Microbial Community Shifts with Soil Properties and Enzyme Activities in Inter-/Mono-Cropping Systems in Response to Tillage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

see comments to the auhtors in the attached pdf-file

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English could profit from a professional language check.

Author Response

Reviewer#1:

 

Reviewer comments to the manuscript entitled: Microbial community shifts with soil properties andenzyme 2 activities in inter-/mono-cropping systems in response to tillage (Manuscript ID: agronomy-262869). The authors conducted a study dealing with the impact of intercropping systems of maize and peawhile focusing on the effects of different tillage strategies on the microbial community structure andenzymatic activities. In general, this topic is of high importance regarding the development ofsustainable future cropping systems. Although this study or better the available data provide a greatpotential, the study has some serious flaws. Here are my major concerns and suggestions forimprovements:

 

The introductions misses the actual importance or reason why cereals are intercropped with legumes such as pea. It is the capacity of legumes to symbiotically interact with bacteria that are enabled to fix molecular nitrogen, transferring it to ammonium (an essential crop available nutrient) and hence incorporate N into the soil-crop-system which intern might reduce the input of external N sources (mineral fertilizer) and potentially mitigate N2O emissions. This fact is not mentioned at all, but it is highly relevant taken into account that tillage might have an impact on this symbiosis. Moreover, there are different types of intercropping which are although not mentioned. Therefore, the introduction needs a profound revision.
Response: Thanks for your comments. We have added this point.

 

The authors have named the goal of the project, but hypotheses are missing. What did the authors expected to happen based on their literature research? What are the scientific gaps to fulfil?

Response: Thanks for your comments. We have added it.

Study site characteristics (line 91/92): what does sandy soil exactly mean? – For example you can write: The soil is characterized as …, with xx% sand, xx% clay, xx% silt, with an organic carbon (Corg) content of xx% and a pH value of x.xx.

Response: Thanks for your comments. We have added the details in the characterization of physical parameters of soil in this part.

Experimental design (line 94): A nine years field experiment cannot be called long-term. long-term means a running field study of more than 20 years. Maybe change “long-term” to “mid-term”.

Response: Thanks for your comments. We have changed the “long-term” to “mid-term”.

 

Experimental design (lines 96/97): here you describe three planting (btw: instead of using “planting”, from an agronomic point of view it would be better to use the term “cropping”) pattern; (1) maize in monoculture, (2) pea in monoculture and (3) intercropping maize and pea. Later on (e.g. lines 111, 189, 210) there are obviously two different intercropping systems; one time “intercropped maize” and one time “intercropped pea” – This is confusing. Please clearify!

Response: Thanks for your comments. First, there is no doubt that the “planting pattern” was correct in this study. Here we had the three planting patterns, (1) maize in monoculture, (2) pea in monoculture and (3) intercropping maize and pea, but soils were sampled in the (1) sole maize, (2) sole pea, (3) the maize strip of intercropping maize and pea, and (4) the pea strip of intercropping maize and pea from conventional tillage (CT) and no-tillage (NT) treatments, with a total of 32 samples (4 patterns x 2 tillage x 4 replicates). We usually sampled the soils and plants from the two crop strips of intercropping system, respectively.

Experimental design (line 107): “In each growing season …” – What does that mean? How many growing season were investigated? And how long that one crowing season lasted?

Response: Thanks for your comments. Only one growing season we conducted per year. Sorry for the misunderstanding. It has been modified.

Experimental design (line 108): The N applications rates seem to be very high. Are these the local typical application rates for maize and pea? If yes, please provide an appropriate reference. There are no information whether the intercropped systems received additional N fertilizer. Please clarify!
Response: Thanks for your comments. We have checked again and added the reference according to your suggestion.

 

Soil sampling (line 114): “…bulk soil cores were taken between the rows …” - Why between the rows? Why not as close as possible to the rhizosphere, particularly as the pea roots are building symbioses with N-fixing bacteria which will have an impact on the systems.

Response: Thanks for your comments. We actually took the soil in rhizosphere of roots also, but it’s sampling time was different. We will have another works to do that.

Soil sampling (line 115): Only one sampling point? It is well known that such experiments should be sampled several times per cropping season, as there will be changes due to the crop development stages! Maybe it would have be better to investigate the soil microbiome diversity by using an amplicon sequencing approach instead of one time (snap shot) metagenome analysis, particularly as the authors did not use the full potential of the generated metagenome data in terms of a profiling the genetically-determined function potential (see also further comments below).

