organic fertilization on soil properties. The manuscript is well structured and written. Some suggestions of improvements are described as follows.

Response: We are grateful for reviewer positive comments and revisions provided that contributed to improve the manuscript. Our responses have been included in blue in this cover letter. Changes in the manuscript were marked in red.

Line 99. The process of composting should be described further since it could affect the compost characteristics. The origin and some characteristics of the raw materials used in composts must also be presented.

Response: In agreement with the suggestion we added more information on the composting process and raw materials (see lines 94-104).

Line 104. I suggest to explicit that the treatments were applied only before the first growing cycles (I could get it only by reading line 392).

Response: Thank for the suggestion, we recognize the information was not explicit. Thus we have added a sentence to clarified it (see lines 121-123).

Line 106-108. I could not follow “all the composts were used (…) double rates (D2)”, since the composts used at double rate do not have “the lowest C/N ratios”.

Response: We recognize that the information was not explicit. What we meant to state was that the composts with the lowest and highest C/N ratios were selected to be applied at double rates (D2), in addition to being applied at single rates (D1). The sentence was revised to improve clarity (see lines 116-117).

Line 108. More details on the soil used as substrate are recommendable (texture, horizon). It seems a soil with low levels of fertility and organic matter.

Response: More details on soil sampling and analysis were provided (see lines 107-112). Soils of this region, usually have low levels of organic matter.

Line 109. I am not familiar with the units P2O5 and K2O for presenting soil analysis.

Response: The laboratories that use these analytical methods all express phosphorus and potassium in the same way, which coincides with how they are expressed in commercial fertilizers.

Line 112. The C/N ratios in Table 1 are from the final composts? How they were measured?

Response: The C/N ratios in Table 1 are indeed from the final composts, and analysis were performed to determinate the content (%) of total organic C (Walkley–Black method) and N (Kjeldahl) in all the samples collected for each compost (3 replicates), to determine the C/N ratio. We recognize the information is lacking and have added a sentence (see lines 114-116).

Line 199. I think that it was not “double rate of leaves”.

Response: We recognized that the idea was not expressed correctly, thus we revised the sentence to “….which was also reflected in the treatment applied at a double rate” (lines 215-216).

Line 324. I suggest “abundance of fungi and bacteria genus”.

Response: In agreement with the suggestion we revised the sentence (lines 343).

Line 363. By Figure 2, it is supposed that the results of diversity indexes were submitted to analysis of variance. It seems an unusual procedure, that is not described in methodology. The index of each pot was considered as replication?

Response: To clarify your doubt, we confirmed that diversity indexes were submitted to analysis of variance, but as you adequately point out, it was not described in the methodology. Hence, we added the lacking information (see lines 207–208). Regarding the question about the procedure used, in this study, samples were collected in triplicate for each treatment, and the abundance of bacteria and fungi was determined in all samples; therefore, the data obtained was used to calculate the diversity indexes.

Line 372. This paragraph could be split.

Response: In agreement with the suggestion the paragraph was split (lines 391; 407).

Lines 406-417 describe again the results and could be synthesized, as well in other parts of Discussion.

Response: In agreement with the suggestion, the results mentioned were revised and synthesized (see lines 427-432), as well as other parts of Discussion (see lines 438 -443; 517-522; 537-539).

Author Response File: Author Response.pdf

Reviewer 2 Report

In the article entitled: " Composts Obtained by Mixing Hop Leaves with Wheat Straw  or Farmyard Manure Improved Soil Properties and Increased Microbial Communities". The authors investigate the effect of composts obtained from mixtures of hop leaves with other organic materials such as wheat straw, farmyard manure and ash from hop stems at different ratios, on soil properties and microbial diversity.

The manuscript is interesting, relevant and current.

In my opinion recommend the manuscript for publication in Horticulturae, but there are some recommendations below for authors:

In materials and methods, the brand of the reagents used in all analyzes must be indicated and also the brand, model and origin of all the equipment used, and the versions and origin of the software used.

On line 128 indicate the meaning of the abbreviations KEC and KEN

In Table 2, 3 and 4 the authors mention that the means are shown but it is not indicated how many experiments they should also show the deviations.

Author Response

Reviewer #2 report:

In the article entitled: " Composts Obtained by Mixing Hop Leaves with Wheat Straw or Farmyard Manure Improved Soil Properties and Increased Microbial Communities". The authors investigate the effect of composts obtained from mixtures of hop leaves with other organic materials such as wheat straw, farmyard manure and ash from hop stems at different ratios, on soil properties and microbial diversity.

