Cultivation of Two Barnyard Varieties Improves Physicochemical Properties of Saline-Alkali Land through Mediating Rhizospheric Microbiome and Metabolome
Round 1
Reviewer 1 Report
The manuscript titled “Cultivation of two barnyard varieties improves physicochemical properties of saline-alkali land through mediating rhizospheric microbiome and metabolome” by Wang et al., has been reviewed for possible publication in Agronomy.
The authors have carried out a study on two varieties of barnyard (Echinochloa frumentacea (Roxb.) link and E. crusgalli var. austro-japonensis, both tolerant to salinity, and that possibly have different bacterial communities in their rhizosphere, as well as different metabolites, this can influence the physicochemical properties of the rhizospheric soil. To know this, the authors have monitored a field trial, developing a large number of techniques based on metagenomics and metabolomics, and measures on soil samples.
The topic is appropriate to this scientific journal (Agronomy), and I think it will be of interest to its readers. In addition, this topic could also provide adequate information (with two years of experimentation in the field), and can generate knowledge of interest from a scientific point of view. We know there are quite a few studies on the plant-soil system, and its microbial biodiversity, and carried out with techniques similar to those proposed in this work. However, a field trial on plant varieties capable of surviving in a saline environment can be considered as original.
But before publishing this manuscript in Agronomy, I think the authors should make some changes to the original manuscript. Some of the changes are described below.
1) In the Introduction, the authors should make an effort to give meaning within the objective of the paper, to the different existing paragraphs; for example, on pg 2, paragraphs 2, 3 and 4 seem to be isolated paragraphs; authors should clarify its importance in relation to the study carried out.
2) Regarding the soil characteristics considered in Table 1, I think parameters linked to soil salinity (for example, chlorides, sulfates, Na+, Mg+2, etc.), should be included. Also, some agronomic parameters such as N content, or available P and K, should be included.
3) The design of the experiment is coherent, but there are some aspects that should be explained. It is not indicated if in the studied crops there is mineral or organic fertilization, or no fertilization. This aspect is of great interest for studies on soil microbial biodiversity.
4) The study carried out on metagenomic is of clear interest, and provides a very good description of the microbial communities involved in the rhizosphere of the considered crops. I think that some measure of microbial activity could also have provided information of relevant interest in this case.
5) The authors have carried out a study on non-targeted metabolomic, but the comments made in the text are very scarce. Even in the conclusions nothing is mentioned about it. The conclusions should be expanded to include aspects of the metabolomics carried out, and try to be more specific in said conclusions.
6) The text should be revised to avoid grammatical errors (for example, see the title: “physiochemical” instead of “physicochemical”).
7) The discussion section is too generalist. It should be focussed in the results and achievements of the work carried out instead of on general assertions and well known concepts.
Author Response
The manuscript titled “Cultivation of two barnyard varieties improves physicochemical properties of saline-alkali land through mediating rhizospheric microbiome and metabolome” by Wang et al., has been reviewed for possible publication in Agronomy.
The authors have carried out a study on two varieties of barnyard (Echinochloa frumentacea (Roxb.) link and E. crusgalli var. austro-japonensis, both tolerant to salinity, and that possibly have different bacterial communities in their rhizosphere, as well as different metabolites, this can influence the physicochemical properties of the rhizospheric soil. To know this, the authors have monitored a field trial, developing a large number of techniques based on metagenomics and metabolomics, and measures on soil samples.
The topic is appropriate to this scientific journal (Agronomy), and I think it will be of interest to its readers. In addition, this topic could also provide adequate information (with two years of experimentation in the field), and can generate knowledge of interest from a scientific point of view. We know there are quite a few studies on the plant-soil system, and its microbial biodiversity, and carried out with techniques similar to those proposed in this work. However, a field trial on plant varieties capable of surviving in a saline environment can be considered as original.
But before publishing this manuscript in Agronomy, I think the authors should make some changes to the original manuscript. Some of the changes are described below.
Author response: Many thanks for your time and valuable suggestions to improve our manuscript. We have revised the manuscript as per your suggestions. Please see below the response to your specific comments.