Response: Thanks for your comments. The results we only provided the taxonomic data on microbial communities based on the metagenomic sequencing. Our title is Microbial community shifts with soil properties and enzyme activities in inter-/mono-cropping systems, that why we only focused on the changes in yields, soil properties, enzyme activities, and the diversity and composition of microbial (archaea, bacterial, eukaryota) community. About your considerations, the functional diversity and the functional genes analysis had posted in another paper. If you are very interested it or have any suggestions, please contact us. Thank you.

Soil sampling (line 116): Why were the samples sieved? By sieving you will lose a respective part of the microbiome, maybe system relevant members.

Response: Exactly, of course, some studies aimed at studying the microbial diversity at the different soil aggregate scale. However, each sampling site contains some roots/ residue or other debris when we sampled, and the soil weight used for DNA extracted only was 0.5 g, so in order to ensure the soil samples homogeneity were tested, the samples sieved is needed.

Line 119/120: “… one was kept fresh for the determination of soil microbial biomass, enzyme
activities” - Is the laboratory for these analyses directly next to the field? If not, this procedure was not a good choice as it is well known that microorganisms will stay active and particularly the measured enzyme activities will be falsified or not the real ones as they was at the sampling time.

Response: Thanks for your comments. The fresh soil generally stored at 4℃ refrigerator. If you are still confused, please see the stored conditions descripted in Lane J M, et al. (2022).

 

Lane J M, Delavaux C S, Van Koppen L, et al. Soil sample storage conditions impact extracellular enzyme activity and bacterial amplicon diversity metrics in a semi-arid ecosystem[J]. Soil Biology and Biochemistry, 2022, 175: 108858.

Matagenomic assembly: This is methodologically very poorly explained. What about the taxonomic assignment? You mentioned QUAST (bwt without providing the reference) for the quality check of you contigs, but maybe you rather used it for the taxonomic assignment? It is highly important to exactly explain what you have done and how, particularly as you only “talk” about the taxonomic diversity later on in the manuscript. Did you make single read analyses for functional profiling? The mentioned program `diamond´ indicates this, but these results are not further shown or later on discussed. What about the generation of metagenome assembled genomes?

Response: Actually, we send soil samples to Novogene Bioinformatics Technology Co. Ltd for DNA extraction, PCR amplification, and metagenomic sequencing. We have descripted main information in Materials and methods part in this manuscript according to the laboratory protocols they supplied. The results of taxonomic assignment only showed in this manuscript, the functional profiling would be shown in further or later.

Statistics: PCA this is not appropriate with biological samples, e.g. see Box 2 in Paliy O, Shankar V. (2016): Application of multivariate statistical techniques in microbial ecology. Mol Ecol. 25(5): 1032-57. doi: 10.1111/mec.13536. Prior to each statistical analyses especially when dealing with biological samples that you should make a test on normal distribution. Often the data, particularly relative abundance data have to be transformed (log transformation, square root transformation. However, for (dis-)similarity analyses it is better to used NMDS, or CCA, or PCoA.

 

Response: Thanks for your suggestions. We have used PCoA instead of PCA in this study.

Results: Why does the authors do not start with a description of the diversity in general and some statistical analyses, e.g. an ANNOVA?

 

Response: Thanks for your comments. We have provided the diversity in general and related relative statistical analyses.

Results: Figure 2 and the respective text part: From a statistical point of view, this is a very sloopy work. Here are two independent statistical methods are mixed. The ANNOVA results have nothing to do with the PCA plots, the ANNOVA does not explain the PCA ordination plot!!! This must be corrected!

Response: Thanks for your comments. Here is Adonis analysis showed in figures, not ANNOVA.

Results (lines 236-240): For only two phyla the relative abundances are provided. What about the other phyla and there abundances?

Response: Thanks for your comments. We have provided the relative abundances for other phyla.

Results: The result descriptions about the microbial diversity are irritating as the authors write about species diversity, but there is no clear explanation on how this assignment was carried out (see above). Additionally, the authors do only provide data at the phylum level (e.g. supplementary tables) and the alpha and beta-diversity indices were calculated based on the absolute abundances at the phylum level, but they are quite often talk about species… Or another example in lines 252/253: “…the abundance of the unclassified species differed substantially between treatments at the class, order, and family levels” This is confusing or implies that the authors are not sure in handling microbiological data.

