Grass-legume mixtures maintain forage biomass under microbial diversity loss via gathering Pseudomonas in root zone soil

ABSTRACT As the most common approach for the restoration of degraded grasslands, the use of grass-legume mixtures has long been recognized for its role in increasing aboveground biomass and resisting grassland degradation. However, whether the legumes in these mixtures can help neighboring plants resist the decline in biomass caused by the loss of soil microbial diversity remains a question worthy of investigation. To address this, we employed a dilution method to create a gradient of decreasing microbial diversity in soil and utilized full-factorial combinations of legumes and two grasses to investigate the crucial role of legumes in the mixture. The results showed that compared to monoculture, the mixture of Medicago sativa L. and Elymus dahuricus Turcz. enhanced the biomass of grass species under conditions of soil microbial diversity loss. We then discovered that a significantly enriched Pseudomonas (ASV53), in the grass-legume mixtures under conditions of microbial diversity loss, was positively correlated with plant biomass and nitrogen-fixing (nifH) gene abundance, implying that it could be a keystone species. In addition, the grass-legume mixture increased the deterministic processes of microbial community enrichment in the root zone soil by enhancing the process of homogeneous selection. Functional predictions revealed that grass-legume mixtures increased the potential abundance of N-related and phototrophy-related microbial communities in the root zone soil. This study provides an important insight into the mechanism underlying the role of legumes in increasing and maintaining grass biomass despite soil microbial diversity loss. IMPORTANCE Grass-legume mixtures are a common practice for establishing artificial grasslands, directly or indirectly contributing to the improvement of yield. In addition, this method helps maintain soil and plant health by reducing the use of chemical fertilizers. The impact of grass-legume mixtures on yield and its underlying microbial mechanisms have been a focus of scientific investigation. However, the benefits of mixtures in the context of soil microbial diversity loss remain a problem worthy of exploration. In this study, we examined different aboveground and belowground diversity combinations to elucidate the mechanisms by which grass-legume mixtures help maintain stable yields in the face of diversity loss. We identified the significantly enriched Pseudomonas genus microbial ASV53, which was gathered through homogeneous selection and served as a keystone in the co-occurrence network. ASV53 showed a strong positive correlation with biomass and the abundance of nitrogen-fixing genes. These findings provide a new theoretical foundation for utilizing grass-legume mixtures to enhance grass yields and address the challenges posed by diversity loss.

In the manuscript titled "Grass-legume mixtures maintain forage biomass under microbial diversity loss via gathering Pseudomonas in root zone soil" authored by Liu and colleagues, the researchers investigated how the legume-grass mixture assist neighboring grass to resist the biomass decline when soil microbial diversity is lost.The experiment was conducted by constructing the soil biodiversity gradient and the full factorial combination of legumes and two grasses.The authors discovered an interesting phenomenon that when grass was monocultured, biomass was lost as soil biodiversity declined, but biomass remained stable when grass was mixed with legume.This research enhances our comprehension of how aboveground biomass is maintained in the face of disturbance and provides a deeping understanding for the role of legumes.The author makes a lot of discussion on microbe especially Pseudomonas, but ignores the plant host in the Discussion.Just as when two grasses are mixed with legumes, only one grass (Elymus dahuricus Turcz.)shows a stable biomass when microbial diversity is lost.Does this mean that it is affected by plant identity?Here authors need to further add the possible differences between the two grasses in the Disscussion and also show the results of Festuca elata Keng. in the Results.I have the following minor suggestions.Line 25: What specific combinations of legumes and grasses were made?Such as full-factorial combinations of legume and two grasses?Line 27: legume and which grass of two grasses?Be specific.Line 31: add (nifH) between nitrogen-fixing and gene.Line 37: remove in the microbial community.Line 54: add reference.Line 77-80: add reference.Line 109: Replace ',' with '.' Line 127: Replace 'under different diversity conditions' with 'across soil dillution gradients' Line 365: Why is 10-2 used here and 102 in the figure?Line 378: add spaces between numbers and units Line 388: RH mean?Line 370: 5 plots mean 5 replicates?Does it contradict 4 replicates in Line 402? Line 421: As far as I know, using data2 to process amplicon data does not need to consider 97% similarity.A deeper understanding is needed about data2 processes.Line 438-482: Active tense is used too much.Except for necessary explanations, all other sentences are changed to the passive tense.Line 682: Replace ',' with '.' Line 683: Which method was used to test for significant differences?It also needs to be shown in the figure legend.The manuscript by Liu et al. aims to examine whether grass-legume mixtures play a positive role in increasing plant biomass under low soil microbial diversity.The authors created a gradient of soil microbial diversity using a dilution method, and then utilized various combinations of legume and grass to investigate the role of legumes in influencing the biomass of neighboring plants.They found that grass-legume mixtures enhanced the biomass of grass under low soil microbial diversity.They revealed a significant enrichment of Pseudomonas (ASV53) in the grass-legume mixtures, which was positively associated with plant biomass and nitrogen-fixing gene abundance.Further functional predictions indicated that grass-legume mixtures may increase the abundance of N-related and phototrophy-related microbial communities in the root zone soil.Investigating the effects of plant-plant and plant-microbiomes interactions on plant performance under different microbial diversity and their associated ecological mechanisms represent a very interesting and promising research topic.Results from this study can offer an important knowledge for engineering microbial functions to improve crop production in a sustainable way.The results of this article are highly interesting, and the authors have employed appropriate bioinformatic and statistical analyses.I just have a few minor comments that might be worth thinking before the publication.Specific comments are provided below: L36-37: I would recommend reconsidering the evidences supporting conclusion on human pathogens, as this isn't the paper's main focus and functional prediction and genus-level identification of pathogens based on 16S is limited in reliability.
L196-198: It would be great if you could provide the similarity between these two sequences (i.e.ASV53 and A1501).I would suggest enlarging the text in Fig. 3c as it looks not very clear.L360: "soil microbial communities" to "the diversity of soil microbial communities" L407-409: Please add more detailed information on the PCR amplification.
L496-498: Please add the accession number for raw sequence data.1.This research enhances our comprehension of how aboveground biomass is maintained in the face of disturbance and provides a deeping understanding for the role of legumes.