1) In the Introduction, the authors should make an effort to give meaning within the objective of the paper, to the different existing paragraphs; for example, on pg 2, paragraphs 2, 3 and 4 seem to be isolated paragraphs; authors should clarify its importance in relation to the study carried out.
Author response: We have added some extra references to link these paragraph to our study objectives and combined the 3rd and 4th paragraph for better understanding.
2) Regarding the soil characteristics considered in Table 1, I think parameters linked to soil salinity (for example, chlorides, sulfates, Na+, Mg+2, etc.), should be included. Also, some agronomic parameters such as N content, or available P and K, should be included.
Author response: We have added agronomic parameters N, P and K in Table 1 as suggested. Moreover, basic parameters linked to soil salinity before start of experiment have been listed in Table S-1 in supplementary material.
3) The design of the experiment is coherent, but there are some aspects that should be explained. It is not indicated if in the studied crops there is mineral or organic fertilization, or no fertilization. This aspect is of great interest for studies on soil microbial biodiversity.
Author response: No fertilizer and mineral supplementation was applied throughout the growth period, and all plots were managed in the same field under uniform management conditions. We have revised the text in the relevant section as suggested.
4) The study carried out on metagenomic is of clear interest, and provides a very good description of the microbial communities involved in the rhizosphere of the considered crops. I think that some measure of microbial activity could also have provided information of relevant interest in this case.
Author response: Yes, you are right. But unfortunately we didn’t examined any measure of microbial acivity in the present study.
5) The authors have carried out a study on non-targeted metabolomic, but the comments made in the text are very scarce. Even in the conclusions nothing is mentioned about it. The conclusions should be expanded to include aspects of the metabolomics carried out, and try to be more specific in said conclusions.
Author response: We agree with your comment. We have added concluding remarks about metabolomic analysis as suggested and also added relevant information in the manuscript. Moreover, we have included more results of metabolomics in the manuscript as suggested (Figure 6). In the present study, metabolomic analysis identified important pathways like arginine and proline metabolism and secondary metabolite, which might have played role in salt stress management.
6) The text should be revised to avoid grammatical errors (for example, see the title: “physiochemical” instead of “physicochemical”).
Author Response: Many thanks for your comments. We have corrected the typos and grammatical errors throughout the manuscript.
7) The discussion section is too generalist. It should be focused in the results and achievements of the work carried out instead of on general assertions and well known concepts.
Author response: We have removed some general information and added more specific information in the discussion section as suggested. Moreover, we have also modified the discussion to interpret our findings.
Reviewer 2 Report
The manuscript submitted by Zhang and colleagues aims at comparing the effect of two salt tolerant plant species (Echinochloa crusgalli and E. frumentacea) on soil properties and fertility in an alkaline soil of China, as well as the microbial communities shaped by each plant species in the rhizosphere and the metabolic profile of the plants. In this regard the paper proposes an interesting and original topic that would probably attract attention from a wide audience. However, I have big concerns with regards to the experimental design of the study, as it is not clear enough. Most important is the fact that the author state that the study was done on two successive years, but samples were taken only in 2019, from each treatment that was replicated 3 times (BZ1 to BZ3 and HNJZ1 to HNJZ3) with 2 blocks in total”…So, what is the experimental design here? Is it a CRD or a CRBD, with 2 reps (blocks?). In other words the size of the N here seems to be very small. Where the barnyard varieties the treatments? The application of treatments is not explained (sowing, management, etc.). The rhizosphere soil is said to be collected from soil adjacent 3 mm from the root, but there is not reference to justify this method (3 mm seems rather thick layer for rhizosphere soil), and soil texture and classification of the study site is totally missing. The authors also state that rhizosphere samples from the same treatments were mixed which would turn this into an un-replicated study. This is worrisome; make this point clear. They report that the 16S rDNA was amplified by PCR targeting the V3-V4, V4-V5 and V5-V7 bacterial regions, which does make little sense as normally sequencing is done on only one of these regions. And if bacterial 16S is targeted, then they can’t get reliable amplification of archaeal 16S. However, there are sequences identified as Thaumarchaeota and Euryarchaeota that are reported in the results. Finally, statistical analysis needs to be soundly used: for instance, using PCA for separation of only 6 (?) samples based on more than 1300 variables (OTU’) is of little value as most of these variables are correlated with each other (violating one of the PCA assumptions).