 

Response: Thanks for your comments. Based on metagenomic sequencing, we got the abundance of many microorganisms at all levels. The results were not accurately classified which is normally, indicating that we need to further explore these different species for analysis, and the changes of its related functional genes.

Table S1: What does “absolute abundance” mean? There are absolute values in terms of the number of detected phyla or the relative abundances, while the calculation of diversity indices makes more sence using relative abundance values. What does “homogenization matrix” means? There is no explanation in the material and method part of the manuscript! Why is the microbial diversity just given at the phylum level? It is well know, that the deeper the phylogenetic resolution, the more distinct the differences are.

Response: Thanks for your comments. We have provided the relative abundances at the phylum analysis results in the supplementary word.

 

General comment: The verification of the impact of soil parameters (or agronomic management
measures such as tillage) on the microbial diversity is only considered at the phylum level? Why not at the most deepest phylogenetic level, the species level (which data seems to be available) as it is well known, that system differences are often first visible at the species level. Maybe have a look at the studies from the Earth-Microbiome-Project.

 

Response: Thanks for your comments. Based on metagenomic sequencing, we got the abundance of many microorganisms at all levels. We know that the significantly differences appear in deeper the phylogenetic resolution (maybe like genus, species), there would found significant differences in some unclassified species at different levels (e.g.  class/order/family/genus/species) in different treatments showed in Figure 3. That is why we only provided the diversity and abundance of archaea, bacteria and Eukaryota at the phylum level, separately.

Lines 341/342: “… tillage practices significantly affected the ß-diversity of bacterial community.” -This is highly questionable as (i) the authors mixed two independent statistical analyses and (ii) the beta-diversity is based on the phylum level, instead of deeper phylogenetic levels, particularly as the authors have obviously data until the species level.

Response: Thanks for your comments. We combined the PCoA and Adonis analysis could explain this sentence. We believed that the diversity of bacterial community at the phylum level would more representation in general, instead of based on other levels.

Lines 343-349: As the calculated Shannon index and Pielou evenness are based on the phylum level, they are not meaningful enough to make conclusions on the steadiness and adaptability of the microbial community against disturbances, while the co-occurrence network analyses might be it they are based at least at the genus level. Despite the valuable indications of network analyses, they should be considered with caution, as they do not (yet) reflect reliable/evident/causal ecological conclusions. For example, one problem is the sample heterogeneity, which can be overcome by applying various filtering methods such as by defining OTUs or ASVs, by filtering out taxa which are low-abundant in a given sample and/or rare across samples or by collapsing taxa to a higher-level hierarchy. The remaining taxa will mainly be members of the core-microbiome (generalists), and the rare taxa (members of the transient-microbiome) will get lost (Karpinets et al. 2018, Front. Microbiol. 9, 297)

Response: Thanks for your comments. The diversity, co-network, abundance of microbial community all were analyzed based on the phylum level. This study aimed to to determine the soil chemical properties, enzyme activities, and the microbial community diversity and composition at phylum level in response to monoculture and intercropping in conventional and no-tillage systems in a semi-arid area.

 

Formats:
Table 1: It is generally hard to read due to the format. Why the authors counted the pods per plant for pea and grains per pod? What is “net area yield”? What does “total of pea and maize” indicate? Total what of pea and maize? And last: “kg hm-2“ is a strange kind of unit. Yield normally is given in kg per hectar; I´ve never seen this kind of unit before.

Response: Thanks for your comments. the pods per plant for pea, grains per pod, number of spikes per square meter, kernel number per spike all was deleted. Additionally, Net area yields of pea were the yield divide by 8/19 according to the proportion of pea area in intercropping., net area yields of maize were the yield divide by 11/19 according to the proportion of maize area in intercropping. We added related information in the study.

Table 2: What all the abbreviations stand for? Tables and figures must be understandable without reading the main text. ! It is really annoying for the reader to scroll back to the material and methods part each time in order to find out what the abbreviations stand for! That means all used abbreviations must be explained either in the table/figure legends or as footnote! This has to be considered for all tables and figures.

Response: Thanks for your comments. We have checked and added it.

Final statement: As there are some serious methodological drawbacks, I did not read the entire
manuscript as the drawbacks first have to be removed. This study has a very high potential if the authors go deeper into the data. I recommend an in-depth revision and I´m pretty sure, this could be a highly valued study in particular as the topic is highly important.