Response and actions taken with respect to the reviewer comments for:
Response: Thank you very much for the positive comments.
2. The author makes a lot of discussion on microbe especially Pseudomonas, but ignores the plant host in the Discussion.Just as when two grasses are mixed with legumes, only one grass (Elymus dahuricus Turcz.)shows a stable biomass when microbial diversity is lost.Does this mean that it is affected by plant identity?Here authors need to further add the possible differences between the two grasses in the Discussion and also show the results of Festuca elata Keng. in the Results.
Response: Thank you very much for providing valuable feedback.As you pointed out, the role of plant hosts is indeed crucial in our experiment.I have incorporated your suggestions into the revised version, addressing why there was no similar increase in biomass for Festuca elata Keng.compared to Elymus dahuricus Turcz.Furthermore, you mentioned the absence of Festuca elata Keng. in the subsequent results analysis.This was due to our observation during biomass analysis that the Medicago sativa L. and Elymus dahuricus Turcz.mixture increased the biomass of Elymus dahuricus Turcz., while the biomass of Festuca elata Keng.did not show an increase when mixed with Medicago sativa L. or grown in monoculture.Therefore, we did not investigate the underlying mechanisms for Festuca elata Keng. in this context.I added the relevant content in lines 278-296, which are shown below: We first compared the variations in biomass across different plant combinations.We observed that the M and P mixture increased the biomass of P, while mixtures had no significant effect on the biomass of G. Similar observations were made with plant combinations GP and MGP.This is the reason why we focused our subsequent analysis exclusively on the individual growth of M and P and their mixed cultivation as MP.The occurrence of these patterns is not only attributed to the crucial role of the soil microbial community, which we have been investigating but also to the inherent characteristics of the plants themselves (1).As commonly used grass species in establishing artificial grasslands, G and P exhibit preferences for specific soil environments and tolerances.Empirical knowledge from livestock farming indicates that P thrives in nutrient-rich soils (2).When P is mixed with M, the nitrogen-fixing capacity of M provides a more abundant nitrogen source, creating a nutrient-rich environment that favors the growth of P. On the contrary, G responds sensitively to soil fertilization, and excessively high nitrogen content in the soil can be detrimental to its growth (3).Thus, a mixture with M does not increase G biomass.In addition, P demonstrates strong adaptability to temperature, while G prefers cooler environments (4).The controlled temperature conditions in our study may not have reached the optimal growth temperature for G. Consequently, there were no significant differences in biomass, whether in monoculture or mixture.
3. What specific combinations of legumes and grasses were made?Such as full-factorial combinations of legume and two grasses?(Line 25).
Response: Yes, our plant combinations included one legume, Medicago sativa L., and two grasses, Elymus dahuricus Turcz.and Festuca elata Keng..These plants were grown individually and in pairwise combinations, as well as all three together, making a total of seven plant combinations.In the revised version, I will incorporate the full-factorial interactions as provided here to elucidate the experimental design.
Line 23-25 "To address this, we employed a dilution method to create a gradient of decreasing microbial diversity in soil, and utilized full-factorial combinations of legume and two grasses to investigate the crucial role of legumes in the mixture." 4. legume and which grass of two grasses?Be specific.(Line 27).
Response: Done.I have clarified the plant combinations that exhibit biomass enhancement to avoid any confusion.the dilution factor applied to the soil suspension.To prevent any potential confusion, I have made a consistent modification to describe it in terms of the dilution factor.12. add spaces between numbers and units.(Line 378).