In case the paper is convincingly revised and resubmitted, the authors should also reduce the extension of results reported in section 3.2 and 3.3 which are mostly descriptive data with little relevance (alpha diversity analysis shows no differences, so can be moved to supplementary information). Figures 1 and 2 should also move to the SM. Table 3 is not necessary and its information can be included in the text.
Typesetting of species names should be corrected using italics.
Reference 94 is not relevant.
Author Response
The manuscript submitted by Zhang and colleagues aims at comparing the effect of two salt tolerant plant species (Echinochloa crusgalli and E. frumentacea) on soil properties and fertility in an alkaline soil of China, as well as the microbial communities shaped by each plant species in the rhizosphere and the metabolic profile of the plants. In this regard the paper proposes an interesting and original topic that would probably attract attention from a wide audience.
Author response: Many thanks for your valuable comments to improve our manuscript. We have revised the manuscript as per your suggestions. Please see below the response to your specific comments.
However, I have big concerns with regards to the experimental design of the study, as it is not clear enough. Most important is the fact that the author state that the study was done on two successive years, but samples were taken only in 2019, from each treatment that was replicated 3 times (BZ1 to BZ3 and HNJZ1 to HNJZ3) with 2 blocks in total”…So, what is the experimental design here? Is it a CRD or a CRBD, with 2 reps (blocks?). In other words the size of the N here seems to be very small. Where the barnyard varieties the treatments?
Author response: Sorry, there is some typing mistake. It’s a basically RCBD design with three blocks. Each block has two varieties having one replicate each. From each replicate, 5 plants were selected for rhizosphere sampling, so for each treatment we collected 15 plants. We have collected data separately for 2018 and 2019, but we presented the data of 2018 as start of the experiment and 2019 as the end of the experiment for physicochemical properties. This is mainly due to the reason that we used the same plots for cultivation of same varieties for two consecutive years and envisioned to elucidate overall effects. Therefore, only rhizosphere sampling of 2019 was used for metagenomic and metabolomic analysis. We have added details about experimental design and methodology in details as suggested. Following text has been added in the materials and method section.
The seeds of Echinochloa frumentacea (Roxb.) Link and E. crusgalli var. austro-japonensis were provided by the School of Ecology and Environment, Ningxia University. The experimental site was divided into three areas (blocks), each area includes two plots. The plot was 7 meters long, 5 meters wide, and an area of ​​35 m2. The area spacing is 1.5 m; plot spacing is 1m. A 5-meter wide protection line was set up to isolate the test area. The experiment was performed with a completely randomized block design with three replications. Before planting, three soil samples (5-cm diameter × 30 cm depth) were collected randomly in each plot before planting. Two forages were planted for two years, HNJZ and BZ seeds were sown at 17th of April in 2018 and 20th of April in 2019, respectively. The experiment ended in August 2019. All seedlings were managed under the general field management methods, and no fertilization was done for the whole growing period.
The rhizosphere samples of Echinochloa frumentacea (Roxb.) Link and E. crusgalli var. austro-japonensis were collected at 20th of August 2019. 5 plants were randomly selected from each plot, and the rhizosphere soils of the 5 plants were mixed (samples of rhizosphere samples of same replicate were mixed not different replicate). The numbers are respectively : (Echinochloa frumentacea (Roxb.) Link: HNJZ1, HNJZ2, and HNJZ3 and; E. crusgalli var. austro-japonensis: BZ1, BZ2, BZ3), The soil was dug about 20cm depth around the roots of both plants after removing the litter and dirt before the sample collection. The soil attached to 1-2mm of the roots of plants was collected and placed in a sterilized 50ml centrifuge tube. The rhizosphere samples from the same replicate were mixed, placed in a dry ice sampling box, and immediately brought back to the real face room. A part of the soil samples was taken out, air-dried, and sieved, and then the soil physicochemical properties were determined according to the "Soil Agrochemical Analysis Method". The remaining samples were stored at -80°C for later use.
The application of treatments is not explained (sowing, management, etc.).