The manuscript is interesting, relevant and current.

In my opinion recommend the manuscript for publication in Horticulturae, but there are some recommendations below for authors:

Response: We are grateful for reviewer positive comments and revisions provided that contributed to improve the manuscript. Our responses have been included in blue in this cover letter. Changes in the manuscript were marked in red.

In materials and methods, the brand of the reagents used in all analyzes must be indicated and also the brand, model and origin of all the equipment used, and the versions and origin of the software used.

Response: The missing information was added (see lines 129-135; and 152-153).

On line 128 indicate the meaning of the abbreviations KEC and KEN

Response: In agreement with the suggestion, the meaning was described (see lines 142-143).

In Table 2, 3 and 4 the authors mention that the means are shown but it is not indicated how many experiments they should also show the deviations.

Response: We acknowledge the reviewer's concern. However, with respect to Tables 2 and 3, since the standard error has already been incorporated, it appears unnecessary to include deviations from our perspective. Regarding Table 4, which presents Spearman correlation coefficients, standard deviation does not apply in this context.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments for author File: Comments.pdf

Author Response

Reviewer #3 report:

Response: We are grateful for the revisions provided that contributed to improve the manuscript. Our responses have been included in blue in this cover letter. Changes in the manuscript were marked in red.

L 108 - The author gives values (pH) without indicating how these characteristics are measured.

Response: In agreement with your recommendation we added the missing information for pH, organic C, total N, and extractable P and K (lines 110-112).

L 122 - Microbial analysis was performed in triplicate for each treatment. For greater study weight, the number of repetitions would need to be increased.

Response: In agreement we recognize that a number of repetitions higher than what was used would increase study weight and consistency of results, which we should take in consideration for further research.

L122 - It would be good to think about the weight of individual indicators. For instance, the amount of biomass indicates the entire mass of organisms (including dead ones), it would be good to include this information only as an additional one and not the first in microbial analyses. On the contrary, little attention is paid to the important indicator of soil basal respiration.

Response: We appreciate the reviewer’s pertinent suggestion and recognize the relevance of soil basal respiration, which unfortunately did not receive significant attention in this study. The reason for this was that the values obtained for this indicator did not varied significantly between treatments and no significant correlation with microbial communities were found. Thus, the lack of variability and correlation did not allow for deeper exploration of its relevance in the context of this specific study.

L 134 - The information to which point the sample was titrated is missing.

Response: In agreement, we added the missing information (lines 148-149).

L 272; Figure 1a) and b) - Since many results have been achieved, it would be useful to make the figures clearer (better color resolution).

Response: In agreement, we have made changes in the figures to improve color resolution, making figures clearer (see Figure 1 at lines 289-290).

L 281 - It would be also appropriate to improve the verbal description. Not simplify it to the mere list of microbial genera and not to repeat the results shown in the graphical representation.

Response: In agreement, we improved the verbal description (see lines 296 – 310).

L556-557 - The study was conducted within the time frame of two growing cycles, which is a short time to draw clear conclusions.

Response: We recognize the limitation of a short-term pot experiment to draw clear conclusions, and thus we removed the sentence in the conclusion section and added a sentence at the end of the discussion to clarify that the results are preliminary, and that further research is needed to confirm the findings (lines 559-563).

Author Response File: Author Response.pdf

Reviewer 4 Report

Specified comments are attached in a pdf file.

Comments for author File: Comments.pdf

Author Response

Reviewer #4 report:

An excellent introduction but there are some awkward phrases in some of the sentences.

Response: We are grateful for reviewer positive comments and revisions provided that contributed to improve the manuscript. Our responses have been included in blue in this cover letter. Changes in the manuscript were marked in red.

L 39 “..times.”

Response: In agreement with your recommendation the phrase was reviewed to “The reconversion and valorization of agricultural waste present significant challenges at the present time.” (lines 39- 40).

L 47 “..from brewing industry.”

Response: In agreement with your recommendation the phrase was reviewed to “…from the brewing industry…” (line 46-47).

L 49 “.. process which allows to manage..”

Response: In agreement with your recommendation the phrase was reviewed to “…is a process that allows the management of agricultural waste…” (line 48).

L 64 “trough”

Response: In agreement with your recommendation the phrase was reviewed to “The decomposition of complex organic compounds through microorganisms…” (line 60-61)

L 70 “..at which occurs..”

Response: In agreement with your recommendation the phrase was reviewed to “…at which this occurs…” (line 66).