RH mean? (Line 388).
Response: I apologize for the oversight.Here, "RH" refers to Relative Humidity, and I have added detailed explanations in the revised version for clarity.14. 5 plots mean 5 replicates?Does it contradict 4 replicates in Line 402? (Line 370).
Response: Yes, the reference to "5 plots" represents 5 replicates.During the experiment, each plant combination under each dilution gradient had 5 replicates.However, based on the growth conditions of the plants and considering the consistency of sample sizes across all groups, we ultimately selected 4 replicates for further analysis.
15.As far as I know, using dada2 to process amplicon data does not need to consider 97% similarity.A deeper understanding is needed about data2 processes.(Line 421).
Response: Thank you for your valuable feedback.In the revised version, I have rewritten the procedure for obtaining the ASV table.
Line 439-446 "DADA2 was used to process raw sequencing reads for each sample (clean data), infer the unique amplicon variant (ASV) through error-corrected reads further quality control through the error model, and filter chimeras using the DADA2 pipeline (5).Subsequently, the sequences were filtered, trimmed, and truncated at 210 bp of forward Then, based on the Bayesian algorithm, we used the SILVA reference database (v.12.8) to classify representative sequences from each ASV (6).
Non-bacterial ASVs (chloroplast, mitochondria, unknown, Archaea and plants) and ASVs with fewer than two reads were also removed."Response: Done.The values indicated in the figure, namely 10 0 , 10 2 , 10 4 , and 10 6 , represent the dilution gradients of the soil suspension.Specifically, 10 0 signifies the undiluted original mother solution, 10 2 corresponds to a 100-fold dilution, 10 4 denotes a 10,000-fold dilution, and 10 6 signifies a 100,000-fold dilution.We have added a detailed description of the soil dilution gradient in the revised version, and the above contents are added to the legend in Figure 1.Response: Done.We performed 16S rDNA amplicon sequencing of the root zone soil samples.For prokaryotic amplicon preparation, the V4-V5 region of the 16S rDNA gene was amplified by PCR using 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGTTT-3') primers.PCR reactions were performed in triplicate in a 15 μL reaction mixture which contained 7.5 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 1 μL of forward and reverse primers (3 mM), 2.5 μL of template DNA (5 ng μL−1), and 4 μL of ddH2O.PCR conditions were: 1 cycle × 98 °C for 1 min; 30 cycles × 98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s; 1 cycle × 72 °C for 5 min.
The quality of amplicons was detected through electrophoresis on 2 % agarose gel, and the purity of amplicons was ensured using Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany).The triplicate PCR reactions for each sample were combined and quantified on a QuantiFluor™-ST Fluorometer (Promega, Madison, WI, USA) following the manufacturer's protocol.16S rDNA sequencing was performed on the Illumina HiSeq® 2500 platform (Illumina Inc., San Diego, CA, USA) at Novogene (Beijing, China) using high-output mode with the paired-end method after library construction (NEB Next® Ultra DNA Library Prep Kit).Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.For your reference, ASM Journals' address is given below.Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication.They will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
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2 : 6 :
Fig. 1ab: Do 'M' in (a) and 'M_mono' in (b) are the same meaning?If yes, keep the same abbreviation.There is no unit in Biomass.Fig. 1c: Soil dilution gradient needs be explained in figures legend.What does 100, 102, 104, 106 mean?Fig.Before showing the ab sub-legend, one sentence as the title of Fig. 2 need be illustrated.Line 694: enriched in monculture and/or in mixture?Line 695: in the corresponding soil dilution gradient?Line 696: The plot called tree plot?Fig. 2a: The indicated taxa are Class level?Fig. 3b: There are no unit in nirH abundance and ASV abundance.Fig. 4a: The color of the negative interaction cannot be seen clearly, so change it to another color.Fig. 4b: The parameter of network topology do not need to be abbreviated, such as positive interaction and edge number.Fig. Remove underline in microbial functions.Reviewer #2 (Comments for the Author): -legume mixtures maintain forage biomass under microbial diversity loss via gathering Pseudomonas in root zone soil Authors: Yu Liu 1 , Wei Yan 1 , Tongyao Yang, Yining An, Xiaomeng Li, Hang Gao, Ziheng Peng, Gehong Wei * , Shuo Jiao * Reviewer #1: abbreviation.There is no unit in Biomass.(Fig.1ab).
21. Before showing the ab sub-legend, one sentence as the title of Fig.2need be illustrated.(Fig.2).revised version.3.It would be great if you could provide the similarity between these two sequences (i.e.ASV53 and A1501).I would suggest enlarging the text in Fig.3cas it looks not very clear..Response: Done.As the sequence information for ASV53 is V4-V5 region derived from high throughput 16S rRNA sequencing data, I selected a segment of the 16S rRNA from the A1501 whole genome sequence and aligned it with ASV53.The results are as follows.The similarity between ASV53 and A1501 was 98.12%.The text in Fig.3cwas enlarged in the revised version.
4. "soil microbial communities" to "the diversity of soil microbial communities".(Line 360).Response: Done.Modify the sentence to "The gradient of the diversity of soil microbial communities was constructed using the dilution gradient method of bacterial suspension." 5. Please add more detailed information on the PCR amplification.(Line 407-409).

6 .
Please add the accession number for raw sequence data.(Line 496-498).Response: Done.The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (70) in the National Genomics Data Center (71), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences, under BioProject accession no.PRJCA018459 and are publicly accessible at https://ngdc.cncb.ac.cn/gsa.-23R1 (Grass-legume mixtures maintain forage biomass under microbial diversity loss via gathering Pseudomonas in root zone soil) Dear Dr. Shuo Jiao: Thank you for submitting your paper to mSystems.