Author response: Before sowing, it is necessary to prepare the land, prepare the seeds, and plant the whole plant. The two types of pastures were sown by drilling. They were managed in a unified manner throughout the growth period, and the inter-cultivation, weeding and irrigation were carried out on time. Details have been added in the manuscript.
The rhizosphere soil is said to be collected from soil adjacent 3 mm from the root, but there is not reference to justify this method (3 mm seems rather thick layer for rhizosphere soil).
Author response: We have corrected this mistake as it was 1-2mm instead of 3mm. While sampling, the roots were dug out, and the extra soil was removed. The soil on the root surface was about 1-2mm.
Soil texture and classification of the study site is totally missing.
Author response: We have added the details regarding soil texture and classification as suggested. The soil in the test area is cracked alkaline soil (Gong et al., 2002)., and the anatomical surface of the soil can be divided into alkaline layer, transition layer and parent material layer from top to bottom. The clay mineral composition of the alkaline layer soil is mainly hydromica, followed by chlorite, kaolinite and smectite, mainly including crust layer and prismatic structure layer. The crust layer is about 1 cm thick, and the upper part is more sticky. The surface of the grain is cracked, and there are a lot of honeycomb pores on the back. The prismatic structure layer is very hard, prismatic or short cylindrical, about 40 cm thick. The downward transition layer structure is mostly block-like. The parent material layer is the bottom sediment of the lake, which is gray in color and has a low degree of alkalinity. Soil ploughing layer organic matter, total nitrogen g·kg-1, alkaline hydrolyzable nitrogen mg·kg-1, available phosphorus mg·kg-1, available potassium mg·kg-1.
The authors also state that rhizosphere samples from the same treatments were mixed which would turn this into an un-replicated study (This is worrisome; make this point clear).
Author response: The rhizosphere samples of 5 plants from each plot (from one replicate of each treatment) were mixed, not from the different replications. So we have three replicates for each treatment (one replicate consisting of 5 plants). We have clearly mentioned this fact in the manuscript as suggested.
They report that the 16S rDNA was amplified by PCR targeting the V3-V4, V4-V5 and V5-V7 bacterial regions, which does make little sense as normally sequencing is done on only one of these regions. And if bacterial 16S is targeted, then they can’t get reliable amplification of archaeal 16S. However, there are sequences identified as Thaumarchaeota and Euryarchaeota that are reported in the results.
Author’s response: Yes, your are right. We have used 16S DNA region V4 (515F and 806R) for identification of bacterial taxa. Now it is corrected accordingly. Additionally, we also used 16S (V4-V5, V8) for the archaeal and bacterial diversity analysis as this region can target both bacterial and archaeal taxa as reported previously (Willis et al., 2019).
Willis, C., Desai, D., & LaRoche, J. (2019). Influence of 16S rRNA variable region on perceived diversity of marine microbial communities of the Northern North Atlantic. FEMS Microbiology Letters, 366(13). doi:10.1093/femsle/fnz152.
Finally, statistical analysis needs to be soundly used: for instance, using PCA for separation of only 6 (?) samples based on more than 1300 variables (OTU’) is of little value as most of these variables are correlated with each other (violating one of the PCA assumptions).
Author’s response: PCA is widely used for grouping of samples based on OTUs as it clusters the samples with similar sequences in groups.
In case the paper is convincingly revised and resubmitted, the authors should also reduce the extension of results reported in section 3.2 and 3.3 which are mostly descriptive data with little relevance (alpha diversity analysis shows no differences, so can be moved to supplementary information). Figures 1 and 2 should also move to the SM. Table 3 is not necessary and its information can be included in the text.
Author response: We have reduced the results in section 3.2 and 3.3 by removing extra details as suggested. Moreover, We have moved alpha diversity results in supplementary material as suggested. Similarly, we have made changes in manuscript according to your suggestion by moving Figures 1 and 2. Moreover, we deleted Table 3 and incorporated its data in the text as suggested.
Typesetting . of species names should be corrected using italics.
Author response: Done
Reference 94 is not relevant.
Author response : Many thanks for your ind effort. We have removed that reference
Round 2
Reviewer 2 Report
Authors have extensively revised their manuscript reaching a satisfactory version.
I feel that the paper can now be accepted, although it might need some English edition prior to acceptance, particularly in the new text added.