L 82 ‘… are able to growth..”

Response: In agreement with your recommendation the phrase was reviewed to “…are able to grow …” (line 76).

Experimental trials – I would like to know more about the pot study. How large were the pots, how

much media was put in them, where did the soil come from, what was its texture, how were the

composts mixed with the soil before the mixture was put into the pots and how frequently were the

pots watered. I assume this experiment was conducted in a greenhouse but that is not mentioned. Also,

soils in the second cycle were kept together by pot, ending up being the same soil as from the first cycle but no new additions of composts were made.

We appreciate the reviewer’s interest in the issue and, in response, provide the following information. We used pots with an approximately 3 L in volume, which were filled leaving 1 cm on top to allow for watering. Each pot was filled with 3 kg of dry and sieved soil (2 mm), sampled from the 0-0.20 m layer of a fallow plot. The initial soil had a sandy clay loam texture (soil separates, 23.9% clay, 21.8% silt and 54.3% sand). To create the experimental mixture, the composts were previously mixed with the soil. Regarding the watering of pots, as the reviewer is aware, there are no specific rules, simply visible management. Depending on the time of year and ambient temperature, and the phenological stage of the lettuces, their evapotranspiration varies greatly. On the other hand, lettuces subjected to different treatments have different development and therefore different water needs. Thus, the pots were regularly watered to maintain optimal moisture, and the amount of water applied varied over time and between pots. The experiment was conducted outdoors under a tile roof. The soils in the second cycle were kept together by pot, being the same soil as from the first cycle, with no new additions of composts.

More details on the pot experiment have been included in line with the reviewer’s questions (see lines 107-123).

Soil analysis – how large were the samples that were collected from each pot? Did freezing of the soils used for microbial analysis have any impact on the populations of microbes?

Response: The soil of each pot was properly homogenized, and a 200 g subsample taken for the analyses. Regarding the freezing of soil, it is a common practice to prevent or minimize potential changes in microbial activity or composition, though some alterations may occur over the time. Therefore, microbial analyses were prioritized and promptly conducted after the collection of soils. 

Results section – Very detailed and well-presented results. I am not a microbiologist but have a strong

soils background and was intrigued with the opportunity to learn something by reviewing this paper. As mentioned I found the results very detailed and overwhelming but well presented. The bottom line

seems to suggest that compost plus straw provided the highest populations of microbes which results in high rates of decomposition of organic matter thus increasing N availability to the plants.

The authors do a good job of explaining the differences in populations of the various microbes based on the state of decomposition of the OM in the soil. One question I would have is how the treatments

were applied to the pots – were they actually mixed with the soil and then put in the posts or were they applied to the top of the soil after the soil had been put in the plots. From a practical standpoint would the later not be the primary way that OM would be added in an agricultural field setting?

Response: Thank you for your positive and pertinent comments. About the application of treatment to the pots, they were mixed with the soil and then put in the pots. As you adequately point out, the application of treatments to the top of the soil would not be the primary way of adding OM in agricultural fields, and the method was chosen taking that into account.

Overall question – is lettuce produced commercially in pots and if not how applicable are the results of a pot study to the field. As requested earlier, was the pot study conducted outside or in a greenhouse and what was the size of the pots used, how frequently were they irrigated, etc. How do the results of the pot study relate to what one my find in a field study. The final sentence of the paper suggests that this study “underscores the importance of organic compost to promote soil health and productivity.”

Depending on how the OM was added in this study – top of pot or mixed with soil before putting it into the pot – may have an effect on the statement. It would be interesting in the closing parts of the

discussion for the authors to relate their pot study results to field results that may be available or to

what they surmise might happen in the field with similar additions of OM.

Response: The question is very pertinent, and we recognize that experiments with lettuce in pots has limitations into the conclusion we may take in relation to what one my find in a field, which aligns with the purpose of in this study. In accordance with your suggestion, we have revised the final part of the discussion to include the relationship between the results obtained from the pot experiment and those anticipated under field conditions (see lines 556 – 563).

We also revised the final sentence of the paper to “… underscore the importance of organic compost to promote soil health and crop productivity, suggesting the potential applicability of hop-leaves based compost in agricultural fields.” (see lines 591 - 593).

Overall an interesting and well-presented paper. The paper needs a careful review of English.

Response: Thank you for the positive comments. We have addressed the language issues in the paper, but as the changes were minor and spread throughout the document, they are not marked.

Author Response File: Author Response.pdf