Response: Thanks for your comments. We appreciate it.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Overall experimental design is in good format and has statistically well described with supporting data. Though, Some minor changes needed:

1. Introduction section needs to improve with the references of soil properties and enzyme activities.

2. Abiotic stress has major impact on to the microbial community shift, hence authors need to add the characterization of physical parameters of soil (Total N, P, K) and needs to mention the previous history of field with respect to fertilizer/pesticide/biofertilizer application in methodology section.

3. In DNA extraction replace the term Raw DNA with MetaDNA

4. DNA quality must me checked by Nanodrop also

Comments on the Quality of English Language

Moderate revision is required. Hence, authors need to take help of a native speaker to revise it.

Author Response

Thank you for your comments. We have revised the manuscript carefully according to your feedback. Below are lists of main things we done:

1. Introduction section needs to improve with the references of soil properties and enzyme activities.

Response:  We have added the new references of soil properties and enzyme activities in the introduction part.

2. Abiotic stress has major impact on to the microbial community shift, hence authors need to add the characterization of physical parameters of soil (Total N, P, K) and needs to mention the previous history of field with respect to fertilizer/pesticide/biofertilizer application in methodology section.

Response: We have added the details in the characterization of the physical parameters of soil in this part.

3. In DNA extraction replace the term Raw DNA with MetaDNA

Response: We have changed the Raw DNA with MetaDNA.

4. DNA quality must me checked by Nanodrop also

Response: We have added the details of DNA quality methods in the Soil DNA extraction and metagenomics sequencing part.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

Thank you for your manuscript submission “Microbial community shifts with soil properties and enzyme activities in inter-/mono-cropping systems in response to tillage.” The manuscript showed the change of soil properties, enzyme activities, and microbial community diversity and composition due to no-tilled cereal-legume intercropping in a semi-arid area under different conditions. Here are my specific comments:

 

-       Abstract should be rewritten as it does not summarize key points from the study. More quantitative results should be indicated in the abstract

-       Are there any equations or assumptions for your experimental conditions and data analysis?

-       A comparison of results in this study with previous papers is expected

-       What are long-term sustainability and long-term effects on soil (e.g., soil health, microbial communities, future plant growth)?

-       The conclusion should focus more on the objectives mentioned in the introduction. More details should be provided in the conclusion

-       Are there any limitations for the method in this study?

-       Future research is missing

 

Comments on the Quality of English Language

The quality of English is good

Author Response

Reviewer3:

Thank you for your manuscript submission “Microbial community shifts with soil properties and enzyme activities in inter-/mono-cropping systems in response to tillage.” The manuscript showed the change of soil properties, enzyme activities, and microbial community diversity and composition due to no-tilled cereal-legume intercropping in a semi-arid area under different conditions. Here are my specific comments:

 

-  Abstract should be rewritten as it does not summarize key points from the study. More quantitative results should be indicated in the abstract.

Response: Thanks for your comments. We have rewritten the abstract.

 

-  Are there any equations or assumptions for your experimental conditions and data analysis?

Response: Thanks for your comments. We have checked and had no any equations or assumptions need for this experimental conditions and data analysis.

 

-  A comparison of results in this study with previous papers is expected

Response: Thanks for your comments. We have tried to do the comparison of results with previous studies.

 

-  What are long-term sustainability and long-term effects on soil (e.g., soil health, microbial communities, future plant growth)?

Response: Thanks for your comments. One of the reviewers suggested that a nine years field experiment cannot be called long-term and believed that long-term means a running field study of more than 20 years, so we have changed the “long-term” to “mid-term”. For the long-term effects on soils, we believed that long-term/mid-term no-tillage and intercropping will have long-term sustainable and significant effects on soil physical, chemical, biological properties, and even the microbial communities in the soils. Soil health also changes, directly affecting crop growth and yields in farmland ecosystems.

 

-  The conclusion should focus more on the objectives mentioned in the introduction. More details should be provided in the conclusion

Response: Thanks for your comments. We have modified the descriptions in details in the part of conclusion.

 

-  Are there any limitations for the method in this study?

Response: Thanks for your comments. Actually, we should take soil sampling in different stages of growth and more locations (e.g. rhizosphere soil, bulk soil), and we analyzed the data that belong to the part of the results by metagenomic sequencing, we did not show the genes results in this study.

-   Future research is missing

Response: Thanks for your comments. We have added it in conclusion.